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This is to certify that the
dissertation entitled

ROLE FOR RECEPTOR ACTIVITY MODIFYING PROTEINS
IN THE TRAFFICKING OF THE ADRENOMEDULLIN
RECEPTOR

presented by
Jennifer Melinda Bomberger

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of the requirements for the

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ROLE FOR RECEPTOR ACTIVITY-MODIFYING PROTEINS
(RAMPs) IN THE TRAFFICKING OF THE
ADRENOMEDULLIN RECEPTOR

By

Jennifer Melinda Bomberger

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Physiology

2005

ABSTRACT

ROLE FOR RECEPTOR ACTIVITY MODIFYING PROTEINS IN THE
TRAFFICKING OF THE ADRENOMEDULLIN RECEPTOR

By
Jennifer Melinda Bomberger

Adrenomedullin (AM) is a vasodilatory peptide with effects in numerous physiological
systems. AM exerts its effects through binding to a heterotrimeric G-protein-coupled receptor
complex (calcitonin receptor-like receptor (CL-R) and single-transmembrane accessory protein,
receptor activity modifying protein (RAMP)). CL-R is unique in that it requires association with
the accessory protein, RAMP, for efﬁcient receptor expression at the plasma membrane and for
determination of receptor phenotype. A heterodimer of CL-R and RAMP] yields a calcitonin
gene-related peptide (CGRPl) receptor, whereas complexing of CL-R with the RAMP2 or
RAMP3 isoforms characterizes a fully functional AM receptor (AMI and AM2 receptors,
respectively). Curiously, although RAMP expression differs from tissue to tissue, the receptor
complex is coupled to Go.s in all systems studied to date, regardless of the RAMP isoform
associated or ligand stimulation (AM or CGRP). Thus, it is the overall hypothesis of this thesis
that although RAMP isoforms do not regulate the type of G-protein coupled to the receptor
complex, the isoform of RAMP associated with CL-R does regulate the life cycle of the receptor
complex.

Thus, this thesis focuses on the role of the RAMPs in regulation of the desensitization,
internalization, recycling and degradation of the CL-R/RAMP receptor complex. In particular,
this study focuses on the differential trafﬁcking of the AMI and AM2 receptor subtypes by
RAMP2 and RAMPB, respectively. Renal cell culture systems, both heterologous expression and
native, are the model systems utilized to study the differential receptor trafﬁcking in this project.

Using mutagenesis approaches, data from this investigation demonstrates that association

of CL-R with RAMP2 or RAMP3 isoforms could result in differential receptor phosphorylation

patterns with prolonged agonist stimulation. The Serine 421 residue on CL-R is critical to the
desensitization and internalization of the CL-R/RAMPZ heterodimer, while Threonine 423 on
CL-R is required for efﬁcient CL-R/RAMP3 receptor complex desensitization and
internalization. While Serine 421 is a putative phosphorylation site for PKA, Threonine 423 is a
putative phosphorylation site for PKC. Selective inhibitors against these kinases also inhibited
CL-R/RAMPZ (by H-89) and CL-R/RAMP3 (by Ro 32-0432) desensitization and internalization.

Differences were also observed in the interaction of AM receptor subtypes with NaVI-F
Exchanger Regulatory Factor-l (NHERF-l). Upon prolonged AM exposure, NHERF-l was
found to inhibit the internalization, but not desensitization, of the CL—R/RAMP3 receptor
complex. The effect of NHERF-l was speciﬁc for the AM2 receptor. This inhibition of
internalization was dependent on PDZ interaction 'of RAMP3 with NHERF-l and NHERF-1
interaction with MERM proteins in the actin cytoskeleton. Finally, a model for differential post-
endocytic targeting of the AM receptor subtypes was proposed from studies in HEK 293, Rat2
ﬁbroblast, and rat mesangial cells. Through adenylate cyclase and CAMP accumulation assays,
whole-cell competition binding, and immunofluorescence microscopy experiments it was
demonstrated that RAMP3 interacts with NSF via a PDZ recognition sequence at the C-terminus
of RAMP3. This was an effect speciﬁc for the CL-R/RAMP3 receptor complex. This RAMP3-
NSF interaction is proposed to target the AM2 receptor complex for a recycling pathway after
agonist-induced internalization, whereas expression of the AMI receptor complex, unable to
interact with NSF, will be targeted for degradation after agonist-induced endocytosis.

These ﬁndings report a novel function for the RAMP3 in post-endocytic receptor
trafﬁcking and provide the ﬁrst difference between the RAMP2 and RAMP3 isoforms in the
trafﬁcking of the AMlR and AMZR. In addition, this data demonstrates a functional difference
between the AMlR (CL-R+RAMP2) and AMZR (CL-R+RAMP3) receptor complexes, in spite

of very similar second messenger systems and the physiological responses thus far identiﬁed.

T 0 Mike and my parents,
for your unwavering support and encouragement,

I am eternally grateful.

iv

ACKNOWLEDGEMENTS

Successful completion of this dissertation is the result of the combined efforts of
many individuals. I would like to express my gratitude to my graduate committee
members: Drs. Nambi Aiyar, Laura McCabe, Kathleen Gallo, Walter Esselman, and
William Spielman. Their continued guidance and assistance during my graduate
education and thesis research proved indispensable. I would like to thank Drs. McCabe,
Gallo, and Esselman for their time dedicated to the review and direction of my thesis
research project. I am particularly indebted to Dr. Aiyar for his training and assistance in
establishing the radioligand binding methodology in our laboratory. I would especially
like to thank Dr. William Spielman, my major advisor, for his continual encouragement
and mentoring during my training.

I would also like to extend a special thanks to my friends and colleagues in the
Spielman lab and the Department of Physiology, particularly Dr. Narayanan
Parameswaran, Dr. Wojciech Nowak, and Carol Hall. I am especially thankful to Nara
for the endless support, critical analysis, and scientiﬁc guidance imparted to me.

Finally, I am very appreciative of the ofﬁce and computer support staff of the
Department of Physiology for the friendly interactions and continual assistance they
provided to me. In particular, I would like to sincerely thank Michelle Duchene for

always taking the time to lend a helping hand.

TABLE OF CONTENTS

List of Tables ........................................................................................ viii
List of Figures ......................................................................................... ix
Key to Abbreviations ............................................................................... xii
1. Introduction ......................................................................................... l
2. Literature Review ................................................................................... 3
2.1. Adrenomedullin....................................................... ................... 3
2.1.1. Adrenomedullin gene and protein structure .............................. 3
2.1.2. Location of adrenomedullin gene and protein expression. . . . . . . . ......4
2.1.3. Biological actions of adrenomedullin .................................... 5
2.1.4. Adrenomedullin expression in pathophysiological states ............. 9
2.1.5. Effects of adrenomedullin gene alteration and adrenomedullin
gene delivery .............................................................. 12
2.2. Adrenomedullin Receptor Complex ................................................ 13
2.2.1. Discovery of adrenomedullin receptor complex ....................... 13
2.2.2. RAMP gene and protein structure ....................................... 17
2.2.3. Tissue and cell speciﬁc RAMP expression ............................. 19
2.2.4. Mechanism of RAMP-receptor interaction ............................. 21
2.2.5. Additional RAMP-interacting receptors ................................ 24
2.2.6. Regulation of RAMP gene expression .................................. 26
2.2.7. Receptor Component Protein (RCP) .................................... 29
2.3. GPCR Life-cycle Regulation ........................................................ 30
2.3.1. GPCR life-cycle ............................................................ 30
2.3.2. Regulation of receptor desensitization .................................. 33
2.3.3. Regulation of receptor intemalization/endocytosis ................... 35
2.3.4. Regulation of receptor recycling vs. degradation ..................... 38
2.3.5. Protein-protein interaction domains/motifs ............................ 41
2.3.6. Na+/H+ Exchanger Regulatory Factor-1 (NHERF-l) ................ 47
2.3.7. N-ethylmaleimide Sensitive Factor (NSF) .............................. 49
3. Characterization of AM-stimulated desensitization response in transfected HEK 293
cells .......................................................................................... 52
3. l . Introduction ............................................................................ 52
3.2. Materials and Methods ............................................................... 53
3.3. Results/ Discussion ................................................................... 57

vi

4. Differential regulation of adrenomedullin receptor desensitization and

internalization by receptor activity-modifying proteins .............................. 68
4.1 . Introduction ............................................................................ 68
4.2. Materials and Methods ............................................................... 70
4.3. Results .................................................................................. 76
4.4. Discussion ............................................................................ 109
5. RAMP isoform-speciﬁc regulation of adrenomedullin receptor trafﬁcking
by NHERF-l .................................................................................. l 13
5. 1 . Introduction ........................................................................... 1 13
5.2. Materials and Methods .............................................................. 115
5.3. Results ................................................................................. 122
5.3.1. Role of NHERF-l in Intemalization of the CL-R/RAMP
complex .................................................................. 122
5.3.2. RAMP isoform-speciﬁc regulation of CL-R/RAMP receptor
complex trafﬁcking ..................................................... 123
5.3.3. Role of PDZ interactions in trafﬁcking of the CL-R/RAMP3
complex .................................................................. 124
5.3.4. Mechanism of inhibition of CL-R/RAMP3 internalization by
NHERF-l ....................................................... 127
5.3.5. NHERF’s role in receptor trafﬁcking in primary human proximal
tubule epithelial cells ................................................... 128
5.4. Discussion ............................................................................ 156
6. Novel function for receptor activity-modifying proteins in post-endocytic
receptor trafﬁcking ........................................................................ 160
6. 1. Introduction ........................................................................... 160
6.2. Materials and Methods .............................................................. 162
6.3. Results ................................................................................. 169

6.3.1. Role of NSF in Resensitization of the CL-R/RAMP complex. . ...169
6.3.2. RAMP isoform-speciﬁc regulation of CL—R/RAMP receptor

complex trafﬁcking ..................................................... 172
6.3.3. Role of PDZ interactions in trafﬁcking of the CL—R/RAMP3

complex .................................................................. 172

6.3.4. RAMP3 and NSF regulation of receptor trafﬁcking in unaltered
cell lines .................................................................. 175
6.4. Discussion ............................................................................ 201
7. Summary and Conclusions ................................................................... 205
7.1. Speciﬁc aims, major hypotheses, and results of the study ..................... 205
7.2. Limitations of the study ............................................................ 208
7.3. Positive outcomes of the study .................................................... 209
References .......................................................................................... 21 1

vii

LIST OF TABLES

Table 1. Biological actions of adrenomedullin .................................................. 8
Table II. RAMP isoform and CL-R receptor expression in renal cell lines ............... 21

Table III. Regulation of expression of RAMP isoforms and CL-R in various
pathophysiologies and animal models .......................................... 28

Table IV. Terminology for the GPCR life-cycle ............................................... 33

Table V. Binding properties for homologous competition binding of rAM on intact
HEK-293T cells expressing CL-R-GF P and RAMP3 +/- NSF or
NHERFI ........................................................................... 67

viii

 

LIST OF FIGURES

Figure l. Depiction of GPCR life-cycle ......................................................... 32
Figure 2. Representative desensitization experiments of AM receptors in HEK 293
Cells ................................................................................. 59
Figure 3. Desensitization dose response of AM receptors in HEK 293 cells ............... 61
Figure 4. Desensitization time course for AM receptors in HEK 293 cells ................ 63
Figure 5. Competition curves for rAM on intact HEK- 293T cells, expressing CL- R
and RAMP3, with [”51] rAM .................................................... 65
Figure 6. Scatchard plot for homologous competition binding of rAM on intact HEK
293T cells, expressing CL—R and RAMP3, with [”51] rAM ................. 66
Figure 7. Desensitization and internalization of the AMI and AM2 receptor in
HEK 293 cells ...................................................................... 84
Figure 8. Illustration of cluster mutations of CL-R to investigate role of
phosphorylation on AM receptor desensitization and internalization. . ....86
Figure 9. Role of phosphorylation on AM] and AM2 receptor desensitization and
internalization (cluster mutations) .............................................. 87
Figure 10. Role of phosphorylation on AM] receptor desensitization and
internalization (point mutations) ................................................ 91
Figure l 1. Role of phosphorylation on AM2 receptor desensitization and
internalization (point mutations) ................................................ 94
Figure 12. Role of phosphorylation of residues in third intracellular loop of CL-R on
AMI and AM2 receptor desensitization ....................................... 97
Figure 13. Role of phosphorylation of RAMPs on AM] and AM2 receptor
desensitization and internalization .............................................. 99
Figure 14. Role of kinases in AMI receptor desensitization and internalization. . . . . 103
Figure 15. Role of kinases in AM2 receptor desensitization and internalization. . . . . 105
Figure 16. Role of kinases in AMI and AM2 receptor desensitization and

internalization ..................................................................... 107

ix

Figure 17.

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.

Figure 24.

Figure 25

Figure 26.

Figure 27.

Figure 28.

Figure 29.

Figure 30.

Figure 31.

Figure 32.

Figure 33

The role of NHERF-l in the trafﬁcking of the AM2R (CL-R+RAMP3) in

HEK 293 cells .................................................................... 131
Localization of CL-R, RAMP3, and NHERF-1 in HEK 293 cells .....l34
The role of NHERF-l in post-endocytic sorting of CGRPI (CL-R+R1)

and AM] receptor ............................................................... 135
The effect of RAMP3 PDZ motif and NHERF-1 PDZ domain mutations

on the trafﬁcking of the AM2R (CL-R+RAMP3) ........................... 139
Localization of CL-R, RAMP3A145-8, and NHERF -1 in HEK 293 cells

during an internalization experiment .......................................... 143
Interaction studies of RAMP3 with NHERF -1 .................................. 144
Mechanism of NHERF-l inhibition of internalization of CL-R/RAMP3

receptor complex ................................................................. 145
Trafﬁcking of AM—R in human proximal tubule cells (hPTE cells) .......... 149

. Effect of RAMP3 and NHERF-1 RNA interference on internalization

of AM2-receptor in human proximal tubule cells ........................... 151

Mechanism of NHERF -1 inhibition of internalization of CL-R/RAMP3

receptor complex in hPTE cells ................................................ 154
Trafﬁcking of AM-R in human proximal tubule cells (hPTE cells) .......... 155
Effect of N-ethylmaleimide, an NSF inhibitor, on resensitization of

CAMP accumulation in rat mesangial cells (RMCs) ........................ 178
The sorting of the AM2R (CL-R+RAMP3) in HEK 293 cells ................ 179

The role of NSF in the sorting of the AM2R (CL-R+RAMP3) in
HEK 293 cells .................................................................... 181

Localization of CL-R, RAMP3, and NSF in HEK 293 cells during
a recycling experiment .......................................................... 183

The role of NSF in post-endocytic sorting of CGRPl (CL-R+Rl)
and AMI receptor ............................................................... 185

. The effect of RAMP3 PDZ motif deletion on the trafﬁcking of the

AM2R (CL-R+RAMP3) ........................................................ 189

Figure 34.

Figure 35.

Figure 36.

Figure 37.

Figure 38.

Figure 39.

Localization of CL-R, RAMP3A145-8, and NSF in HEK 293 cells .......... 191

Effect of PDZ motif substitution on the C-terminus of RAMP2 on
recyclingresensitization of CL-R/RAMP complex ......................... 192

Effect of RAMP3 PDZ motif point mutations on the
recycling/resensitization of the CL-R/RAMP3 complex in the

presence of NSF .................................................................. 194
Interaction studies of RAMP3 with NSF .......................................... 196
Effect of RAMP3 RNA interference on resensitization of AM2-R in rat

mesangial cells ................................................................... 197
AM-R trafﬁcking in Rat2 ﬁbroblast cells ........................................ 200

xi

HEK 293
HIF
HRE

KEY TO ABBREVIATIONS

Adrenomedullin

Adrenomedullin receptor (composed of CL-R+RAMP2)*
Adrenomedullin receptor (composed of CL-R+RAMP3)*
a-amino-3-hydroxy—5-methyl-isoxazolepropionate
Adrenomedullin receptor (generic)

Amylin

Amylin receptor (composed of CTR+RAMP1)*
Amylin receptor (composed of CTR+RAMP3)*
Angiotensin II

Arginine-vasopressin

Bz-adrenergic receptor

Adenosine 3’,5’ cyclic monophosphate

Calcitonin gene-related peptide

CGRP receptor (composed of CL-R+RAMP1)*
Cystic ﬁbrosis transmembrane conductance regulator
Cycloheximide

Calcitonin receptor-like receptor *

Calcitonin

Calcitonin receptor

Cytochalasin D

Deoxycorticosterone acetate

8-opioid receptor

Ezrin/moesin/radixin domain

Endothelin- l

Drosophila enabled/vasodilator-stimulated phosphoprotein homology 1
Glyceraldehyde-3-phosphate dehydrogenase

G protein accessory sorting protein

Glomerular ﬁltration rate

G protein-coupled receptor

GPCR-regulated kinase

Human aortic endothelial cells

Human embryonic kidney 293 cells

Hypoxia inducible factor

Hypoxia response element

 

* Nomenclature recommended by International Union of Pharmacology (IUPHAR) XXXII (as
described by Poyner et al. Pharmacol Rev 54: 233-246, 2002.) This naming system extends to all
of the established receptors for the calcitonin family of peptides.

xii

HUVEC
KOR
L-NAME
LPS

LV

MAPK
NEM
NHE
NHERF
NSF
NO
PAMP
PDGF
PDGF-R
sz
PG

PI
P13K
PKA
PKC
PLCB
PP2A
PSD-95
PTH
PTH-R
Q-PCR
RAEC
RAMP
RBF
RCP
RMC
RTK
RT-PCR
SH2/3
SHR
SNAP
SNARE
UTRS
VSMC

Human umbilical vein cells

tc-opioid receptor

Nco-nitro-L-arginine methyl ester
Lipopolysaccharide

Left ventricle

Mean arterial pressure

Mitogen-activated protein kinase
N-ethylmaleimide

Na+/H+ exchanger

Na+/H+ exchanger regulatory factor
N-ethylmaleimide sensitive factor

Nitric oxide

Proadrenomedullin N-tenninal 20 peptide
Platelet-derived grth factor

PDGF receptor

PSD-95/Discs Large/Zona occludens-l
Prostaglandin

Phosphotidyl inositol

Phosphotidyl inositol 3-kinase

Protein kinase A (CAMP protein kinase)
Protein kinase C

Phospholipase CB

Protein phosphatase 2A

Post-synaptic density-95 protein
Parathyroid hormone

Parathyroid hormone receptor
Quantitative-polymerase chain reaction
Rabbit aortic endothelial cells

Receptor activity-modifying protein
Renal blood ﬂow

Receptor component protein

Rat mesangial cells

Receptor tyrosine kinase

Reverse transcriptase- polymerase Chain reaction
Src Homology domain 2/3
Spontaneously hypertensive rat

Soluble N-ethylmaleimide-sensitive factor attachment protein
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor
Untranslated regions

Vascular smooth muscle cells

xiii

1. Introduction

Recent discovery of receptor activity modifying proteins (RAMPS) has enhanced
our understanding of the mechanisms known to regulate the signaling of the G protein-
Coupled receptor (GPCR) superfamily. RAMPS (1-3) have been demonstrated to
associate with the calcitonin (CTR) and calcitonin receptor-like (CL-R) receptors and
differentially regulate the ligand selectivity of the receptors. Although RAMPS enable
differential receptor phenotypes, the signaling elicited through receptor activation is quite
similar, namely Guts activation to activation adenylate cyclase enzymes, leading to CAMP
accumulation and protein kinase A activation. Similar biological responses are even
seen, regardless of ligand stimulation. For this reason, it has been hypothesized that the
RAMP isoforms have additional regulatory roles, thus far unidentiﬁed. This thesis
research investigates the role of RAMPS in the regulation of the CL-R/RAMP receptor
complex after agonist exposure and receptor activation, in particular, during the processes
of desensitization, internalization, and receptor trafﬁcking from the endosomes.

This brief introductory Chapter is followed by a comprehensive literature review
in the second chapter. The literature review provides an in-depth discussion of the past

and current scientiﬁc literature surrounding the topic of this thesis project, including the
biology of adrenomedullin, the Characterization of the CL-R/RAMP receptor complex,

the protein-protein interactions involved in GPCR Signaling, and the regulation of the
GPCR life-cycle. Chapters three through ﬁve are organized according to the proposed
Speciﬁc aims of this study, and each consist of an introduction, experimental data, and
Speciﬁc discussion relevant to the individual aim. Chapter three investigates if

phosphorylation of the receptor complex regulates the process of agonist-stimulated

 

desensitization and internalization of the receptor complex. An emphasis is placed on
whether the desensitization and internalization are differentially regulated by RAMP2 vs.
RAMP3 in complex with the receptor. The fourth Chapter examines protein-protein
interactions that regulate the internalization of the CL-R/RAMP receptor complex. The
role of NHERF-l in regulation of the CL-R/RAMP complex internalization and the
mechanism for this regulation are investigated. The ﬁfth chapter describes protein-
protein interactions that regulate the trafﬁcking of the CL-R/RAMP complex after
agonist-stimulated receptor endocytosis. This Chapter illustrates differential receptor
complex trafﬁcking by the RAMP2 and RAMP3 isoforms, suggesting a mechanism for
the differential trafﬁcking of the CL-R/RAMP complex observed in different cell lines.
A model for RAMP expression determining receptor fate after agonist-induced
internalization is proposed. Concluding this thesis, chapter six, offers a list of the major
hypotheses tested and the experimental results obtained in each study. Also discussed are
the limitations of this study and the positive outcomes of the thesis work, focusing on the

contributions of these ﬁndings to the ﬁeld of receptor biology and future directions.

2. LITERATURE REVIEW:

2.1. Adrenomedullin.

2.1.1. Gene and protein structure.

Adrenomedullin (AM) was initially isolated and Characterized by Kitamura and
colleagues from a human pheochromocytoma [1]. It was found to have the ability to
increase cyclic AMP levels in rat platelets and exerted strong hypotensive effects through
vasodilatory activity in the resistance vessels. AM levels were also measured in the
circulation with radioimmunoassays. Later, the same laboratory published the sequence
of the AM gene [2]. The AM gene is located in a single locus of Chromosome 11, is
comprised of 4 exons and 3 introns, and is ﬂanked at the 5’ end by RNA polymerase II
responsive TATA, CAAT, and GC boxes. The AM gene also encodes binding sites for
activator protein-2 (AP-2) [3], a cyclic AMP-regulated enhancer [4], nuclear factor— KB
[5], hypoxia-inducible factor-l (HIF -1) [6], hypoxia response elements (HRES) [7], and a
binding site for steroidogenic factor-1 (SF-1) [8]. This multitude of binding sites for
regulatory factors indicates a complex participation of the factors in the regulation of the
AM gene. The AM gene encodes a 185 amino acid precursor protein termed
preproadrenomedullin (preproAM), which is processed to yield the AM polypeptide and
another bioactive molecule, proadrenomedullin N-terminal 20 peptide (PAMP) [9].

AM is a member of the CGRP family of peptides because of its structural
homology to calcitonin gene-related peptide (CGRP), a potent vasodilator in the central
nervous system and peripherally acting neurotransmitter. AM has low sequence

homology with CGRP, but strong structural homology. The 52 amino acid AM peptide

shares an N-terminal ring structure, formed by one intramolecular disulﬁde bond
(between residues 16 and 21), and an amidated carboxy-terminal end with the additional
members of the CGRP family of peptides: calcitonin (CT), CGRP, amylin (AMY), and
newly discovered interrnedin [10, 11]. The structural integrity of the members of this
family of peptides is crucial for their biological activity [12, 13]. For example, the
carboxy-tenninal portion of the peptides, lacking the N-terminal ring structure, serves as
selective peptidyl competitive antagonists to the respective ﬁtll-length counterparts.
Peptide fragment AM22-52 serves as a competitive blocker for AM, while a-CGRP3-37 and
[3- CGRP3-37 are receptor blockers for CGRP receptors [13-15]. Given that these
antagonists are peptide fragments and the limitations peptide inhibitors can impose with
experimental manipulations, much effort is being placed in the development of non-
peptide, selective antagonists. Thus far, BIBN4096BS, WO98/11128 (Compound 1), and
SB-273779 have been Characterized as highly selective, non-peptide antagonists for
subtypes of the CGRP receptor [16-18]. Similar antagonists have yet to be developed for

the AM receptor.

2.1.2. Location of adrenomedullin gene and protein expression

In the current literature, AM expression has been established to be ubiquitous in a
wide diversity of tissues. Demonstrated with high-sensitivity radioimmunoassays and/or
by immunohistochemical studies, AM expression has been reported in the adrenal
medulla, heart, aorta, kidney, lung, brain, pancreas, skin, and additional tissues [1, 19-
24]. AM protein and/or mRNA expression has been detected in a multitude of cell types,

as well. Some examples are cardiac myocytes [6], vascular smooth muscle cells [25, 26],

endothelial cells [27], renal mesangial cells [28-30], renal proximal, distal, and collecting
tubular cells [31-33], pulmonary cells [34], and various human tumor cell lines [7, 35-

37]. Expression of AM in such diverse tissues suggests the multifunctional role for AM.

2.1.3. Biological actions of adrenomedullin

To date, the best-described biological actions of adrenomedullin are in the
cardiovascular system. Most well-Characterized is surely the hypotensive effect of AM,
which is both very potent and long-lasting. However, AM’S additional biological actions
are very diverse, acting in almost any system tested, and therefore extend beyond the
scope of discussion in this literature review. I will focus on the actions of AM in the
cardiovascular and renal systems, the systems of particular interest to the laboratory of
my thesis research. Following this discussion can be found a chart with a brief
description of AM’S wide assortment of actions in additional systems (Tablel).

In the vascular beds of humans, rats, cats, dogs, and sheep, AM studies have
Shown a relaxation of the resistance vessels to attain a long-term drop in the mean arterial
blood pressure (MAP) [38-43]. In most instances, this vasodilation is capable of
attenuation by L-NAME, suggesting an involvement of a nitric oxide (N O)-dependent
pathway for the effect [44-46]. Some variations have been reported in cases of different
species or localization of AM stimulation [47-49]. In addition to decreasing blood
pressure, AM administration will increase heart rate. NO-dependent mechanisms have
also been found to be utilized by AM to dilate the renal vasculature and to mediate
diuretic and natriuretic responses in the kidney [50]. In terms of the microvasculature,

AM’S effects are very similar to those in the larger resistance vessels, with AM acting on

CGRP or AM receptors to elicit relaxation via either activation of adenylate cyclase,
release of NO, or activation of potassium channels.

In addition to its potent vasomotor activity, AM plays critical roles in cell growth
regulation. Its effects on cell growth and apoptosis depend predominantly on the cell
type and the experimental conditions under examination. For example, AM has been
well-Characterized to promote proliferation in a number of human tumor cell lines, as
well as Swiss 3T3 ﬁbroblasts, human oral keratinocytes, rat gastric epithelial cells, and
human retinal pigment epithelial cells [51-55]. Conversely, in several cardiorenal cell
types AM has been reported to be anti-proliferative. These cell types include rat vascular
smooth muscle cells, hypertrophy in cultured myocytes and ﬁbroblasts, rat mesangial
cells, and human proximal tubule epithelial cells [56-59]. In parallel to reports for the
vascular cell types, AM is expressed by a wide variety of renal cells where it is capable of
yielding numerous biological effects. Based on reverse transcriptase- polymerase chain
reaction (RT-PCR) studies in the rat nephron segments, Owada et al. have detected AM
expression in the glomerulus, cortical collecting duct, outer medullary collecting duct,
and intermedullary collecting duct, but not in the proximal convoluted tubule or
medullary thick ascending limb [30]. In opposition, a different study, reported by Jensen
et al., describes mRNA expression for AM and AM-R colocalized in renal vessels,
glomeruli, and inner medullary collecting ducts. AM mRNA was also detected in
proximal tubules, whereas AM-R mRNA was found in distal convoluted tubules [60].
The previous study also detected high levels of expression of AM in the rat mesangial

cells, as well.

Based on the wide expression patterns of AM in the kidney, it is not surprising
that AM exerts profound effects on renal function. Studies by Ebara and colleagues
demonstrated that intrarenal infusion of AM at concentrations suboptimal for heart rate
and blood pressure alterations resulted in an increase in renal blood ﬂow (RBF), total
urine output, and urinary sodium excretion. At higher concentrations of AM, MAP
shows marked decreases, while GFR is now increased, vasodilation of efferent and
afferent arterioles occurs, and distal tubular sodium reabsorption is further decreased,
further increasing sodium excretion [30, 32, 45, 61]. Later studies established these AM-
stimulated renal vasodilatory and natriuretic effects were NO-dependent [45, 46, 62]. In
contrast to these studies and accepted AM actions in the kidney, Leclerc et al. described a
CAMP-dependent sodium-sparing capacity of AM, by a mechanism of regulating the

Na+/H+ exchangers of the distal tubules [63].

Generalized Biological Actions of AM:

Vascular Effects:
> AM administration results in sustained hypotension via NO,
CAMP, and/or PG generation (depends on vascular bed) [13, 38, 44, 64]
> Positive ionotrophic and Chronotrophic effects on coronary artery [65-67]
dilation

Renal Effects: .
> Intrarenal infusion of AM produces increases in RBF, GFR, Na+ [45, 68, 69]
excretion, and urine ﬂow

> Inhibits PDGF-induced (MAPK-dependent) mesangial cell [70, 71]
proliferation
> Stimulates intrarenal renin release [72]

Effects in Bone:
> Promotes osteoblast grth and protein synthesis

(yielding increased area of mineralized bone) [73, 74]
Effects in Lung:
> Inhaled AM reduces histamine-, acetylcholine-, and
antigen-induced bronchoconstriction [12, 64]

Effects in Endocrine System:

> Inhibits ACTH release [75, 76]
> Inhibits insulin secretion (increases blood glucose) [23]
> Inhibits aldosterone production/secretion [77]

Effects in Central Nervous System:
> Central stimulation of the sympathetic nervous system

(increases blood pressure and heart rate) [78, 79]
> AM administration inhibits thirst drive and salt appetite,
attenuates AVP release, and is pro-anorexic [80-82]

Table 1. Biological actions of adrenomedullin.

2.1.4. Adrenomedullin expression in pathophysiological states

Because of AM’S diverse biological actions, acting in almost any system tested, it
is not surprising that AM’s role in pathophysiologies is quite extensive and extends
beyond the scope of discussion in this literature review. I will focus on the involvement
of AM in pathologies of the cardiovascular and renal systems, the systems of particular
interest to the laboratory of my thesis research.
Hypertension

Plasma levels of AM have been demonstrated to be elevated in patients with
primary arterial hypertension, with even higher levels in patients with complications of
hypertension, such as left ventricular hypertrophy and nephrosclerosis [83]. AM gene
expression is increased in the Dahl salt-sensitive rats fed a high Na+ diet [84], but the
most dramatic elevation of AM gene expression is in the spontaneously hypertensive rats
(SHR) treated with deoxycorticosterone acetate (DOCA) and fed a high Na+ diet
(DOCA-salt SHR) and the stroke-prone SHR [85-87]. These two animal models also
Show increased CL-R and RAMP2/3 gene expression. It has been suggested that the up-
regulation of the cardiac AM system in hypertension is a protective mechanism, acting to
decrease myocardial overload with the vasodilatory and natriuretic properties of AM, as
well as limiting further myocardial hypertrophy and remodeling due to the proliferative
regulation by AM.
Heart Failure

AM plasma concentration is increased in patients with congestive heart failure,
and this elevation is correlative with disease severity [88, 89]. AM concentration in

myocardial tissue obtained from heart transplant recipients with severe heart failure is

higher than in donors, suggesting that increased plasma AM levels in heart failure
patients is a result of increased myocardial production of AM [90]. In addition, AM, CL-
R, RAMP2, and RAMP3 gene expression is markedly up-regulated in different animal
models of heart failure (induced by volume or pressure overload) [91-95]. AM up-
regulation may be acting in a protective manner, in this situation, to increase myocardial
contraction due to its positive ionotrophic properties, or as a compensatory mechanism to
decrease cardiac preload and afterload. Additional protective aspects of AM expression
are a decrease in myocardial remodeling, due to AM’S role in attenuating myocyte
hypertrophy, proliferation of myocardial ﬁbroblasts, and production of extracellular
matrix. Finally, AM acts in accordance with natriuretic peptides to counteract the
vasoconstriction and sodium retention by renin-angiotensin-aldosterone, endothelin, and
the sympathetic nervous system. AM infusion in heart failure patients has had beneﬁcial
effects, including an increase in cardiac output and natriuresis, reduction of peripheral
resistance, pulmonary capillary wedge pressure, left ventricular end diastolic pressure and
plasma aldosterone, and an increase in ejection fraction [96]. Some studies have
suggested, though, that the vasodilatory and natriuretic properties of AM are impaired in
heart failure, with no mechanism for the abated function apparent at the present time [41,
97].
Atheroscierosﬁ

AM levels in the plasma of patients with chronic ischemic stroke are elevated and
correlate with the extent of carotid artery atherosclerosis [98]. In addition, AM has been
detected in macrophages found within atherosclerotic plaques [99]. Due to AM’s

inhibitory effect on migration and proliferation of vascular smooth muscle cells,

10

my: .

inhibition of endothelial cell apoptosis, and anti-inﬂammatory activity, it could be
hypothesized that AM could have beneﬁcial effects in atherosclerosis[100]. AM’S role in
atherosclerosis, in Classic models of the disease, has yet to be tested. Data does exist that
indirectly suggests AM may have atheroprotective roles, including enhanced intimal
thickening observed following arterial injury in AM” mice and attenuation of restenosis
with AM gene overexpression following balloon-induced or cuff-induced arterial injury
in rodents [101-103].
Myocardial Infarction

During the acute phase of myocardial infarction, plasma AM levels have been
demonstrated to be increased, reaching their maximum after 2-3 days and returning to
baseline after a duration of about 3 weeks [104]. Increased AM levels are associated with
hemodynamic impairment. Evidence for this is a positive correlation between plasma
AM and central venous pressure, left ventricular and diastolic pressure, and pulmonary
capillary wedge pressure, and negative correlations of plasma AM with ventricular
ejection fraction [105]. AM, CL-R, and RAMP2 expression increases in ischemic and
non-ischemic myocardium following coronary artery ligation in the rat [106, 107]. AM
gene expression is also shown to be increased with hypoxia, due in part, to an oxidative
Stress mechanism [108]. AM production by myocytes may also be stimulated in the
infracted region by mechanical stretch, angiotensin II, and proinﬂammatory cytokines.
Due to AM’S effects of local coronary vasodilation and reduction of oxidative stress-
induced myocardial cell injury, it is believed that AM is playing a protective role in
myocardial ischemia [109, 110]. Supporting this concept, AM overexpression before

ischemia/reperfusion injury will decrease the superoxide anion generation in the

11

hypoperfused myocardium, decrease the infart area, inhibit apoptosis of cardiomyocytes,
and reduce the number of ventricular ﬁbrillation incidents [111]. Additionally, when AM
is infused at suboptimal levels for effect on blood pressure, it retains the ability to inhibit
cardiac remodeling following experimental myocardial infarction in the rat [112].
Renal Diseases

In patients with Chronic renal failure, plasma AM levels gradually increase. This
is thought to be due to decreased peptide Clearance, but an elevation due to chronic
volume overload cannot be disqualiﬁed. In various types of glomerulonephritis, while
plasma AM levels are increased, AM excretion is decreased [113, 114]. Hypoxia is
known to up-regulate AM gene expression, suggesting that AM may protect the kidney
from ischemia-reperfusion injury [115]. In fact, it has been shown that in AM +/' mice,
the AM deﬁciency aggravates histological lesions and functional impairment in
experimental ischemic acute renal failure, while AM overexpression has been shown to

be protective against these effects [116].

2.1.5. Effects of adrenomedullin gene alteration and adrenomedullin gene delivery
Two labs have produced AM knockout mice, and while some differences exist,
AM’S role in development is very evident. Both labs demonstrated that homozygosity of
the AM knockout is embryonically lethal. The ﬁrst report disrupted the AM gene so that
neither AM or PAMP were produced [117, 118]. The homozygous AM knockout
embryos showed poorly developed vitelline vessel vasculogenesis, hemorrhages,
myocardial hypertrophy, and hydrops fetalis. These mice typically died between

embryonic day 13.5 and 14.5. The heterozygous AM +/' mice are able to survive to

12

adulthood and are fertile, but they are characterized arterial hypertension due to NO
deﬁciency.

The second group to generate AM knockout mice did so by placing a stop codon
at the starting point for the AM coding sequence in the preproAM gene [119]. This
resulted in mice that did not express AM, but Show normal levels of PAMP. While the
homozygous AM mutation in these mice was also lethal (although these AM-/- mice did
not show the same placental and vascular defects or hydrops fetalis), some differences
were seen in the heterozygous knockout mice. These animals also reached adulthood and
were fertile, but in this case, the AM +/- mice showed normal blood pressure regulation.
In experimental hypertension induced with angiotensin H administration and high-salt
diet, the AM+/- mice showed similar increases in blood pressure as wild-type mice, but
did Show greater organ damage, due to greater hypertrophy of the left ventricle and
coronary arteries, and higher vascular oxidative stress. This data indicates a role for AM
in protecting against end-organ damage in Na+-induced hypertension, independent of BP
regulation. When comparing the two animal models of AM knockout, it could be
hypothesized that PAMP plays a primary role in blood pressure regulation (through NO-
dependent mechanisms), but it must be considered that both animal models were

generated using different genetic backgrounds.

2.2. Adrenomedullin Receptor Complex

2.2.1. Discovery of adrenomedullin receptor complex,
The numerous biological activities of AM (discussed previously, Table 1) had

been suggested to be mediated by a cell-surface receptor. Until recently, identiﬁcation of

13

this receptor had been quite elusive, due to conﬂicting reports in both pharmacological
inhibition and Cloning studies. While AM’S vasodilatory response was inhibited in some
tissues with the CGRP antagonist, CGRP3-37, in other tissues no effect was seen, even at
concentrations that potently inhibited the CGRP-mediated vasodilatory response [42,
120-122]. This observation led to the hypothesis that an AM-speciﬁc receptor existed.
The AM-speciﬁc vasodilation was observed, for example, in guinea pig pulmonary
artery, hypotensive effects in Long-Evans rats, control of aldosterone production in rat
adrenals, and other AM-mediated actions [123-126]. Meanwhile, other groups
demonstrated AM-stimulated responses that were sensitive to both CGRP and AM
peptidal inhibitors (CGRP3-37 and AM22-52, respectively) [127].

Unfortunately, the molecular cloning attempts of the AM receptor initially yielded
data equally confusing as the pharmacological inhibition studies. The AM receptor was
ﬁrst reported to have been Cloned from the rat lung by Kapas et al. This receptor was
capable of binding 125I-AM and eliciting an elevation in CAMP in response to AM
stimulation when transfected into COS-7 cells [128]. This receptor and a canine receptor,
identiﬁed a few months later by Kapas and Clark, RDC-l, showed considerable
homology to a previously described orphan receptor, termed L1 or GIOd [129-131]. Part
of the confusion arose when subsequent attempts by other laboratories to replicate and
further these studies, resulted in an inability of these labs to reproduce the previously
reported observations [ 132-134].

Later an alternative for the AM receptor was proposed in studies by Aiyar and
colleagues. They identiﬁed a previous orphan receptor, now termed calcitonin receptor-

like receptor (CL-R), which exhibited well-characterized CGRP1 receptor pharmacology

14

with a weak AM cross-reactivity. When this human CL—R was expressed in human
embryonic kidney (HEK 293) cells, a robust increase in CGRP-stimulated CAMP
accumulation was observed, as was Speciﬁc ”SI-CGRP binding and dose-dependent
inhibition of CAMP production by CGRP3-37 [135]. Similar results were seen for rat and
porcine CL-R [136, 137]. Propagating the classiﬁcation of CL-R as an orphan receptor,
additional labs were unable to reproduce the results of Aiyar and colleagues in different
cell lines [43, 138, 139]. CL-R was Characterized as a seven transmembrane-Spanning G-
protein-coupled receptor, but its native ligand remained unidentiﬁed. Based on structural
and amino acid homology, CL-R was Classiﬁed as a family B GPCR (Secretin family of
GPCRS). Other members of this GPCR family include receptors for secretin, gastric
inhibitory peptide, glucagon, pituitary adenylate cyclase activating hormone, vasoactive
intestinal peptide, grth hormone releasing hormone, parathyroid hormone, and
calcitonin. CL-R is comprised of 461 and 464 amino acids and shares 50% and 54%
amino acid sequence identity with the rat and human calcitonin receptors, respectively
[43, 139].

Reforming the study of this receptor system, a critical study was published in
1998 by McLatchie et al. that Clariﬁed the elusiveness of the AM and CGRP receptors,
while also providing an entirely novel form of GPCR regulation. Utilizing a Xenopus
oocyte system that expressed endogenous CGRP receptor and an exogenous CAMP-
sensitive cystic ﬁbrosis transmembrane regulator (CFTR), they systematically screened
complementary RNAs derived from the SK-N-MC (human neuroblastoma cell line
endogenously expressing CGRP receptor characteristics) cell’s DNA library [134, 140].

This screen yielded Cloning of a 148 amino acid protein, termed receptor activity

15

modifying protein-l (RAMP-l), that was capable of signiﬁcant CGRP-mediated CAMP
accumulation in the oocytes. Yet when expressed in HEK 293T, COS-7, or Swiss 3T3
cells, this protein was unable to elicit a CGRP-stimulated CAMP response. Dual
expression of CL-R and RAMP-1 was found to restore the CGRP-mediated CAMP
production in oocytes and HEK 293T cells. This presumably was the result of these cells
not expressing endogenous CL-R, so both the receptor and RAMP expression were
required for the CAMP response to be observed. Database search identiﬁed two
additional isoforms of RAMPS (RAMP-2 and RAMP-3), which together the RAMP
proteins only Show approximately 31% sequence identity. Repeating similar experiments
with RAMP-2/3 in oocytes, as performed for RAMP-1, yielded a quite surprising result.
For the ﬁrst time, a GPCR showed a differing receptor phenotype when expressed with
different accessory proteins. Namely, when CL-R was expressed with RAMP-1, a CGRP
receptor was produced, whereas when CL-R was expressed with RAMP-2 or RAMP-3
the receptor was responsive to AM (AMI receptor and AM2 receptor, respectively)
[134]. These results were conﬁrmed by a number of additional labs in human endothelial
and vascular smooth muscle cells, rat osteoblast-like UMR-106 and COS-7 cells, and
Drosophila Schneider 2 cells [141-143]. This was a revolutionizing concept for the
GPCR ﬁeld.

Notably, while RAMP2 and RAMP3 share only approximately 30% sequence
identity, they generate virtually pharmacologically and biologically identical AM
receptors when CO-expressed with CL-R in HEK 293T cells [144]. Until recently
published by our laboratory, no other laboratories detected differences in receptor

phenotype or signaling between the AMlR and AM2R. Additionally, it was presumed

16

that differences in receptor regulation must exist between the two receptor isoforms. A
report from our lab recently described the differential regulation of RAMP2 and RAMP3
expression by platelet-derived grth factor (PDGF) and lipopolysaccharide (LPS) in rat
glomerular mesangial cells [145, 146]. Moreover, RAMP expression studies in animal
disease models Show variable RAMP2 and RAMP3 mRNA expression (see section

2.2.6).

2.2.2. RAMP gene and protein structure

RAMP-1 and RAMP-2 were initially cloned from human neuroblastoma cell (SK-
N-MC) DNA library, whereas RAMP-3 was isolated from the human spleen.
Comparison of sequences of the RAMPS with the genomic map predicted their
Chromosomal location, as well as gene organization. RAMP-1 was shown to reside on
Chromosome 2, the RAMP-2 gene on chromosome 17, and RAMP-3 gene is on
chromosome 7 [134]. A scan of the human genome revealed no more sequences similar
to the RAMPS [147]. RAMP-1 and -3 share some similarities in their gene composition,
being comprised of three exons divided by large introns. The RAMP-l and -3 genes are
large in comparison to that of RAMP-2 (approximately 24 kilobases vs. 5 Kb,
respectively). RAMP-2 has four exons, but shorter introns regions. All three genes have
similar localization of the 5’UTR and the signal peptide sequence on the ﬁrst exon and
the C-terminal and transmembrane domains on the last exon of the corresponding RAMP
genes [148].

Despite the relatively low amino acid sequence identity between the RAMP

isoforms (approximately 30%), the hydrophobicity plot analysis suggested a substantial

l7

similarity of protein structure. The sequence similarity of the RAMP isoforms between
species is quite well conserved, at approximately 90% between mouse and rat. Sequence
identity between the rodent and human sequences is 70, 65, and 85% for the RAMP],
RAMP2, and RAMP3, respectively [149, 150]. RAMP-l and RAMP-3 are 148 amino
acid proteins, while RAMP2 is composed of 175 amino acids, but all isoforms are made
up of large extracellular domains, an approximately 20 amino acid transmembrane
domain, and a roughly 10 amino acid intracellular domain [134]. The RAMPS have a
molecular weight of only approximately 14-17 molecular weight. Several sequences
and/or residues are conserved over the RAMP isoforms and different species, indicating
an importance in regulatory functions or preservation of secondary structure. Included
are four cysteine residues located in the extracellular domain and two sequences localized
to the N-terminal and C-terrninal regions, respectively: DPPXX and LVVWXSK [134].
Several consensus Sites for N-glycosylation have also been identiﬁed on RAMP2 and
RAMP3. Four N-glycosylation Sites identiﬁed on human, mouse and rat RAMP3 are
conserved in the mouse RAMP2. Suggesting differential post-translational modiﬁcations
of the RAMP isoforms, RAMP] has no consensus sites for N-glycosylation [151].
Protein kinase A and C phosphorylation consensus sites also are present on the C-
terrninal intracellular domains of RAMP] and RAMP3, but not RAMP2 [150, 152]. Our
laboratory has also recently reported a function for the previously-identiﬁed PDZ
recognition motif on the extreme C-terminus of RAMP3, but not RAMP] or RAMP2,
that is responsible for protein-protein interactions important in the regulation of

trafficking of the receptor complex [153, 154]. Further investigation is Clearly required

18

to determine the functional consequences of the discussed conserved regulatory

sequences.

2.2.3. Tissue and cell Speciﬁc RAMP expression

RAMP mRNA distribution has been analyzed in several species to date, including
mouse, rat, and human, with differences existing, but general expression patterns have
been established. RAMP-l has shown predominant expression in the heart, brain,
skeletal muscle, thymus, spleen, fat, and kidney; RAMP-2 expression is abundant in the
heart, aorta, kidney, spleen, fat, and lung. RAMP-3 shows the widest distribution, but
tends to exhibit prevalent expression in the kidney, heart, brain, and lung [134, 148-150,
155,156]

In addition to tissue distribution, the RAMPS’ expression has been studied in a
variety of cell types. Differential expression of RAMP isoforms in cell types has been
reported, especially in the initial characterization of the CGRP and AM receptors, leading
to the identiﬁcation of CL-R, a previous orphan receptor capable of Signaling through
both ligands, depending on RAMP expression. Endogenous expression of RAMPS in a
particular cellular background has been well-documented to affect the functional
character of the observed receptor subtype. Endogenous expression of the RAMPS also
affects the functional receptor observed when overexpressing RAMP isoforms in a cell
type. For example, rabbit aortic endothelial cells (RAECS) endogenously express
RAMP-2, but not RAMP-l, and are selectively responsive to AM and blockade by AMzz-
52, but not CGRP3-37. While transfection of hRAMP-l into RAECs will yield a CGRP

response that is selectively inhibited by CGRP3-37, overexpression of hRAMP-l and

19

hRAMP-3 into the RAECS results in decrease of CGRP-mediated CAMP response. This
work suggests a greater afﬁnity of the rabbit CL-R for RAMP-3 as compared to RAMP-
]. However, the overexpression of hRAMP-3 does not alter the responsiveness of the
CL-R in RAECS to AM, suggesting an intraspecies preference for RAMP isoforms [157].
Variable RAMP isoform-CL receptor afﬁnity has been demonstrated in additional cell
types, namely UMR 106-06 cells and COS-7 cells [139, 142].

RAMP distribution has also been elucidated in several organ systems to date,
namely the central nervous system and the kidney. In the central nervous system, using
in situ hybridization to detect expression, RAMP] mRNA was predominantly expressed
in the cortex, caudate putamen and olfactory tubercles; RAMP2 mRNA was most
abundant in hypothalamus; and RAMP3 expression was restricted to the thalamic nuclei
[158]. Notably, in speciﬁc brain areas only a single RAMP isoform was often detected,
suggesting mutual exclusivity in expression. Of particular interest to our laboratory,
renal RAMP mRNA expression has been evaluated. RAMP-2 and RAMP-3 mRNA has
been detected in abundance in the rat kidney, showing similar expression patterns in both
the cortical and medullary parts of the kidney [159]. Using quantitative RT-PCR,
Totsune and colleagues measured the mRNA expression levels of RAMP-2, RAMP-3,
and CL-R in the kidney of normal Munich-Wistar rats. RAMP-2 expression was
26.5i1.9 mmol per mole of GAPDH and RAMP-3 mRNA levels were 7.73:0.4 mmol per
mole of GAPDH. CL-R was detected in the kidney in this study, but at signiﬁcantly
lower concentrations [160]. Our lab has characterized RAMP and CL-R mRNA
expression in several renal cell lines. Please see the chart below for RAMP isoform and

CL-R distribution (mRNA expression) in the various renal cell lines. The differential

20

expression of the RAMPS (especially RAMP-2 and RAMP-3) in the different regions of

the kidney suggests additional, yet to be identiﬁed, regulatory roles for the RAMPS.

 

 

 

 

 

CELL LINE RAMP-l RAMP-2 RAMP-3 CL—R
Human primary proximal tubule cells + + + +
Rat mesangial cells + + + +
Rat kidney ﬁbroblasts + + +
Rabbit cortical collecting duct cells + +

 

 

 

 

 

 

 

Table II. RAMP isoform and CL-R receptor expression in renal cell lines.

2.2.4. Mechanism of RAMP-receptor interaction

Several studies have now established that RAMPS initially interact with their
receptor partners in the ER/Golgi and maintain this association throughout the life-cycle
of the receptor complex. The RAMP and receptor are trafﬁcked together to through the
endocytic pathway to the recycling endosome or lysosome after agonist activation of the
receptor [153, 161-164].

The interaction of the RAMP isoform with CL-R results in modiﬁcation of the
terrnina] glycosylation of the receptor [134, 163]. RAMP-l and RAMP-2 are both known
to modulate CL-R through glycosylation, although only the RAMP-2/CL-R complex
requires glycosylation for expression of the receptor complex at the cell surface [163].
While glycosylation may determine cell surface expression for the receptor complex,

glycosylation is not required for determination of receptor phenotype by the RAMPS.

2]

 

 

 

 

Experiments in insect cells, where glycosylation state of the receptor is not altered, Show
no alteration in ligand recognition or receptor activation, as compared to similar studies
in mammalian cell lines [14]].

Evidence exists that indicates that the N-terminal, extracellular domain of the
RAMP is critical to determination of receptor phenotype, while the transmembrane
domain is important in the stabilization of the RAMP-receptor interaction [144].
Chimera studies with the RAMP-1 and RAMP-2 extracellular domains suggest that the
N-terminal regions of the RAMPS determined ligand selectivity of CL-R [165]. This
work surprisingly suggests that the extracellular domains of RAMPS are sufficient to
maintain a fully functional receptor for CGRP. Chimera of the extracellular domain of
RAMP-1 with the transmembrane and intracellular domains of PDGF-R only showed a
ten-fold decrease in potency for CGRP signaling and binding. However, the extracellular
domain alone showed a greatly diminished (approximately 4000-fold) responsiveness to
CGRP. These studies aren’t entirely consistent with data reported by Steiner et al. with
respect to the transmembrane domain of RAMP-1 [166]. Steiner and colleagues reported
that deletion of only a portion of the RAMP-1 transmembrane domain caused a dramatic
decrease in the potency of CGRP, regardless of functional trafﬁcking of the receptor to
the cell surface. Further work will be needed, possibly studying the extracellular and
transmembrane interactions of RAMP-2 and -3 as well, to determine the interactions
necessary for receptor trafﬁcking, activation, and heterodimer stability.

AS mentioned above, a role has been indicated for the transmembrane domain of
RAMPS in the stabilization of the RAMP interaction with CL-R and the calcitonin

receptor (CTR). Chimera experiments with RAMP-1 and RAMP-2 deﬁned the

22

extracellular domain as having the responsibility for receptor phenotype determination.
Because of a selective recognition of amylin by RAMP-1 or RAMP-3 complexed with
the CTR, RAMP-1 and -2 chimeras is an excellent tool to delineate the domains
responsible for agonist binding, receptor activation, and heterodimer stability. Levels of
amylin binding, induced with the wild-type RAMPS, were paralleled by the chimeras
according to the transmembrane domain/C-terminus present in the heterodimer [140].
This led to the theory that the transmembrane domain of the RAMPS was the primary
interaction site for the RAMP and receptor (at least for the CTR). This model may be
rather simplistic or receptor-speciﬁc, though, in light of additional recent studies on the
N-terrninus of RAMP-1 that have identiﬁed a stretch of aromatic residues within RAMP-
] that are probably important in the interaction between RAMP-1 and the CL-R. These
N-terrninal residues (F93, YIOO, or F101) act independently of determination of receptor
phenotype [167]. While not altering the ECSO value for the CGRP-mediated CAMP
response, mutation of any of the above aromatic residues to alanine rendered the receptor
complex unable to express at the cell surface and bind agonist (CGRP). In addition,
recent work by Sexton and colleagues, with a more diverse set of RAMP-U2 chimeras,
indicates a strong induction of amylin binding, even in the presence of the
transmembrane domain of RAMP-2 [168]. Further work is required to establish the
regions responsible for interaction between the RAMP and receptor, with differences
possibly existing for different receptor partners of the RAMPS.

Segments of all three RAMPS have been identiﬁed that are required for generation
of a functional receptor phenotype. On RAMP-2 the extracellular region from amino

acid 77 to 101, in particular residues 86-92, are critical for adrenomedullin receptor

23

phenotype when the RAMP is expressed with CL-R [169]. The equivalent region on
RAMP-3, residues 59-65, is also required for AM receptor phenotype, although no
homology between the crucial sequences of RAMP-2 and RAMP-3 exists. This suggests
that this sequence plays a structural role in the RAMPS or this region possesses an
allosteric effect on CL-R conformation. Data from RAMP-l deletion studies suggests a
role for these short sequences in the extracellular region on the proper conformation of
CL-R to bind ligand [167]. Deletion of the short N-tennina] sequence from RAMP-1,
residues 101-103, abolishes the induction of CGRP receptor phenotype by the receptor
complex, but if the residues are replaced with alanine residues, the potency of CGRP
remains unaltered.

Disulﬁde bond formation also appears critical to ligand binding and receptor
activation for the RAMPS and their heterodimeric partners (CL-R and CTR). While four
cysteine residues are conserved between all three RAMP isoforms, RAMP-1 and RAMP-
3 contain an additional 2 conserved cysteine residues [170]. Data from Flahaut et al.
suggests that all cysteine residues on RAMP-3 are crucial for ligand binding and receptor »
activation. It could be speculated that the additional disulﬁde bond formation on RAMP-
] and RAMP-3 allow for the increased afﬁnity of the RAMP-l/CL-R and RAMP-3/CL-R

(to a lesser extent) receptor complexes for CGRP.

2.2.5. Additional RAMP-interacting receptors
One of the ﬁrst questions raised after the discovery of RAMPS and their role in
trafﬁcking and ligand selectivity of CL-R was if additional receptors can interact with the

RAMPS. Given the Signiﬁcant sequence identity between the CTR and CL-R, the CTR

24

was an obvious ﬁrst Choice. Strong evidence exists to establish that RAMP association is
not required for CTR binding and activity induced by calcitonin [161, 17]]. However,
RAMP-l and RAMP-3 isoforms were found, by several independent laboratories, to
interact with the CTR to form a high afﬁnity amylin receptor. These results were
conﬁrmed in exogenous systems in COS-7 and rabbit aortic endothelial cells and with an
endogenous CTR in Chinese hamster ovary (CHO)—Kl cells [161, 172].

RAMPS have a relatively ubiquitous distribution, with at least one RAMP
expressed in most tissues examined to date. Noteworthy, this distribution extends beyond
that of the Characterized receptor partners (CL-R and CTR), suggesting a more
widespread role for RAMPS in receptor regulation [134, 148, 149]. The discovery of the
RAMPS led to an interest in the potential of the RAMPS to interact with additional
GPCRS to determine cell surface expression or receptor phenotype, especially for
receptors that had been thus far difﬁcult to Characterize. RAMPS were thought to maybe
he the missing link in pairing numerous orphan receptors with their ligands. A recent
study by Christopoulos et al. identiﬁed additional interacting receptors for the RAMPS by
screening the capacity of the receptors to trafﬁc the RAMPS to the cell surface [134, 161,
172, 173]. Screened in this study were 10 of the GPCRS of the family B of GPCRS, the
family to which CL-R and CTR belong. Of the 10 GPCRS tested, 6 of the receptors were
capable of trafﬁcking at least one of the RAMP isoforms to the cell surface [174]. In
addition to CL-R and CTR, the newly identiﬁed RAMP-interacting receptors include the
vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide (VPAC)-l
receptor, parathyroid hormone receptor (-1 and -2), and the glucagon receptor. While the

CL-R, CTR, and VPAC-l receptors interact strongly with all three RAMP isoforms, the

25

PTHIR and glucagon receptor only interact with RAMP-2, and PTH2R only interacts
with RAMP-3. The VPAC2, glucagon-like peptide (-1 and -2), and growth honnone-
releasing hormone receptors were unable to interact with any of the RAMP isoforms
[174]. This study, while suggesting a more widespread role for the RAMPS in receptor
regulation, also reveals additional speciﬁcity of RAMP-receptor interactions.

Of note, with the exception of CL-R, RAMP association with interacting
receptors is not required for Chaperoning of the receptors. The most compelling
consequence of the newly-discovered RAMP-receptor interactions is the augmentation of
VPAC] receptor-mediated signaling [174]. RAMP-2 interaction with VPACl-R does
not alter the agonist binding properties or CAMP signaling of the receptor, however
RAMP-2 association with the VPACl-R does enhance the receptor-mediated
phosphoinositol hydrolysis pathway. While no Change is seen in the EC50 for the agonist-
mediated PI hydrolysis, the maximal VPACI-mediated PI signaling (Emax) is increased.
The physiological relevance of this observation has yet to be deﬁned.

The data reported in this project suggests additional roles RAMPS may be
fulﬁlling with their interacting receptors. Beyond roles in cell surface trafﬁcking and
receptor phenotype determination, results from this study propose roles for RAMPS in

sorting of receptors after ligand activation to determine the receptor’s fate.

2.2.6. Regulation of RAMP gene expression
The Changes in RAMP gene expression have been studied under many disease
models, physiological Changes, and drug treatment. Below is a table summarizing the

ﬁndings of these studies, focusing on the cardiovascular and renal system Changes (Table

26

III adapted from review by Udawela et al.) [168]. Dramatic expression pattern
alterations are seen, indicating an important role for dynamic RAMP regulation in these
systems. These results suggest the potential for RAMP regulation as a means of
modulating some of the pathophysiological conditions associated with the RAMP-

interacting GPCRS.

27

32:25.35; - m -

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Model Tissue R1 R2 R3 CL-R AM Ref.
Exp. Exp. Exp. Exp. Exp.
Heart Failure (HF) in Atrium N/D T T N/D TT TT [94]
rats (coronary artery Ventricle N/D T N/D T TTT
ligation) Kidney N/D -- N/D -- --
Rat ischemic heart Non-ischemic N/D T N/D N/D T [ 107]
failure (left coronary left ventricle
ligation) Ischemic LV N/D T T T N/D N/D T T T
LV hyptertrophy to LV at N/D T T T T T T T T T [93]
heart failure in rat (KCl hypertrophy
Lnjection) LV at HF N/D TT T TT T T T TT T
Chronic HP in rats Atria T -- TT T -- -- [91]
(aortic banding) Ventricles T T -- T T -- --
Myocardial ischemia in Myocardium N/D T N/D N/D TTT [175]
rats (isoproterenol Aorta N/D T N/D N/D T T
induced)
Blood pressure changes PVN N/D ll N/D N/D -- [176]
in rat Nucleus of N/D ll N/D N/D --
Increase by solitary tract
phenylephrine PVN N/D -- N/D N/D I I
Decrease by Nucleus of MD TT T N/D N/D --
nitroprusside solitary tract
Hypertensive rat [85]
Salt loaded Renal cortex N/D 111 TT 1 --
Renal medulla N/D I I -- -- --
DOCA treated- salt Renal cortex N/D 1 -- -- --
loaded Renal medulla N/D 11 T T -- T T
DOCA- salt loaded LV N/D T T T T [86]
Chronic salt loading in Adrenal gland T T T -- T T T T [177]
rat Kidney -- -- n t n
SHR LV N/D T -- T T [87]
Ang 11 treatment in rat Cardiomyocytes TT -- TT -- N/D [178]
Endothelin treatment Cardiomyocytes -- l L T T [179]
Hypoxic rat Lung T T T -- T -- N/D [180]
Hypoxia [MR-32 N/D 111 N/D N/D T T [18 1]
NB-96 N/D -- N/D N/D T T
Renal injury in rat Kidney (remnant N/D -- 11 11 -- [160]
(mass ablation) tissue)
Renal injury in rat Kidney T T T T T -- T T -- [150]
(obstructive
nephropathy)
Renal ﬁbrosis in rat Kidney T T T LT -- T -- [1 82]

 

 

 

 

 

 

 

 

 

Table III. Regulation of expression of RAMP isoforms and CL-R in various pathophysiologies

and animal models. (N/D, not done; --, no Change).

28

2.2.7. Receptor Component Protein (RCP)

During the process of trying to Clone the CGRP receptor, Dickerson and
colleagues identiﬁed a novel 148 amino acid protein that appeared to play a role in the
regulation of CGRP signaling [183]. This protein was identiﬁed with a system Similar to
that used to identify the receptor activity modifying proteins, a system utilizing the PKA
activation requirement of CFTR to register CAMP responses to particular ligands [134].
In this case, CGRP stimulation allowed the Cloning of a protein later named CGRP
receptor component protein (RCP). RCP, now cloned in several species, Shows highly
conserved (82%) protein sequence homology between species thus far sequenced [184,
185]. While RCP is detected primarily in membrane fractions prepared from cells, it
shows no amino acid structure for hydrophobic regions or consensus motifs for lipid
attachment [186]. This data concluded that RCP is a cytosolic, yet peripheral membrane
protein. RCP has also been shown to be immunoprecipitated with RAMP] and CL-R in
complex from NIH3T3 cells or lysates of the cerebellum [186, 187]. There are no
conserved glycosylation sites in the RCP sequence, but several conserved protein
phosphorylation sites do exist [184, 185]. This raises the possibility of regulation of RCP
function in an in vivo setting.

RCP expression has been studied in several in vivo settings, with expression
correlating to tissues known to contain CGRP receptors. CGRP is present in the spinal
cord in primary afferent ﬁbers and mediates nociception [188]. In these tissues, RCP
expression is found juxtaposed to that of the CGRP receptors [189]. CGRP receptors are

also known to be expressed on the walls of blood vessels, in this situation mediating

29

vasodilation, and again RCP expression is found in this tissue, correlating with a role for
RCP in CGRP signaling and biological effects [184].

The role of RCP was tested in NIH3T3 cells, a cell line that endogenously
expresses the CGRP receptor system, as well as RCP. RCP expression was inhibited in
these cells, to test the affect, with antisense constructs for RCP. The antisense inhibited
the expression of RCP to that below detection with Western blot. Loss of RCP in the
NIH3T3 cells did not affect either the afﬁnity or the receptor density of CGRP receptor in
the antisense cells, as determined by radioligand binding with 125I-CGRP [186, 187].
Interestingly, the RCP antisense cells showed a 70% reduction in CAMP response, as
compared to wild-type NIH3T3 cells. Recent results from the Dickerson laboratory have
suggested a role for RCP in adrenomedullin signaling, as well [190]. Lack of RCP
expression did not appear to have any effect on the signal transduction for other GPCRS,
including the Bz-adrenergic receptor or the A21, adenosine receptor [186, 187]. These
results suggested that RCP was not working in a Chaperoning capacity, but had more
inﬂuence on coupling of the receptor to its signaling pathway.

The model proposed by Dickerson et al. for a functional CGRP receptor includes
the CL-R receptor, RAMP], and now RCP. More work is needed to establish the role of

RCP in the coupling of the CGRP receptor to its respective G protein.

2.3. GPCR Life-cycle Regulation

2.3.1. GPCR life-cycle
G protein-coupled receptors (GPCRS) are known to convey environmental signals

to the intracellular environment via heterotrimeric G proteins to affect the cellular

30

behavior in response to a stimulus. Their form of regulation had in the past been thought
of as Short-term control, whereas more recently they have been shown to play roles in
longer-terrn regulation of such physiological processes as proliferation, apoptosis,
cellular migration, and hypertrophy [191, 192]. Their long-term regulation is partially a
result of the desensitization and endocytosis processes and the role of these processes in
the intracellular signaling. This attenuation of signaling and removal of the receptor from
the plasma membrane can decrease the receptor numbers capable of signaling for minutes
to hours, depending on the cell type and receptor. Thus, desensitization is the process of
an attenuation of Signaling as a result of prolonged exposure of the receptor to its agonist
(see Table IV and Figure 1 below). Desensitization of a receptor often signals for the
removal of the receptor from the plasma membrane by the pinching off of the plasma
membrane and internalization of the receptor, termed endocytosis. Many forms of
regulation control these processes, some of which being phosphorylation of the receptor
intracellularly, interaction of the receptor with additional proteins (caveolin, arrestin
molecules), and interaction of the receptor with the endocytic machinery (Clathrin,
dynamin, adaptor protein-2 (AP-2), etc.). The receptors, regardless of the type, are often
endocytosed into the early endosomes where their fate is determined. From this point
receptors are targeted for recycling to the plasma membrane or sorted for degradation in
the lysosomes. Protein-protein interactions and post-translational modiﬁcations are being
elucidated that serve as sorting Signals from the early endosome, to target proteins for
either recycling to the plasma membrane to promote receptor Signaling or shuttling of the

receptor to the lysosomes for proteolytic degradation and a down-regulation of the

31

receptor. The factors that determine the regulation of the GPCR life-cycle will be

discussed in the following sections.

 

Figure 1. Depiction of GPCR life-cycle (modeled for prototypical GPCR life-cycle of the B2-

adrenergic receptor) [193].

32

 

 

 

 

 

 

 

 

 

 

Term: Deﬁnition:

Desensitization Attenuation in receptor Signaling in response to prolonged agonist
exposure

Endocytosis Uptake by a cell of material from the environment by invagination
(infolding) of its plasma membrane

Intemalization Transportation of cells or soluble material into the cell via a
vacuole/vesicle

Resensitization Return of unbound receptor to plasma membrane, ready for ligand
stimulation and receptor Signaling

Recycling Targeting of receptor from endosome for return pathway to plasma
membrane for continued signaling

Degradation Targeting of receptor from endosome for pathway to
lysosomes/proteosomes to be degraded

Down-regulation Trafﬁcking of receptor for internalization and degradative pathway to
promote decrease of available receptors at plasma membrane

 

Table IV: Terminology for steps in GPCR life-cycle.

2.3.2. Regulation of receptor desensitization

A prolonged exposure of cells to a particular ligand results in an attenuation of
responsiveness to subsequent stimulation with that ligand. This phenomenon is called
desensitization, which can be homologous or heterologous. Homologous desensitization
is Classiﬁed as a decrease in the response that is speciﬁc for the stimulated receptor.
Because G-protein regulated kinases (GRKS) are activated and only phosphorylate
GPCRS in the agonist-bound state, GRKS are capable of attenuating receptor signaling by
homologous desensitization [194, 195]. An attenuation of receptor signaling that is the

result of second messenger Signaling, and in principal not speciﬁc for the activated

33

 

receptor, is termed heterologous desensitization. Protein kinase A and C are kinases
activated through second messenger signaling of GPCRS, and therefore can activate
heterologous receptor desensitization [196].

The most comprehensive study of desensitization, thus far, has been in elucidating
the molecular mechanisms of this process for the B-adrenergic receptors [195, 197].
Using concepts from the examination of desensitization of the B-adrenergic receptors, it
has been Shown in numerous membrane-bound receptor systems, particularly GPCRS,
that phosphorylation is an integral step in the attenuation of receptor signaling, termed
desensitization. Virtually all GPCRS thus far studied have speciﬁc serine, threonine,
and/or tyrosine amino acid residues in the third intracellular loop or C-terminus of the
receptor that require phosphorylation for the efﬁcient desensitization of the receptor. In
most cases, phosphorylation of these key residues allows for interaction with non-visual
arrestins. This interaction disrupts the coupling of the receptor to the G-protein, thus
halting signaling of the receptor through the G-protein uncoupling. In addition, B-arrestin
interaction with the GPCR promotes Clathrin-mediated receptor internalization. Without
phosphorylation of required residues on the third intracellular loop or C-terminus of the
GPCR, signaling will be maintained and desensitization of receptors is attenuated, or
prevented.

The mechanism of desensitization of CL-R has been studied in several cell lines
to date. Desensitization of CL-R was found to involve protein kinase A activation in rat
mesangial cells (RMCS) stimulated with AM, SK-N-MC (neuroblastoma cell line) cells
stimulated with CGRP, and vascular smooth muscle cells (VSMCS) stimulated with

CGRP [198-200]. Whereas, in HEK 293 cells, desensitization of the CGRP receptor

34

(CL-R/RAMPI) has been shown to be dependent on the activity of G-protein receptor
kinase(GRK)-6 [201]. In HEK 293 cells, CL-R has been shown to be phosphorylated
when stimulated with agonist (AM or CGRP) via in vivo phosphorylation assays [202].
Given the published data for the B-adrenergic receptor and additional GPCRS supporting
a phosphorylation-dependent mechanism for receptor desensitization, and the above-
described studies with CL-R, it is hypothesized that phosphorylation of CL-R is required
for receptor desensitization after prolonged agonist exposure. The studies in this project
will focus on the requirement of phosphorylation in the desensitization process for the
AM receptor subtypes.

With respect to physiological effects of the desensitization process, the inhibition
of the process of desensitization for particular GPCRS has been Shown to elicit protective
therapeutic effects. Leﬂcowitz et. al. have Shown that the inhibition of B-adrenergic
receptor kinase (kinase that phosphorylates the B-adrenergic receptor to cause
desensitization) in the heart can delay the development of heart failure in multiple animal
models, in some cases even restoring cardiac function [203, 204]. Others have also
shown inhibition of desensitization of the p-opioid receptor to be beneﬁcial in preventing
morphine tolerance [205, 206]. Because of the potential therapeutic effect of prolonged
AM signaling in a system where it exerts protective effects, understanding the regulation

of the desensitization process for the AM receptor is crucial.

2.3.3. Regulation of receptor internalization/endocytosis

Predominantly, endocytosis of G-protein coupled receptors is enhanced by agonist

binding, whereas many nutrient receptors, such as the transferring and LDL receptors are

35

constitutively endocytosed regardless of ligand occupancy status [207-209]. The best-
characterized pathway for receptor internalization is mediated by Clathrin-coated vesicles.
Often, clathrin-mediated internalization of mammalian GPCRS requires agonist
stimulation and interaction with [3-arrestin molecules. The B-arrestin proteins not only
disrupt G-protein coupling with the receptor, but also serve as adaptors to link the
receptor to the endocytic machinery [210, 211]. Clathrin-mediated endocytosis generally
also requires the activity of dynamin GTPaseS for proper vesicle formation and cleavage
from the plasma membrane [212].

A second pathway for receptor internalization is now emerging. Data now
conﬁrms that receptors are internalized via non-coated vesicles, such as the ﬂask-shaped
caveolae and other pinocytic mechanisms [207, 212]. Caveolin-mediated internalization
also requires the activity of dynamin, but internalization mechanisms independent of
dynamin have even been suggested [212, 213]. In fact, internalization of a GPCR by
different endocytic pathways in single cell has even been demonstrated for the
Cholecystokinin (CCK) receptor [214]. Caveolin-mediated endocytosis involves receptor
complex localization in a lipid raft domain enriched in caveolin proteins. These domains
are termed caveolae. Intemalization of receptors by the different pathways may be
determined by the phosphorylation state of the receptor. The [3 l-adrenergic receptor has
been demonstrated undergo agonist-stimulated internalization via caveolae when
phosphorylated by protein kinase A and internalization by Clathrin-Coated pit endocytosis
when phosphorylated by GRKS during the desensitization process [215]. The mechanism
of internalization may also have an important effect on the endocytic sorting of the

receptors. The receptor tyrosine kinase, TGF-B, is degraded when internalized by a

36

caveolae-mediated mechanism, but is recycled to promote receptor signaling when
internalized by Clathrin-Coated pit endocytosis [216].

NHERF-l, PDZ domain-containing protein discussed previously, has been shown
to associate with the epidermal growth factor receptor (EGFR), a receptor tyrosine
kinases (RTK) to regulate receptor internalization. In this case, NHERF-l association
acts to stabilize the receptor at the plasma membrane and decrease the endocytosis rate of
the receptor [217]. NHERF-l has also been shown to attenuate the constitutive
endocytosis of the PTHl-R by a mechanism of tethering the receptor to the actin
cytoskeleton through protein-protein interactions [218]. Using an endogenous system, I
have also shown in this project that NHERF-l is essential to ‘hold’ the receptor-complex
at the membrane, the absence of which leads to internalization of the receptor complex.
These studies suggest a critical role for protein-protein interactions and NHERF-1 in
regulation of receptor internalization and maintenance of receptor numbers at the plasma
membrane.

While many GPCRS utilize similar mechanisms for endocytosis, the functional
consequence of endocytosis varies from receptor to receptor. Intemalized receptors that
are trafﬁcked through a rapid recycling pathway are restored to the plasma membrane in
a functional state to achieve resensitization. On the other hand, receptors that are
internalized and targeted to late endosomes and lysosomes experience proteolytic
degradation, thus promoting attenuation of receptor signaling and down-regulation of the

receptor.

37

2.3.4. Regulation of receptor recycling vs. degradation

After internalization, factors inﬂuencing the sorting of receptors in the early
endosome are largely unknown, but some of the critical players are beginning to be
identiﬁed for the GPCRS. Receptor ubiquitination, interaction of the receptor with PDZ
domain-containing proteins, and/or interaction of the receptor with additional, newly-
identiﬁed proteins has been shown to be required in some GPCR systems for efﬁcient
targeting of the GPCR for either degradation or recycling pathways [219-221].

Agonist-induced endocytosis is a means of regulating signaling for a multitude of
membrane-bound receptors, particularly for G-protein coupled receptors. For some
receptors in distinct cell backgrounds, internalization is a means of rapid recycling,
characterized by dephosphorlyation and dissociation from agonist in intracellular vesicles
for restoration at the plasma membrane in a functional state to achieve resensitization.
On the other hand, some receptors that are internalized and targeted to late endosomes
and lysosomes experience proteolytic degradation, thus promoting down-regulation of the
receptor at the plasma membrane and attenuation of receptor signaling.

Much effort is currently being placed on the signal that sorts the internalized
receptors for recycling or degradation. Ubiquitination of GPCRS has recently been
shown to contribute to the sorting fate of the receptors for degradation. Ubiquitination is
a process that involves the addition of multiple ubiquitin molecules on targeted lysine
residues of a protein marked for degradation by an ubiquitinating enzyme complex. This
addition of ubiquitin subunits to a particular protein acts as a degradation sorting signal
for the degradation machinery [219, 222]. Much of the mechanism in the recognition of

the degradation signal is unknown, but the mechanism for the addition of ubiquitin

38

molecules to targeted lysine residues on proteins has been determined. Ubiquitin
addition to a protein is a result of the coordinated activity of three enzymes: the ubiquitin-
activating enzyme (E1), ubiquitin—carrying enzyme (E2), and the ubiquitin ligase (E3).
Marchese and Benovic have recently shown that ubiquitination of the Chemokine
receptor, CXCR4, by the ubiquitin E3 ligase, atrophin-interacting protein 4 (AIP4), is
mediated by lysine residues in the C-terminus of the receptor. In this study, mutation of
lysine residues to arginine on the C-terminus of the receptor inhibits ubiquitination and
subsequent receptor degradation, but did not alter the internalization of the receptor upon
agonist binding [219, 223]. Irnmunoprecipitation with an ubiquitin antibody also showed
that the receptor was ubiquitinated, as well. In addition to the CXCR4 receptor, the [32-
AR has been shown to be targeted for degradation upon ubiquitination of the receptor.
Leﬂcowitz’s group also showed that rapid ubiquitination of associated B-arrestin plays a
role in the internalization of the ubiquitinated BZ-AR. Mutations of lysine residues on the
C-terminus of the receptor not only inhibited the degradation of the receptor, but also
promoted the recycling of the receptor back to the plasma membrane after agonist-
induced internalization [220].

It has been shown in several GPCR systems that interactions with PSD-95/Discs-
large/ZO-l homology (PDZ) domain containing proteins are responsible for the efﬁcient
targeting of the receptor after internalization [224-226]. This is a relatively new area of
research in the GPCR ﬁeld, but it is currently thought that the presence of a PDZ
recognition sequence on the C-terminus of the receptor is the sorting Signal for the
interaction of the receptor with the PDZ domain-containing protein and targeting of the

receptor for recycling to the plasma membrane from the early endosome. Recycling of

39

the [32-Adrenergic receptor (BZ-AR) is dependent on the interaction of B2-AR, via its
PDZ motif, with a protein called N-ethylmaleimide sensitive factor (NSF) [224, 225]. In
addition, via the PDZ domain, PDZ domain-containing protein called Na+/H+ Exchange
Regulatory Factor (NHERF) interaction with the tc-opioid receptor has recently been
Shown to increase the recycling efﬁciency of this particular GPCR [226]. It has also been
shown that the 8-opioid receptor (DOR) is targeted for degradation following agonist-
induced internalization, yet when a Chimera of the DOR and the C-terminal PDZ motif
from the BZ-AR are treated with DOR agonist, the receptor is rapidly recycled to the
plasma membrane aﬁer endocytosis [221, 227]. This suggests that the lack of the PDZ
recognition motif in the C-terminus of the DOR inhibits binding of the DOR with
proteins to target the receptor for recycling, and subsequently the receptor is degraded.
Recent data suggests that the C-terminus of the DOR interacts with a novel protein,
termed G-protein accessory sorting protein (GASP), to target the receptor for degradation
[22]], suggesting that sorting for degradation is not the default pathway for GPCRS, but
is also a tightly regulated decision. While for most GPCRS the PDZ motif seems to
predominantly be required for receptor recycling after agonist-induced endocytosis, the
CXCR4 receptor requires an intact PDZ motif on the receptor to be targeted to the
lysosomes for degradation. This PDZ recognition motif falls in the region found to be
required for proper receptor ubiquitination for degradation. Disruption of this domain
decreases the targeting of the receptor to the lysosomes from the early endosomes after
internalization [219]. Clearly, more work is required to begin to understand the complex
regulation of the sorting of GPCRS for the endosome after agonist-induced

internalization.

4O

As described, the fate of the GPCR is quite variable after agonist-induced
internalization, with multiple GPCRS shown to both recycle and degrade, depending on
the cell type and agonists interacting with the receptor. In the case of CL-R, the receptor
fate differs, depending on the cell type where the receptor is expressed. It has been
shown that CL-R/RAMP complex will degrade upon prolonged agonist exposure and
receptor endocytosis in HEK 293 cells and Rat2 ﬁbroblast cells, while in rat mesangial
cells (RMCS) the receptor complex is effectively recycled after internalization [162, 198,
228]. The mechanism that regulates the pathway to which the receptor complex is
targeted after agonist-induced internalization was previously unknown. Work from this
project has proposed a model that speciﬁc RAMP isoform expression characterizes the
cell to either recycle or degrade the AM receptor complex after prolonged agonist

stimulation.

2.3.5. Protein-protein interaction domains/motifs

Protein-protein interactions are being Shown routinely in to regulate numerous
biological processes, tying together crucial partners in signaling complexes. This method
of regulation has become commonplace the GPCR ﬁeld. The number of protein-protein
interaction domains is ever-growing and for the purpose of this literature review, I will
focus on the most common interactions that have been shown to regulate GPCRS (i.e.
8H2, SH3, EVHI, WW domains). I will concentrate, in particular, on the post-synaptic
density 95, discs large, zonula occludens-l (PDZ) domain interactions. This dissertation
project focuses, in large part, on the role of PDZ interactions in the regulation of the life-

CyCle and trafﬁcking of the adrenomedullin receptor.

41

SrC-homology 2 (SH2) domains are modules of ~100 amino acids that bind to
Speciﬁc phosphor-tyrosine (pY)-containing peptide motifs. Conventional SH2 domains
have a conserved pocket that recognizes pY, and a more variable pocket that binds 3-6
residues C-terrninal to the pY, granting speciﬁcity. Phosphopeptides of optimal sequence
bind to SH2 domains with dissociation constants of ~50-500 nM [229, 230]. The SH2
domain is embedded in a wide variety of metazoan proteins that regulate functionally
diverse processes, and for this reason, SH2 domains must display sufﬁciently high off-
rates for rapid and reversible signal transduction. SH2 domain interactions are
responsible for the regulation of such varied processes as scaffolding, kinases,
cytoskeletal regulation, phosphatases, phosphoinositide signaling, transcription, and
ubiquitination, to name just a few [229]. Ste plays an active role in the agonist-induced
desensitization of betaZ-adrenergic receptors via SH2 domain interactions. Src binds, via
its SH2 domain, to the beta2-adrenergic receptor on an agonist-mediated phosphor-
tyrosine residue, thereby allowing Src to phosphorylate and activate G-protein-coupled
receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization [231].

Src-homology 3 (SH3) domains generally bind to Pro-rich peptides that form a
left-handed helix, with the minimal consensus Pro-X-X-Pro. Each Pro is usually preceded
by an aliphatic residue. Each of these aliphatic-Pro pairs binds to a hydrophobic pocket
on the SH3 domain. From this, two Classes of SH3 domains have been deﬁned (Class I
and Class 2) which recognize RKXXPXXP and PXXPXR motifs, respectively. The
ligand can, in principle, bind in either orientation. Such peptides usually bind to the SH3

domain with a Kd in the mM range [232, 233]. Dopamine (D4) receptor has recently

been reported to bind SH3 domains of proteins, such as Grb2, at its proline-rich putative

.42

third cytoplasmic loop. GPCRS have also been demonstrated to bind via internal SH3
domains, namely the P2Y(2) receptor, binds Src via an agonist-dependent SH3 domain
interaction that facilitates Src activation, which recruits the EGF R into a protein complex
with the P2Y(2) receptor and allows Src to efﬁciently phosphorylate the EGFR. The
functional signiﬁcance of these interactions is presently unknown [234].

WW domains are small 38 to 40 amino acid residue modules that have been
implicated in binding to Pro-rich sequences. WW domains and SH3 domains can
potentially bind overlapping sites, with both binding a left-handed poly-proline type II
helix. In addition, the Pin] WW domain functions as a phosphoserine- or
phosphothreonine-binding module. In its function, the WW domain shares elements of
SH3 and SH2 domains by recognizing proline-rich ligands and, in some cases, by being
regulated by phosphorylation. The domain name is derived from two conserved Trp
residues spaced 20 to 22 residues apart within the consensus sequence. The dissociation
constants (Kd) for WW—ligand complexes lie in the high nM to low mM range for
proline-rich ligands, and in the low mM range for phospho-SP- or phospho-TP-
containing ligands [233, 235]. W domains have attracted attention because the
signaling complexes they mediate have been implicated directly or indirectly in several
human diseases including muscular dystrophy, Alzheimer’s and Huntington’s diseases,
and, more recently, cancer.

Drosophila enabled/vasodilator-stimulated phosphoprotein homology 1 (EVHl)
domains are 115 residue protein-protein interaction modules which provide essential
links for their host proteins to various signal transduction pathways. Like Src homology

3, WW and GYF domains and proﬁlin, EVH] domains recognize and bind speciﬁc

43

proline-rich sequences [233]. The binding is of low afﬁnity, but tightly regulated by the
high speciﬁcity encoded into residues in the proteinzpeptide interface [236]. Many
EVHl-containing proteins are associated closely with actin-based structures and are
involved in re-organization of the actin cytoskeleton. EVH] domains are also present in
proteins enriched in neuronal tissue, thus implicating them as potential mediators of
synaptic plasticity, linking them to memory formation and learning. For example, homer,
an EVH] domain protein forms an adaptor system that regulates coupling of group 1
metabotropic glutamate receptors with intracellular inositol trisphosphate receptors and is
modiﬁed by neuronal activity [237].

The discovery of the PDZ domain was based on the recognition of sequence
repeats in several proteins. The ﬁrst initial of these three proteins (Postsynaptic density
95, Discs large, Zonula occludens-l) fashioned the name PDZ domain. PDZ domains are
quite widespread throughout the metazoans, yet the several yeast species thus far
sequenced Show surprisingly few PDZ domains.

Classiﬁcation of PDZ domains, based on their structure and their recognition
peptide sequences, has been more difﬁcult than originally thought. Bezprozvanny and
Maximov proposed Classifying PDZ domains into 25 groups, based on the nature of the
residues in two positions, the (18] position and the position immediately after the [3B
strand [238]. Instead, classiﬁcation of PDZ domains has been focused primarily on their
speciﬁcity for C-tenninal peptides. Class I PDZ domains recognize a C-tenninal
sequence of X-S or T-X-V or L. Examples of proteins containing these interaction sites
include the NMDAZAB receptor, the BZ-adrenergic receptor, 5-Catenin. The PDZ domain-

containing proteins in this Class, sharing this interaction include PSD-95, Erbin, and

44

NHERF [239-241]. The second Class of PDZ domains recognizes a C-terrninal peptide
with the sequence of X-w-X-w. Interacting proteins with Class II PDZ domains include
neurexin, syndecan, and EphB2. Examples of members of the PDZ Class II domain-
containing proteins are CASK, PICK], and syntenin [242-245]. The ﬁnal Class of PDZ
domains is Class III and classiﬁed by a X-D or E-X-w recognition sequence. The
melatonin receptor interacts with the Class III PDZ domain-containing protein, nNOS
[246].

In many cases, as we have observed, PDZ domain interactions are constitutive,
with binding afﬁnities in the 1 to 10 pM range. There are demonstrations for regulation
of this interaction, as well. For example, the interaction of the Bz-adrenergic receptor
with NHERF has been shown to be dependent on the receptor activation by agonist [240].
In addition, phosphorylation has been shown to regulate some PDZ domain interactions.
For example, the interaction between the inwardly rectifying 1K+ Channel Kit 2.3 and
PSD-95 is disrupted by PKA-dependent, serine phosphorylation of the channel at the -2
position [247]. Also, the Bz-adrenergic receptor, when serine-phosphorylated at its -2
position by GRKS, is unable to interact with NHERF [224].

Solving the structural basis of the PDZ domain interaction was critical in
determining the speciﬁcity with which the PDZ domain-containing proteins bound their
ligands. PDZ domains are comprised of 80 to 90 amino acids that make up Six [3 strands
(BA-BF) and two a-helices (CIA and (1B), arranged in a compact globular structure.
Several PDZ domains, both complexed with their ligands and ligand-free, have been
solved for their three-dimensional structure to clarify the mechanism of the PDZ domain

interactions. In short, the binding of the peptide ligand to the PDZ domain takes place in

45

an elongated surface groove as an antiparallel B strand interacts with the BB strand and
the (1B helix [248]. PDZ domains have been shown in interact with their peptides in four
different types of interactions: recognition of C-terminal motifs in peptides, recognition
of internal motifs in peptide binding partners, PDZ-PDZ interactions, and recognition of
lipids [249].

PDZ domains were originally thought to simply scaffold signaling complexes
and/or receptors at the cell surface. It has become evident that PDZ domain interactions
regulate a multitude of functions in cells of many types. A few examples to be discussed
brieﬂy in this review are: adaptors for tyrosine kinase receptors, epithelial polarity, and
mediators of protein networks. PDZ interactions of Na+/H+ Exchanger Regulatory
Factor (NHERF) have been shown to regulate the Clustering and autophosphorylation of
the platelet-derived growth factor (PDGF) receptor. More potent receptor
autophosphorylation is seen when the receptor is complexed with NHERF, leading to a
more robust activation of MAPK signaling [250]. In terms of epithelial polarity, NHERF
interactions also determine the expression of the Na+/H+ Exchanger-3 (NHE3) in the
polarized proximal tubule cells and tissue. NHE3 is an antiporter responsible for the
majority of the Na+ reabsorption in the proximal tubule, with the Na+/H+ exchange
dependent on proper NHERF interactions to localize the transporter on the correct side of
the epithelial cell for Na+ reabsorption [251]. Protein networks maintained by PDZ
domain interactions are demonstrated many times over at the neural synapse. For
example, through PDZ interactions, the NMDA receptor is tied to the Ca2+-ATPase
Channel, the kainate receptor, the Shaker K+ Channel, and the metabotropic glutamate

receptor. To portray the complexity of these interactions, in the literature the

46

postsynaptic NMDA receptor has been reported to have 17 primary interactions leading
to 385 interactions with secondary proteins [249] . A major Challenge for the future will
be to build models of multiprotein networks and explain the complexity and regulation of

these “multiprotein machines”.

2.3.6. Na+/H+ Exchanger Regulatory Factor-l (N HERF-l)

NHERF-1 was initially identiﬁed as a regulator of the Na+/H+ exchanger (N HE),
mediating the activity of the transporter through CAMP-induced inhibition of NHE3.
Through the identiﬁcation of numerous targets of NHERF-1, some membrane-bound
receptors and transporters, the role for this protein has been expanded to that of a
scaffolding protein in membrane physiology, as well. NHERF-1 has been characterized
as a 55 KDa protein containing two tandem PDZ domains, type I and type H domains,
respectively [251]. NHERF-1 has also been shown to associate with members of the
ezrin/radixin/moesin family of actin-binding proteins, and thus has the ability to link PDZ
domain-interacting proteins to the actin cytoskeleton [252]. The two PDZ domains and
MERM domain also allow NHERF to facilitate the formation of multiprotein signaling
complexes. For example, NHERF-l is known to bind NHE3 and the PTHl-receptor, and
through interactions with cytoskeletal proteins ties NHE3 to an activated PKA that
phosphorylates and inactivates NHE3. Disruption of any of the previous interactions will
abate NHE3 inactivation and allow improper Na+/H+ exchange [253].

Yeast two-hybrid experiments identiﬁed an additional protein that bound the C-
terrninal region of NHE3. This protein shared structural homology with NHERF-1, and

was thus termed NHE3 kinase A-regulated protein, or NHERF-2. Differential expression

47

of NHERF-1 and -2 has been observed throughout the kidney. NHERF-1 was detected in
the proximal tubules, while NHERF-2 was detected in the glomeruli, peritubular
capillaries, collecting duct principal cells, and low levels in the proximal tubules [254].
The differential expression suggests distinct physiological roles for each protein in the
kidney. NHERF expression has been detected in tracheal, pancreatic, intestinal, and
kidney epithelia.

Determined by immunoﬂuorescence microscopy, MERM cytoskeletal proteins
are typically localized at the apical membranes of polarized cells. Because of their
interactions with MERM proteins, both NHERF-1 and -2 are localized primarily in the
apical membranes of kidney cells [255]. This apical localization is critical for the
scaffolding of signaling complexes to function properly in a physiological setting. For
example, Changes in the cystic ﬁbrosis transmembrane regulator (CFTR) trafﬁcking are
seen in the majority (~90%) of humans with cystic ﬁbrosis. Mutants of CFTR lacking
the C-terminal PDZ interaction motif are unable to interact with NHERF and therefore
Show altered trafﬁcking of the mutant CF TR to basolateral membranes, in contrast to the
wild-type CFTR expression at apical membranes [256]. Studies have implicated the
interaction of CFTR with NHERF-l with the correct apical localization and proper
intracellular trafﬁcking of CFTR, and lack of NHERF-l interaction with contributing to
the disease process.

Several studies have shown NHERF-1 to play differing roles in various cellular
processes, including receptor trafﬁcking [257, 25 8]. NHERF-1 has been demonstrated to
bind the extreme carboxy-tenninus of several G-protein coupled receptors (GPCRS),

namely the Bz-adrenergic receptor, the K-opioid receptor, PTH-R, and the P2Y purinergic

48

receptor [154, 259]. Agonist exposure promotes NHERF-l association with the B2-
adrenergic and the Ic-opioid receptors. NHERF-1 association with these receptors
enhances the recycling of the receptors after agonist stimulation [240, 259]. In addition
to GPCRS, NHERF-1 has also been shown to associate with the epidermal growth factor
receptor (EGFR), a receptor tyrosine kinases (RTK). In this case, NHERF-1 association
acts to stabilize the receptor at the plasma membrane and decrease the endocytosis rate of
the receptor [217]. While utilizing different mechanisms, collectively these data suggest
that NHERF-l interaction with both RTKS and G-protein-coupled receptors is mandatory

to enhance the portion of receptors present at the cell surface.

2.3.7. N-etbylmaleimide Sensitive Factor (NSF)

NSF is a 76 KDa protein with functions in vesicle exocytosis and membrane
fusion events. NSF is composed of three domains: an N-terminal NSF-N domain
followed by two ATP-binding domains, NSF-D] and NSF-D2. The N-terminal NSF-N
domain is critical for binding to the SNAP-SNARE complex during vesicle fusion and
exocytosis. Contained in the two ATP-binding domains is a 230-250 amino acid motif
that is Characteristic of the ATPaseS Associated with various cellular Activities (AAA)
family of ATPases, and the orientation of this domain classiﬁes NSF as a Class II AAA
protein [260]. NSF forms a homo-hexamer and requires binding of ATP for its active
conformation.

NSF is commonly known to interact with aSNAP (soluble NSF attachment
protein) and SNARE (soluble N-ethy]maleimide-sensitive factor attachment protein

receptor) proteins to form the 208 particle, a complex that plays a critical role in

49

intracellular membrane fusion and exocytosis [261-263]. Monomeric a-SNAP binds to
NSF only upon binding SNARES. The C-terminal 45 amino acids of a-SNAP are
required for binding and for stimulation of NSF’S ATPase activity [264-266]. The
ATPase activity of NSF is responsible for the dissociation of a-SNAP and the SNARES
to complete the fusion of a vesicle with the cell membrane.

Factors inﬂuencing the sorting of receptors in the early endosomes are largely
unknown, but interactions with PSD-95/Discs-large/ZO-l homology (PDZ) domain
proteins are being suggested to regulate the receptor-targeting after internalization [259,
267, 268]. The life cycle of the B2-adrenergic receptor (B2-AR) was reported to be
altered in the presence of NSF [268]. It has been shown that the B2AR interacts with
NSF via a PDZ type I recognition motif (-DSLL) at its extreme C-terminus. Binding of
the B2AR with NSF enhances the recycling of the B2AR alter agonist-stimulated receptor
internalization. In addition, binding of NSF to the Glu2 subunits of the AMPA receptor
was demonstrated to be crucial for the recycling of the a-amino-3-hydroxy-5-methyl-
isoxazolepropionate (AMPA) receptor [269, 270]. NSF binds the Gluch subunit, but
binding is enhanced by binding of another PDZ domain protein, PICK]. PICK], Gluch,
and NSF form a tripartite complex that also binds a-SNAP to increase the disassembly of
the complex upon vesicle fusion with the membrane [271]. NSF plays a Chaperoning role
for SNARES in the majority of membrane fusion events in a cell, but when targeting
membrane receptors for recycling, NSF acts independently of the SNARE complex to
promote rapid resensitization of the receptors at the plasma membrane [261-263]. A
report, published from our laboratory recently, demonstrated a role for NSF in the sorting

of the AM2 receptor at the endosome for a recycling pathway. In this case, NSF ’s

50

interaction with an accessory protein (RAMP3) directs the trafﬁcking of a receptor from
the sorting endosome [153].

NSF has been shown to interact with another protein critical to receptor
trafficking, the B-arrestin molecule. B-arrestin is responsible for two steps in the
desensitization process for many GPCRS. First, the B-arrestin binds the receptor, upon
receptor phosphorylation, and disrupts further signaling by the G-protein. Second, the B-
arrestin molecule interacts with proteins to target many receptors for Clathrin-mediated
endocytosis [272, 273] . B-arrestin binding to NSF appears to play a role in the turnover
of the receptor at the plasma membrane. Overexpression of NSF causes an increase in
the receptor Clearance from the plasma membrane after agonist-stimulation of the B2-

adrenergic receptor [274].
At the present time, there is no obvious sequence that could be described as the
NSF binding motif. It will be important to determine, through mutational analysis, the

domains on NSF important for these protein-protein interactions.

51

3. Characterization of AM-stimulated desensitization response in

transfected HEK 293 cells

3.1. Introduction

The recent discovery of receptor activity modifying proteins (RAMPS) new
alternatives in the regulation of G-protein coupled receptors (GPCRS). RAMPS are
characterized as single transmembrane-spanning accessory proteins required for the
function of an orphan GPCR, now termed the calcitonin receptor-like receptor (CL-
R)[275]. Through structural homology, three RAMP isoforms (1-3) have been identiﬁed
and are synthesized as distinct gene products. RAMPS are required for the cell surface
expression, as well as for ligand selectivity of CL-R [275, 276]. A heterodimer of
RAMP] and CL-R yields a calcitonin gene-related peptide-l (CGRP-1) receptor, whereas
RAMP2 or -3 coexpressed with CL-R produces adrenomedullin receptors, AM] and
AM2 receptors, respectively [277, 278]. Both AM and CGRP are multi-functional
peptides with many overlapping functions, ranging from potent vasodilation to
proliferation regulation to regulation of salt and water balance [279]. It has been
suggested in the literature that differential expression of RAMP isoforms regulates both
physiological and pathological states.

Upon agonist binding, the CL-R/RAMP receptor complex causes cyclic AMP
activation in most systems, regardless of whether the ligand is AM or CGRP. In addition,
the receptor complex is phosphorylated by protein kinases and subsequently undergoes
desensitization and internalization in response to a prolonged agonist stimulation [280].
Intemalization of all three RAMP isoforms with CL-R has been reported to be clathrin-

mediated and dynamin-dependent. In addition, the CL-R/RAMP] receptor complex is

52

known to internalize with agonist pretreatment in a complex with the B-arrestin molecule
and all three components colocalize to the endosomes. From this stage, the receptor
complex is targeted for a recycling or degradation pathway, depending on the cell type
and RAMP expression proﬁle.

The ﬁndings in this study characterized the AM-stimulated CAMP accumulation
and desensitization of the two AM receptor subtypes in response to AM treatment. These
experiments represent pilot studies performed to determine agonist doses and time points

for remaining studies in this thesis.

3.2. Materials and Methods

3.2.1. Materials: Adrenomedullin was purchased from Bachem Bioscience, Inc. (King
of Prussia, PA). 125I-labeled adrenomedullin was purchased from Amersham Biosciences
Corp. (Piscataway, NY). Cell culture media, fetal bovine serum, penicillin/streptomycin,
trypsin-EDTA were purchased from GibcoBRL® (Grand Island, NY). All other reagents

were of highest quality available.

3.2.2. Cell Culture and Transfection protocols: HEK-293T cells (obtained ﬁ'om
ATCC) are maintained in DMEM low glucose media containing 10% FBS, 1%
penicillin-streptomycin. Transfection of HEK293T was performed using Lipofectamine
Plus protocol (Invitrogen Life Technologies, Carlsbad, CA). Cells were transfected with
the DNA and Lipofectamine Plus as per manufacturer’s protocol. Cells were collected for

assays after 48 hours of transfection.

53

3.2.3. RAMP cloning and expression: Full length CDNA of human RAMPS], 2 and 3
and bovine CL-R were described before [281, 282]. CL-R, Cloned into Nl-EGFP and

also in pCDNA3.l expression vectors, was used for transfection in HEK 293T cells.

3.2.4. Desensitization assays: 48 hours post-tranfection cells were pretreated with or
without 10 nM AM in DMEM containing 0.2% BSA for indicated time periods (up to 4
hr). After agonist exposure, cells were washed three times with Dulbecco’s phosphate
buffered saline (dPBS, Gibco BRL) containing 0.2% BSA and either frozen for
membrane preparation for adenylate cyclase assays or used immediately for intact-cell

radioligand binding.

 

Desensitization Protocol:

Removal of AM,

 

 

Cells AM pretreatment followed by washing
transfected1 starts 1H\
R pt cti ity
ass or a v assay
Mrs 1 h" (CAMP accumulation) and
receptor binding
No Pretreat
(Full Stimulation) 1 hr AM
Pretreat
(Desensltlzatlon)

 

 

 

3.2.5. Receptor binding: Homologous competition radioligand binding assays were
performed as described by Aiyar et a1 and as established in our laboratory [283]. HEK

293T cells were transfected and approximately 200,000 cells/well seeded in poly-D-

54

lysine precoated 24-well plates (BD Biosciences, Palo Alto, CA). 48 hours post-
tranfection cells were treated for desensitization or resensitization assays as described
above. After agonist exposure, cells were washed three times with dPBS buffer
containing 0.2% BSA then incubated with increasing concentrations (1 pM to 100 nM) of
competing ligand (rAM) and 175-250 pM 12“’I-rAM for 30 min at 37°C. After incubation,
plates were washed three times with ice-cold assay buffer and the reactions were
terminated by the addition of 2M NaOH. Cells were then harvested and associated
radioligand activity is counted on a y-counter. All binding assays were performed in
duplicate, with each experiment repeated at least three times. Nonspeciﬁc binding was
determined in the presence of 100 nM of unlabeled rAM. Data was analyzed by
LIGAND (assuming radioligand and competitor both bind reversibly to a single binding
site; MacLigand, Version 4.97, NIH, Bethesda, MD) using the following equation:

Bmax 0 [hot ligand]

 

KD + [hot ligand] + [cold ligand]

Where B represents speciﬁc binding, [hot ligand] the single concentration of [1251]rAM
studied, [cold ligand] the concentration of unlabeled rAM competing with the radiolabe]
for AM receptor binding, Bmax the maximum number of binding Sites and KD the
equilibrium dissociation constant (the equation was solved where the “cold ligand” ICso =
[hot ligand] + KD). Analysis of all binding data was performed by computer-assisted
nonlinear least square ﬁtting using GraphPad PRIZM version 4 (GraphPad Software, San
Diego, CA). Binding sites/cell was calculated and data was expressed as percent of the

control.

55

3.2.6. Adenylate cyclase assays: Cyclase activity was done as described before with
slight modiﬁcations [146, 284]. Cells were harvested from P100 or P60 plates and
homogenized in Tris HCl (10mM)/EDTA (10mM) buffer. Membranes were prepared by
homogenization and centrifugation in Tris HC] (50mM)/MgCl (10mM) buffer. Fina]
concentration of 20 pg of protein/assay tube was obtained. Membranes were incubated
for 15 min at 30°C with appropriate concentrations of drugs (lOOnM AM, 10nM
Forskolin) and assay mix containing ATP regeneration system and a32P-ATP. After the
reaction was stopped (with stop solution containing 3H-cAMP) contents of the assay
tubes were passed through Dowex and subsequently through alumina columns to separate
the degradation products of ATP, by washing the dowex with water and alumina with
imidazole. Elution proﬁle was done to determine the amount of water and imidazole
needed to wash and elute the products. Product eluted from alumina column was counted
for the presence of 3H-CAMP and GBZP-CAMP in a B-counter. Each experiment was done
in triplicates and repeated at least 3 times. Data is expressed as percent maximal
response, % forskolin. Basal cyclase activity and forskolin stimulation did not Show

statistically signiﬁcant differences between treatments.

3.2.7. CAMP accumulation assays: HEK 293 cells were seeded on a 24-well plate until
reaching 80-90% conﬂuency, then incubated in serum-free media overnight before
experiment. Desensitization experiments were carried out as described in Materials and
Methods section, with cells pretreated with 10nM rAM and subsequently Challenged for

10 min at 37°C with appropriate concentrations of drugs (IOOnM AM, 10nM Forskolin)

in the presence of 200uM 3-isobutyl-1-methylxanthine. Determination of CAMP level

56

d.

all

(163

was measured using the biotrak CAMP enzyme immunoassay system (Amersham
Biosciences) according to the manufacturer's instructions. CAMP levels in HEK 293 cells
were calculated using a standard curve ranging from 10 to 10 4 frnol of CAMP. Each
experiment was done in duplicate and repeated at least 3 times. Data is expressed as
percent maximal response, % forskolin. Basal cyclase activity and forskolin stimulation

did not Show statistically signiﬁcant differences between treatments.

3.2.8. Statistics: Data are presented as mean i S.E.M. Single group comparisons

exercised a paired Student’s t-test method. Statistical signiﬁcance was set at P<0.05.

3.3. Results/Discussion

We and other groups have previously Shown that the AM] receptor (CL-R +
RAMP2) and AM2 receptor (CL-R + RAMP3 complex) undergo agonist-stimulated
desensitization, internalization, and degradation in HEK 293 cells [153, 276, 285]. An
initial 100 nM AM Challenge to HEK 293 cells expressing CL-R and RAMP2 or RAMP3
results in an approximate 9 and 8-fold increase, respectively, in CAMP accumulation over
basal. Pretreatment of HEK 293 cells expressing CL-R and RAMP2 or RAMP3 with 10
nM AM for one hour and subsequent challenge with 100 nM AM resulted in
desensitization of the CAMP accumulation response. AM pretreatment did not
signiﬁcantly alter the basal or forskolin-stimulated (10 nM) CAMP accumulation (Figure
2A, B). Because AM pretreatment was not shown to alter the basal or FK-Stimulated
CAMP accumulation, the remaining CAMP accumulation and adenylate cyclase activity

data throughout the thesis will be expressed as percent maximal response, or percent

57

..a‘

forskolin stimulation. Dose response experiments experiments for receptor
desensitization indicate that maximal desensitization is observed with a 10 nM AM
pretreat and 100 nM AM Challenge (Figure 3A, B). These will be the AM doses used for
pretreatment and challenge in the desensitization and resensitization assays employed
throughout the remaining Chapters of the thesis. The time course for desensitization
shows a maximal attenuation of CAMP accumulation between one and two hours of
agonist pretreatment (Figure 4A, B), and for this reason a one hour agonist pretreatment
is used throughout the remaining thesis Chapters to measure receptor desensitization.
Homologous competitive binding experiments were optimized by altering ligand
and competing ligand concentrations, temperature, and duration of binding experiments.
Figure 5 depicts a representative competition curve for the whole-cell, homologous
competitive binding experiments employed throughout this thesis. Receptor binding sites
per cell were determined from scatchard plots, as described in Materials and Methods
section. A representative scatchard plot is shown in Figure 6. Table V shows receptor
afﬁnity and receptor density calculations for HEK 293 cells expressing CL-R and
RAMP3 with NSF or NHERF-l expression. This table depicts a representative
experiment and indicates no change in receptor afﬁnity with agonist pretreatment or with
expression of additional proteins (NSF or NHERF-l). In addition, co-expression of NSF
or NHERF-1 with CL-R/RAMP3 does not signiﬁcantly alter receptor density in the HEK
293 cells. For the remaining homologous competitive binding experiments, these

calculations will be followed and data expressed as percent of control.

58

 

- No Pretreat

 
 

 

 

 

 

DAM Pretreat
c 250-
.0 ‘E
g 5 200- l
a ,
g :2; 1504
u U)
2 :5, 100-
n- 0
° 0. _ I __
53‘ “8“ “0

Figure 2A: Representative desensitization experiment of AM] receptor in HEK 293 cells,
measured by CAMP accumulation. HEK 293 cells were transiently transfected with CL-R and
RAMP2 and pretreated with 10 nM AM for one hour. After agonist pretreatments, cells were
washed repeatedly, and CAMP accumulation in response to 100 nM AM challenge was measured.
Determination of CAMP level was measured using the Biotrak CAMP enzyme immunoassay
system (Amersham Biosciences) according to the manufacturer's instructions. CAMP levels in
transfected HEK 293 cells were calculated using a standard curve ranging from 10 to 10 " frnol of

CAMP. CAMP accumulation is shown on the left y-axis, raw data shown. Representative

experiment is shown in ﬁgure.

59

 

 

- No Pretreat
300- D AM Pretreat

200'

100-

cAMP Accumulation
(pmollmg protein)

 

 

 

 

6353‘ ”A (‘00 “\N

(\D 9‘“

CK

Figure 2B: Representative desensitization experiment of AM2 receptor in HEK 293 cells,
measured by CAMP accumulation. HEK 293 cells were transiently transfected with CL-R and
RAMP3 and pretreated with 10 nM AM for one hour. After agonist pretreatments, cells were
washed repeatedly, and CAMP accumulation in response to 100 nM AM Challenge was measured.
Determination of CAMP level was measured using the Biotrak CAMP enzyme immunoassay
system (Amersham Biosciences) according to the manufacturer's instructions. CAMP levels in
transfected HEK 293 cells were calculated using a standard curve ranging from 10 to 104 frnol of
CAMP. CAMP accumulation is shown on the left y-axis, raw data Shown. Representative

experiment is shown in ﬁgure.

60

+ No Pretreat
+AM Pretreat

O O
n I

CAMP Accumulation
(nmollmg protein)
We Forskolin]

'63

 

 

1 0-
G I I U I I
\ «A ‘m (a 9%
a9 e o e e
9 03° “*0 “o 030

Figure 3A: Desensitization dose response of AM] receptor in HEK 293 cells. CL-R/RAMP2
complex (AMI receptor) desensitizes in HEK 293 cells after prolonged agonist stimulation. HEK
293 cells transiently transfected with CL-R and RAMP2 were treated for one hour with AM (10
nM), then washed repeatedly, and after repeated wash steps, cells were re-Challenged with 100
nM rAM for 15 minutes and plates were frozen. Determination of CAMP level was measured
using the Biotrak CAMP enzyme immunoassay system (Amersham Biosciences) according to the
manufacturer's instructions. CAMP levels in transfected HEK 293 cells were calculated using a
standard curve ranging from 10 to 10 4 frnol of CAMP. CAMP accumulation is shown on the left
y-axis, expressed as percent maximal response (% forskolin stimulation). Representative

experiment shown in ﬁgure.

6]

-I- No Pretreat
+AM Pretreat

 

 

Dd) ..
<3?”
CI“.—
33,3

_ x .
age“
0:0
or?“
532110-
>‘E

C

33 c
<

Figure 3B: Desensitization dose response of AM2 receptor in HEK 293 cells. CL-R/RAMP3
complex (AM2 receptor) desensitizes in HEK 293 cells after prolonged agonist stimulation. HEK
293 cells transiently transfected with CL-R and RAMP3 were treated for one hour with AM (10
nM) and then washed and receptor desensitization (adenylate cyclase activity) was measured.
Adenylate cyclase activity is shown on the left y-axis, expressed as percent maximal response (%

forskolin stimulation). n 2 3 experiments.

62

[% Forskolin]
N w -h
R R R

.3
C
I

l

l

cAMPAccumulation
(nmollmg protein)

 

 

0.
\ 6 D 6 0 D
0““0 l‘ '5 A 6 «7. I

AM Pretreat (min.)

 

Figure 4A: Desensitization time course for AM] receptor in HEK 293 cells. CL-R/RAMP2
complex (AMl receptor) desensitizes in a time-dependent manner in HEK 293 cells after agonist
stimulation. HEK 293 cells transiently transfected with CL-R and RAMP2 were treated for one
hour with AM (10 nM), then washed repeatedly, and after repeated wash steps, cells were re-
challenged with 100 nM rAM for 15 minutes and plates were frozen. Determination of CAMP
level was measured using the Biotrak CAMP enzyme immunoassay system (Amersham
Biosciences) according to the manufacturer's instructions. CAMP levels in transfected HEK 293
cells were calculated using a standard curve ranging from 10 to 10 4 fmol of CAMP. CAMP
accumulation is shown on the left y-axis, expressed as percent control (no pretreatment) sample.

Representative experiment shown in ﬁgure.

63

CAMP Accumulation
(nmollmg protein)
[% Control]

 

 

 

AM Pretreat (min)

Figure 4B: Desensitization time course for AM2 receptor in HEK 293 cells. CL-R/RAMP3
complex (AM2 receptor) desensitizes in a time-dependent manner in HEK 293 cells after agonist
stimulation. HEK 293 cells transiently transfected with CL-R and RAMP3 were treated for one
hour with AM (10 nM), then washed repeatedly, and after repeated wash steps, cells were re-
challenged with 100 nM rAM for 15 minutes and plates were frozen. Determination of CAMP
level was measured using the Biotrak CAMP enzyme immunoassay system (Amersham
Biosciences) according to the manufacturer's instructions. CAMP levels in transfected HEK 293
cells were calculated using a standard curve ranging from 10 to 10 ’ fmol of CAMP. CAMP
accumulation is shown on the left y-axis, expressed as percent control (no pretreatment) sample.

Representative experiment shown in ﬁgure.

64

 

 

125-
2’
'5 100.
.E
In 75.
5.3 .
g 50-
o.
”3 251
o\
c I I r I I I I
13 12 11 1O 9 8 7 6
log rADM

Figure 5: Competition curves for rAM on intact HEK-293T cells expressing CL-R and RAMP3
with [”51] rAM as radioligand were generated as described in Materials and Methods section.
Representative experiment for homologous competition binding employed throughout remaining

thesis Chapters.

65

P
a
C:

  
 

Bound/Free
O
'o
'9

P

O

‘
l

 

 

P
o
0

0:1 0.2 0.3 0.4

P
c

Figure 6: Scatchard plot for homologous competition binding of rAM on intact HEK-293T cells
expressing CL-R and RAMP3, with [”51] rAM as radioligand, generated as described in Materials
and Methods section. Representative experiment for homologous competition binding employed

throughout remaining thesis Chapters.

66

 

 

 

 

 

 

 

 

 

 

 

Experlment: Kd (nM) Ki (nM) Bmax cellslwell Binding % control
[ﬁnal/assay) siteslcell
CL-R+RAMP3 J
Control 4.00 2.66 0.122 200000 368123 100.00
1 hr Pretreat 3.44 2.29 0.078 231500 203848 55.37
4 hr Recovery 3.73 2.49 0.071 246800 173477 47.12
CL-R+RAMP3+N8F
Control 3.44 2.29 0.093 209300 267894 100.00
1 hr Pretreat 5.35 3.57 0.031 189400 97324 36.33
4 hr Recovery 4.45 2.97 0.076 177200 259451 96.85
CL-R-I-RAMP3-1-NHERF1
Control 3.80 2.62 0.129 230000 336335 100.00
1 hr Pretreat 2.44 1.68 0.138 220000 363389 108.04
4 hr Recovery 2.76 1.90 0.108 200000 323876 96.30

 

Table V: Binding properties for homologous competition binding of rAM on intact HEK-293T

cells expressing CL-R and RAMP3 +/- NSF or NHERF], with [”51] rAM as radioligand,

generated as described in Materials and Methods section. No statistically Signiﬁcant changes in

receptor afﬁnity or receptor density observed in homologous competitive binding experiments.

Representative experiment for homologous competition binding to determine binding sites per

cell, employed throughout remaining thesis Chapters.

67

 

4. Differential regulation of adrenomedullin receptor desensitization
and internalization by receptor activity-modifying proteins.

4.1. Introduction

The recent discovery of receptor activity modifying proteins (RAMPS) has raised
new possibilities for modes of regulation of G-protein coupled receptors (GPCRS).
RAMPS are Characterized as single transmembrane-spanning accessory proteins requisite
to the function of an orphan GPCR, now termed the calcitonin receptor-like receptor (CL-
R)[275]. Through structural homology, three RAMP isoforms (1-3) have been identiﬁed
as distinct gene products. RAMPS are required for the cell surface expression, as well as
for ligand selectivity of CL-R [275, 276]. A heterodimer of RAMP] and CL-R yields a
calcitonin gene-related peptide-1 (CGRP-1) receptor, whereas coexpression of RAMP2
or -3 with CL-R produces adrenomedullin receptors, AM] and AM2 receptors,
respectively [277, 278]. Both AM and CGRP are multi-functional peptides with many
overlapping functions, ranging from potent vasodilation .to proliferation regulation to
regulation of natriuresis and diuresis [279]. It has been suggested in the literature that
differential expression of RAMP isoforms plays a regulatory role in both physiological
and pathophysiological disease states. Moreover, the recent identiﬁcation of RAMP
interactions with additional members of the Class II GPCR family and RAMP expression
in cell lines lacking CL-R have raised the possibility of additional functions for RAMPS
in GPCR regulation [286]. In addition, we have recently reported novel roles for
RAMPS, through protein-protein interactions with NSF and NHERF, in the trafﬁcking of

the AM receptor subtypes.

68

Upon agonist binding, the CL-R/RAMP receptor complex causes cyclic AMP
activation in most systems, regardless of whether the ligand is AM or CGRP. In addition,
the receptor complex is phosphorylated by protein kinases and subsequently undergoes
desensitization and internalization in response to a prolonged agonist stimulation [280].
Intemalization of all three RAMP isoforms with CL-R has been reported to be Clathrin-
mediated and dynamin-dependent. In addition, the CL-R/RAMP] receptor complex is
known to internalize with agonist pretreatment in a complex with the B-arrestin molecule
and all three components colocalize to the endosomes. From this stage, the receptor
complex is targeted for a recycling or degradation pathway, depending on the cell type
and RAMP expression proﬁle.

Phosphorylation has been demonstrated to be a requisite step in the
desensitization process of the majority of GPCRS. The phosphorylation of the receptor,
upon agonist exposure, allows interaction of the receptor with the B-arrestin molecules to
promote an attenuation of receptor signaling, or receptor desensitization. Receptor
desensitization can occur by two mechanisms, termed homologous and heterologous
desensitization. Homologous desensitization is Classiﬁed as desensitization speciﬁc for
the agonist-occupied receptor. This occurs as a result of G-protein coupled receptor
kinase (GRK) speciﬁcity for only agonist-occupied receptors. On the other hand,
heterologous desensitization occurs by activation of second messenger kinases, PKA and
PKC for example, and has no Speciﬁcity for agonist-occupied receptors. Heterologous
desensitization raises the possibility of “cross-desensitization” for unoccupied receptors.

Upon ligand binding, it has been reported that CL-R undergoes phosphorylation,

while the RAMP partner remains unphosphorylated. Moreover, PKA has been shown to

69

regulate the phosphorylation and desensitization of the CL-R/RAMP] receptor complex
in SK—N-MC and vascular smooth muscle cells, while GRK-6 was reported to
phosphorylate and promote desensitization of the CL-R/RAMP] complex in HEK 293
cells [200-202]. Kinases responsible for the phosphorylation of the CL-R/RAMP2 or -3
receptor complexes have yet to be speciﬁcally Characterized.

The ﬁndings in this study reveal that different RAMP isoform association with
CL—R may lead to differential phosphorylation of the receptor complex. The Ser 421
residue (a putative PKA phosphorylation site) on CL-R was found to be critical for the
desensitization and internalization of the CL-R/RAMP2 receptor complex, whereas the
Thr 423 residue (a putative PKA phosphorylation site) on the C-terminus of CL-R was
found to be crucial for the CL-R/RAMP3 receptor desensitization and internalization.
CL-R-R2 complex desensitization and internalization was blocked by H89, a PKA
inhibitor, while CL-R-R3 complex desensitization and internalization was blocked by RO
32-0432, a PKC inhibitor. Taken together, these results suggest that in addition to the
various roles of RAMPS in the regulation of CL-R/RAMP complex as reported by us and
others, the isoform of RAMPS could also be involved in the differential phosphorylation

of CL-R.

4.2. Materials and Methods

4.2.1. Materials: Adrenomedullin was purchased from Bachem Bioscience, Inc. (King
of Prussia, PA). 1251-labeled adrenomedullin was purchased from Amersham

Biosciences Corp. (Piscataway, NY). Cell culture media, fetal bovine serum,

70

penicillin/streptomycin, trypsin-EDTA were purchased from GibcoBRL® (Grand Island,
NY). RAMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-rabbit Cy3 secondary antibody was from Jackson Immunoresearch
Laboratories (West Grove, PA). PKA (H-89) and PKC (Ro 32-0432) inhibitors were
purchased from Sigma (St. Louis, MO). All other reagents were of highest quality

available.

4.2.2. Cell Culture and Transfection protocols: HEK-293T cells (obtained from
ATCC) are maintained in DMEM low glucose media containing 10% F BS, 1%
penicillin-streptomycin. Rat-2 ﬁbroblast cells (obtained from ATCC) are maintained in
DMEM high glucose media containing 10% FBS, 1% penicillin-streptomycin.
Transfection of HEK293T and Rat-2 ﬁbroblast cells was performed using Lipofectamine
Plus protocol (Invitrogen Life Technologies, Carlsbad, CA). Cells were transfected with
the DNA and Lipofectamine Plus as per manufacturer’s protocol. Cells were collected for

assays aﬁer 48 hours of transfection.

4.2.3. RAMP cloning and expression: Full length CDNA of human RAMPS], 2 and 3
and bovine CL-R were described before [281, 282]. CL-R, cloned into Nl-EGFP and

also in pcDNA3.1 expression vectors, was used for transfection in HEK 293T cells.

4.2.4. Desensitization assays: 48 hours post-tranfection cells were pretreated with or

without 10 nM AM in DMEM containing 0.2% BSA for indicated time periods (up to 4

71

hr). After agonist exposure, cells were washed three times with Dulbecco’s phosphate
buffered saline (dPBS, Gibco BRL) containing 0.2% BSA and either frozen for
membrane preparation for adenylate cyclase assays or used immediately for intact-cell

radioligand binding.

 

Desensitization Protocol:

Removal of AM,
Cells AM Pretreatment [- followed by washing

.......1 .... 7

¥ Y I \ Receptor activity assay
48 ours 1 hr

 

/ (CAMP accumulation) and
receptor binding
No Pretreat 1
(Full Stimulation) 1 hr AM
Pretreat
(Desensitization)

 

 

 

4.2.5. Receptor binding: Homologous competition radioligand binding assays were
performed as described by Aiyar et al and as established in our laboratory [283]. HEK
293T cells were transfected and approximately 200,000 cells/well seeded in poly-D-
lysine precoated 24-well plates (BD Biosciences, Palo Alto, CA). 48 hours post-
tranfection cells were treated for desensitization or resensitization assays as described
above. After agonist exposure, cells were washed three times with dPBS buffer
containing 0.2% BSA then incubated with increasing concentrations (1 pM to 100 nM) of
competing ligand (rAM) and 175-250 pM 125I-rAM for 30 min at 37°C. After incubation,

plates were washed three times with ice-cold assay buffer and the reactions were

72

terminated by the addition of 2M NaOH. Cells were then harvested and associated
radioligand activity is counted on a y-counter. All binding assays were performed in
duplicate, with each experiment repeated at least three times. Nonspeciﬁc binding was
determined in the presence of 100 nM of unlabeled rAM. Data was analyzed by
LIGAND (assuming radioligand and competitor both bind reversibly to a Single binding
site; MacLigand, Version 4.97, NIH, Bethesda, MD) using the following equation:

Bmax 0 [hot ligand]

 

KD + [hot ligand] + [cold ligand]

Where B represents speciﬁc binding, [hot ligand] the single concentration of [1251]rAM
studied, [cold ligand] the concentration of unlabeled rAM competing with the radiolabel
for AM receptor binding, Bmax the maximum number of binding sites and KD the
equilibrium dissociation constant (the equation was solved where the “cold ligand” ICso =
[hot ligand] + KD). Analysis of all binding data was performed by computer-assisted
nonlinear least square ﬁtting using GraphPad PRIZM version 4 (GraphPad Software, San
Diego, CA). Binding sites/cell was calculated and data was expressed as percent of the

control.

4.2.6. Adenylate cyclase assays: Cyclase activity was done as described before with
slight modiﬁcations [146, 284]. Cells were harvested from P100 or P60 plates and
homogenized in Tris HC] (10mM)/EDTA (10mM) buffer. Membranes were prepared by
homogenization and centrifugation in Tris HCl (50mM)/MgCl (10mM) buffer. Final

concentration of 20 pg of protein/assay tube was obtained. Membranes were incubated

for 15 min at 30°C with appropriate concentrations of drugs (100nM AM, lOpM

73

Forskolin) and assay mix containing ATP regeneration system and a32P-ATP. After the
reaction was stopped (with stop solution containing 3H-CAMP) contents of the assay
tubes were passed through Dowex and subsequently through alumina columns to separate
the degradation products of ATP, by washing the dowex with water and alumina with
imidazole. Elution proﬁle was done to determine the amount of water and imidazole
needed to wash and elute the products. Product eluted from alumina column was counted
for the presence of 3H-CAMP and a32P-CAMP in a B-Counter. Each experiment was done
in triplicates and repeated at least 3 times. Data is expressed as percent maximal
response, % forskolin. Basal cyclase activity and forskolin stimulation did not Show

statistically signiﬁcant differences between treatments.

4.2.7. CAMP accumulation assays: HEK 293 cells were seeded on a 24-well plate until
reaching 80-90% conﬂuency, then incubated in serum-free media overnight before
experiment. Desensitization experiments were carried out as described in Materials and
Methods section, with cells pretreated with 10nM rAM and subsequently Challenged for
10 min at 37°C with appropriate concentrations of drugs (100nM AM, 10nM Forskolin)
in the presence of ZOOpM 3-isobutyl-l-methylxanthine. Determination of CAMP level
was measured using the biotrak CAMP enzyme immunoassay system (Amersham
Biosciences) according to the manufacturer's instructions. CAMP levels in HEK 293 cells
were calculated using a standard curve ranging from 10 to 10 4 frnol of CAMP. Each
experiment was done in duplicate and repeated at least 3 times. Data is expressed as
percent maximal response, % forskolin. Basal cyclase activity and forskolin stimulation

did not Show statistically signiﬁcant differences between treatments.

74

4.2.8. Mutagenesis procedure: Site-directed mutagenesis was performed using a PCR-
based strategy that employs the pfu Turbo polymerase (Stratagene, La Jolla, CA). A pair
of complementary oligonucleotides containing the appropriate point mutations in the
sequence of RAMP or a premature stop codon at position 145 or 147 codon of RAMP-3
for deletion mutants were synthesized (Michigan State University Macromolecular

structure facility). The PCR for the mutation was as follows: 94°C for 2 minutes; 30

cycles of 94°C for 30 sec., 50°C for 30 seC., 68°C for 8 min.; ﬁnal cycle of 68°C for 8
minutes. PCR product was digested for 4 hours with DpnI enzyme (lnvitrogen) and
transformed in to DH50t cells. Mutations were conﬁrmed by automated sequencing
(Michigan State University Genomic Technology Support Facility). Putative
phosphorylation sites and kinase consensus sites were identiﬁed with NetPhosK Server

1.0 database (http://www.cbs.dtu.dk/services/NetPhosK).

 

4.2.9. Immunoﬂuorescence microscopy: HEK 293 cells were transfected as described
above and seeded at 24hr post-transfection onto collagen type I-Coated coverslips.
Resensitization assays were performed as described and reactions were stopped by ﬁxing
cells in 4% paraforrnaldehyde for 30min. at room temperature. Samples were
perrneablized with 0.1% v/v Triton X-100 in PBS and blocked overnight in 0.1% v/v
Triton X-100 in PBS + 10% goat serum. Samples were incubated in primary antibody in
blocking buffer for 2h at room temperature (NSF at 1:250 and RAMP3 at 1:200).
Appropriate secondary antibodies were applied for 1h at room temperature (Goat anti-

mouse Cy3 at 1:500 and Goat anti-rabbit Cy5 at 1:400). Coverslips were mounted in

75

Shandon Permaﬂuor mounting medium and slides stored at 2-8°C until analysis. Cells
were visualized on a Zeiss 210 laser confocal microscope at a zoom of 2. Images
presented are representative single optical sections of a z-series taken from at least twenty
ﬁelds per experiment and at least three individual experiments. Images in this

thesis/dissertation are presented in color.

4.2.10. Statistics: Data are presented as mean i S.E.M. Single group comparisons

exercised a paired Student’s t-test method. Statistical signiﬁcance was set at P<0.05.

4.3. Results

We and other groups have previously shown that the AM2 receptor (CL-R +
RAMP3 complex) undergoes agonist-stimulated desensitization, internalization, and
degradation in HEK 293 cells [153, 276, 285]. Furthermore, we have recently shown that
in the presence of NSF, the AM2 receptor complex undergoes recycling, instead of
following a degradation pathway, in HEK 293 cells [153]. We have also demonstrated
that the presence of the adaptor protein, NHERF, in HEK 293 cells can inhibit the
internalization, but not the desensitization, of the AM2 receptor complex through protein-
protein interactions. Given the recognized role of RAMPS in the trafﬁcking of the AM
receptor subtypes, in this study we have examined the role of RAMPS in regulating the
phosphorylation of the AM receptor subtypes in the desensitization and internalization
processes.

Pretreatment of HEK 293 cells expressing CL-R and RAMP2 or RAMP3 with 10

nM AM for one hour resulted in desensitization of the CAMP accumulation response

76

from approximately 40% (of forskolin stimulation) in untreated cells to 15% in AM-
treated cells (Figure 7A, B). In addition, as shown in FigureIOC and 11C, respectively,
CL-R/RAMP2 or CL-R/RAMP3 complex underwent agonist-induced internalization as
determined by receptor binding (Figure 7A, B) and immunoﬂuorescence microscopy.
These ﬁndings are in agreement with those of Kuwasako et al. and as reported by us
recently [153, 276]. Using site-directed mutagenesis, we mutated Clusters of putative
phosphorylation residues in the C-terminus of CL-R to alanine residues and tested the
effect of these mutations on desensitization and internalization of the receptor complexes
(Figure 8). Mutant CL-R/RAMP receptor complexes showed similar levels of receptor
stimulation and receptor complex expression levels as compared to wild-type CL-
R/RAMP receptor complex, as determined by CAMP accumulation and whole-cell ligand
binding experiments, respectively (Figure 9A, C; data not shown). Additionally, the CL-
R Cluster mutants showed similar receptor stimulation and receptor complex expression
when co-expressed with RAMP2 or RAMP3 (determined by CAMP accumulation and
whole-cell binding assays, respectively). Interestingly, when desensitization assays were
performed on the Cluster mutants of CL-R in complex with RAMP2 or RAMP3, the same
cluster mutant of CL-R showed reduced levels of receptor desensitization when
complexed with RAMP2 or RAMP3 (Figure 9A, C, respectively). Cluster mutant #2 of
CL-R, when in complex with RAMP2 or RAMP3, showed no signiﬁcant receptor
desensitization with AM pretreatment, as compared to wild-type CL-R and Cluster
mutants #1 and #3. In addition, when receptor complex internalization was measured
with whole-cell binding experiments, AM pretreatment also failed to promote signiﬁcant

internalization of the Cluster mutant #2 of CL-R when it was complexed with RAMP2 or

77

RAMP3 (Figure 93, D, respectively). Wild-type CL-R and Cluster mutants #1 and #3
showed similar levels of receptor internalization with one hour AM pretreatment.
Individual residues within the second Cluster of CL-R were then examined for their role
in regulation of receptor desensitization and internalization of the CL-R/RAMP receptor
complex.

Site-directed mutagenesis was again employed to create the Single mutations of
putative phosphorylation residues within the second Cluster of CL-R, determined to be
critical for agonist-stimulated receptor complex desensitization and internalization,
regardless of RAMP in association with CL-R. Point mutants of putative
phosphorylation residues to alanine residues were CO-expressed with RAMP2 or RAMP3
in HEK 293 cells and desensitization and internalization assays were performed. Point
mutants of CL-R when complexed with RAMP2 or RAMP3 Showed similar levels of
receptor stimulation and receptor complex expression as compared to wild-type CL-
RfRAMP complexes (determined by CAMP accumulation and whole-cell binding assays,
respectively). Additionally, no differences in receptor stimulation or receptor complex
expression were seen when CL-R point mutants were expressed with RAMP2 or
RAMP3. Using CAMP accumulation assays to determine receptor desensitization, only
Ser 421 mutant of CL—R, when complexed with RAMP2, Showed a loss of receptor
desensitization as compared to wild-type CL-R/RAMP2 receptor complex (Figure 10A).
Whole-cell binding and immunoﬂuorescence microscopy determined Ser 421 of CL-R to
also be critical to the agonist-stimulated internalization of the CL-R/RAMP2 receptor
complex (Figure 10B, C). The additional point mutants of CL—R, when co-expressed

with RAMP2, showed no signiﬁcant Changes in agonist-induced receptor desensitization

78

or internalization as compared to wild-type CL-R/RAMP2 receptor complex (determined
by CAMP accumulation and whole-cell binding assays, respectively).

Meanwhile, co—expression of the point mutants of CL-R with RAMP3 showed a
different putative phosphorylation residue critical to receptor desensitization and
internalization. While all point mutants of CL-R, when complexed with RAMP3,
showed similar levels of receptor stimulation and receptor complex expression as
compared to wild-type CL—R/RAMP3 complexes (determined by CAMP accumulation
and whole-cell binding assays, respectively), the Thr 423 mutant of CL-R showed a lack
of signiﬁcant receptor desensitization when pretreated with AM, compared to an
approximate 50% attenuation of receptor signaling seen for the additional point mutants
and wild-type CL-R co-expressed with RAMP3 (as measured by CAMP accumulation
assays) (Figure 11A). In addition, mutation of Thr 423 of CL-R to alanine inhibited the
agonist-stimulated receptor internalization when co-expressed with RAMP3 in HEK 293
cells (as measured by whole-cell binding and immunoﬂuorescence microscopy) (Figure
11B, C, respectively). The additional point mutants of CL-R, when co-expressed with
RAMP3, showed no signiﬁcant Changes in agonist-induced receptor desensitization or
internalization as compared to wild-type CL—R/RAMP3 receptor complex (determined by
CAMP accumulation and whole-cell binding assays, respectively). These ﬁndings
indicate that the two RAMP isoforms differentially regulate the CL-R receptor in the
process of receptor desensitization and internalization.

To determine if mutation of putative phosphorylation residues on the third
intracellular loop of CL-R altered receptor desensitization or internalization when

complexed with RAMP2 or RAMP3, Site-directed mutagenesis of these residues to

79

alanine was performed. Desensitization assays, measured by CAMP accumulation,
showed no effect of third intracellular loop mutations on desensitization of CL-
R/RAMP2 or CL-R/RAMP3 receptor complex. The receptor activity was attenuated to
levels similar to wild-type CL-R, when mutant receptors were CO-expressed with RAMP2
or RAMP3 (Figure 12A, B). Third intracellular loop mutants of CL—R, in complex with
RAMP2 or RAMP3, showed no difference in receptor expression levels at the plasma
membrane (as measured with whole-cell binding) in untreated cells, as compared to wild-
type CL-R/RAMP2 or CL-R/RAMP3 complex (data not shown). These results indicate
that amino acid residues on the third intracellular loop of CL-R are not required for
desensitization or internalization of the CL—R/RAMP2 or CL-R/RAMP3 receptor
complex.

It was then important to determine if putative phosphorylation residues on the
RAMP isoforms contributes to the regulation of receptor complex desensitization and/or
internalization. Putative phosphorylation residues on the C-terminus of the RAMP2 and
RAMP3 isoforms were mutated to alanine residues and desensitization/internalization
assays were performed. The phosphorylation mutant RAMPS co-expressed with wild-
type CL-R showed no difference in receptor stimulation or receptor complex expression
levels at the plasma membrane (as measured with CAMP accumulation and whole-cell
binding, respectively) in untreated cells, as compared to wild-type CL-R/RAMP2 or CL-
R/RAMP3 complex (Figure 13A-D). Desensitization of the receptor complex was
unaltered with the RAMP phosphorylation mutants, in comparison to wild-type RAMP2
or RAMP3 in complex with CL-R (as measured by CAMP accumulation) (Figure 13A,

C). In addition, the RAMP phosphorylation mutations were unable to modify the

80

internalization of the CL-R/RAMP2 or CL-R/RAMP3 receptor complexes (Figure 13B,
D). No signiﬁcant differences were seen in the desensitization or internalization patterns
of the phosphorylation mutant RAMPS, when compared to wild-type RAMP2 or RAMP3
complexed with CL-R. These ﬁndings indicate that serine/threonine amino acid residues
of the RAMPS do not appear to play a role in regulation of the desensitization and
internalization processes of the CL-R/RAMP receptor complex.

Data presented so far supports the hypothesis that different CL-R/RAMP isoform
associations could result in differential phosphorylation of the AM receptor subtypes as a
means of regulating desensitization and internalization of the receptor complexes. It was
then important to identify the kinases regulating the desensitization and internalization of
the different CL-R/RAMP2 and CL-R/RAMP3 receptor complexes. Sequence analysis
of CL-R in the NetPhosK database (http://www.Cbs.dtu.dk/serviceS/NetPhosK) predicted
consensus sites for PKA and PKC phosphorylation on amino acid residues Ser 421 and
Thr 423 of CL-R, respectively. These amino acid residues were identiﬁed in earlier
experiments within this study to regulate desensitization and internalization of the CL-
R/RAMP2 and CL—R/RAMP3 receptor complexes, respectively. Therefore, it was
hypothesized that inhibition of these kinases would block receptor desensitization and
internalization of the appropriate AM receptor complex.

The Ser 421 amino acid residue of CL-R that was determined to be critical to the
agonist-stimulated desensitization and internalization of the CL-R/RAMP2 receptor
complex was predicted to be regulated by protein kinase A. In order to test if this kinase
was regulating the CL-R/RAMP2 receptor complex desensitization and internalization, a

speciﬁc protein kinase A (PKA) inhibitor, H—89, was used to perform desensitization and

8]

internalization assays. As a control, a protein kinase C (PKC) inhibitor, RO 32-0432,
was also tested. Cells pretreated with the PKC inhibitor and AM showed similar levels of
receptor desensitization and internalization as cells pretreated with only AM (Figure 14A,
B). On the other hand, cells pretreated with the PKA inhibitor in the presence of AM
showed an inhibition of both receptor desensitization and internalization of the CL-
R/RAMP2 receptor complex (Figure 14A, B). No Signiﬁcant differences were observed
in receptor CAMP stimulation and receptor complex expression levels in cells co-
expressing CL-R and RAMP2 that were pretreated with or without the PKA and PKC
inhibitors. These ﬁndings indicate that PKA, but not PKC, is involved in CL-R/RAMP2
desensitization and internalization, presumably by phosphorylating Ser 421 of CL-R.

PKC and PKA inhibitors were then used to test their effects on the desensitization
and internalization patterns of the CL-R/RAMP3 receptor complex. Because the Thr 423
on CL-R was a PKC consensus site, it was predicted that the PKC inhibitor would alter
the desensitization and internalization of the receptor complex. CAMP accumulation and
whole-cell binding assays showed that while the PKA inhibitor had no effect on the
desensitization and internalization of the CL-R/RAMP3 receptor complex, the PKC
inhibitor completely blocked the desensitization and internalization of the CL-R/RAMP3
complex (Figure 15A, B). These studies suggest that PKC, but not PKA, is involved in
the desensitization and internalization of CL-R/RAMP3 receptor complex, seemingly by
phosphorylation of Thr 423 of CL-R.

To further Characterize the role of PKA and PKC in the desensitization and
internalization of the AM] and AM2 receptors, respectively, the kinases were

individually activated and tested for their ability to induce receptor desensitization and/or

82

internalization. No signiﬁcant alterations were seen in the receptor expression levels or
basal or forskolin-stimulated CAMP accumulation when transfected HEK 293 cells were
pretreated with forskolin (10 TAM) or phorbol-l2-myristate-13-acetate (PMA, 1 nM) for
the given time periods (data not shown). As would be predicted, activation of PKA with
forskolin pretreatment resulted in efﬁcient desensitization and internalization of the
AMI, but not AM2 receptors (Figure 16A, B). PKA activation was capable of achieving
levels of desensitization and internalization comparable to that induced by AM in cells
transfected with CL-R and RAMP2. When transfected HEK 293 cells were pretreated
with PMA, to activate PKC, desensitization and internalization was observed for the
AM2, but not AM] receptors (Figure 16A, B). The PKC-induced desensitization and
internalization observed for cells expressing CL-R and RAMP3 was comparable to that
stimulated by AM. These ﬁndings are in agreement with the observations from the
kinase inhibitor studies (Figures 14, 15). This data conﬁrms a role for PKA in the
desensitization and internalization processes for the AM] receptor (CL-R+RAMP2), and

a role for PKC in the desensitization and internalization of the AM2 receptor.

83

- CAMP Accumulation

D Binding
— -100
-75
i
* ~50

I
N
0|

 

 

 

CAMP (nmollmg protein)
[°/o Forskolin]
licence %1
("ea/saus) xeui Bugpuga

 

 

 

 

 

 

 

No Pretreat AM Pretreat

Figure 7A: Desensitization and internalization of the AM] and AM2 receptor in HEK 293 cells.
CL-R/RAMP2 complex (AMI receptor) desensitizes and intemalizes in HEK 293 cells after
prolonged agonist stimulation. HEK 293 cells transiently transfected with CL-R and RAMP2
were treated for one hour with AM (10 nM) and then washed and receptor desensitization (CAMP
accumulation) and internalization (radioligand binding) were measured. CAMP accumulation is
shown on the left y-axis, expressed as percent maximal response (% forskolin stimulation), and
radioligand binding is shown on the right y-axis, expressed as percent control. * p S 0.05; n 2 4

experiments.

84

- CAMP Accumulation

   

 

 

 

 

 

 

 

 

DBinding
E 40- —~ -100 Q.
s a
O H —-
5g 30- -75 g”:
as i as
=0- 9 20- -50 g x
E .2 :75
= <1 .3: '7"
:9. 10- -25 “3
E 8
o 0' 0 5

No Pr'etreat AM Pfetreat

Figure 7B: Desensitization and internalization of the AM] and AM2 receptor in HEK 293 cells.
CL-R/RAMP3 complex (AM2 receptor) desensitizes and intemalizes in HEK 293 cells after
prolonged agonist stimulation. HEK 293 cells transiently transfected with CL-R and RAMP3
were treated for one hour with AM (10 nM) and then washed and receptor desensitization (CAMP
accumulation) and internalization (radioligand binding) were measured. CAMP accumulation is
shown on the left y-axis, expressed as percent maximal response (% forskolin stimulation), and
radioligand binding is shown on the right y-axis, expressed as percent control. * p S 0.05; n 2 4

experiments.

85

 

@renomedullin)

 

 

 

    
   

Plasma

membrane
T324 53293 3/841'416 RAMP

S419
S421
CLR Cluster #1 T423
34254426 Cluster #3
S428
Cluster #2 T431

8439
T450

Figure 8: Illustration of Cluster mutations of CL-R to investigate role of phosphorylation on AM
receptor desensitization and internalization. Site-directed mutagenesis techniques, described in
Materials and Methods section, were employed to mutate putative phosphorylation residues on C-
tenninus of CL-R to alanine residues. Cluster mutants (1-3), as depicted in ﬁgure, are used in

experiments reported in Figure 9A-D.

86

- CL-R+R2

D CL-R Cluster mutant 1
E21 CL-R Cluster mutant 2
D CL-R Cluster mutant 3

 

 

 

%‘ 60-

’6 .

A?“ ”I

21% 40"

2.2” w *
=\°20-I * *
'L

2

5 0-

 

No Prbtreat AM Pfetreat

Figure 9A: Role of phosphorylation on AM] and AM2 receptor desensitization and
internalization (Cluster mutations). Amino acid residues 421-428 are critical to desensitization of
AM] receptor. HEK 293 cells transiently transfected with CL-R/CL-R Cluster mutants and
RAMP2 were seeded in 48-well plates and treated for one hour with AM (10 nM) and then
washed and receptor desensitization (CAMP accumulation) was measured. After repeated wash
steps, cells were re-Challenged with 100 nM rAM for 15 minutes and plates were frozen.
Determination of CAMP level was measured using the Biotrak CAMP enzyme immunoassay
system (Amersham Biosciences) according to the manufacturer's instructions. CAMP levels in
transfected HEK 293 cells were calculated using a standard curve ranging from 10 to 104 fmol of
CAMP. Each experiment was done in duplicate and repeated at least 3 times. Data is expressed

as percent maximal response, % forskolin. * p S 0.05; n 2 3 experiments.

87

- CL-R+RAMP2

I: CL-R Cluster mutant 1
1:11 CL-R cluster mutant 2
D CL-R cluster mutant 3

100- F — F l

75-

50-

N
01
l

 

Binding max (sites/cell)
[% Control]
31'
3(-

 

 

 

 

 

 

 

 

 

No Pr'etreat AM Pfetreat

Figure 93: Role of phosphorylation on AM] and AM2 receptor desensitization and
internalization (Cluster mutations). Amino acid residues 421-428 are critical to AM] receptor
internalization. HEK 293 cells were CO-transfected with CL-R or CL-R cluster mutants and
RAMP2. 48h post-transfection, cells were pretreated with AM (10 nM) for one hour, wasth as
described in Materials and Methods, and receptor internalization was measured with whole-cell
binding using ”SI-rAM as the ligand and cold rAM as the competitor. Number of binding

sites/cell was estimated with GRAPHPAD PRISM software. * p S 0.05; n 2 3.

88

- CL-R+R3

E CL-R cluster mutant 1

D CL-R cluster mutant 2
. D CL-R Cluster mutant 3

010)
?O

 

co 45
O O
1 n

CAMP (nmollmg protein)
[% Forskolin]
N
R

 

jiiil

No Pretreat AM Pretreat

Figure 9C: Role of phosphorylation on AM] and AM2 receptor desensitization and
internalization (Cluster mutations). Amino acid residues 421-428 are critical to desensitization of
AM2 receptor. HEK 293 cells transiently transfected with CL-R/CL-R Cluster mutants and
RAMP3 were seeded in 48-well plates and treated for one hour with AM (10 nM) and then
washed and receptor desensitization (CAMP accumulation) was measured. After repeated wash
steps, cells were re-Challenged with 100 nM rAM for 15 minutes and plates were frozen.
Determination of CAMP level was measured using the Biotrak CAMP enzyme immunoassay
system (Amersham Biosciences) according to the manufacturer's instructions. CAMP levels in
transfected HEK 293 cells were calculated using a standard curve ranging from 10 to 104 fmol of
CAMP. Each experiment was done in duplicate and repeated at least 3 times. Data is expressed

as percent maximal response, % forskolin. * p S 0.05; n 2 3 experiments.

89

- CL-R+RAMP3

D CL-R Cluster mutant 1
1:1 CL-R Cluster mutant 2
D CL-R Cluster mutant 3

 

 

 

g 125- l
E H 100
177.9
g g 75' * *
E U 50 *
\° -1
EL
2 251

 

No Prbtreat AM Pfetreat

Figure 9D: Role of phosphorylation on AM] and AM2 receptor desensitization and
internalization (Cluster mutations). Amino acid residues 421-428 are critical to AM2 receptor
internalization. HEK 293 cells were co-transfected with CL-R or CL-R Cluster mutants and
RAMP3. 48h post-transfection. cells were pretreated with AM (10 nM) for one hour, washed as
described in Materials and Methods. and receptor internalization was measured with whole-cell
binding using ”SI-rAM as the ligand and cold rAM as the competitor. Number of binding

sites/cell was estimated with GRAPHPAD PRISM software. * p S 0.05; n 2 3.

90

h
o
I
I—1

- WT CL-R+R2
CL-R S421A

  
      
    

     
 

30' E i: CL-R Y422A
5;; i: CL-R T423A
its: :3 CL-R S425A

"4 mm CL-R T426A

A
O
I

CL-R S428A

  

 

 

 

’///////////////////////////A

FWWFZZ‘?‘
ﬁwﬁ‘hm ,v , L2...

 

CAMP (nmollmg protein)
[% Forskolin]
N
R

NoiPretreat AM Pretreat

Figure 10A: Role of phosphorylation on AM] receptor desensitization and internalization (point
mutations). Residue Ser 421 is crucial to desensitization of AM] receptor. HEK 293 cells
transiently transfected with CL-R/CL-R point cluster mutants and RAMP2 were seeded in 48-
well plates and treated for one hour with AM (10 nM) and then washed and receptor
desensitization (CAMP accumulation) was measured. After repeated wash steps, cells were re-
challenged with 100 nM rAM for 15 minutes and plates were frozen. Detemtination of CAMP
level was measured using the Biotrak CAMP enzyme immunoassay system (Amersham
Biosciences) according to the manufacturer's instructions. CAMP levels in transfected HEK 293
cells were calculated using a standard curve ranging from 10 to 10 " fmol of CAMP. Each
experiment was done in duplicate and repeated at least 3 times. Data is expressed as percent

maximal response, % forskolin. * p S 0.05; n 2 3 experiments.

91

100-

N
‘1'

[% Control]
0|
R

25d

Binding max (sites/cell)

 

   

1 :‘
.7 h. z .
. ‘ .1, 44‘
., 1;. f:
"'-. . l
is» ‘. i. ,
> *
4 1 ‘43; '5.
'H : l I
1. l l ‘
' i 1 I} .
'3‘ 4
'4. .I: .0 .
. . 1' ,9;
2 . 3.: ..
.4, ll.‘ 1 .
1'4, [pp
, ii 4
.. 2: I1 :"‘
.14 u ; -.
,; il l ».,
7: ill '-'
_.,. . w .
_/ l l w .
.‘9 1 ,l -§ , .
.;. i = , a.
7 ‘l‘. '4:
7 :» ‘v . J
r. e 1. .2
1 .

No Pretreat AM Pretreat

- WT CL-R+ R2
1:31 CL-R S421A
E CL-R Y422A
E: CL-R T423A
- CL—R S425A
E21 CL-R T426A
:21 CL-R S428A

Figure 108: Role of phosphorylation on AM] receptor desensitization and internalization (point

mutations). Residue Ser 421 is critical to AM] receptor internalization. HEK 293 cells were co-

transfected with CL-R or CL-R point mutants and RAMP2. 48h post-transfection, cells were

pretreated with AM (10 nM) for one hour, washed as described in Materials and Methods, and

receptor internalization was measured with whole-cell binding using ”SI-rAM as the ligand and

cold rAM as the competitor. Number of binding sites/cell was estimated with GRAPHPAD

PRISM software. * p S 0.05; n z 3.

92

WT CL-R RAMP2 ()Vcrluy

No
Prctrcat

AM
Pretreat

 

 

CL-R S421A RAMP2 ()\Crla_\'
No

Pretreat

AM
Pretreat

   

Figure 10C: Role of phosphorylation on AM] receptor desensitization and intemalization (point
mutations). lmmunoﬂuorescence microscopy shows failure of CL-R S421A/RAMP2 receptor
complex to internalize after agonist stimulation. HEK 293 cells transfected with CL-R S421A-
GFP and RAMP2 were pretreated with 10nM AM for lh. After pretreatment with AM, cells
were washed, ﬁxed, and components were visualized using anti-RAMP2 antibody (1:150) and
detected with Cy3 secondary antibody (1:250), and CL-R is detected with an EGFP tag; overlays
of staining patterns are shown in the far right panels. Images shown are representative of at least
twenty ﬁelds imaged per experiment from at least three experiments. Bar scales on all images

represent lOOum.

93

 

 

 

 

 

 

 

E 40-

3 - WT CL-R+R3
g? 30. p f i:iCL-R S421A
m .3 ;,,_ i:i CL-R Y422A
E if m CL-R T423A
T: o 20‘ :iCL-R S425A
g “t CL-R T426A
IE- 10- - CL-R S428A
2

<

o .

No Pretreat AM Pretreat

Figure 11A: Role of phosphorylation on AM2 receptor desensitization and internalization (point
mutations). Residue Thr 423 is crucial to desensitization of AM2 receptor. HEK 293 cells
transiently transfected with CL-R/CL-R point mutants and RAMP3 were seeded in 48-well plates
and treated for one hour with AM (10 nM) and then washed and receptor desensitization (CAMP
accumulation) was measured. After repeated wash 5th5, cells were re-Challenged with 100 nM
rAM for 15 minutes and plates were frozen. Determination of CAMP level was measured using
the Biotrak CAMP enzyme immunoassay system (Amersham Biosciences) according to the
manufacturer's instructions. CAMP levels in transfected HEK 293 cells were calculated using a
standard curve ranging from 10 to 10 ’ fmol of CAMP. Each experiment was done in duplicate
and repeated at least 3 times. Data is expressed as percent maximal response, % forskolin. * p

5 0.05; n 2 3 experiments.

94

           

% 100- , g
u _
E _ E -WT CL-R+ R3
,3 = 75. - g: * IZICL-R S421A
3.2 - * i: CL-R Y422A
a: 8 50. EZICL-R T423A
E g 1g ‘ - CL-R S425A
EL- ‘ ; m CL-R T426A
'2 25' :i 1:! CL-R S428A
is

04 ‘

 

No Pretreat AM Pretreat

Figure 11B: Role of phosphorylation on AM2 receptor desensitization and internalization (point
mutations). Residue Thr 423 is critical to AM2 receptor internalization. HEK 293 cells were co-
transfected with CL-R or CL-R point mutants and RAMP3. 48h post-transfection, cells were
pretreated with AM (10 nM) for one hour. washed as described in Materials and Methods, and
receptor internalization was measured with whole-cell binding using 125l—rAM as the ligand and
cold rAM as the competitor. Number of binding sites/cell was estimated with GRAPHPAD

PRISM software. * p S 0.05; n 2 3.

95

WT (‘L-R RAMP3 ()\ Ci‘lziy

No
Pretreat

CL-R T423A

 

AM
Pretreat

   
 

No
Pretreat

AM
Pretreat

Figure 11C: Role of phosphorylation on AM2 receptor desensitization and internalization (point
mutations). lmmunoﬂuorescence microscopy shows failure of CL-R T423A/RAMP3 receptor
complex to internalize after agonist stimulation. HEK 293 cells transfected with CL-R T423A-
GFP and RAMP3 were pretreated with 10nM AM for 1h. After pretreatment with AM, cells
were washed, ﬁxed, and components were visualized using anti-RAMP3 antibody (1:150) and
detected with Cy3 secondary antibody (1:250), and CL-R is detected with an EGFP tag; overlays
of staining patterns are shown in the far right panels. Images shown are representative of at least
twenty ﬁelds imaged per experiment from at least three experiments. Bar scales on all images

represent lOOum.

96

- No Pretreat

 

 

 

 

 

 

 

 

 

       

1:21AM Pretreat
A 40-
c
E
-' 30
> at
'4: x *
2 E. g 20- :r. * .1. *
2 g IL H I [t
B E- 10-
E l
9.
OJ _ l — d _—
v? 019 v
939)“ ix" *9)“ is" *9}: is" *0“

Figure 12A: Role of phosphorylation of residues in third intracellular loop of CL-R on AM] and
AM2 receptor desensitization. Phosphorylation of third intracellular loop residues of CL-R not
vital in AM 1 receptor desensitization. HEK 293 cells transiently transfected with CL-R/CL-R
point mutants and RAMP2 were seeded in 48-well plates and treated for one hour with AM (10
nM) and then washed and receptor desensitization (CAMP accumulation) was measured. After
repeated wash steps, cells were re-Challenged with 100 nM rAM for 15 minutes and plates were
frozen. Determination of CAMP level was measured using the Biotrak CAMP enzyme
immunoassay system (Amersham Biosciences) according to the manufacturer's instructions.
CAMP levels in transfected HEK 293 cells were calculated using a standard curve ranging ﬁ'om
10 to 10 4 ﬁrm] of CAMP. Each experiment was done in duplicate and repeated at least 3 times.

Data is expressed as percent maximal response, % forskolin. * p S 0.05; n 2 3 experiments.

97

- No Pretreat
D AM Pretreat

[°/o Forskolin]
w e.
? 9'

AC Activity
(pmollminllmg protein)
25

 

 

 

 

 

 

 

 

0.:
W"

«P “V3

9.9.

‘5
9.?
“a: k k 93‘ :c

0“”

Figure 128: Role of phosphorylation of residues in third intracellular loop of CL-R on AM] and
AM2 receptor desensitization. Third intracellular loop residues of CL-R not phosphorylated in
desensitization of AM2 receptor. HEK 293 cells transiently transfected with CL-R/CL-R point
mutants and RAMP3 were seeded in 48-well plates and treated for one hour with AM (10 nM)
and then washed and receptor desensitization (CAMP accumulation) was measured. After
repeated wash steps, cells were re-Challenged with 100 nM rAM for 15 minutes and plates were
frozen. Determination of CAMP level was measured using the Biotrak CAMP enzyme
immunoassay system (Amersham Biosciences) according to the manufacturer's instructions.
CAMP levels in transfected HEK 293 cells were calculated using a standard curve ranging from
10 to 10 4 fmol of CAMP. Each experiment was done in duplicate and repeated at least 3 times.

Data is expressed as percent maximal response, % forskolin. * p _<_ 0.05; n 2 3 experiments.

98

- CL-R+RAMP2
E CL-R+RAMP2AS168,171A

 

CAMP (nmollmg protein)
[% Forskolin]
N
‘P

 

 

 

if]

No Pr'etreat AM Pretreat

 

0d

 

Figure 13A: Role of phosphorylation of RAMPS on AM] and AM2 receptor desensitization and
internalization. Phosphorylation of residues on RAMP2 not essential to desensitization of AM]
receptor. HEK 293 cells transiently transfected with CL-R and RAMP2/RAMP2 phosphorylation
mutants were seeded in 48—well plates and treated for one hour with AM (10 nM) and then
washed and receptor desensitization (CAMP accumulation) was measured. After repeated wash
steps, cells were re-Challenged with 100 nM rAM for 15 minutes and plates were frozen.
Determination of CAMP level was measured using the Biotrak CAMP enzyme immunoassay
system (Amersham Biosciences) according to the manufacturer's instructions. CAMP levels in
transfected HEK 293 cells were calculated using a standard curve ranging from 10 to 10 " fmol of
CAMP. Each experiment was done in duplicate and repeated at least 3 times. Data is expressed

as percent maximal response, % forskolin. * p S 0.05; n 2 3 experiments.

99

- CL-R+RAMP2
D CL-R+RAMP2$168,171A

100-

*

1'.

Binding max (sites/cell)
[°/o Control]
0|
8’

 

 

 

 

 

 

 

 

 

O
I

No Pr'etreat AM Pretreat

Figure 138: Role of phosphorylation of RAMPS on AM] and AM2 receptor desensitization and
internalization. Phosphorylation of RAMP2 is not critical to AMI receptor internalization. HEK
293 cells were co-transfected with CL-R and RAMP2/RAMP2 phosphorylation mutants. 48h
post-transfection, cells were pretreated with AM (10 nM) for one hour, washed as described in
Materials and Methods, and receptor internalization was measured with whole-cell binding using
12sl-rAM as the ligand and cold rAM as the competitor. Number of binding sites/cell was

estimated with GRAPHPAD PRISM software. "' p S 0.05; n 2 3.

100

- CL-R+RAMP3
D CL-R+RAMP3AS141T144,146A

h
C
I

N
O
n

E'— .

A
O .
n

 

 

 

 

 

CAMP (nmollmg protein)
[°/o Forskolin]
N
‘P

if]

No Pr'etreat AM Pretreat

O
I

Figure 13C: Role of phosphorylation of RAMPS on AM] and AM2 receptor desensitization and
internalization. Phosphorylation of residues on RAMP3 not required for desensitization of AM2
receptor. HEK 293 cells transiently transfected with CL-R and RAMP3/RAMP3 phosphorylation
mutants were seeded in 48-well plates and treated for one hour with AM (10 nM) and then
washed and receptor desensitization (CAMP accumulation) was measured. Aﬂer repeated wash
steps, cells were re-Challenged with l00 nM rAM for 15 minutes and plates were frozen.
Determination of CAMP level was measured using the Biotrak CAMP enzyme immunoassay
system (Amersham Biosciences) according to the manufacturer's instructions. CAMP levels in
transfected HEK 293 cells were calculated using a standard curve ranging from ID to 104 frnol of

CAMP. Each experiment was done in duplicate and repeated at least 3 times. Data is expressed

as percent maximal response, % forskolin. * p S 0.05; n 2 3 experiments.

10]

- CL-R+RAMP3
E CL-R+RAMP3S141T144,146A

100-

   

Binding max (sites/cell)
[°/o Control]
$

 

 

 

 

 

 

 

O
I

No Pfetreat AM Pretreat

Figure 13D: Role of phosphorylation of RAMPS on AM] and AM2 receptor desensitization and
internalization. Phosphorylation of RAMP3 is not vital for AM2 receptor internalization. HEK
293 cells were co-transfected with CL-R and RAMP3/RAMP3 phosphorylation mutants. 48h
post-transfection, cells were pretreated with AM (10 nM) for one hour, washed as described in
Materials and Methods, and receptor internalization was measured with whole-cell binding using
I2SI-rAM as the ligand and cold rAM as the competitor. Number of binding sites/cell was

estimated with GRAPHPAD PRISM software. * p S 0.05; n 2 3.

102

- CL-R+RAMP2
El CL-R+RAMP2 (H-89)
CL-R+RAMP2 (R0 32-0432)

h
C
I

.T. I.

09
O
I

A
O
I

 

  

 

 

 

 

 

 

No Pretreat AM Pretreat

CAMP (nmollmg protein)
[% Forskoli ]
N
C

Figure 14A: Role of kinases in AMI receptor desensitization and internalization.
Phosphorylation of residues of CL-R for desensitization and internalization of AMI receptor is
sensitive to H-89 in HEK 293 cells. Cells transiently transfected with CL-R and RAMP2 were
seeded in 48-well plates and treated for one hour with AM (10 nM) with and without H-89 (1 pM)
and Ro 32-0432 (3 uM) treatment, then washed, and receptor desensitization (CAMP
accumulation) was measured. Aﬁer repeated wash steps, cells were re-Challenged with 100 nM
rAM for 15 minutes and plates were frozen. Determination of CAMP level was measured using
the Biotrak CAMP enzyme immunoassay system (Amersham Biosciences) according to the
manufacturer's instructions. CAMP levels in transfected HEK 293 cells were calculated using a
standard curve ranging from 10 to 10 4 fmol of CAMP. Each experiment was done in duplicate
and repeated at least 3 times. Data is expressed as percent maximal response, % forskolin. * p

S 0.05; n 2 3 experiments.

103

- CL-R+RAMP2
D CL-R + RAMP2 (H-89)
IE: CL-R + RAMP2 (R0 32-0432)

125-
I.

§

7, 100' r-
g:

tag 75-

x c

u o

E: 50.

3°“

:3 25-

m

 

  

 

 

 

 

 

l {31“}

"'l' -r-
No Pretreat AM Pretreat

Figure 143: Role of kinases in AMI receptor desensitization and internalization.
Phosphorylation of residues of CL-R for internalization of AMI receptor is sensitive to H-89 in
HEK 293 cells. HEK 293 cells were co-transfected with CL-R and RAMP2. 48h post-
transfection, cells were pretreated with AM (l0 nM) for one hour with and without H-89 (luM)
and Ro 32-0432 (3uM) treatment, washed as described in Materials and Methods, and receptor
internalization was measured with whole-cell binding using ”SI-rAM as the ligand and cold rAM

as the competitor. Number of binding sites/cell was estimated with GRAPHPAD PRISM

software. * p S 0.05; n 2 3.

104

- CL-R+RAMP3
E CL-R+RAMP3 (H—89)
CL-R+RAMP3 (R0 32-0432)

A
O
I

I.

U
C
I

.x
O
I

     

 

 

 

 

CAMP (nmollmg protein)
[% Forskolin]
N
c.’

O
I_

No Pretreat AM Pretreat

Figure 15A: Role of kinases in AM2 receptor desensitization and internalization.
Phosphorylation of residues of CL-R for desensitization and internalization of AM2 receptor is
sensitive to RO 32-0432 in HEK 293 cells. Cells transiently transfected with CL-R and RAMP3
were seeded in 48-well plates and treated for one hour with AM (10 nM) with and without H-89
(luM) and Ro 32-0432 (3},LM) treatment, then washed, and receptor desensitization (CAMP
accumulation) was measured. After repeated wash steps, cells were re-Challenged with 100 nM
rAM for IS minutes and plates were frozen. Determination of CAMP level was measured using
the Biotrak CAMP enzyme immunoassay system (Amersham Biosciences) according to the
manufacturer's instructions. CAMP levels in transfected HEK 293 cells were calculated using a
standard curve ranging from l0 to 10 4 fmol of CAMP. Each experiment was done in duplicate
and repeated at least 3 times. Data is expressed as percent maximal response, % forskolin. * p

S 0.05; n 2 3 experiments.

105

- CL-R+RAMP3

 

 

 

 

 

 

[:1 CL-R+RAMP3 (RO 32-0432)
n CL-R+RAMP3 (H-89)
A 125-
g l
7, 100- —~
g ..."'.."
£3 75-
X C
a O *
E 3 5o.
3"“
E 25-
m ,
' '1‘ -I-
No Pretreat AM Pretreat
Figure 158: Role of kinases in AM2 receptor desensitization and internalization.

Phosphorylation of residues of CL-R for internalization of AM2 receptor is sensitive to R0 32-
0432 in HEK 293 cells. HEK 293 cells were co-transfected with CL-R and RAMP3. 48h post-
transfection, cells were pretreated with AM (IO nM) for one hour with and without H-89 (luM)
and Ro 32-0432 (3uM) treatment, washed as described in Materials and Methods, and receptor
internalization was measured with whole-cell binding using I25I-rAM as the ligand and cold rAM
as the competitor. Number of binding sites/cell was estimated with GRAPHPAD PRISM

software. "‘ p S 0.05; n 2 3.

106

- CL-R+RAMP2
D CL-R+RAMP3

II

N u
o o
l I

11

[°/o Forskolin]
2;;

CAMP Accumulation
(nmollmg protein)

 

 

 

 

 

   

04 _
633‘ 6°" C9?“ C0"

goq‘e le‘v‘e «3‘6 “P‘Q‘e
V

Figure 16A: Role of kinases in AM2 receptor desensitization and internalization.
Desensitization and internalization of AMI receptor is dependent on PKA activation, while the
AM2 receptor requires PKC activation. Cells transiently transfected with CL-R and RAMP2 or
RAMP3 were seeded in 48-well plates and treated either for one hour with AM (10 nM) or for 30
min. with forskolin (10 nM) or for 10 min. with PMA (1 uM), then washed, and receptor
desensitization (CAMP accumulation) was measured. After repeated wash steps, cells were re-
Challenged with 100 nM rAM for IS minutes and plates were frozen. Determination of CAMP
level was measured using the Biotrak CAMP enzyme immunoassay system (Amersham
Biosciences) according to the manufacturer's instructions. CAMP levels in transfected HEK 293
cells were calculated using a standard curve ranging from 10 to 10 4 fmol of CAMP. Each
experiment was done in duplicate and repeated at least 3 times. Data is expressed as percent

maximal response, % forskolin. "‘ p S 0.05; n 2 3 experiments.

107

- CL-R+RAMP2
E3 CL-R+RAMP3

125-
100- )—

 

an N
O 0|
I I

Binding Sites/Cell
[°/o Control]
«'3

 

 

 

 

 

 

 

 

 

O
k

Figure 168: Role of kinases in AM2 receptor desensitization and internalization.
Desensitization and internalization of AMI receptor is dependent on PKA activation, while the
AM2 receptor requires PKC activation. Cells transiently transfected with CL-R and RAMP2 or
RAMP3 were seeded in 48-well plates and treated either for one hour with AM (10 nM) or for 30
min. with forskolin (IO uM) or for IO min. with PMA (1 uM), washed as described in Materials
and Methods, and receptor internalization was measured with whole-cell binding using ”SI-rAM
as the ligand and cold rAM as the competitor. Number of binding sites/cell was estimated with

GRAPHPAD PRISM software. * p S 0.05; n 2 3.

108

4.4. Discussion

The process of agonist-promoted receptor desensitization and internalization is an
indispensable self-regulation mechanism of the GPCRS. These processes allow receptor
signaling to be Closely-monitored, providing a mechanism to promote receptor signaling
or receptor down-regulation. Receptors preserve this maintenance in many different
ways, with phosphorylation of the receptor ranking as one of the most common [196].
Receptor phosphorylation during the process of desensitization is thought to enable [3-
arrestin molecule interaction with the receptor, thus uncoupling the receptor from its
obligate G-protein and signaling for the assembly of the endocytic machinery for receptor
internalization. In this study we examined the role of phosphorylation in the
desensitization and internalization processes of the AM receptor subtypes. Putative
phosphorylation residues were found to play a vital role in the process of desensitization
and internalization of both AM receptor complexes. Notably, the two AM receptor
subtypes were regulated by different amino acid residues on CL—R. Namely, the serine
residue 421 of CL-R and threonine residue 423 of CL—R were responsible for the efﬁcient
receptor desensitization and internalization of the CL—R/RAMPZ and CL-R/RAMP3
receptor complexes, respectively. Protein kinase A inhibitor blocked only CL-R/RAMPZ
desensitization and internalization, while a PKC inhibitor blocked only CL-R/RAMP3
desensitization and internalization, suggesting different kinases are involved in the
phosphorylation of CL—R when different RAMP isoforms are associated. In addition, it
was demonstrated the phosphorylation of the RAMP proteins or the third intracellular
loop of CL-R was not required for AM receptor complex desensitization or

internalization.

109

A prolonged exposure of cells to a particular ligand results in an attenuation of
responsiveness to subsequent stimulation with that ligand. This phenomenon is called
desensitization, which can be homologous or heterologous. Homologous desensitization
is Classiﬁed as a decrease in the response to a particular ligand that is speciﬁc for the
stimulated receptor. Because G-protein regulated kinases (GRKS) are activated and only
phosphorylate GPCRS in the agonist-bound state, GRKS are capable of attenuating
receptor signaling by homologous desensitization [194, 195]. An attenuation of receptor
signaling that is the result of activation of second messenger signaling, and in principal
not speciﬁc for the agonist-bound receptor, is termed heterologous desensitization.
Protein kinase A and C are common kinases activated through second messenger
signaling of GPCRS, and therefore can activate heterologous receptor desensitization
[196]. While past studies in HEK 293 cells have identiﬁed homologous desensitization
as a means of regulating CL-R, this was only Characterized for the CL-R/RAMPI
receptor complex (CGRP-1 receptor) [20]]. Meanwhile, this study has identiﬁed
heterologous desensitization to play a crucial role in the regulation of AM signaling in the
HEK 293 cells, through both AM receptor subtypes.

The current literature characterizes the CL-R/RAMP receptor complex to undergo
heterologous desensitization in the majority of cell lines studied. Desensitization of CL-
R was found to involve CAMP-dependent protein kinase (PKA) activation in rat
mesangial cells (RMCS) stimulated with AM, SK-N-MC (neuroblastoma cell line) cells
stimulated with CGRP, Rat2 ﬁbroblast cells stimulated with AM, and vascular smooth
muscle cells (V SMCs) stimulated with CGRP [198-200, 287]. No studies to date have

reported regulation of desensitization of the CL-R/RAMP receptor complex by PKC.

110

Although, this kinase is found to promote heterologous desensitization of numerous other
GPCRS, for example the D2 dopamine [288], H1 histamine [289], serotonin 5-HT(2A)
[290], thromboxane receptor alpha [291], and alpha(2)-adrenergic receptors [292]. Data
from this manuscript demonstrates a requirement in HEK 293 cells of PKA
phosphorylation for AMI receptor desensitization/intemalization and PKC
phosphorylation for AM2 receptor desensitization/internalization.

Further studies are needed to deﬁne the implications of differential receptor
phosphorylation to the AM signaling pathway. It is tempting to hypothesize that the
different phosphorylation sites on CL-R when complexed with RAMP2 or RAMP3 may
allow different protein-protein interactions that dictate down-stream receptor signaling or
trafﬁcking. It could be predicted that the differential phosphorylation of the AMI and
AM2 receptors allow protein-protein interactions that allow the documented targeting of
the two receptor complexes for different pathways after endocytosis, degradative vs.
recycling pathways, respectively [153].

Understanding the mechanism of AM receptor desensitization is of great
importance given the documented protective role of AM in various cardiovascular and
renal disorders. In the cardiorenal disease states where AM is protective, circulating
plasma levels of AM have been shown to be increased. For example, in Chronic
glomerulonephritis, type I diabetes, and type II diabetes plasma AM levels are elevated
[293-295]. In addition, AM delivery through adenoviral injection has been shown to
decrease cardiac hypertrophy and renal damage in rat models of hypertension and
improves cardiac function and prevents renal damage in streptozotocin-induced diabetic

rats [296-300]. Because AM levels are chronically elevated in many of the above

111

cardiovascular and renal disorders, targeting receptor desensitization is a possible therapy
for these pathophysiologies. The inhibition of the process of desensitization for
additional GPCRS has been shown to elicit protective therapeutic effects for the disease

states associated with the receptors. Leﬂcowitz et. al. have shown that the inhibition of [3-

adrenergic receptor kinase (kinase that phosphorylates the B-adrenergic receptor to cause
desensitization) in the heart can delay the development of heart failure in multiple animal
models, in some cases even restoring cardiac function [203, 204]. Others have also
shown inhibition of desensitization of the u—opioid receptor to be beneﬁcial in preventing
morphine tolerance [205, 206].

This is the ﬁrst study to show that the RAMP isoforms are capable of dictating
differential phosphorylation of CL-R to regulate the AM receptor complex agonist-
stimulated desensitization and internalization. In addition, it has not previously been
shown that different kinases are responsible for the phosphorylation of the two AM
receptor subtypes (PKA for AMI receptor and PKC for AM2 receptor). This report
indicates yet another novel form of regulation of the AM receptor life-cycle by the
RAMP proteins. Given the recent report of RAMPS ability to interact with other
receptors in the family 11 of GPCRS, RAMPS may be playing a more prevalent role in the

regulation of GPCR life-cycle [286].

112

5. RAMP isoform-speciﬁc regulation of adrenomedullin receptor
trafﬁcking by NHERF-1.

5.1 Introduction

The recent discovery of receptor activity modifying proteins (RAMPS) has
broadened the ﬁeld of G-protein coupled receptor (GPCR) regulation. RAMPS were
discovered as required accessory proteins to an orphan GPCR, now termed the calcitonin
receptor-like receptor (CL-R) [275]. The three RAMP isoforms (1-3) are products of
three distinct genes and yield unique single transmembrane accessory proteins. RAMPS
are required for the plasma membrane expression and determination of receptor
phenotype for CL-R [275, 276]. RAMPS have recently been found to associate with
additional members of the Class 11 family of GPCRS [286]. Co-expression of RAMP]
with CL-R yields a functional calcitonin gene-related peptide (CGRP) receptor, while co-
expression of RAMP2 or -3 with CL-R produces a receptor responsive to adrenomedullin
(AM) (AMI-R and AM2-R, respectively) [277, 278]. AM and CGRP are multi-
functional peptides with many overlapping functions, ranging from potent vasodilation to
proliferation to regulation of salt and water balance [279]. The RAMP isoforms have
shown a differential expression patterns in different organ systems and in different
pathophysiological states, suggesting a regulatory role for RAMPS in both physiological
and pathophysiological situations. Furthermore, the identiﬁcation of RAMP interactions
with additional members of the Class II GPCR family and RAMP expression in cell lines
lacking CL-R have raised the possibility of novel functions for RAMPS in GPCR

regulation.

113

It has been shown in other GPCR systems that interactions with PSD-95/Discs-
large/ZO-l homology (PDZ) domain proteins are responsible for altering the receptor-
trafﬁcking after agonist stimulation [259, 267, 268]. In particular, a protein called
Na+/H” Exchange Regulatory Factor-1 (NHERF-1) has been shown to regulate the
trafﬁcking of the BZ-AR, K-OR, and PTH-R after agonist activation [240, 276, 301].
NHERF-1 is an adapter protein that is thought to tether membrane receptors to
cytoskeletal proteins through PDZ interactions and interactions with MERM family of
cytoskeletal proteins [257].

Comparable to the C-terminus of BZ-AR, PTH-R, CFTR, and PDGF-R, human
RAMP3 C-terminus contains a type-I PDZ recognition motif, whereas CL-R, RAMPl or
RAMP2 do not contain any PDZ recognition sequences [153, 154, 240, 250, 259, 302].
We hypothesized that RAMP3, via its interaction with NHERF-1, can regulate the
trafﬁcking of the CL-R/RAMP3 complex. We show here that while CL-R/RAMPI and
CL-R/RAMP2 complexes do not interact with NHERF-1, CL—R/RAMP3 complex
interacts with NHERF-l via the PDZ domain of NHERF-l [303]. Moreover, we show
here that over-expression of NHERF-l in HEK-293 cells alters the trafﬁcking pattern of
the receptor complex to block the receptor’s internalization by tethering the receptor
complex to the actin cytoskeleton via interactions between RAMP3 and NHERF-1
through a type I PDZ domain. Furthermore, we also demonstrate that in primary human
proximal tubule cells (which express endogenous NHERF-l and CL-R/RAMPB), the CL-
R/RAMP3 complex does not internalize upon agonist stimulation. Knocking down

NHERF-1 or RAMP3 expression with RNAi causes the receptor to undergo

114

internalization upon agonist treatment, suggesting critical roles for both NHERF and

RAMP3 in receptor internalization.

5.2. Materials and Methods

5.2.1. Materials: Adrenomedullin was purchased from Bachem Bioscience, Inc. (King
of Prussia, PA). 1251-labeled adrenomedullin was purchased from Amersham
Biosciences Corp. (Piscataway, NY). DMEM media, fetal bovine serum,
penicillin/streptomycin, trypsin-EDTA were purchased from GibcoBRL® (Grand Island,
NY). RAMP3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA)
and NHERF-1 (EBPSO) antibody was from Afﬁnity Bioreagents (Golden, CO). Alexa
488-phalloidin was purchased from Molecular Probes (Eugene, OR). Anti-mouse Cy3
and anti-rabbit Cy5 secondary antibodies were from Jackson Immunoresearch

Laboratories (West Grove, PA). All other reagents were of highest quality available.

5.2.2. Cell Culture and Transfection protocols: HEK-293T cells were obtained from
ATCC and are maintained in DMEM low glucose media containing 10% FBS,
1%penicillin-streptomycin. Transfection of HEK293T cells was performed using
Lipofectamine Plus protocol (Invitrogen). Cells were transfected with the DNA and
Lipofectamine Plus as per manufacturer’s protocol. Cells were collected for assays after
48 hours of transfection. Human proximal tubule epithelial (hPTE) cells were acquired
from Mediatech and maintained in appropriate media. Transfection of d-siRNA into
hPTE cells was performed with Lipofectamine 2000 (Invitrogen) and cells incubated for

48 hours before assaying.

115

5.2.3. RAMP Cloning and expression: Full length CDNA of human RAMPS], 2 and 3
and bovine CL-R were described before [281, 282]. CL-R, Cloned into Nl-EGFP and

also in pcDNA3.l expression vectors, was used for transfection in HEK 293T cells.

5.2.4. Desensitization and Resensitization assays: 48 hours post-tranfection cells were
pretreated with or without 10 nM AM in DMEM containing 0.2% BSA for indicated time
periods (up to 4 hr). After agonist exposure, cells were washed three times with
Dulbecco’s phosphate buffered saline (dPBS, Gibco BRL) chtaining 0.2% BSA and
either frozen for membrane preparation for adenylate cyclase assays or used immediately
for intact-cell radioligand binding. For receptor resensitization assays, after agonist
exposure, cells were washed and incubated for indicated time periods in DMEM

containing 0.2% BSA and 5 11ng cycloheximide to allow receptor recovery.

 

Desensltizatioanesensitlzation Protoc I:

 

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5.2.5. Receptor binding: Homologous competition radioligand binding assays were
performed as described by Aiyar et a1 and as established in our laboratory [283]. HEK

293T cells were transfected and approximately 200,000 cells/well seeded in poly-D-

116

lysine pre-coated 24-well plates (BD Biosciences, Palo Alto, CA). 48 hours post-
transfection cells were treated for desensitization or resensitization assays as described
above. After agonist exposure, cells were washed three times with dPBS buffer
containing 0.2% BSA then incubated with increasing concentrations (1 pM to 100 nM) of
competing ligand (rAM) and 175-250 pM 125I-rAM for 30 min at 37°C. After incubation,
plates were washed three times with ice-cold assay buffer and the reactions were
terminated by the addition of 2M NaOH. Cells were then harvested and associated
radioligand activity is counted on a y-counter. All binding assays were performed in
duplicate, with each experiment repeated at least three times. Nonspeciﬁc binding was
determined in the presence of 100 nM of unlabeled rAM. Data was analyzed by
LIGAND (assuming radioligand and competitor both bind reversibly to a single binding
site; MacLigand, Version 4.97, NIH, Bethesda, MD) using the following equation:

Bmax 0 [hot ligand]

 

KD + [hot ligand] + [cold ligand]

Where B represents speciﬁc binding, [hot ligand] the single concentration of [1251]rAM
studied, [cold ligand] the concentration of unlabeled rAM competing with the radiolabel
for AM receptor binding, Bmax the maximum number of binding sites and KD the
equilibrium dissociation constant (the equation was solved where the “cold ligand” ICso =
[hot ligand] + KD). Analysis of all binding data was performed by computer-assisted
nonlinear least square ﬁtting using GraphPad PRIZM version 4 (GraphPad Software, San
Diego, CA). Binding sites/cell was calculated and data was expressed as percent of the

control.

117

5.2.6. Adenylate cyclase assays: Cyclase activity was done as described before with
slight modiﬁcations [282, 284]. Cells were harvested from P100 or P60 plates and
homogenized in Tris HCI (10mM)/EDTA (10mM) buffer. Membranes were prepared by
homogenization and centrifugation in Tris HCl (50mM)/MgCl (10mM) buffer. Final
concentration of 20 pg of protein/assay tube was obtained. Membranes were incubated
for 15 min at 30°C with appropriate concentrations of drugs and assay mix containing
ATP regeneration system and a32P-ATP. After the reaction was stopped (with stop
solution containing 3H-CAMP) contents of the assay tubes were passed through Dowex
and subsequently through alumina columns to separate the degradation products of ATP,
by washing the dowex with water and alumina with imidazole. Elution proﬁle was done
to determine the amount of water and imidazole needed to wash and elute the products.
Product eluted from alumina column was counted for the presence of 3H-CAMP and
(13 2P-CAMP in a B-Counter. Each experiment was done in triplicates and repeated at least
3 times. Data is expressed as percent maximal response, % forskolin. Basal cyclase
activity and forskolin stimulation did not show statistically signiﬁcant differences

between treatments.

5.2.7. CAMP accumulation assays: Human proximal tubule cells were seeded on a 24-
well plate until reaching 80-90% conﬂuency, then incubated in serum-free media
overnight before experiment. Desensitization experiments were carried out as described

in Materials and Methods section, with cells pretreated with 10nM rAM and subsequently

Challenged for 10 min at 37°C with appropriate concentrations of drugs (100nM AM,

118

lOuM Forskolin) in the presence of ZOOuM 3-isobutyl-l-methylxanthine. Determination
of CAMP level was measured using the Biotrak CAMP enzyme immunoassay system
(Amersham Biosciences) according to the manufacturer's instructions. CAMP levels in
human proximal tubule cells were calculated using a standard curve ranging from 10 to
10 4 fmol of CAMP. Each experiment was done in duplicate and repeated at least 3 times.
Data is expressed as percent maximal response, % forskolin. Basal cyclase activity and

forskolin stimulation did not show statistically signiﬁcant differences between treatments.

5.2.8. RNA Interference analysis: Gene-speciﬁc d-siRNA for lacZ (control), NHERF-I
and RAMP3 were generated and puriﬁed using BLOCK-iT Dicer RNAi kit from
Invitrogen (Carlsbad, CA). hPTE cells were transfected with d-siRNAs using
Lipofectamine 2000 as per manufacturer’s instructions (Invitrogen). 48 hours after
transfection cells were frozen for mRNA analysis, or used for CAMP accumulation assays

or immunoﬂuorescence microscopy.

5.2.9. Reverse transcriptase PCR (RT-PCR) analysis: RT-PCR analysis performed as
described before [282].Total RNA was isolated from hPTEs using Trizol reagent
(GIBCO BRL). After sodium acetate-ethanol precipitation and several ethanol washes,
RNA was used as a template in a reverse transcriptase PCR ampliﬁcation procedure. The
RT-PCR reaction was carried out using Superscript One-Step RT-PCR with Platinum T aq
(GIBCO BRL), in accordance with the manufacturer's speciﬁcations. Reactions were
carried out, with a Perkin-Elmer model 9600 thermal cycler, in 50 ul of total reaction

volumes subjected to the following conditions: 1) 94°C for 2 min (1 cycle); 3) 94°C for

119

 

30 s, 50°C for 30 s, 68°C for 30 s (30 cycles); and 4) 68°C for 7min (1 cycle). Products
were separated by gel electrophoresis and subsequently visualized by ethidium bromide

staining and ultraviolet illumination. Photographs of the gels were taken and digitalized

with a UMAX Astra 2000P ﬂat-bed scanner.

5.2.10. Mutagenesis procedure: Site-directed mutagenesis was performed using a PCR-
based strategy that employs the pfu Turbo polymerase (Stratagene, La Jolla, CA). A pair
of complementary oligonucleotides containing the appropriate point mutations in the
sequence of RAMP/CL-R or a premature stop codon at position 145 or 147 codon of
RAMP3 for deletion mutants were synthesized (Michigan State University
Macromolecular structure facility). The PCR for the mutation was as follows: 94°C for 5
minutes; 30 cycles of 94°C for 30 sec., 50°C for 30 sec., 68°C for 8 min.; ﬁnal cycle of
68°C for 8 minutes. PCR product was digested for 4 hours with DpnI enzyme
(Invitrogen) and transformed in to DHSG. cells. Mutations were conﬁrmed by automated

sequencing (Michigan State University Genomic Technology Support Facility).

5.2.11. Immunoﬂuorescence microscopy: HEK293 and hPTE cells were transfected as
described above and seeded at 24hr post-transfection onto collagen type I-Coated
coverslips. Desensitization assays were performed as described and reactions were
stopped by ﬁxing cells in 4% paraformaldehyde for 30min. at room temperature.
Samples were perrneablized with 0.1% v/v Triton X-100 in PBS and blocked overnight in
0.1% v/v Triton X—100 in PBS + 10% goat serum. Samples were incubated in primary

antibody in blocking buffer for 2h at room temperature (NHERF-1 at 1:250 and RAMP3

120

at 1:200). Appropriate secondary antibodies were applied for 1h at room temperature
(Goat anti-mouse Cy3 at 1:500 and Goat anti-rabbit Cy5 at 1:400). Cytoskeletal staining
was carried out using Alexa 488-phalloidin antibody at 1:75 (Molecular Probes).
Coverslips were mounted in Shandon mounting medium and slides store at 2-8°C until
analysis. Cells were visualized on a Zeiss 210 laser confocal microscope at a zoom of 2.
Images presented are representative single optical sections of a z-series taken from at
least twenty ﬁelds per experiment and at least three individual experiments. Images in

this thesis/dissertation are presented in color.

5.2.12. Fusion protein overlays and western blotting: Overlay assays and western
blotting performed as described before [153]. 10 pg of GST-fusion proteins were
resolved on a 10% SDS-PAGE gel and transferred to nitrocellulose ﬁlters. Filters were
blocked with 5% w/v fat-free milk powder in Tris-buffered saline with Tween 20 (TTBS:
20 mM Tris, pH 7.4, 500 mM NaCI, 0.1% v/v Tween 20) and incubated overnight at 4 °C
in lysates of HEK293 cells with or without overexpression NHERF-l. Blots were then
washed three times with TTBS buffer and incubated with anti-EBPSO (NHERF-1)
monoclonal antibody for 2 h at room temperature. After three washes with TTBS, ﬁlters
were incubated for 1h with horseradish peroxidase—conjugated goat anti-mouse secondary
antibody (Gibco BRL®, Grand Island, NY), washed again with TTBS, soaked in
Supersignal West Pico chemiluminescent substrate (Pierce) and exposed to x-ray ﬁlm.
Same protocol, with the exception of the overnight incubation with cell lysate, was

followed for immunoblot analysis of RAMP3.

121

II-*— ——- r -I

5.2.13. Statistics: Data are presented as mean i S.E.M. Single group comparisons

exercised a paired Student’s t-test method. Statistical signiﬁcance was set at P<0.05.

5.3. Results
5.3.1. Role of NHERF-1 in Intemalization of the CL-R/RAMP complex

We and others have previously shown that the AM2 receptor (CL-R + RAMP3
complex) undergoes agonist-stimulated desensitization, internalization, and degradation
[153, 276, 285]. Furthermore, we have recently shown that in the presence of NSF, the
AM2 receptor complex undergoes recycling, instead of following a degradation pathway,
in HEK 293 cells [153]. In this study, we have examined the role of another protein,
namely NHERF-1, on agonist-induced trafﬁcking of the CL-R/RAMP3 complex.

Pretreatment of HEK 293 cells expressing CL-R and RAMP3 with 10 nM AM for
one hour resulted in desensitization of the adenylate cyclase response from 50% (of
forskolin stimulation) in untreated cells to 28% in AM-treated cells (Figure 17A). In
addition, as shown in Figure 178 and 18, CL-R/RAMP3 complex underwent agonist-
induced internalization as determined by receptor binding and immunoﬂuorescence
microscopy. These ﬁndings are in agreement with those of Kuwasako et al. and as
reported by us recently [153, 276]. However, over-expression of NHERF-1 with CL-
R/RAMP3 resulted in a remarkable change in the agonist-induced receptor complex
trafﬁcking in HEK293 cells.

HEK293 cells transfected with CL-R/RAMP3 and NHERF-1 showed similar
levels of adenylate cyclase activity and desensitization patterns as compared to CL-

R/RAMP3 alone (Figure 17A). But in the presence of NHERF-1, the receptor complex

122

failed to internalize with agonist pretreatment (Figure 17B, 18). To determine if
NHERF-1 co-expression had changed the kinetics of internalization, agonist pretreatment
was carried out for a time course extending to 4 hours. Four hours of agonist
pretreatment still yielded a complete inhibition of receptor complex internalization in
HEK293 cells co-expressing NHERF-l and the CL-R/RAMP3 complex, as compared to
a continued internalization of the receptor complex in HEK293 cells lacking NHERF-l
over-expression (Figure 17C). These results indicate that over-expression of NHERF-l
alters the trafﬁcking of the CL—R/RAMP3 receptor complex after AM-stimulated

desensitization.

5.3.2. RAMP isoform-specific regulation of CL-R/RAMP receptor complex
trafficking

To determine if this effect of NHERF-1 was speciﬁc for RAMP3, the additional
RAMPS (RAMPI or -2) were tested for their ability to act with NHERF-1 to alter the
receptor complex trafﬁcking. Interestingly, in contrast to RAMP3, presence of NHERF-I
did not alter the internalization pattern of the CL-R/RAMPI or -2 receptor complexes.
No signiﬁcant differences were seen in the receptor numbers from whole-cell binding
when CL-R was co-expressed with RAMPI, 2, or 3 (with and without NHERF-1).
Desensitization patterns in cells transfected with CL-R+RAMP1 or CL—R+RAMP2 also
remained unchanged in the absence or presence of NHERF-l (Figure l9A-D). These
results indicate that RAMP3 must contain a molecular feature distinct from the other

RAMPS that allowed its interaction and action with NHERF-1.

123

5.3.3. Role of PDZ interactions in trafficking of the CL-R/RAMP3 complex

We have observed before that NSF regulated the CL-R/RAMP3 recycling by
interacting speciﬁcally with the PDZ motif present at the extreme C-terminus of RAMP3.
Neither CL-R, nor the other two RAMP isoforms (RAMPl or RAMP2), contain PDZ
recognition motifs. To test the hypothesis that this domain is critical for interaction of the
CL-R/RAMP3 complex with NHERF-l, the PDZ motif (-DTLL) on RAMP3 was deleted
(RAMP3A145-8) and an internalization assay was performed. Deletion of this domain
did not affect basal adenylate cyclase activity or the desensitization response of the CL-
R/RAMP3 complex in response to AM, even in the presence of NHERF-l. In addition,
the RAMP3 PDZ motif mutant (RAMP3AI45-8) showed no difference in receptor
expression levels at the plasma membrane (as measured with whole-cell binding), as
compared to wild-type CL-R/RAMP3 complex (in the presence/absence of NHERF-1).
Unlike CL-R/RAMP3 complex, the CL—R/RAMP3A145-8 receptor complex was now
capable of AM-induced internalization, as measured by whole-cell receptor binding and
immunoﬂuoresence microscopy (Figure 20A and 21).

To further test the hypothesis that the absence of the PDZ motif on the RAMP2
accounts for the lack of ability of NHERF-1 to interact and therefore inhibit
internalization, the PDZ motif of RAMP3, the amino acids -DTLL, were substituted on
the C-terminus of RAMP2, in exchange for its original four C-terminal amino acids (-
EAQA). The CL—R/RAMPZADTLL mutant complex expression levels were comparable
to that of CL-R/RAMP2 complex, as determined by whole-cell binding and adenylate
cyclase assays. Additionally, in the absence of NHERF-1, both CL-R/RAMPZ and CL-

R/RAMPZADTLL showed similar levels of AM-stimulated internalization. However, C0-

124

expression of NHERF-1 with CL-R/RAMPZADTLL was now capable of inhibiting the
internalization of the receptor complex with agonist pretreatment, as compared to wild-
type CL-R/RAMPZ complex (Figure ZOB). These ﬁndings provide additional evidence
that the PDZ motif on the RAMP3 interacts with NHERF-1, causing an inhibition of
receptor internalization, despite normal desensitization.

To identify the critical amino acids in the PDZ binding sequence that regulate the
RAMP3/NHERF-1 interaction, site-directed mutagenesis was performed to mutate the
individual amino acids of the PDZ motif to alanine. Mutations of the individual amino
acids in the PDZ motif of RAMP3 did not affect the basal levels of receptor expression
and function, as measured by whole-cell binding experiments and adenylate cyclase
assays, respectively. In addition, the desensitization and internalization in the absence of
NHERF-l were also similar between the wild-type and mutant CL-R/RAMP3 complexes.
However, in the presence of NHERF-l, the CL-R/RAMP3T146A complex now
underwent agonist-stimulated internalization, similar to when expressed without NHERF-
] (Figure 20C). The other point mutant RAMP3/CL-R complexes behaved like wild-type
in the presence of NHERF-l, indicating Thr146 in the PDZ domain is critical for the PDZ
interaction between RAMP3 and NHERF-1.

As described before, NHERF-1 contains two PDZ domains through which it
interacts with numerous proteins [258]. To determine which PDZ domain of NHERF-1
is responsible for the interaction with RAMP3, the two PDZ domains of NHERF-1 were
deleted individually and agonist-induced internalization assays, employing whole-cell
receptor binding, were performed. Cells expressing wild-type or mutant NHERF-1 with

CL-R/RAMP3 receptor complex showed comparable levels of receptor expression and

125

function, as assessed by whole-cell receptor binding and adenylate cyclase assays,
respectively. In addition, they showed similar desensitization patterns. However,
internalization assays (with whole—cell binding) showed that the ﬁrst PDZ domain of
NHERF-l is responsible for the interaction with RAMP3. HEK 293 cells expressing the
mutant NHERF-IAPDZI (lacking only the ﬁrst PDZ domain) and CL-R/RAMP3
complex underwent AM-stimulated internalization compared to cells expressing the wild-
type NHERF-1 (Figure 20D). Deletion of the second PDZ domain of NHERF-l
(NHERF-IAPDZZ) had no effect on the internalization pattern of CL-R/RAMP3, as
compared to wild-type NHERF-1 (Figure 20D). These ﬁndings further conﬁrm that
RAMP3 and NHERF-1 are interacting via their PDZ domains to inhibit internalization of
the CL-R/RAMP3 receptor complex.

To examine if the PDZ domain on RAMP3 is physically interacting with NHERF-
], overlay assays were performed. This was accomplished using GST-RAMP3 fusion
proteins in an overlay assay with cell lysates of HEK293 cells overexpressing NHERF-l.
Control experiments run with GST protein showed no detectable bands when incubated
with NHERF-1 lysates and probed with an NHERF-1 antibody (Figure 22A).
Irnportantly, GST- RAMP3 fusion proteins showed signiﬁcant interaction with NHERF-l
in the cell lysates of HEK293 cells overexpressing NHERF-1 in the overlay assay (Figure
22A). As a control, lysates of HEK 293 cells not over-expressing NHERF-1 showed no
detectable bands when run with GST-RAMP3 in the overlay assay (Figure 22B).
Additionally, when GST-RAMP3A145-8 fusion proteins were tested for interaction with
NHERF-l using the above described overlay assay, no bands were detected, in contrast to

wild-type GST-RAMP3 (Figure 22A). When blots used in the overlay assay were

126

stripped and probed for RAMP3, a band for RAMP3 was detected at the exact location as
that of the NHERF-1 band detected in the overlay assay (Figure 22C). This data
demonstrates a physical interaction between RAMP3 and NHERF-1, an interaction that is
dependent on PDZ domain interactions of the two proteins and is capable of regulating

CL-R/RAMP3 (AM2R) complex trafﬁcking.

5.3.4. Mechanism of inhibition of CL-R/RAMP3 internalization by NHERF-1

The ERM domain of NHERF-1 is known to interact with MERM cytoskeletal
proteins, allowing NHERF-1 to tether proteins to the actin cytoskeleton [304]. Hence, we
hypothesized that the CL—R/RAMP3 receptor complex internalization is regulated by
NHERF-1 through interactions of NHERF-l with cytoskeletal proteins. To test this
hypothesis, we employed a mutant of NHERF-l with a deletion of its ERM domain
[252]. Control experiments with the ERM domain mutants of NHERF-1 co-transfected
with CL-R/RAMP3 showed similar levels of receptor expression and function, as
measured by whole-cell binding and adenylate cyclase. As hypothesized, the ERM
domain mutant of NHERF-l, when co-expressed in HEK 293 cells with CL-R/RAMP3
complex, was now capable of internalization after agonist pretreatment, differing from
the wild-type NHERF-1 that inhibits receptor internalization (Figure 23A). These
experiments were repeated and conﬁrmed with confocal immunoﬂuorescence
microscopy, using phalloidin to label the actin cytoskeleton and test for NHERF-1 co-
localization. While wild-type NHERF-l colocalized with the actin cytoskeleton (to
presumably tether RAMP3 and therefore CL-R to the plasma membrane after agonist

pretreatment), the ERM domain mutant of NHERF-l showed disrupted actin

127

cytoskeleton co-localization and therefore, did not block the internalization of RAMP3
(and CL-R) with agonist pretreatment (Figure 23B). These ﬁndings indicate that
NHERF-1 inhibits the internalization of the CL-R/RAMP3 receptor complex by acting as
an adaptor to tether the receptor complex to the actin cytoskeleton.

To further test this mechanism for inhibiting internalization, an actin
depolymerization agent (Cytochalasin D) was employed to disrupt the actin cytoskeleton
and the ability of NHERF-1 to tether the CL-R/RAMP3 complex to the plasma
membrane. Treatment of HEK293 cells expressing CL-R/RAMP3 in the presence and
absence of NHERF-l had no effect on the adenylate cyclase activity or desensitization
pattern of the receptor complex (Figure 23C). After treatment with Cytochalasin D,
HEK293 cells expressing CL-R/RAMP3 and NHERF-1 were now capable of
internalization. The Cytochalasin D experimental data further supports that the
mechanism of inhibition of internalization of the CL-R/RAMP3 complex by NHERF-1
by tethering of the receptor complex to the plasma membrane by NHERF-1’s interaction

with the actin cytoskeleton.

5.3.5. NHERF’s role in receptor trafficking in primary human proximal tubule
epithelial cells

To examine if our observations using the over-expressed system in HEK293 cells
could be translated to a more physiological cell type, we Chose human proximal tubule
cells to test our hypothesis. Because of the many roles of adrenomedullin and NHERF-1
in the kidney, we chose a human primary proximal tubule cell line to perform these

studies. The human proximal tubule epithelial cells (hPTEs) were determined to express

128

 

CL-R, RAMP2, RAMP3, and NHERF-1 and show high levels of speciﬁc adrenomedullin
binding and receptor stimulation by AM (data not shown). When pretreated with AM for
a period of one hour, desensitization was observed with adenylate cyclase assays (Figure
24A). However, as we would predict, internalization was not observed (by whole-cell
receptor binding assays) (Figure 24B). Intemalization was not observed when hPTE cells
were pretreated with AM for up to four hours, indicating desensitization and
internalization were not simply showing different kinetics. In order to test our hypothesis
that RAMP3 and NHERF-1 were critical to the inhibition of internalization of the CL-
R/RAMP3 receptor complex, RNA interference technology was employed to individually
knockdown RAMP3 and NHERF-1. In both mRNA and protein expression studies,
RAMP3 and NHERF-1 dramatically decreased in d-siRNA treated samples, while control
experiments using lacZ knockdown showed no signiﬁcant alteration in RAMP3 or
NHERF-1 expression when compared to wild-type hPTE cells (Figure 25A, B; data not
shown). Intemalization assays and whole-cell receptor binding assays were performed to
determine the effect of RAMP3 and NHERF-1 RNA interference on receptor
internalization in hPTE cells. Strikingly, when pretreated with agonist, hPTE cells with
RAMP3 or NHERF-l RNA interference showed a regained ability to internalize the
receptor complex, unlike the wild-type hPTE cells were internalization was inhibited
(Figure 25C). This ﬁnding demonstrates that RAMP3 and NHERF-1 are both critical for
the receptor trafﬁcking of the CL-R/RAMP3 complex in hPTE cells, an unaltered cell
line absent the issues of overexpression.

To determine if the mechanism of inhibition of internalization in hPTE cells was

similar to that in the HEK 293 cells, actin cytoskeletal tethering was examined in the

129

r“ A‘L l1

hPTE cells. hPTE cells were treated with Cytochalasin D, as was used in HEK 293 cells
to disrupt the actin cytoskeleton, and internalization assays were performed.
Cytochalasin D treatment did not alter the receptor expression levels in the hPTE cells, as
measured by whole-cell receptor binding, from the levels in untreated cells.
Interestingly, when cells were treated with Cytochalasin D and pretreated with AM for
one hour, the receptor complex was now capable of internalization (Figure 26). This data
supports the proposed model that NHERF-1 inhibits the internalization of the CL-
R/RAMP3 receptor complex after agonist stimulation by tethering the receptor complex
to the actin cytoskeleton via NHERF-1’s ERM domain interactions with the cytoskeleton
and PDZ domain interactions with RAMP3.

Finally, it was important to determine the effect of the inhibition of internalization
on AM signaling in the hPTE cells. To this end, a resensitization assay was performed
and CAMP accumulation was measured. AM pretreatment of the hPTE cells results in an
attenuation of CAMP accumulation from 35% to 15% of maximal response, whereas
allowed recovery time in the absence of agonist after the AM pretreatment was shown to
recover AM signaling to similar levels as untreated cells. While it was shown in a
previous Figure that AM pretreatment causes desensitization of the AM receptor
signaling in the hPTE cells, the resensitization assay now showed that resensitization
occurs in the hPTE cells, even in the absence of receptor internalization (Figure 24A, 27).
This ﬁnding suggests the inhibition of internalization of the AM receptor in the hPTE
cells is a mechanism for receptor resensitization, not requiring receptor internalization as

a prerequisite.

130

'q

F‘m ‘. “u
I

50. - No Pretreatment
CIAM Pretreatment

6F!

E:

33

gr, *

it —"'—~ *
VL- a;

Adenylate cyclase activi

 

 

 

 

 

 

 

 

CL-R-I-RAMP3 CL-R+RAMP3+NHERF1

Figure 17A: The role of NHERF-l in the trafﬁcking of the AM2R (CL-R+RAMP3) in HEK 293
cells. NHERF-1 does not alter desensitization of AM2R in transfected HEK 293 cells. HEK 293
cells transiently transfected with CL-R and RAMP3 with or without NHERF-l. At 48h post-
transfection, cells were treated for one hour with AM (10 nM) and then washed and adenylate
cyclase activity was measured. Alter agonist pretreatments, membranes were extracted and AC
activity in response to 100 nM AM was measured. NHERF-l overexpression with AM2R had no
signiﬁcant effect on receptor desensitization. Experiments performed in triplicates and data

expressed as percent maximal stimulation (% Forskolin). * p S 0.05; n 2 4.

131

- No Pretreatment
D AM Pretreatment

 

 

E5 ...1°°'

? 'o'

.9 h 75-

.6 8

go 50- *
‘6 $3 ”
.5 " 25-

m

 

 

 

 

 

 

CL-R-I-RAMP3 CL-R-I-RAMP3-I-NHERF1

Figure 173: The role of NHERF-l in the trafﬁcking of the AM2R (CL-R+RAMP3) in HEK 293
cells. NHERF-l inhibits internalization of AM2R in HEK 293 cells. HEK 293 cells transfected
and pretreated with agonist as described in 17A. Receptor internalization measured by whole-cell
competition binding assays using ”SI-rAM as ligand (cold rAM served as the competitor) and
number of binding sites/cell was estimated using the GRAPHPAD PRISM software. “No
pretrea ” represents samples at maximal radioligand binding that were not pre-incubated with
agonist. “1h pretreat” represents samples pretreated with AM (10 nM) for one hour, washed as
indicated in Methods section, and tested immediately after wash steps for radioligand binding.
NHERF-l overexpression in cells expressing AM2R caused altered receptor trafficking to inhibit

internalization of the receptor complex. * p S 0.05; n z 4 experiments.

132

.CL-R+RAMP3+NHERF1
150. IIICL-R+RAMP3

 

 

 

 

3
2A
“3100- r—
05
:2
38
c * *
--\°
22,50- *
a

A l

0 1 2 4

Pretreatment time (hrs)

Figure 17C: The role of NHERF-l in the traﬁicking of the AM2R (CL-R+RAMP3) in HEK 293
cells. Time course internalization of AM2-R in presence/absence of NHERF-l. Lengthening of
agonist pretreatment does not alter inhibition of internalization of CL-R/RAMP3 complex with
NHERF-l co-expression. HEK 293 cells transfected and pretreated with agonist for varying time
points, as described in 17A. Receptor internalization measured by whole-cell competition binding
assays using ”SI-rAM as ligand (cold rAM served as the competitor) and number of binding

sites/cell was estimated using the GRAPHPAD PRISM software. * p S 0.05; n 2 3 experiments.

133

RAMP3 OVERLAY

RAMP3 NHERF1

No
Pretreat

AM
Pretreat

    
 

No
Pretreat

AM
Pretreat

 

Figure 18: Localization of CL-R, RAMP3, and NHERF-1 in HEK 293 cells during an

internalization experiment. After lh AM pretreatment. CL-R and RAMP3 are internalized in
absence of NHERF-l co-expression. In cells co-expressing NHERF-l with CL-R/RAMP3
complex. internalization of receptor complex is blocked alter AM pretreatment. HEK 293 cells
transfected with CL-R-GFP, RAMP3, and NHERF-1 were pretreated with 10nM ADM for 1h.
Note: “1 hr pretreatment” indicates time just after AM pretreatment and wash steps. Cells were
ﬁxed and components were visualized using anti-RAMP3 antibody (1:200) and anti-NHERF-l
antibody (1:250) with Cy5 anti-rabbit secondary antibody (1:400, in blue) and Cy3 anti-mouse
secondary antibody (1:500, in red), respectively; CL-R-GFP is detected with an EGFP tag and
shown in green; overlays of staining patterns are shown in the far right panels. Images shown are
representative of at least twenty ﬁelds imaged per experiment from at least three experiments.

Bar scales on all images represent lOOum.

134

- CL-R+RAMP1
D CL-R+RAMP1+NHERF1

   

 

 

 

 

 

 

 

3% 75‘ .I.
o .
< .9. _ 30
0 2 .5
8 3,3
— x .
a: 2 2°
0 .5 E
E o
3% 2.10:
2‘s
35 o-
< No Pretreat CGRP Pretreat

Figure 19A: The role of NHERF-l in post-endocytic sorting of CGRP] (CL-R+Rl) and AMI
receptor. NHERF-l overexpression does not alter CGRP receptor desensitization after agonist
stimulation. HEK 293 cells were transfected and pretreated as in ﬁgure 17A and then membranes
were extracted. Adenylate cyclase activity was measured in membranes stimulated with 100 nM
CGRP for 15 min. Experiments performed in triplicates and expressed as percent maximal

stimulation (% Forskolin). * p S 0.05; n 2 3.

135

- CL-R+RAMP1

   

 

 

 

 

 

D CL-R+RAMP1+NHERF1
_ 100« ﬁ
3
£ 2
g e
"7’ E 50« *
5’0
.E o
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.E V
m

0'

No pretr'eatment CGRP Pretreatment

Figure 198: The role of NHERF-l in post-endocytic sorting of CGRPl (CL-R+Rl) and AM]
receptor. NHERF-l overexpression does not alter the trafficking of the CGRP receptor after
agonist stimulation. HEK 293 cells were transfected with CL-R and RAMP], with or without
NHERF-l. 48h post-transfection, cells were pretreated with CGRP (I 0 nM) for one hour, washed
as described in Materials and Methods, and receptor internalization was measured with whole-
Cell competition binding using l25l-rCGRP as the ligand and cold rCGRP as the competitor.
Number of binding sites/cell was estimated with GRAPHPAD PRISM software. "‘ p _<_ 0.05; n 2

3.

136

- CL-R+RAMP2
D CL-R+RAMP2+NHERF1

I.

«h
C
I

[% Forskolin]
N
‘3

   

 

 

 

 

 

 

 

 

Adenylate Cyclase Activity
(pmollminlmg protein)

No Pr'etreat AM Pr'etreat

Figure 19C: The role of NHERF-l in post-endocytic sorting of CGRPI (CL-R+Rl) and AMI
receptor. NHERF-I overexpression does not alter AMI receptor desensitization after agonist
induction. HEK 293 cells were transfected and pretreated as in ﬁgure 17A and then membranes
were extracted. Adenylate cyclase activity was measured in membranes stimulated with 100 nM
AM for 15 min. Experiments performed in triplicates and expressed as percent maximal

stimulation (% Forskolin). * p S 0.05; n 2 3.

137

- CL-R+RAMP2
E CL-R+RAMP2+NHERF 1

   

8

873 i
:15

in .

5,8 50

CE °

'59\

5"

m

 

 

 

 

 

 

 

No Pretfeatment AM Preti'eatment

Figure 19D: The role of NHERF-l in post-endocytic sorting of CGRP] (CL-R+Rl) and AMI
receptor. NHERF-l overexpression does not alter internalization of AMI receptor after receptor
activation. HEK 293 cells were transfected with CL-R and RAMP2, with or without NHERF-1.
48h post-transfection, cells were pretreated with AM (10 nM) for one hour, washed as described
in Materials and Methods, and receptor internalization was measured with whole-cell competition
binding using ”SI-rAM as the ligand and cold rAM as the competitor. Number of binding

sites/cell was estimated with GRAPHPAD PRISM software. "' p S 0.05; n 2 3.

138

- CL-R+RAM P3
E CL-R+RAM P3+NHERF1
E3 CL-R+RAMP3A1 45-8+NHERF1

% 1oo- — l
2 2 F
W O
3 1'.“- 75' *
Ia c
0
2’0 50.. *
‘Q 2‘:
a 25‘

 

 

 

 

 

 

 

 

No Pretfeatment AM Preti'eatment

Figure 20A: The effect of RAMP3 PDZ motif deletion on the trafﬁcking of the AM2R (CL-
R+RAMP3). Deletion of RAMP3 PDZ motif allowed internalization of the CL-R/RAMP3
complex when co-expressed with NHERF-l. HEK 293 cells were co-transfected with CL-R,
wild-type RAMP3 or RAMP3AI45-8, and NHERF-1. 48h post-transfection, cells were
pretreated with AM (10 nM) for one hour. washed as described in Materials and Methods, and
receptor internalization was measured with whole-cell binding using ”SI-rAM as the ligand and
cold rAM as the competitor. Number of binding sites/cell was estimated with GRAPHPAD

PRISM software. "‘ p S 0.05: n 2 3.

139

- CL-R+RAMP2
E CL-R+RAMP2+NHERF1
CL-R+RAMP2ADTLL+NHERF1

     

 

 

 

 

= 100-

8

a ‘0‘ 8°“

8 b

'6 g 60-

2’0

:5 °\° 40'

.E V

m 20‘
01

No pretr'eatment AM pretreatment

Figure 208: Effect of PDZ motif substitution on the C-terminus of RAMP2 on internalization of
CL—R/RAMP complex. Substitution of PDZ motif (-DTLL) on the C—terminus of RAMP2
caused a Change in receptor trafficking to inhibit internalization of the receptor complex when co-
expressed with NHERF-l. HEK 293 cells were co-transfected with CL-R, wild-type RAMP2 or
RAMP2ADTLL, and NHERF-1. 48h post-transfection, cells were pretreated with AM (10 nM)
for one hour, washed as described in Materials and Methods, and receptor internalization was
measured with whole-cell binding using ”SI-rAM as the ligand and cold rAM as the competitor.
Number of binding sites/cell was estimated with GRAPHPAD PRISM software. * p S 0.05; n 2

3.

140

- No pretreatment

 

 

 

 

 

 

DAM pretreatment
— 1oo- ¥
5. l
E? 8°‘ *
'5 = 60-
g8
E 20' l
0" I I I I
'5 :6 v v '5 :b v~ v~
$9§°Q€§£s§ QvQI} °\°9«\§)
e e «9’ e e a

Figure 20C: Effect of RAMP3 PDZ motif point mutations on the internalization of the CL-
R/RAMP3 complex in the presence of NHERF-l. Only point mutant RAMP3TI46A was critical
in the inhibition of the internalization of the CL-R/RAMP3 complex seen when co-expressed
with NHERF-l. HEK 293 cells were co-transfected with CL-R, wild-type RAMP3 or RAMP3
point mutants, and NHERF-1. 48h post-transfection. cells were pretreated with AM (10 nM) for
one hour. washed as described in Materials and Methods, and receptor internalization was
measured with whole-cell binding using l25l-rAM as the ligand and cold rAM as the competitor.
Number of binding sites/cell was estimated with GRAPHPAD PRISM software. * p S 0.05; n 2

3..

141

- NHERF

 

ENHERFAPDZ‘I
ENHERFAPDZZ
125'
g =. 100- l
m 0 w ‘,_l
:3 b 75-
m c ,‘ ..-.
c» o * ‘
c U 50.
"5 E: ’ :j
.E 25- -' 2‘
m * ;

 

 

 

 

 

No Pr'etreat 1 hr Pietreat

Figure 20D: The effect of NHERF-l PDZ domain deletion on the trafﬁcking of the AM2R (CL-
R+RAMP3). Deletion of NHERF-l PDZl domain allowed internalization of the CL-R/RAMP3
complex. HEK 293 cells were co-transfeCted with CL-R, wild-type RAMP3, and wild-type or
PDZ domain mutants of NHERF-l. 48h post-transfection, cells were pretreated with AM (10
nM) for one hour, wasth as described in Materials and Methods, and receptor intemalization
was measured with whole-cell binding using ”SI-rAM as the ligand and cold rAM as the
competitor. Number of binding sites/cell was estimated with GRAPHPAD PRISM software. * p

S 0.05; n 2 3.

142

RAMP3Af45-8 NHERF1 OVERLAY

Figure 21: Localization of CL-R, RAMP3A14S-8. and NHERF-1 in HEK 293 cells during an

  

No
Pretreat

AM
Pretreat

 

internalization experiment. Untreated and after one hour ADM (10nM) pretreatment, CL-R and
RAMP3AI45-8 internalize similarly to the wild-type CL-R/RAMP3 complex in the absence of
NHERF-l. Experiments performed as described in Figure 18. Fixed cells were stained with anti-
RAMP3 antibody (1:200) and anti-NHERF-l antibody (1:250) with CyS anti-rabbit secondary
antibody (1:400, in blue) and Cy3 anti-mouse secondary antibody (1:500, in red), respectively;
CL—R-GFP is shown in green: overlays of staining patterns are shown in the far right panels.
Images shown are representative of at least twenty ﬁelds imaged from at least three experiments.

Bar scales on all images represent lOOum.

143

' (M- 8' (.us'r- C' (.‘ST-
(N. mun (.s'r- RAMP3 (.‘s'r- RAMP3
csr RAMP3 mm on RAMP3 ANS-8 (zs’r RAMP3 nus-s

70 KDa

50 KDa

Figure 22: Interaction studies of RAMP3 with NHERF-l. Fusion proteins of GST-RAMP3
demonstrate a physical interaction with NHERF-l in an overlay assay and western blot analysis.
lOug of GST, GST-RAMP3. and GST-RAMP3AI45-8 protein was run and separated on an SDS-
PAGE gel and transferred to a nitrocellulose ﬁlter. The nitrocellulose ﬁlter was incubated
overnight in lysate of HEK293 cells transfected with or without NHERF-I at 4°C. Filter was
then washed and probed by immunoblot for NHERF-l (1:250). Identical ﬁlters were probed by
immunoblot for RAMP3 (12400). A. GST. GST-RAMP3, and GST-RAMP3AI 45-8 overlay assay
incubated in NHERF-l-transfected HEK 293 lysates and probed for NHERF-l. B, GST, GST-
RAMP3, and GST-RAMP3AI45-8 overlay assay incubated in non-transfected HEK 293 lysates
and probed for NHERF-l. C. lmmunoblot of GST, GST-RAMP3. and GST-RAMP3A145-8

probed for RAMP3. Shown are representative blots of at least four experiments.

144

- CL-R+RAMP3+NHERF1
D CL-R+RAMP3+NHERF1AERM

 

_ 100-

a T

a ‘3 3°“

3 .5

la 5 60" *
5’0

% °\° 40'

.E V

m 20-

 

 

 

 

No pretr'eatment AM preﬁeatment

Figure 23A: Mechanism of NHERF-l inhibition of internalization of CL-R/RAMP3 receptor
complex. ERM domain mutant of NHERF-I blocks the inhibition of internalization of CL-
R/RAMP3 complex seen with wild-type NHERF-l. HEK 293 cells were transfected with CL-R,
RAMP3, and wild-type NHERF-l or ERM domain mutant NHERF-l. 48h post-transfection,
cells were pretreated with AM (10 nM) for one hour, washed as described in Materials and
Methods, and receptor internalization was measured with whole-cell binding using ”SI-rAM as
the ligand and cold rAM as the competitor. Number of binding sites/cell was estimated with

GRAPHPAD PRISM software. "‘ p S 0.05; n 2 3.

145

OVERLAY

PHALLOIDIN

 

No
Pretreat

AM
Pretreat

 

RAMP-3 NHERFAERM PHALLOIDIN OVERLAY
No

Pretreat

AM
Pretreat

 

I46

Figure 238: Mechanism of NHERF-l inhibition of internalization of CL-R/RAMP3 receptor
complex. Localization of RAMP3, NHERF-l (or NHERF-IAERM), and actin cytoskeleton in
HEK 293 cells during an internalization experiment. Untreated cells show RAMP3 and wild-type
NHERF-l distributed at the plasma membrane with NHERF-l colocalizing with the actin
cytoskeleton, and after AM pretreatment, all component remain in the same distribution. ERM
domain mutant of NHERF-l shows poor co-Iocalization with the actin cytoskeleton in both
untreated and AM-pretreated conditions, and therefore, shows internalization of RAMP3 with
agonist pretreatment, HEK 293 cells were transfected with CL-R, RAMP3, and wild-type or
ERM domain mutant NHERF-l. Experiments performed as described in Figure 18. Fixed cells
were stained with anti-RAMP3 antibody (1:200) and anti-NHERF-l antibody (1:250) with Cy5
anti-rabbit secondary antibody (1:400, in blue) and Cy3 anti-mouse secondary antibody (1:500, in
red), respectively; actin cytoskeleton is stained with Alexa 488-phalloidin and is shown in green;
overlays of staining patterns are shown in the far right panels. Images shown are representative of

at least twenty ﬁelds imaged from at least three experiments. Bar scales on all images represent

lOOum.

147

- Basal
E Cyto D treated

100'

0|
C,
l

 

Binding max (sites/cell)
[% Control]

 

 

 

 

 

 

No Pretreat AM Pretreat

Figure 23C: Mechanism of NHERF-l inhibition of internalization of CL-R/RAMP3 receptor
complex. Cytochalasin D treatment blocks the inhibition of internalization of CL-R/RAMP3
complex. HEK 293 cells were transfected with CL-R, RAMP3, and wild-type NHERF-1. 48h
post-transfection, cells were pretreated Cytochalasin D (IOuM) for 15 min., then with AM (10
nM) for one hour, washed as described in Materials and Methods, and receptor internalization
was measured with whole-cell binding using ”SI-rAM as the ligand and cold rAM as the
competitor. Number of binding sites/cell was estimated with GRAPHPAD PRISM software. * p

S 0.05; n 2 3.

148

A
O
I

[% Forskolin]
N
C.’

CAMP Accumulation
(nmollmg protein)

 

I

     

No Pretreat 1 hr AM Pretreat

Figure 24A: Trafﬁcking of AM-R in human proximal tubule cells (hPTE cells). In hPTE cells,
AM-R signaling desensitizes with AM pretreatment. hPTE cells seeded in 24-well plates were
pretreated with IOnm rAM for lh were washed extensively to remove residual agonist and plates
were frozen. Determination of CAMP level was measured using the Biotrak CAMP enzyme
immunoassay system (Amersham Biosciences) according to the manufacturer's instructions.
CAMP levels in hPTE cells were calculated using a standard curve ranging from 10 to 104 fmol of
CAMP. Each experiment was done in duplicate and repeated at least 3 times. Data is expressed

as percent maximal response, % forskolin. “ p S 0.05; n 2 4 experiments.

149

 

 

E 125-
a:

2
3%,100-
£3 75.
x c

a: O

a: 50-
“L.

c

:5 25]
.E

m 0

e “I “‘ gt“

 

AM Pretreat

Figure 248: Trafﬁcking of AM-R in human proximal tubule cells (hPTE cells). AM-R fails to
internalize when incubated in AM for varying time points. hPTE cells were grown as described
in Figure 24A. Receptor internalization measured by whole-cell competition binding assays
using ”SI-rAM as ligand (cold rAM served as the competitor) and number of binding sites/cell
was estimated using the GRAPHPAD PRISM software. “No pretreat” represents samples at
maximal radioligand binding that were not pre-incubated with agonist. “1,2, and 4h pretreat”
represents samples pretreated with AM (10 nM) for according time period, washed as indicated in
Methods section, and tested immediately after wash steps for radioligand binding. * p S 0.05; n 2
3 experiments. In cells endogenously expressing AMR, internalization is inhibited, but

desensitization in unaltered.

150

RAMP3 NHERF1
WT RNAi WT RNAi

      

PCR: RAMP3 NHERF1

Figure 25A: Effect of RAMP3 and NHERF-1 RNA interference on internalization of AM2-R in
human proximal tubule cells. RAMP3 and NHERF-1 RNA interference disrupts the inhibition of
internalization of AM-R after agonist pretreatment. hPTE cells seeded in 48-well plates were
transfected with d-siRNA of RAMP3 or NHERF-l and incubated for 48 hr to allow RNA
knockdown. mRNA analysis by Q-PCR showed dramatically decreased levels of RAMP3 and
NHERF-1 mRNA in RAMP3 and NHERF-1 RNA interference samples (respectively) as
compared to wild type, while lacZ knockdown had no effect on RAMP3 or NHERF-l message
levels (data not shown). RNA isolation and RT-PCR performed as described in Experimental

Procedures section. n=3 experiments.

151

Wildelype hPTE cells

NHERF Knockdown

      

RAMP-3 Knockdown

   

Figure 25B: Effect of RAMP3 and NHERF-1 RNA interference on internalization of AM2-R in
human proximal tubule cells. RAMP3 and NHERF-1 RNA interference disrupts the inhibition of
internalization of AM-R after agonist pretreatment. hPTE cells seeded in 48-well plates were
transfected with d-siRNA of RAMP3 or NHERF-l and incubated for 48 hr to allow RNA
knockdown. lmmunoﬁuorescence microscopy of hPTEs with RAMP3 or NHERF-l RNA
interference demonstrated greatly decreased levels of RAMP3 and NHERF-1 protein expression
in RAMP3 and NHERF-1 RNA knockdown cells, as compared to wild-type hPTE cells. lacZ
knockdown had no effect on RAMP3 or NHERF-l protein expression in hPTEs (data not shown).
Cells prepared as described in Experiment procedures section for immunoﬂuorescence
microscopy. Fixed cells were stained with anti-RAMP3 antibody ( 1:200) or anti-NHERF-I
antibody (1:250) and detected with a Cy3 anti-rabbit secondary antibody (1:500). Images shown

are representative of at least three experiments.

152

- Wild-type
El RAMP-3 RNAi

  

  

 

 

DNHERF RNAi
‘=‘ 125-
a:
2
g H 100'
a _
-- 0
19,35 75.
x c *
«I 0 *
E 2 50-
m\
.E L 25..
1:
.E j
oi oi

No Pr'etreat AM Pfetreat

Figure 25C: Effect of RAMP3 and NHERF-1 RNA interference on internalization of AM2-R in
human proximal tubule cells. RAMP3 and NHERF-1 RNA interference disrupts the inhibition of
internalization of AM-R alter agonist pretreatment. hPTE cells seeded in 48-well plates were
transfected with d-siRNA of RAMP3 or NHERF-l and incubated for 48 hr to allow RNA
knockdown. AM-R is capable of internalization when RAMP3 or NHERF-l are knocked down
with RNA interference. hPTE cells were grown as described in Figure 24A. Receptor
internalization measured by whole-cell competition binding assays using ”SI-rAM as ligand (cold
rAM served as the competitor) and number of binding sites/cell was estimated using the
GRAPHPAD PRISM software. "No pretreat" represents samples at maximal radioligand binding
that were not pre-incubated with agonist. "l pretreat” represents samples pretreated with AM (10
nM) for according time period, washed as indicated in Methods section, and tested immediately

after wash steps for radioligand binding. * p S 0.05; n 2 3 experiments.

153

-Wild-type hPTE
DCyto D treated hPTE

   

x ”100' r—
:23
C, 5 cl
5322' 75
.s '0 °
2%‘3 50-
"Vi.
m 25-

 

 

 

 

No Pretr'eatment AM Pretfeatment

Figure 26: Mechanism of NHERF-I inhibition of internalization of CL-R/RAMP3 receptor
complex in hPTE cells. Cytochalasin D treatment blocks the inhibition of internalization of CL-
R/RAMP3 complex. hPTE cells were grown as described in Figure 24A. hPTE cells were
pretreated Cytochalasin D (lOuM) for 15 min., then with AM (10 nM) for one hour, washed as
described in Materials and Methods, and receptor internalization was measured with whole-cell
binding using ”SI-rAM as the ligand and cold rAM as the competitor. Number of binding

sites/cell was estimated with GRAPHPAD PRISM software. "‘ p S 0.05; n 2 3.

154

$

30

  
 

15-

CAMP Accumulation
(nmollmg protein)
[% Forskolin]

 

OJ

‘6.
3““9
“c We“?! “Wm“

Figure 27: Trafﬁcking of AM-R in human proximal tubule cells (hPTE cells). In hPTE cells,
AM-R signaling desensitizes with AM pretreatment, but also resensitizes when then incubated in
the absence of agonist. hPTE cells seeded in 24-well plates were pretreated with lOnm rAM for
1h were washed extensively to remove residual agonist, incubated for 4h in serum-free media in
the absence of agonist and presence of Sug/ml cycloheximide, and plates were frozen.
Determination of CAMP level was measured using the Biotrak CAMP enzyme immunoassay
system (Amersham Biosciences) according to the manufacturer's instructions. CAMP levels in
hPTE cells were calculated using a standard curve ranging from 10 to 10 4 fmol of CAMP. Each
experiment was done in duplicate and repeated at least 3 times. Data is expressed as percent

maximal response, % forskolin. * p S 0.05; n 2 4 experiments.

155

5.4. Discussion

Like other GPCRS, the adrenomedullin and CGRP receptors mediate various
physiological actions in different cell types using a variety of mechanisms [305, 306].
The receptor undergoes classical lifecycle wherein it is phosphorylated upon agonist
stimulation, leading to desensitization, internalization and either recycling or degradation
depending on the cell type. However, unlike other GPCRS, the AM and CGRP receptors
are regulated by single transmembrane accessory protein called RAMPS (RAMP1-3). In
addition to the original observation by Foord’s group that RAMPS are necessary for the
cell surface expression and speciﬁcity of CL-R to the ligands CGRP and AM, our
observations reported recently demonstrated that RAMPS (particularly RAMP3) also has
other roles that may regulate the lifecycle of the receptor complex. Speciﬁcally, RAMP3
interaction with NSF was found to be important for recycling the receptor after
internalization. Absence of RAMP3 or inhibition of NSF resulted in receptor complex
degradation [153]. The present study was undertaken to investigate other binding partners
and additional roles for RAMP3 in CL-R/RAMP lifecycle. We show here using both
heterologous expression, as well as endogenous systems, that RAMP3 interaction with
NHERF-l is essential for regulating the internalization of the CL—R/RAMP3 complex. In
addition, similar to our NSF study, the PDZ motif in RAMP3 is critical for the interaction
with NHERF-l. In particular, we have found that the Thr146 in RAMP3 is critical for this
interaction. Whether RAMP3 is phosphorylated at this site by any of the kinases, and if
this is essential for NHERF-1 interaction, is not known.

Several studies have shown NHERF-l to play differing roles in various cellular

processes, including receptor trafﬁcking [257, 258]. NHERF-1 has been demonstrated to

156

bind the extreme COOH terminus of several G-protein coupled receptors (GPCRS),
namely the Bz-adrenergic receptor, the K-OpIOid receptor, and the P2Y purinergic receptor
[154, 259]. Agonist exposure promotes NHERF-l association with the fig-adrenergic and
the K-opioid receptors. Unlike our study, NHERF-1 association with these receptors
enhances the recycling of the receptors after agonist stimulation [240, 259]. The DSLL
motif in the C-terminus of adrenergic receptor was found to the critical for NHERF-l
interaction. In the present study, we also found that the DTLL motif in RAMP3 is
essential for the interaction with NHERF-l. Similar to the Thr in the DTLL motif, the Ser
in the DSLL motif was found to be essential for the interaction with NHERF-1 [240]. In
addition to GPCRS, NHERF-1 has also been shown to associate with the epidermal
growth factor receptor (EGFR), a receptor tyrosine kinases (RTK). In this case, NHERF-
1 association acts to stabilize the receptor at the plasma membrane and decrease the
endocytosis rate of the receptor [217]. Using an endogenous system we have also shown
here that NHERF-l is essential to ‘hold’ the receptor-complex at the membrane, the
absence of which leads to internalization of the receptor complex. While utilizing
different mechanisms, collectively these data suggest that NHERF-l interaction with both
RTKS and G protein-coupled receptors and RAMP3 is mandatory to enhance the portion
of receptors present at the cell surface.

Recent data suggests that RAMP3 can interact with receptors other than CL-R. It
is important to determine if this novel role of RAMP3 in receptor trafﬁcking is speciﬁc
for CL-R or also for the other receptors RAMPS interact with, namely VPAC, PTHl- and
2-R, and glucagon receptors [286]. NHERF—l and -2 have been reported to interact with

the PTHlR in a scaffolding capacity and tether the receptor to phospholipase C-B (PLCB)

157

and the actin cytoskeleton [301, 302]. In opossum kidney (OK) cells, NHERF-l
mediates PTH-stimulated entry of extracellular calcium by a mechanism that is apically
localized, PLC-dependent, pertussis toxin-sensitive, and requires an intact actin
cytoskeleton. NHERF1 was also shown in inhibit the activation-independent
internalization of the PTHIR in kidney distal tubule cells (when stably expressed) [218].
Given the ability of RAMP2 and RAMP3 to interact with the PTHlR and PTH2R,
respectively, RAMP3 may be interacting with NHERF-l to play a trafﬁcking role in
these systems, as well.

In addition to AM, Roh et al. have reported that interrnedin, a newly discovered
peptide from the calcitonin gene peptide superfamily, can also bind the CL-R/RAMP3
complex [307]. It remains to be determined if this reported function for RAMP3 'is
speciﬁc for AM, or if another peptide like intermedin could yield similar results. In our
studies of interaction of RAMP3 with NSF and NHERF-1, the PDZ motif of RAMP3 was
found to be critical for the interaction. Whether this motif binds additional proteins in a
cell type speciﬁc manner remains to be examined. One could hypothesize that these
predicted cell-type speciﬁc interactions would lead to regulation of the various events in
the receptor life cycle. In addition to RAMP3, RAMPl and RAMP2 also regulate the
expression of CL-R at the plasma membrane. Whether RAMP] and 2 bind other proteins

similar to RAMP3 also remains to be examined.

Conclusions: This study has shown that one of CL-R’s heterodimeric partners, RAMP3,

is capable of altering the trafﬁcking of the receptor complex after agonist stimulation by

interacting with NHERF-l via its PDZ motif, and thus the actin cytoskeleton. We have

158

demonstrated here for the ﬁrst time that NHERF-RAMP3 interaction dissociates receptor
desensitization from internalization of the CL-R/RAMP complex. Additionally, this
reveals a novel function for the RAMP accessory proteins in receptor trafﬁcking and an
additional difference between the AMlR and AM2R. With recent reports of RAMPS
complexing and regulating GPCRS other than CL-R, future studies will focus on
additional binding partners of RAMPS and how they regulate the various events in the

GPCR life-cycle.

159

 

6. Novel function for receptor activity-modifying proteins in post-
endocytic receptor trafﬁcking.

6.1. Introduction

The recent discovery of receptor activity modifying proteins (RAMPS) has raised
new possibilities for modes of regulation of G-protein coupled receptors (GPCRS).
RAMPS were discovered as accessory proteins indispensable to the function of an orphan
GPCR, now termed the calcitonin receptor-like receptor (CL-R)[275]. Three RAMP
isoforms (1-3) have been identiﬁed as distinct gene products that yield single
transmembrane-spanning proteins. RAMPS are required for the plasma membrane
expression, as well as for determination of receptor phenotype for CL-R (selective ligand
recognition)[275, 276]. Coexpression of RAMP-1 with CL-R yields a calcitonin gene-
related peptide-l (CGRP-l) receptor, while coexpression of RAMP—2 or -3 with CL-R
produces adrenomedullin receptors, AM-l and AM-2 receptors, respectively [277, 278].
AM and CGRP are multi-functional peptides with many overlapping functions, ranging
from potent vasodilation to proliferation regulation to regulation of salt and water balance
[279]. Differential expression of RAMP isoforms has been hypothesized to play a
regulatory role in both physiological and pathophysiological disease states. Moreover,
the recent identiﬁcation of RAMP interactions with additional members of the Class II
GPCR family and RAMP expression in cell lines lacking CL-R have raised the
possibility of novel functions for RAMPS in GPCR regulation [286].

Upon activation, the CL-R/RAMP receptor complex causes cyclic AMP
activation in most systems, irrespective of whether the ligand is AM or CGRP. In

addition, the receptor complex undergoes desensitization and internalization (via Clathrin-

160

mediated endocytosis) in response to a prolonged agonist stimulation [280]. Once
internalized, the receptor complex either undergoes degradation or recycling, depending
on the cell type. In HEK 293 cells the CL-R/RAMP complex has been shown to be
targeted to the lysosomes for degradation, while in rat mesangial cells, the CL—R/RAMP
receptor complex is sorted for dephosphorylation and resensitization (and presumably
recycling) as a fully functional receptor [276, 285]. The mechanism that regulates the
pathway to which the receptor complex is targeted after agonist-induced internalization
remains unknown.

Factors inﬂuencing the sorting of receptors in the early endosomes are largely
unknown, but some of the critical players are beginning to be identiﬁed for the GPCRs.
It has been shown in other GPCR systems that interactions with PSD-95/Discs-large/ZO-
l homology (PDZ) domain proteins are responsible for altering the receptor-targeting
after internalization [259, 267, 268]. The life cycle of the BZ—adrenergic receptor ([52-
AR) was reported to be altered in the presence of a protein termed N-ethylmaleimide
sensitive factor (NSF) [268]. It has been shown that the BZAR interacts with NSF via a
PDZ type I domain (-DSLL) at its extreme C-terminus. In addition, binding of NSF to the
Glu2 subunits of the AMPA receptor was also demonstrated to be crucial for the
recycling of the a-amino-3-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptor
[269, 270]. NSF is a hexameric ATPase that plays a Chaperoning role for SNARES in the
majority of membrane fusion events in a cell, but when targeting membrane receptors for
recycling, NSF acts independently of the SNARE complex to promote rapid

resensitization of the receptors at the plasma membrane [261-263].

161

Similar to the C-terminus of B2-AR, human RAMP3 C-terminus has a type-I PDZ
recognition sequence (-DTLL motif). CL-R, RAMP] or RAMP2 do not, however,
contain any PDZ motifs. We hypothesized that RAMP3, via its interaction with NSF,
regulates the trafﬁcking of the CL-R/RAMP3 complex. We show here that while CL-
R/RAMP] and CL-R/RAMP2 complexes do not interact with NSF, CL-R/RAMP3
complex interacts with NSF via the PDZ motif of RAMP3 [153]. Moreover, we
demonstrate that over-expression of NSF in HEK-293 cells alters the life cycle of CL-
R/RAMP3 complex from a degradative to recycling pathway via interactions of the PDZ
motif of RAMP3 and NSF. These ﬁndings demonstrate that RAMP3, in addition to
determining the receptor phenotype and allowing receptor membrane expression, is also

signiﬁcantly involved with the regulation of the turnover of the CL-R/RAMP complex.

6.2. Materials and Methods

6.2.]. Materials: Adrenomedullin was purchased from Bachem Bioscience, Inc. (King
of Prussia, PA). 1251-labeled adrenomedullin was purchased from Amersham
Biosciences Corp. (Piscataway, NY). N-ethylmaleimide was purchased from Sigma (St.
Louis, MO). Cell culture media, fetal bovine serum, penicillin/streptomycin, trypsin-
EDTA were purchased from GibcoBRL® (Grand Island, NY). RAMP3 antibody was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and NSF antibody was from
Calbiochem® (La Jolla, CA). Anti-mouse Cy3 and anti-rabbit Cy5 secondary antibodies
were from Jackson Immunoresearch Laboratories (West Grove, PA). All other reagents

were of highest quality available.

162

 

6.2.2. Cell Culture and Transfection protocols: Rat mesangial cells were obtained
from ATCC and are maintained in RMPI 1640 media containing 15% FBS and 1%
penicillin-streptomycin. HEK-293T cells (obtained from ATCC) are maintained in
DMEM low glucose media containing 10% FBS, 1% penicillin-streptomycin. Rat-2
ﬁbroblast cells (obtained from ATCC) are maintained in DMEM high glucose media
containing 10% FBS, 1% penicillin—streptomycin. Transfection of HEK293T and Rat-2
ﬁbroblast cells was performed using Lipofectamine Plus protocol (Invitrogen Life
Technologies, Carlsbad, CA). Cells were transfected with the DNA and Lipofectamine
Plus as per manufacturer’s protocol. Cells were collected for assays after 48 hours of

transfection.

6.2.3. RAMP cloning and expression: Full length CDNA of human RAMPS], 2 and 3
and bovine CL-R were described before [281, 282]. CL—R, Cloned into Nl-EGFP and

also in pCDNA3.l expression vectors, was used for transfection in HEK 293T cells.

6.2.4. Desensitization and Resensitization assays: 48 hours post—tranfection cells were
pretreated with or without 10 nM AM in DMEM containing 0.2% BSA for indicated time
periods (up to 4 hr). After agonist exposure, cells were washed three times with
Dulbecco’s phosphate buffered saline (dPBS, Gibco BRL) containing 0.2% BSA and
either frozen for membrane preparation for adenylate cyclase assays or used immediately
for intact-cell radioligand binding. For receptor resensitization assays, after agonist
exposure, cells were washed and incubated for indicated time periods in DMEM

containing 0.2% BSA and 5 jig/ml cycloheximide to allow receptor recovery.

163

 

 

 

Resensitization Protocol:
Removal of AM. followed by
Celia AM PNUOOUMM (’ washing, followed by incubation without AM
transfected atarte j
1 - I I i 1
X Y W Receptor activity assay
(adenylate cyclase) and
‘8 0"" 1 hr 2" h" ti receptor binding at
(recovery me) different time polnta
No Pretreat 1 h AM
(Full Stimulation) '
Pretreat 2'4 h'
(Desensitization) R°°°V°W
(Resensitization)

 

 

 

6.2.5. Receptor binding: Homologous competition radioligand binding assays were
performed as described by Aiyar et a1 and as established in our laboratory [283]. HEK
293T cells were transfected and approximately 200,000 cells/well seeded in poly-D-
lysine pre-coated 24-well plates (BD Biosciences, Palo Alto, CA). 48 hours post—
transfection cells were treated for desensitization or resensitization assays as described
above. After agonist exposure, cells were washed three times with dPBS buffer
containing 0.2% BSA then incubated with increasing concentrations (1 pM to 100 nM) of
competing ligand (rAM) and 175-250 pM 125I-rAM for 30 min at 37°C. After incubation,
plates were washed three times with ice-cold assay buffer and the reactions were
terminated by the addition of 2M NaOH. Cells were then harvested and associated
radioligand activity is counted on a y-counter. All binding assays were performed in
duplicate, with each experiment repeated at least three times. Nonspeciﬁc binding was

determined in the presence of 100 nM of unlabeled rAM. Data was analyzed by

164

LIGAND (assuming radioligand and competitor both bind reversibly to a single binding
site; MacLigand, Version 4.97, NIH, Bethesda, MD) using the following equation:

Bm,x - [hot ligand]

 

KD + [hot ligand] + [cold ligand]

Where B represents speciﬁc binding, [hot ligand] the single concentration of [1251]rAM
studied, [cold ligand] the concentration of unlabeled rAM competing with the radiolabel
for AM receptor binding, Bmax the maximum number of binding sites and KD the
equilibrium dissociation constant (the equation was solved where the “cold ligand” ICso =
[hot ligand] + KD). Analysis of all binding data was performed by computer-assisted
nonlinear least square ﬁtting using GraphPad PRIZM version 4 (GraphPad Software, San
Diego, CA). Binding sites/cell was calculated and data was expressed as percent of the

control.

6.2.6. Adenylate cyclase assays: Cyclase activity was done as described before with
slight modiﬁcations [282, 284]. Cells were harvested ﬁ'om P100 or P60 plates and
homogenized in Tris HCl (10mM)/EDTA (10mM) buffer. Membranes were prepared by
homogenization and centrifugation in Tris HCI (50mM)/MgC1 (10mM) buffer. Final
concentration of 20 pg of protein/assay tube was obtained. Membranes were incubated
for 15 min at 30°C with appropriate concentrations of drugs (100nM AM, lOpM
Forskolin) and assay mix containing ATP regeneration system and (1.3 2P-ATP. After the
reaction was stopped (with stop solution containing 3H-CAMP) contents of the assay
tubes were passed through Dowex and subsequently through alumina columns to separate

the degradation products of ATP, by washing the dowex with water and alumina with

165

imidazole. Elution proﬁle was done to determine the amount of water and imidazole
needed to wash and elute the products. Product eluted from alumina column was counted
for the presence of 3H-CAMP and a32P-CAMP in a B-counter. Each experiment was done
in triplicates and repeated at least 3 times. Data is expressed as percent maximal
response, % forskolin. Basal cyclase activity and forskolin stimulation did not show

statistically signiﬁcant differences between treatments.

6.2.7. CAMP accumulation assays: Rat mesangial cells were seeded on a 48-well plate
(and Rat2 ﬁbroblast cells on 24-well plates) until reaching 80-90% conﬂuency, then
incubated in serum-free media overnight before experiment. Resensitization experiments
were carried out as described in Materials and Methods section, with cells pretreated with
10nM rAM and subsequently challenged for 10 min at 37°C with appropriate
concentrations of drugs (100nM AM, IOpM Forskolin) in the presence of 200pM 3-
isobutyl-l-methylxanthine. Determination of CAMP level was measured using the
Biotrak CAMP enzyme immunoassay system (Amersham Biosciences) according to the
manufacturer's instructions. CAMP levels in cells were calculated using a standard curve
ranging from 10 to 10 4 fmol of CAMP. Each experiment was done in duplicate and
repeated at least 3 times. Data is expressed as percent maximal response, % forskolin.
Basal cyclase activity and forskolin stimulation did not show statistically signiﬁcant

differences between treatments.

6.2.8. RNA Interference analysis: Gene-speciﬁc d-siRNA for lacZ (control) and

RAMP3 were generated and puriﬁed using BLOCK-iT Dicer RNAi kit from Invitrogen

166

(Carlsbad, CA). RMCs were transfected with d-siRNAs using Lipofectamine 2000 as per
manufacturer’s instructions (Invitrogen). 48 hours after transfection cells were frozen for
mRNA analysis, or used for CAMP accumulation assays or immunoﬂuorescence

microscopy.

6.2.9. Quantitative PCR analysis: Total RNA was isolated from RMCs using Trizol
reagent (GIBCO BRL). After sodium acetate-ethanol precipitation and several ethanol
washes, RNA was used as a template in a quantitative PCR ampliﬁcation procedure.
Quantitative PCR analysis was carried out with the LUX (Light Upon Extension)
fluorogenic primer method, following the protocol in the manufacturer’s manual, as

described by Nazarenko et al .

6.2.10. Mutagenesis procedure: Site-directed mutagenesis was performed using a PCR-
based strategy that employs the pfu Turbo polymerase (Stratagene, La Jolla, CA). A pair
of complementary oligonucleotides containing the appropriate point mutations in the
sequence of RAMP or a premature stop codon at position 145 or 147 codon of RAMP-3
for deletion mutants were synthesized (Michigan State University Macromolecular
structure facility). The PCR for the mutation was as follows: 94°C for 2 minutes; 30
cycles of 94°C for 30 sec., 50°C for 30 sec., 68°C for 8 min.; ﬁnal cycle of 68°C for 8
minutes. PCR product was digested for 4 hours with DpnI enzyme (Invitrogen) and
transformed in to DHSa cells. Mutations were conﬁrmed by automated sequencing

(Michigan State University Genomic Technology Support Facility).

167

6.2.1]. Immunoﬂuorescence microscopy: HEK 293 cells were transfected as described
above and seeded at 24hr post-transfection onto collagen type I-Coated coverslips.
Resensitization assays were performed as described and reactions were stopped by ﬁxing
cells in 4% paraformaldehyde for 30min. at room temperature. Samples were
perrneablized with 0.1% v/v Triton X-100 in PBS and blocked overnight in 0.1% v/v
Triton X-100 in PBS + 10% goat serum. Samples were incubated in primary antibody in
blocking buffer for 2h at room temperature (NSF at 1:250 and RAMP3 at 1:200).
Appropriate secondary antibodies were applied for 1h at room temperature (Goat anti-
mouse Cy3 at 1:500 and Goat anti-rabbit Cy5 at 1:400). Coverslips were mounted in
Shandon Permaﬂuor mounting medium and slides stored at 2-8°C until analysis. Cells
were visualized on a Zeiss 210 laser confocal microscope at a zoom of 2. Images
presented are'representative single optical sections of a z-series taken from at least twenty
ﬁelds per experiment and at least three individual experiments. Images in this

thesis/dissertation are presented in color.

6.2.12. Fusion protein overlays and western blotting: 10 pg of GST-fusion proteins
were resolved on a 10% SDS-PAGE gel and transferred to nitrocellulose ﬁlters. Filters
were blocked with 5% w/v fat-ﬂee milk powder in Tris-buffered saline with Tween
20 (TTBS: 20mM Tris, pH 7.4, 500mM NaCl, 0.1% v/v Tween 20) and incubated
overnight at 4 °C in lysates of HEK293 cells with or without overexpression NSF. Blots
were then washed three times with TTBS buffer and incubated with anti-NSF monoclonal
antibody for 2 h at room temperature. After three washes with TTBS, ﬁlters were

incubated for 1h with horseradish peroxidase-conjugated goat anti-mouse secondary

168

antibody (Gibco BRL®, Grand Island, NY), washed again with TTBS, soaked in
Supersignal West Pico chemiluminescent substrate (Pierce) and exposed to x-ray ﬁlm.
Same protocol, with the exception of the overnight incubation with cell lysate, was

followed for immunoblot analysis of RAMP3.

6.2.13. Statistics: Data are presented as mean i S.E.M. Single group comparisons

exercised a paired Student’s t-test method. Statistical signiﬁcance was set at P<0.05.

6.3. Results

6.3.]. Role of NSF in Resensitization of the CL—R/RAMP complex

Our lab has previously published that rat mesangial cells (RMCS) endogenously
express the AMlR (CL-R + RAMP2) and the AM2R (CL-R + RAMP3) (8). This data
was repeated and NSF expression was conﬁrmed in the RMCs with RT-PCR and
immunocytochemistry (data not shown). Our laboratory has also reported that
pretreatment of rat mesangial cells (RMCS) with AM leads to an agonist-stimulated
desensitization and internalization of the CL-R/RAMP complex. Phosphatase-dependent
resensitization of AM responsiveness was also demonstrated after agonist-stimulated
desensitization [285]. Measuring CAMP accumulation we repeated these results in this
study (Figure 28). As a preliminary test to determine if NSF is involved in the
resensitization of AM responsiveness, we used a pharmacological inhibitor of NSF, N-
ethylmaleimide (NEM). In RMCs treated for 45 seconds with 50 pM NEM during the

resensitization experiment, resensitization was blocked, as measured by CAMP

169

accumulation (Figure 28). NEM, however, did not affect basal CAMP accumulation or
the desensitization response when compared to untreated cells, an important ﬁnding
given the ability of NEM to interfere with Ga subunits (Figure 28). These results
indicate that NSF plays a role in the sorting of the CL-R/RAMP complex following
agonist-induced internalization in this endogenous CL-R/RAMP system where the
receptor complex is recycled. To fully evaluate the molecular mechanisms of this
observation, we used HEK 293 cells to examine the interaction of the CL—R/RAMP
complex with NSF and the impact of this interaction on receptor trafﬁcking.

In contrast to RMCs, HEK 293 cells express very low endogenous levels of
RAMPS. Kuwasako et al. have demonstrated that in HEK 293 cells overexpressing the
CL-R/RAMP complex, agonist-induced internalization leads to receptor trafﬁcking to a
degradation pathway [276]. In this study the internalized CL-R/RAMP complexes were
colocalized with LAMP-1, a lysosomal marker, to show the targeting of the receptor for
the degradation pathway. Utilizing adenylate cyclase activity assays, whole-cell ligand
binding, and immunoﬂuorescence microscopy we conﬁrmed these ﬁndings (Figure
29A,B). Pretreatment of HEK 293 cells transfected with CL-R and RAMP3 with 10 nM
AM for one hour resulted in desensitization of the adenylate cyclase response from 50%
(of forskolin stimulation) in untreated cells to 28% in AM-treated cells (Figure 29A, left
axis). Even after the removal of agonist and incubation with buffer alone for indicated
times through 4h, the adenylate cyclase response remained desensitized (Figure 29A, left
axis), indicating a lack of resensitization. Consistent ﬁndings were obtained with whole-
cell binding and immunoﬂuorescence microscopy experiments (Figure 29A, right axis,

29B).

170

To determine if NSF overexpression could alter the receptor trafﬁcking in this cell
system, NSF was co-transfected with CL-R and RAMP-3, and resensitization and
recycling assays were performed. Resensitization and recycling were monitored by
adenylate cyclase activity assays and whole-cell competition binding, respectively. In
addition, visualization of the trafﬁcking of the receptor complex was performed by
immunoﬂuorescence microscopy. In the absence of NSF, pretreatment with AM for one
hour resulted in desensitization of the adenylate cyclase response and internalization of
the receptor complex. Upon removal of agonist and incubation with buffer alone for 4h,
the adenylate cyclase response remained desensitized and the receptor complex remained
internalized, indicating a lack of resensitization (Figure 30A, B). In contrast, when NSF
was co-transfected in the cells, although the desensitization response (i.e. response after
1h agonist treatment) was not altered, the cells now underwent time-dependent
resensitization (i.e. response after 1, 2, or 4h agonist removal) in response to AM (Figure
30A). Consistent ﬁndings were obtained with whole-cell binding and
immunoﬂuorescence microscopy experiments (Figure 30B, 31). Time course
experiments indicated the time course for complete resensitization and recycling of the
CL-R/RAMP3 receptor complex to be 4b in HEK 293 cells, as measured by adenylate
cyclase, whole-cell binding, and immunoﬂuorescence microscopy experiments (Figure
31). All subsequent experiments in HEK 293 cells use the 4h time point to determine
receptor complex recycling and resensitization. These results indicate that the presence
of NSF alters the intracellular sorting of the CL-R/RAMP3 receptor complex after AM-

stimulated endocytosis.

171

6.3.2. RAMP isoform-specific regulation of CL-R/RAMP receptor complex
trafficking

To determine if this effect of NSF was speciﬁc for RAMP3, the additional
RAMPS (RAMP—l or -2) were tested for their ability to act with NSF to alter the receptor
complex life cycle. Interestingly, in contrast to RAMP3, presence of NSF did not alter
the resensitization response or recycling pattern of the CL-R/RAMP-l or -2 receptor
complexes. Both the activity and receptor number remained at desensitized levels in cells
transfected with CL-R+RAMP1 or CL-R+RAMP2 (along with NSF) (Figure 32A-D).
Both CL-R/RAMP] and CL-R/RAMP2 complexes showed no difference in receptor
expression levels at the plasma membrane (as measured with whole-cell binding) in
untreated cells, as compared to CL-R/RAMP3 complex. These results indicate that
RAMP3 must contain a molecular determinant distinct from the other RAMPS that

allowed its interaction and activity with NSF.

6.3.3. Role of PDZ interactions in trafﬁcking of the CL-R/RAMP3 complex

We hypothesized that the unique characteristic of RAMP3 that allowed its
interaction with NSF to change receptor trafﬁcking was a PDZ motif on its extreme C-
terrninus. In order to establish if this domain is critical for interaction of the CL-
R/RAMP3 complex with NSF, the PDZ motif (-DTLL) on RAMP3 was deleted.
Deletion of this domain did not affect basal adenylate cyclase activity or the
desensitization response of the CL-R/RAMP3 complex in response to AM, even in the
presence of NSF (Figure 32). In contrast, the deletion of the PDZ motif (-DTLL)

signiﬁcantly affected the resensitization and recycling of the CL-R/RAMP3 receptor

172

BL“-

complex in the presence of NSF. Both radioligand binding and adenylate cyclase assays
showed a loss of recycling and resensitization, respectively, of the receptor complex
when RAMP3A145-8 was expressed in the presence of NSF, as compared to the wild-
type RAMP3 with NSF (Figure 33A, B). Mutant RAMP3 (RAMP3A145-8) showed no
difference in receptor expression levels at the plasma membrane (as measured with
whole-cell binding) in untreated cells, as compared to wild-type CL—R/RAMP3 complex.
Additionally, results were conﬁrmed when a recycling assay with RAMP3A145-8 was
performed with immunoﬂuorescence microscopy (Figure 34).

To further test the hypothesis that the absence of the PDZ motif on the RAMP2
accounts for the lack of interaction of the CL-R/RAMP2 complex with NSF, and hence
the inability of the CL-R/RAMP2 complex to follow a recycling pathway, the PDZ motif
of RAMP3, the amino acids -DTLL, were substituted on the C-terminus of RAMP2, in
exchange for its original four C-terminal amino acids (-EAQA). The RAMPZADTLL
mutant showed similar levels of adenylate cyclase activity and whole-cell radioligand
binding without pretreatment and after desensitization, as compared to wild type RAMP2
in control experiments (Figure 35). The RAMPZADTLL mutant also showed similar
receptor expression levels at the plasma membrane (as measured with whole-cell
binding) in untreated cells, as with the wild-type CL-R/RAMP2 complex.
Resensitization assays (measured with adenylate cyclase activity) and recycling assays
(measured by whole-cell binding) were performed in HEK 293 cells were transfected
with CL-R, RAMPZADTLL, and NSF, as described previously. Similar to the CL-
R/RAMP3 complex, the CL-R- RAMPZADTLL complex underwent resensitization and

recycling in the presence of NSF, as assessed by adenylate cyclase and whole-cell

173

binding (Figure 35). These ﬁndings provide additional evidence that the PDZ motif on
the RAMP3 is the site of interaction of the receptor complex with NSF, causing a Change
in receptor trafﬁcking, from a degradative to a recycling pathway.

To further identify the critical amino acids in the PDZ recognition sequence
which regulate the RAMP3/NSF interaction, point mutations of the amino acids of the
RAMP3 PDZ motif to alanine were performed. The functional effects of the point
mutations were analyzed with resensitization assays, measured by adenylate cyclase
activity, and recycling assays, measured by whole-cell binding, as described before. Our
results indicate that mutation of D145, T146, or L148 to alanine disrupted the RAMP3
interaction with NSF and inhibited the resensitization and recycling of the CL-R/mutant
RAMP3 complex after AM-induced endocytosis in HEK 293 cells (Figure 36). Mutation
of L147 to alanine had no effect on the resensitization or recycling of the receptor
complex in the presence of NSF (Figure 36). Control experiments with the point
mutations co-transfected with CL-R showed similar levels of adenylate cyclase activity
and whole-cell binding as compared to wild-type RAMP3 without pretreatment and after
desensitization. RAMP3 point mutants also showed no difference in receptor expression
levels at the plasma membrane (as measured with whole-cell binding) in untreated cells,
as compared to wild-type CL-R/RAMP3 complex.

To examine if the PDZ motif on RAMP3 is interacting with NSF, overlay assays
were performed. This was accomplished using GST-RAMP3 fusion proteins in an
overlay assay with cell lysates of HEK 293 cells overexpressing NSF. Control
experiments run with GST protein showed no detectable bands when incubated with NSF

lysates and probed with an NSF antibody (Figure 37A). Importantly, wild-type RAMP3

174

fusion proteins showed interaction with NSF in the cell lysates of HEK 293 cells
overexpressing NSF in the overlay assay (Figure 37A). In addition, RAMP3A145-8
fusion proteins, lacking the PDZ motif on RAMP3, showed no detectable bands when
incubated with NSF lysates and probed with an NSF antibody (Figure 37A). Lysates of
HEK 293 cells not over—expressing NSF showed no detectable bands when run with
GST-RAMP3 in the overlay assay and probed for NSF (Figure 37B). When blots used in
the overlay assay were stripped and probed for RAMP3, a band was detected in the GST—
RAMP3 lane in the exact location as in the overlay assay when probed for NSF (Figure
37C). This data demonstrates an interaction between RAMP3 and NSF via the PDZ
motif on RAMP3, an interaction that is capable of regulating CL-R/RAMP3 (AM2R)

complex trafﬁcking.

6.3.4. RAMP3 and NSF regulation of receptor trafﬁcking in unaltered cell lines

It was important to establish if our observations in the HEK 293 cells was
transferable to unaltered cells lines. Reexamining the rat mesangial cells used in the ﬁrst
set of experiments, we employed RNA interference technology to knockdown RAMP3
expression. Having demonstrated the requirement of NSF in the cells for efﬁcient
receptor resensitization (Figure 28), the RAMP3 RNA interference experiment would
determine if RAMP3 was also required for effective receptor resensitization. In both
mRNA and protein expression studies, RAMP3 expression dramatically decreased, while
control experiments using lacZ knockdown showed no signiﬁcant alteration in RAMP3
expression when compared to wild-type cells (Figure 38A, B; data not shown).

Resensitization assays were performed and CAMP accumulation was measured to

175

&\\"'r '

determine the effect of RAMP3 RNA interference on receptor resensitization in rat
mesangial cells. RNA interference for RAMP3 showed similar levels of CAMP
accumulation as compared to wild-type RMCs without pretreatment and after
desensitization (Figure 38C). Strikingly, when allowed sufﬁcient recovery time in the
absence of agonist following desensitization, RMCs with RAMP3 RNA interference
showed an inability to resensitize, unlike the wild-type RMCs (Figure 38C). This ﬁnding
demonstrates that NSF and RAMP3 are both critical for receptor targeting for
resensitization in rat mesangial cells, an unaltered cell line absent of the issues of
overexpression.

As a further test of our proposed model, we employed a cell line that does not
express RAMP3, but does express the AMlR (CL-R and RAMP2). We hypothesized
that, with the absence of RAMP3, this cell line would fail to resensitize following agonist
pretreatment, and that expression of RAMP3 in these cells would allow a switch in
receptor targeting for resensitization. Rat2 ﬁbroblast cells have been shown by Choksi et
al. to lack RAMP3 expression, but do express the AM1R(CL-R and RAMP2) [308]. We
repeated this ﬁnding and conﬁrmed the expression of NSF in Rat2 ﬁbroblast cells with
RT-PCR and immunocytochemistry (data not shown). We performed resensitization
assays with the Rat2 ﬁbroblast cells and measured resensitization by CAMP
accumulation. The Rat2 ﬁbroblast cells exhibited a decrease in CAMP accumulation after
agonist pretreatment and failed to resensitize, as predicted, when allowed to recover in
the absence of agonist for four hours. Interestingly, consistent with our model, RAMP3
expression in the Rat2 cells showed no alteration in CAMP accumulation without

pretreatment and after desensitization when compared to wild-type Rat2 cells, but now

176

showed resensitization when allowed recovery time in the absence of agonist (Figure 39).
Furthermore, expression of the RAMP3 PDZ motif mutant in the Rat2 cells was unable to
cause receptor resensitization, while showing similar levels of CAMP accumulation as
compared to wild-type Rat2 cells without pretreatment and following desensitization
(Figure 39). RAMP3 and RAMP3A145-8 showed similar levels of transfection
efﬁciency in the Rat2 cells, as measured by immunocytochemistry (data not shown).
This data further conﬁrms the crucial role of RAMP3 in the targeting of the AM2R for
resensitization/recycling after agonist-stimulated desensitization. Additionally, it
suggests that differential RAMP expression in cells may determine the sorting of the

AM-R from the endosomes after internalization.

177

-Control

 

40- er; 50 pM NEM
E
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5E
a: O
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Figure 28: Effect of N-ethylmaleimide, an NSF inhibitor, on resensitization of CAMP
accumulation in rat mesangial cells (RMCS). The lh pretreatment time point shows
desensitization of CAMP accumulation response in cells with or without treatment with NEM.
The 2 hr recovery time point shows resensitization is inhibited in cells treated with NEM, as
compared to untreated RMCs. This indicates a role for NSF in the resensitization observed in
RMC cells. RMC cells seeded in 24-well plates were pretreated with IOnm rAM for 1h were
washed extensively to remove residual agonist and treated with 50 pM NEM for 45 seconds.
Following NEM treatment. cells were washed repeatedly and incubated in serum-free media with
Spg/ml cycloheximide for indicated times. After recovery time, RMC cells were re-Challenged
with 100 nM rAM for 15 minutes and plates were frozen. Determination of CAMP level was
measured using the Biotrak CAMP enzyme immunoassay system (Amersham Biosciences)
according to the manufacturer's instructions. CAMP levels in rat mesangial cells were calculated
using a standard curve ranging from 10 to 10 4 frnol of CAMP. Each experiment was done in
duplicate and repeated at least 3 times. Data is expressed as percent maximal response, %

forskolin. * p S 0.05; n 2 3 experiments.

178

 

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Recovery time (hr)

Figure 29A: The sorting of the AM2R (CL-R+RAMP3) in HEK 293 cells. CL-R/RAMP3
complex does not resensitize/recycle in HEK 293 cells after agonist-stimulated
‘ " " ‘ " " HEK 293 cells transiently transfected with CL-R and RAMP3
were treated for one hour with AM (10 nM) and then washed and receptor resensitization
(adenylate cyclase activity) and recycling (radioligand binding) were measured after indicated
recovery times in the absence of agonist. Adenylate cyclase activity is shown on the left y-axis,
expressed as percent maximal response (% forskolin stimulation), and radioligand binding is

shown on the right y-axis, expressed as percent control. * p S 0.05; n 2 3 experiments.

179

  
 

1hr

Pretreat O

4 hr
Recovery

Figure 298: The sorting of the AM2R (CL-R+RAMP3) in HEK 293 cells. lmmunoﬂuorescence
microscopy shows failure of CL-R/RAMP3 receptor complex to recycle after agonist-stimulated
internalization. HEK 293 cells transfected with CL—R-GFP and RAMP3 were pretreated with
10nM AM for lh. After pretreatment with AM, cells were washed and incubated in serum-free
media with Spg/ml cycloheximide to allow receptor recycling for indicated times. Cells were
ﬁxed and components were visualized using anti-RAMP3 antibody (1:200) and detected with
Cy5 secondary antibody (1:200), and CL-R is detected with an EGFP tag; overlays of staining
patterns are shown in the far right panels. Images shown are representative of at least twenty
ﬁelds imaged per experiment from at least three experiments. Bar scales on all images represent

50pm.

180

- CL-R+RAMP3
D CL-R+RAMP3+NSF

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 30A: The role of NSF in the sorting of the AM2R (CL-R+RAMP3) in HEK 293 cells.
NSF causes recycling of AM2R in HEK 293 cells. HEK 293 cells transiently transfected with
CL-R and RAMP3 with or without NSF. At 48h post-transfection, cells were treated for one
hour with AM (10 nM) and then washed and receptor recycling was measured after indicated
recovery times in the absence of agonist. Receptor recycling measured by whole-cell competition
binding assays using ”SI-rAM as ligand (cold rAM served as the competitor) and number of
binding sites/cell was estimated using the GRAPHPAD PRISM software. “No pretreat”
represents samples at maximal radioligand binding that were not pre-incubated with agonist. “1h
pretreat” represents samples pretreated with AM (10 nM) for one hour, washed as indicated in
Methods section, and tested immediately after wash steps for radioligand binding. “1 , 2, and 4 hr
recovery” samples were pretreated with AM (10 nM) for one hour, washed, and allowed to
recover for indicated times in media without agonist plus Spg/ml cycloheximide, then analyzed
for radioligand binding. NSF overexpression in cells expressing AM2R caused altered receptor

trafficking ﬁom degradation to recycling pathway. * p S 0.05; n 2 3 experiments.

181

- CL-R+RAMP3
:1 CL-R+RAMP3+NSF

 

 

 

 

 

 

 

 

 

 

 

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Figure 30B: The role of NSF in the sorting of the AM2R (CL-R+RAMP3) in HEK 293 cells.
NSF causes recycling of AM2R in HEK 293 cells. NSF causes resensitization of AM2R in
transfected HEK 293 cells. HEK 293 cells transfected and pretreated with agonist as described in
29A. After agonist pretreatments, membranes were extracted and AC activity in response to 100
nM AM was measured. NSF overexpression with AM2R allowed time-dependent receptor
resensitization. Experiments performed in triplicates and data expressed as percent maximal

stimulation (% Forskolin). " p S 0.05; n 2 4.

182

No Pretreatment 1 hr Pretreatment 4 hr Recovery

CL-R ..-
RAMP3...-
NSF
I

Overlay‘
I

 
 

,I

183

Figure 3]: Localization of CL-R, RAMP3, and NSF in HEK 293 cells during a recycling
experiment. After lh AM pretreatment, CL-R and RAMP3 are internalized and show co-
localization with NSF intracellularly. After 4h recovery time post-AM pretreatment, CL-R and
RAMP3 show distribution at the plasma membrane of the cell, demonstrating recycling of the
receptor complex. HEK 293 cells transfected with CL-R-GF P, RAMP3, and NSF were pretreated
with 10nM AM for lh. After pretreatment with AM, Cells were washed and incubated in serum-
free media with Spg/ml cycloheximide to allow receptor recycling for indicated times. Note: “1
hr pretreatment” indicates time just after AM pretreatment and wash steps, with no recovery time.
Cells were ﬁxed and components were visualized using anti-RAMP3 antibody (1:200) and anti-
NSF antibody (1:250) with Cy5 anti-rabbit secondary antibody ( 1:400, in blue) and Cy3 anti-
mouse secondary antibody (1:500, in red), respectively; CL-R-GFP is detected with an EGFP tag
and shown in green; overlays of staining patterns are shown in the far right panels. Images shown
are representative of at least twenty ﬁelds imaged per experiment ﬁ'om at least three experiments.

Bar scales on all images represent lOOpm.

184

- CL-R+RAMP1
E: CL-R+RAMP1+NSF

 

 

 

 

 

 

 

 

 

 

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=9.
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Figure 32A: The role of NSF in post-endocytic sorting of CGRP] (CL-R+Rl) and AM]
receptor. NSF overexpression does not alter the trafﬁcking of the CGRP receptor after
internalization. HEK 293 cells were transfected with CL-R and R], with or without NSF. 48h
post-transfection, cells were pretreated with CGRP (10 nM) for one hour, washed as described in
Materials and Methods, allowed indicated recovery times in agonist-free media, and receptor
recycling was measured with whole-cell competition binding using l2"'I-rCGRP as the ligand and
cold rCGRP as the competitor. Number of binding sites/cell was estimated with GRAPHPAD

PRISM software. * p S 0.05; n 2 3.

185

- CL-R+RAMP1
D CL-R+RAMP1+NSF

l ,

N I»
O O
I I

% Forskolin
3:3

AC Actiwty
(pmollminlmg protein)

 

 

 

 

 

 

 

 

O
I

 

Figure 328: The role of NSF in post-endocytic sorting of CGRPI (CL-R+R1) and AMI
receptor. NSF overexpression does not cause CGRP receptor resensitization after agonist-
stimulated endocytosis. HEK 293 cells were transfected and pretreated as in ﬁgure 29A and then
membranes were extracted. Adenylate cyclase activity was measured in membranes stimulated
with 100 nM CGRP for 15 min. Experiments performed in triplicates and expressed as percent

maximal stimulation (% Forskolin). * p S 0.05; n 2 3.

186

- CL-R-I-RAMPZ
1:1 CL-R+RAMP2+NSF

 

 

 

 

 

 

 

 

 

 

 

 

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3
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Figure 32C: The role of NSF in post-endocytic sorting of CGRP] (CL-R+Rl) and AM]
receptor. NSF overexpression does not cause recycling of AMI receptor after internalization.
HEK 293 cells were transfected with CL-R and R2, with or without NSF. 48h post-transfection,
cells were pretreated with AM (10 nM) for one hour, washed as described in Materials and
Methods, allowed indicated recovery times in agonist-free media, and receptor recycling was
measured with whole-cell competition binding using I25I-rAM as the ligand and cold rAM as the
competitor. Number of binding sites/cell was estimated with GRAPHPAD PRISM software. * p

S 0.05; n 2 4.

187

 

161

in

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- CL-R+RAMP2
E CL-R+RAMP2+NSF

 

 

 

 

 

 

 

 

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Figure 32D: The role of NSF in post-endocytic sorting of CGRP] (CL-R+Rl) and AM]

receptor. NSF overexpression does not cause AMI receptor resensitization after agonist-induced

internalization. HEK 293 cells were transfected and pretreated as in ﬁgure 29A and then

membranes were extracted. Adenylate cyclase activity was measured in membranes stimulated

with 100 nM AM for 15 min. Experiments performed in triplicates and expressed as percent

maximal stimulation (% Forskolin). * p S 0.05; n _>_ 3.

188

 

 

- CL-R+RAMP3+NSF
[:1 CL-R+RAMP3A145-8+NSF

 

 

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Figure 33A: The effect of RAMP3 PDZ motif deletion on the trafﬁcking of the AM2R (CL-
R+RAMP3). Deletion of RAMP3 PDZ motif blocked the recycling of the CL-R/RAMP3
complex when co-expressed with NSF. HEK 293 cells were co-transfected with CL-R, wild-type
RAMP3 or RAMP3AI45-8, and NSF. 48h post-transfection, cells were pretreated with AM (10
nM) for one hour, washed as described in Materials and Methods, allowed 4h recovery times in
agonist-free media, and receptor recycling was measured with whole-cell binding using I25I-rAM
as the ligand and cold rAM as the competitor. Number of binding sites/cell was estimated with

GRAPHPAD PRISM software. * p S 0.05; n 2 3.

189

 

- CL-R+RAMP3+NSF
E CL-R+RAMP3A145-8+NSF

*
*
0' I I
6“

Figure 333: The effect of RAMP3 PDZ motif deletion on the trafﬁcking of the AM2R (CL-

50-

w 1k
C O
1 a
3(-

% Forskolin
N
‘P

AC Activity
(pmollminlmg protein)

 

 

 

 

R+RAMP3). Deletion of RAMP3 PDZ motif blocked the resensitization of the CL-R/RAMP3
complex aﬁer agonist-stimulated desensitization when co-expressed with NSF. HEK 293 cells
were transfected and pretreated as in ﬁgure 29A and then membranes were extracted as described
in Materials and Methods section. Adenylate cyclase activity was measured in membranes
stimulated with 100 nM AM for 15 min. Experiments performed in triplicates and expressed as

percent maximal stimulation (% Forskolin). "‘ p S 0.05; n 2 3.

190

No Pretreatment 1 hr Pretreatment 4 hr Recovery

Overlay

 

Figure 34: Localization of CL-R, RAMP3AI45-8, and NSF in HEK 293 cells during a recycling
experiment. Untreated and aﬂer one hour AM (10nM) pretreatment, CL-R and RAMP3A145—8
internalize similarly to the wild-type CL-R/RAMP3 complex. Aﬁer a 4h recovery time in the
absence of agonist, CL-R and RAMP3A145-8 fail to recycle to the plasma membrane.
Experiments performed as described in Figure 31. Fixed cells were stained with anti-RAMP3
antibody (1:200) and anti-NSF antibody (1:250) with Cy5 anti-rabbit secondary antibody (1 :400,
in blue) and Cy3 anti-mouse secondary antibody (1:500, in red), respectively; CL-R-GFP is
shown in green; overlays of staining patterns are shown in the far right panels. Images shown are
representative of at least twenty ﬁelds imaged from at least three experiments. Bar scales on all

images represent lOOpm.

191

- CL-R+RAMP2+NSF
E CL-R+RAMP2ADTLL+NSF

l

Bmax (binding sites/cell)
% Control
0|
‘?

 

 

 

 

 

 

Figure 35A: Effect of PDZ motif substitution on the C-terminus of RAMP2 on
recycling/resensitization of CL-R/RAMP complex. Substitution of PDZ motif (-DTLL) on the C-
tenninus of RAMP2 cause a change in receptor trafficking phenotype from degradation to
recycling of the receptor complex. HEK 293 cells were co-transfected with CL-R, wild-type
RAMP2 or RAMPZADTLL, and NSF. 48h post-transfection, cells were pretreated with AM (10
nM) for one hour, washed as described in Materials and Methods, allowed 4h recovery times in
agonist-free media plus Sug/ml cycloheximide, and receptor recycling was measured with whole-
cell binding using ”SI-rAM as the ligand and cold rAM as the competitor. Number of binding

sites/cell was estimated with GRAPHPAD PRISM soﬁware. “‘ p S 0.05; n 2 4.

192

- CL-R+RAMP2+NSF
E CL-R+RAMP2ADTLL+NSF

 

 

 

 

 

 

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.. l
2 .E 30
.— Q— -
O
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Figure 358: Effect of PDZ motif substitution on the C-terminus of RAMP2 on

recycling/resensitization of CL-R/RAMP complex. PDZ motif substituted on the C-terminal tail
of RAMP2 cause resensitization of adenylate cyclase activity. HEK 293 cells were transfected
and pretreated as in ﬁgure 29A and then membranes were extracted as described in Materials and
Methods section. Adenylate cyclase activity was measured in membranes stimulated with 100
nM AM for 15 min. Experiments performed in triplicates and expressed as percent maximal

stimulation (% Forskolin). "‘ p S 0.05; n 2 4.

193

- No Pretreatment
D 1 hr Pretreatment
[:34 hr Recovery Time

Bmax (binding sites/cell)

 

 

 

Figure 36A: Effect of RAMP3 PDZ motif point mutations on the recycling/resensitization of the
CL-R/RAMP3 complex in the presence of NSF. All point mutants within the PDZ motif on
RAMP3, except R3Ll47A, blocked the recycling of the CL-R/RAMP3 complex seen when co-
expressed with NSF. HEK 293 cells were co-transfected with CL-R, wild-type RAMP3 or
RAMP3 point mutants, and NSF. 48h post-transfection, cells were pretreated with AM (10 nM)
for one hour, washed as described in Materials and Methods, allowed 4h recovery times in
agonist-free media, and receptor recycling was measured with whole-cell binding using I25I-rAM
as the ligand and cold rAM as the competitor. Number of binding sites/cell was estimated with

GRAPHPAD PRISM soﬁware. "‘ p S 0.05; n 2 3.

I94

 

- No Pretreatment
D 1 hr Pretreatment
m 4 hr Recovery Time

 

 

 

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Figure 368: Effect of RAMP3 PDZ motif point mutations on the recycling/resensitization of the
CL—R/RAMP3 complex in the presence of NSF. Conﬁrming the whole-cell binding data, all
point mutations to the PDZ motif of RAMP3, except R3Ll47A, inhibited the resensitization of
adenylate cyclase activity seen with wild-type RAMP3-CL-R complex and co-expression of NSF.
HEK 293 cells were transfected and pretreated as in ﬁgure 29A and then membranes were
extracted as described in Materials and Methods section. Adenylate cyclase activity was
measured in membranes stimulated with 100 nM AM for 15 min. Experiments performed in
triplicates and expressed as percent maximal stimulation (% Forskolin). * p S 0.05 two-tail; # p5

0.05 one-tail ; n 2 3-4.

195

a. b. c.
GST

GSTr GST-
GST- RAMP3 GST RAMP} GST- RAMP3
GST RAMP3 .\145-8 GST RAMP3 A145-8 GST RAMP3 A145-8
70 KDa
- --
so KDa

Figure 37: Interaction studies of RAMP3 with NSF. Fusion proteins of GST-RAMP3
demonstrate interaction with NSF in an overlay assay and western blot analysis. lOug of GST,
GST-RAMP3, and GST-RAMP3AI45-8 protein was run and separated on an SDS-PAGE gel and
transferred to a nitrocellulose ﬁlter. The nitrocellulose ﬁlter was incubated overnight in lysate of
HEK293 cells transfected with or without NSF at 4°C. Filter was then washed and probed by
immunoblot for NSF (1:250). Identical ﬁlters were probed by immunoblot for RAMP3 (1:400).
A) GST, GST-RAMP3, and GST-RAMP3AI45-8 overlay assay incubated in NSF-transfected
HEK 293 lysates and probed for NSF. B) GST, GST-RAMP3, and GST-RAMP3A145-8 overlay
assay incubated in non-transfected HEK 293 lysates and probed for NSF. C) lmmunoblot of

GST, GST-RAMP3, and GST-RAMP3A145-8 probed for RAMP3. Shown are representative

blots of at least three experiments.

196

- Wild-type
200. D RAMP3 Knockdown

..L

Cl

C
e

I.

[% Forskolin]
3
C.’

cAMP Accumulation
(nmollmg protein)
0|
‘?

 

 

 

 

 

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Figure 38A: Effect of RAMP3 RNA interference on resensitization of AM2-R in rat mesangial
cells. RAMP3 RNA interference disrupts the ability of AM—R to resensitize aﬁer agonist
pretreatment. RMC cells seeded in 24-well plates were transfected with d-siRNA of RAMP3 and
incubated for 48 hr to allow RNA knockdown. Wild-type and d-siRNA transfected cells were
pretreated with IOnm rAM for 1h and were washed extensively to remove residual agonist.
Following washes, cells were incubated in serum-free media with 5 ug/ml cycloheximide for 2
hr. After recovery time, RMC cells were re-challenged with 100 nM rAM for 15 minutes and
plates were frozen. cAMP levels were measured using the Biotrak cAMP enzyme immunoassay
system (Amersham Biosciences) according to the manufacturer's instructions. cAMP levels in rat
mesangial cells were calculated using a standard curve ranging from 10 to 10 4 fmol of cAMP.

Each experiment was done in duplicate and repeated four times. Data is expressed as percent

maximal response, % forskolin. * p S 0.05; n 2 4

experiments.

I97

 

 

 

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Figure 38B: Effect of RAMP3 RNA interference on resensitization of AM2-R in rat mesangial
cells. RAMP3 RNA interference disrupts the ability of AM-R to resensitize aﬁer agonist
pretreatment. RMC cells seeded in 24-well plates were transfected with d-siRNA of RAMP3 and
incubated for 48 hr to allow RNA knockdown. mRNA analysis by RT-PCR showed dramatically
decreased levels of RAMP3 mRNA in RAMP3 RNA interference sample as compared to wild
type, while lacZ knockdown had no effect on RAMP3 message levels (data not shown). RNA
isolation and Q-PCR performed as described in Experimental Procedures section. GAPDH

expression measured for all samples for normalization. n=3 experiments.

198

Control RAMP3 Knockdown

Anti-RAMP3

   

Figure 38C: Effect of RAMP3 RNA interference on resensitization of AM2-R in rat mesangial
cells. RAMP3 RNA interference disrupts the ability of AM-R to resensitize after agonist
pretreatment. RMC cells seeded in 24-well plates were transfected with d-siRNA of RAMP3 and
incubated for 48 hr to allow RNA knockdown. lmmunoﬂuorescence microscopy of RMCs with
RAMP3 RNA interference demonstrated greatly decreased levels of RAMP3 protein expression
in RAMP3 RNA knockdown cells, as compared to wild-type RMC cells. LacZ knockdown had
no eﬁect on RAMP3 protein expression in RMCs (data not shown). Cells prepared as described
in Experiment procedures section for immunoﬂuorescence microscopy. Fixed cells were stained
with anti-RAMP3 antibody (1:200) and detected with a Cy3 anti-rabbit secondary antibody
(1 :500, in red). Images shown are representative of at least three experiments. Bar scales on all

images represent 20pm.

199

- No transfection
E RAMP3
-RAMP3A145-8

15-

cAMP Accumulation
(nmollmg protein)
[%Forskolin]

 

 

 

 

 

   

-r- '1-
No pretreat 1hr pretreat 4hr recovery

Figure 39: AM-R trafﬁcking in Rat2 ﬁbroblast cells. Rat2 ﬁbroblast cells show a lack of
receptor resensitization aﬁer agonist pretreatment, whereas when RAMP3 is expressed in Rat2
cells, receptor resensitization is now observed. Expression of PDZ motif mutant of RAMP3 in
Rat2 cells is unable to alter the receptor trafficking. Rat2 cells seeded in 24-well plates were
pretreated with 100 nm rAM for 1h and were washed extensively to remove residual agonist.
Following washes, cells were incubated in serum-free media with Sag/ml cycloheximide for
indicated times. After recovery time, Rat2 cells were re-challenged with 1 mM rAM for 15
minutes and plates were frozen. Determination of cAMP level was measured using the Biotrak
cAMP enzyme immunoassay system (Amersham Biosciences) according to the manufacturer's
instructions. cAMP levels in Rat2 ﬁbroblast cells were calculated using a standard curve ranging
from ID to 10 4 frnol of cAMP. Each experiment was done in duplicate and repeated at least 3
times. Data is expressed as percent maximal response, % forskolin. * p S 0.05; n 2 3

experiments.

200

6.4. Discussion:

While many GPCRS utilize similar mechanisms for endocytosis, the functional
consequences of endocytosis vary from receptor to receptor. Intemalized receptors that
are trafﬁcked through a rapid recycling pathway are restored to the plasma membrane in
a functional state to achieve resensitization. On the other hand, receptors that are
internalized and targeted to late endosomes and lysosomes experience proteolytic
degradation, thus promoting attenuation of receptor signaling and down-regulation of the
receptor. The CL-R/RAMP system shows sorting of the receptor complex by both
trafﬁcking pathways in different cell lines. That is, in RMCs the CL-R/RAMP complex
follows a post-endocytic recycling pathway, while in HEK 293 cells and Rat2 ﬁbroblast
cells the receptor complex undergoes degradation [276, 285]. Findings in this report
demonstrate some of the cellular and molecular mechanisms responsible for the CL-
R/RAMP receptor complex’s trafﬁcking. We have shown that the PDZ type I motif at
the C-terrninus of RAMP3 interacts with NSF to target the CL-R/RAMP3 complex for
recycling after internalization. NSF is commonly known to interact with aSNAP (soluble
NSF attachment protein) and SNARE (soluble N-ethylmaleimide-sensitive factor
attachment protein receptor) proteins to form the 208 particle, a complex that plays a
critical role in intracellular membrane fusion and exocytosis [261-263]. While other labs
have demonstrated involvement of NSF with the trafﬁcking of receptors, including the
BZAR and AMPA receptors, this study is the ﬁrst demonstration of NSF acting in concert
with a heterodimeric partner of a receptor to alter the trafﬁcking of the GPCR [268, 269].

Targeting of the receptor complex after agonist-stimulated internalization

demonstrates a novel role for RAMP3. This new function for RAMP3 may explain the

201

 

 

 

altered RAMP expression patterns in certain animal models of disease. RAMP3
expression has been shown to be increased during the transition from left ventricular
hypertrophy to heart failure in Dahl salt-sensitive and deoxycorticosterone acetate
(DOCA)-salt spontaneously hypertensive rats and in the myocardium of rats with chronic
heart failure, where recycling of the receptor complex would be advantageous for
continued ligand responsiveness [309-311]. In fact, in various cardiorenal disease states
where AM is protective, circulating plasma levels of AM have been shown to be
increased. For example, in chronic glomerulonephritis, type I diabetes, and type II
diabetes plasma AM levels are elevated [293-295]. In addition, AM delivery through
adenoviral injection has been shown to decrease cardiac hypertrophy and renal damage in
rat models of hypertension and improves cardiac function and prevents renal damage in
streptozotocin-induced diabetic rats [296-300]. If indeed, AM is exerting protective
effects against these diseases, then overcoming desensitization of the receptor complex
would be important. It is of interest to determine if NSF expression or localization is
altered in these disease states as well. Furthermore, studies identifying additional
molecular mechanisms of CL—R/RAMP trafﬁcking may be valuable for therapeutic
targeting.

While not only discovering a novel function for the RAMPS in post-endocytic
trafﬁcking, these ﬁndings also demonstrate a ﬁrnctional difference between the AMIR
(CL-R+RAMP2) and AM2R (CL-R+RAMP3) receptor complexes, in spite of very
similar second messenger systems and the physiological responses thus far identiﬁed.
This study demonstrates the ﬁrst difference between the RAMP2 and RAMP3 isoforms

in the trafﬁcking of the AMIR and AM2R. We suggest that this novel difference in

202

AMI and AM2 receptors may lead to either or both of the following two consequences:
1) there could be yet discovered roles of AM that differentially act through AMI and
AM2R because of this novel role of RAMP3 or 2) in disease conditions, expression of
RAMP3 may be preferentially stimulated to effect recycling of CLéR/RAMPB complex
to affect physiological consequences of AM. This could result in a beneﬁcial or harmful

effect depending on the system in question (cardiorenal diseases vs. cancer, respectively).

It is important to address the possibility of a species-speciﬁc effect, as the
prototypic class I PDZ motif is present only in the human RAMP3 and not other species
thus far sequenced. The human RAMP3 contains the classical PDZ class I motif (-
DTLL), while the two rodent species sequenced (rat and mouse) have a'C-terminal amino
acid sequence of -DRLL. The resensitization experiments in the rat mesangial cells
demonstrate the importance of the rat RAMP3 for efﬁcient receptor targeting for
resensitization. Furthermore, the resensitization experiments in the Rat2 ﬁbroblast cells
illustrate the ability of human RAMP3 to alter receptor targeting in a rat cell line to
promote receptor resensitization/recycling after agonist pretreatment. Taken together,
these data suggest that the lack of complete conservation between species of the C-

terrninal PDZ motif on RAMP3 does not alter our proposed model.

Finally, recent data suggests that RAMP3 can interact with receptors other than
CL-R. It is important to determine if this novel role of RAMP3 in receptor trafﬁcking is
speciﬁc for CL-R or also for the other receptors RAMPS interact with, namely VPAC,
PTHI- and 2-R, and glucagon receptors [286]. In addition to AM, Roh et al. have
reported that intermedin, a newly discovered peptide from the calcitonin gene peptide

superfamily, can also bind the CL-R/RAMP3 complex [307]. If this reported function for

203

 

 

 

RAMP3 is speciﬁc for AM, or if another peptide like intermedin could cause this

phenomenon, remains to be tested.

This study has shown that CL-R’s heterodimeric partner, RAMP3, is capable of
altering the trafﬁcking of the receptor complex after endocytosis by interacting with NSF
via its PDZ domain. This demonstrates a novel function for the RAMP accessory
proteins and the ﬁrst difference between the AMIR and AM2R. With recent reports of
RAMPS complexing and regulating additional GPCRS, these ﬁndings may reveal a more

widespread form of regulation of the GPCR life-cycle.

204

7. Summary and Conclusions

7.1. Speciﬁc aims, major hypotheses, and results of the study

The major aim of this study was to investigate the role of the different RAMP
isoforms in the regulation of the AM receptor complex life-cycle. It was hypothesized
that through post-translational modiﬁcations and protein-protein interactions, the three
RAMP isoforms differentially regulate the trafﬁcking and signaling of the AM receptor.
Hence, as summarized below, the following speciﬁc aims are addressed and the various
hypotheses within these aims were tested, and the corresponding experimental results

were obtained:

Speciﬁc Aim #1: Phosphorylation of amino acid residues of AM receptor complex is

 

involved in regulation of receptor desensitization and internalization.

Hypothesis 1: Speciﬁc serine/threonine residues in the C-terminus or 3rd intracellular

loop of CL-R are required for AM-R desensitization and internalization.

Hypothesis 2: Speciﬁc serine/threonine residues in the intracellular domain of RAMPS

are required for desensitization and internalization of AM-R.

Hypothesis 3: Based on the isoform of RAMP associated with CL-R, different amino

acid residues are important for receptor phosporylation, desensitization, and

internalization.

205

 

 

 

Hypothesis 4: Different protein kinases regulate the phosphorylation of CL-R in

presence of different RAMP isoforms.

Results:

a. Serine/threonine amino acid residues (Ser 421 for AMI receptor and Thr 423 for
AM2 receptor) on the C-terminal tail of CL-R are required for CL-R/RAMP
complex desensitization and internalization.

b. Serine and threonine residues in the third intracellular loop of CL-R or
intracellular domain of RAMPS are not required for receptor desensitization and
internalization.

c. Protein kinase A inhibitor blocks only CL-R/RAMPZ desensitization and
internalization, while a PKC inhibitor blocks only CL-R/RAMP3 desensitization
and internalization, suggesting different kinases are involved in the

phosphorylation of CL—R when different RAMP isoforms are associated.

Speciﬁc Aim #2: Protein-protein interactions of RAMPS with other proteins are involved

in the regulation of agonist-stimulated internalization of the AM receptor.

Hypothesis 1: RAMP3 (but not RAMP] or RAMP2) interacts with NHERF-1 to inhibit

agonist-induced receptor internalization.

Hypothesis 2: RAMP3 interacts with NHERF-l via a PDZ recognition motif on RAMP3

and a type I PDZ domain on NHERF-1.

206

 

 

 

Hypothesis 3: NHERF-1 inhibits internalization of the AM2 receptor (CL-R+RAMP3)

via interactions with MERM proteins in the actin cytoskeleton.

Results:
a. NHERF-l speciﬁcally interacts with RAMP3 and causes an inhibition of agonist-
induced receptor internalization, without affecting receptor desensitization.
b. RAMP3 interacts with NHERF-l via a PDZ recognition motif on the extreme C-
terminus of RAMP3 and a type I PDZ domain on the N-terminus of NHERF-l.
c. NHERF inhibits internalization of AM2 receptor through interactions with the

MERM actin cytoskeletal proteins via a C-terminal MERM domain on NHERF-1.

Speciﬁc Aim #3: Protein-protein interaction of the RAMPS with various other proteins is

involved in the regulation of AM receptor trafﬁcking from the sorting endosome.

Hypothesis 1: RAMP3, but not RAMPI or RAMP2, interacts with NSF to target the

receptor complex for recycling pathway after agonist-stimulated internalization.

Hypothesis 2: RAMP3 interacts with NSF via a PDZ recognition motif on RAMP3.

Results:

3. RAMP3 (but not RAMP] or RAMP2) interacts with NSF to target AM receptor

complex for a recycling pathway after agonist-induced internalization.

207

 

 

 

b. RAMP3 was determined to interact with NSF via a PDZ recognition motif on the

extreme C-terminus of RAMP3.

7.2. Limitations of the study.

I.

The primary limitation of this study is that all experiments were performed
in cell culture system, as opposed to using a whole-animal model system.
The practicality of deﬁning detailed molecular mechanisms required the
use of cell culture systems.

The cell culture systems utilized that were derived from the rat may not
necessarily reﬂect the same regulatory mechanisms in human cells. The
rat cell lines were chosen because of the speciﬁc RAMP isoform
expression levels. Obtaining and maintaining human cell lines, both from
the standpoint of cost and labor, was not practical.

All changes in receptor signaling were elicited by exogenous
administration of AM. Concentrations of AM used were within the
established circulating levels of AM, yet it remains unknown what
concentrations mesangial cells and proximal tubule cells are exposed to in
vivo. Until physiological and pathological concentrations are determined
in the vicinity of these two cell types, no arguments can be made regarding
this issue.

Changes in receptor trafﬁcking were characterized using cAMP

accumulation as the endpoint. Measuring the role of receptor trafﬁcking

208

 

 

 

changes in regulation of a more physiological parameter (such as
proliferation or migration) may provide additional information.

5. Chemical and pharmacological inhibitors were used to examine the roles
of NSF and protein kinases. Given the limited speciﬁcity of chemical and
pharmacological inhibitors, the results from these studies should be
interpreted with some degree of caution. Nonetheless, it should be noted
that the concentrations used in the studies have been published by others
and characterized as speciﬁc within that range. In addition, where

possible, supplementary controls were tested.

7.3. Positive outcomes of the study.

This thesis research sought to examine differences in AM receptor signaling,
dictated by the co-expression of the different RAMP isoforms. The three individual
studies within the project characterized roles for the RAMP isoforms in differentially
regulating the post-translational modiﬁcations and protein-protein interactions that

regulate AM receptor complex signaling.

While phosphorylation of CL-R had been demonstrated with prolonged agonist
exposure, this study was the ﬁrst to report speciﬁc amino acids of the receptorcritical for
desensitization and internalization. In addition, it was not previously known that
association of CL-R with different RAMP isoforms generated different phosphorylation

patterns on the receptor, regardless of exposure to the same ligand (AM). It could be
envisioned that this differential regulation of the receptor, directed by the RAMP

isoforms, allows further protein-protein interactions that regulate receptor signaling and

209

 

 

trafﬁcking. Future research investigating the interaction of differentially phosphorylated
AM receptor complexes with different arrestin molecules could clarify a role for
phosphorylation in the targeting of the receptor complex after agonist stimulation.

The last two studies deﬁned a role for the RAMPS in protein-protein interactions
that regulate the trafﬁcking and life-cycle of the receptor complex. Interactions of
NHERF-1 and NSF with RAMP3 have been demonstrated, in different ways, to promote
AM receptor signaling. The major ﬁnding of this thesis research that could potentially
contribute to the greater ﬁeld of receptor biology is the concept of RAMP expression
dictating the receptor fate after agonist-stimulated desensitization. In light of the recent
report of RAMPs’ interaction with other GPCRS, our observations may be applicable to
additional receptor systems. Future studies investigating the role of RAMPS in the
trafﬁcking of other GPCRS could help establish this idea of more widespread receptor
regulation. Given the documented expression of RAMPS in tissues devoid of CL-R
expression, the possibility of additional regulatory roles for the RAMPS has been
proposed.

This thesis research has deﬁned novel roles for the RAMP proteins in regulation
of the AM receptor signaling and endocytic trafﬁcking. Considering the established
beneﬁcial and harmful effects of AM signaling in different physiological systems
(cardiorenal disease vs. cancer, respectively), it is important to have speciﬁc mechanisms
to promote or repress AM signaling. In review of this study, we believe differential

RAMP isoform expression provides a form of this indispensable receptor regulation.

210

 

 

 

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