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This is to certify that the
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ROLE OF RECEPTOR ACTIVITY MODIFYING PROTEIN-3 IN ERK-
MEDIATED PROLIFERATION OF MESANGIAL CELLS

presented by
Carolyn S. Hall

has been accepted towards fulfillment
of the requirements for the

 

 

 

 

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ROLE OF RECEPTOR ACTIVITY-MODIFYING PROTEIN-3 IN ERK-MEDIATED
PROLIFERATION OF MESANGIAL CELLS
By

Carolyn S. Hall

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of
DOCTOR OF PHILOSOPHY
Department of Physiology

2007

 

 

 

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ABSTRACT
ROLE OF RECEPTOR ACTIVITY-MODIFYING PROTEIN-3 IN ERK-MEDIATED
PROLIFERATION OF MESANGIAL CELLS
By

Carolyn S. Hall

Receptor activity-modifying proteins (RAMPS) are single transmembrane accessory .
proteins critical to various G-protein coupled receptors for plasma membrane expression
and receptor phenotype. A functional receptor for the ligand, adrenomedullin (AM), is
comprised of RAMP2 (R2) or RAMP3 (R3) and calcitonin receptor-like receptor
(CRLR). Although R2 and R3 share only 30% homology, when CRLR is expressed with
R2 or R3 virtually identical (pharmacologically and biologically) AM receptors (AM-1R,
AM2-R, respectively) are produced. Recent studies from our laboratory have
demonstrated that R3 regulates the recycling of the AM2 receptor through protein
interactions involving a PSD-95/Discs-large/ZO-l homology (PDZ) domain located at
the extreme C-terminus of R3. Differential RAMP gene expression has been studied
under many disease models, physiological changes, and drug treatments. Several reports
have indicated that growth associated conditions can result in specific upregulation of R3
gene expression, including a study from our laboratory that demonstrated increased R3
expression in rat mesangial cells following platelet-derived grth factor beta
(PDGFBB) treatment. The mechanisms of R3 involvement in proliferation have not been
thoroughly investigated. Uncontrolled mesangial cell proliferation is a general

characteristic of a variety of progressive glomerular diseases. PDGFBB and basic

fibroblast growth factor (FGF-2) are potent mesangial cell mitogens that play critical
roles in the progression of renal disease. Both exert proliferative responses by activation
of a mitogen-activated protein kinase (MAPK) signal transduction cascade, namely
extracellular signal-regulated kinase -1/2 (ERK). The major aim of this study was to
determine the role of R3:ERK interaction in PDGFBB and F GF -2 stimulated mesangial
cell proliferation. Employing GST fusion protein overlay assays; we discovered that R3
could physically interact with ERK protein. We also observed that R3 gene knockdown
resulted in a complete inhibition of PDGFBB and FGF-2 stimulated proliferation in
mesangial cells. Accordingly, R3 introduction into a normal rat fibroblast cell line, NRK,
resulted in an increased proliferative response following FGF-2 treatment. Mutating
specific amino acid residues within a putative N-terminal ERK docking domain of R3
identified residues mediating the R3:ERK interaction. These mutations also resulted in
decreased FGF-2 proliferation of NRK following FGF-2 treatment. Subcellular
fractionation and confocal microscopy revealed that R3:ERK interaction occurs at the
endoplasmic reticulum. Finally, CO-immunoprecipitation experiments identified
interaction between R3 and pERK in a supernatant fraction from a 100,000G spin, which
probably represents a cytosolic cellular fraction. This interaction was increased
following PDGF-BB treatment. R3 was detected only in the lO0,000G pellet fraction
(representing membrane) when co-immunoprecipitated with the N-terminal Flag epitope.
Further experiments are warranted to determine the fate of R3’s signal peptide. Taken
together, these results suggest a novel role for R3 in ERK-mediated proliferation in NRK

and RMC.

 

 

To

To my sons and my parents for their unwavering love,
and
To the Department of Physiology for providing the opportunity to pursue my dream.

iv

ACKNOWLEDGEMENTS

I would like to express my gratitude to my committee members: Dr. Julia Busik,
Dr. Gregory Fink, Dr. Kathy Gallo, Dr. Laura McCabe, and Dr. William Spielman for
their guidance and assistance throughout my graduate career. I would like to thank Drs.
Busik, Gallo, and McCabe for their continual encouragement as well as the time they
devoted in reviewing this dissertation and helping me prepare my thesis defense seminar.
I would like to thank Dr. Fink for his guidance regarding statistics, and for sharing his
advice regarding my findings. I would especially like to thank my major advisor, Dr.
William Spielman, for providing unwavering support and friendship for so many years. I
would not have been able to accomplish my dream without your help. I would also like
to extend my thanks to Dr. Harvey Sparks, for hiring me as his technician twenty years
ago. The scientific skills I learned in your laboratory remain with me today.

My laboratory life was greatly enriched on countless occasions by my good
friends in the Physiology Department. I’m particularly thankfirl to the immeasurable
kindness and friendship offered to me by Dr. Narayanan Parameswaran. Nara was
always there for me, in good times and bad.

I would also like to thank my good friends in the Physiology Department office,

Barbara Burnett, Michelle Duchene, and Tanja Brady, for their love and support.

TABLE OF CONTENTS

List of Tables .................................................................................................................... viii
List of Figures .................................................................................................................... ix
Key to Abbreviations ........................................................................................................ .xi
1. Introduction ................................................................................................................... 1
2. Literature Review .......................................................................................................... 3
2.1 Mesangial Cells ....................................................................................................... 3
2.1.1 Mesangial cell functions .......................................................................... 3
2.1.2 Mesangial cells in disease ....................................................................... 4
2.1.3 Animal Models of Glomerulonephritis ................................................... 4
2.2 PDGFBB .................................................................................................................. 5
2.2.1 PDGFBB Introduction ............................................................................ 5
2.2.2 Interaction between PDGF isoforms and PDGF receptors .................... 6
2.2.3 PDGFBB and renal disease ..................................................................... 7
2.3 FGF-2 ..................................................................................................................... 8
2.3.1 FGF-2 Introduction ............................................................................... 8
2.3.2 FGF-2 SignalmgIO
2.3.3 FGF-2 and renal disease ...................................................................... 12
2.4 RAMPs and the AM signaling system ................................................................. 13
2.4.1 The AM signaling system .................................................................... 13
2.4.2 RAMP gene and protein structure ....................................................... 14
2.4.3 Role of RAMP N-terminal and transmembrane domains ................... 16
2.4.4 Role of RAMP C-terminal domains ................................................... 17
2.4.5 RAMP interaction with other class B and C receptors ....................... 20
2.4.6 RAMPs are a regulated component of the AM signaling system ....... 21
2.4.7 Growth related mediators of R3 expression ........................................ 23
2.5 MAPK Cell Signaling ........................................................................................ .25
2.5.1 MAPK Introduction... ..25
2.5.2 ERKl/2 Properties... ... ...26
2.5.3 Scaffolding Proteins are ERK-actmty modulatOrs ....................... 28
2.5.4 ERK- Docking Domains are ERK-activity modulators .................. 29
2.5.5 Plasma membrane and ERK signaling........................................32
2.5.6 Endosomes and ERK signaling ........................................................... 34
2.5.7 Golgi membrane associated ERK signaling ....................................... 35
2.5.8 ERK signaling at the ER ..................................................................... 38
2.5.9 Potential roles of compartment-specific ERK signaling ..................... 39

vi

. In Vitro identification and characterization of the ERKDD amino acid sequence in

R3 ............................................................................................. 41
3.1 Introduction... .... .....41
3. 2 Materials and Methods 45
3.3 Results ................................................................................ 47
3.4 Discussion .......................................................................... 52

. The role of R3 in ERK phosphorylation and cellular proliferation following growth

factor stimulation ................................................................................................... .54
4.1 Introduction... .... ......54

4. 2 Materials and Methods 57

4.3 Results ............................................................................... 64

4.4 Discussion ......................................................................... 85

. Identification of the subcellular site(s) of R3:ERK interaction ........................... 90
5.1 Introduction... .... .....90
5.2 Materials and Methods 95
5.3 Results ............................................................................... 99

5.4 Discussion ........................................................................ 112

. The effects of FGF- 2 treatment on membrane R3 expression in RMC

114

6.1 Introduction... .... ........114
6. 2 Materials and Methods 115
6.3 Results .............................................................................. 116
6.4 Discussion ........................................................................ 119
. Summary and Conclusions ...................................................................... 120
7.1 Specific aims, major hypotheses, and results of the study .................. 120
7.2 Limitations of the study ......................................................... 123
7.3 Positive outcomes of the study ................................................. 124
7.4 Future studies ..................................................................... 128
. References ................................................................................................................ .131

vii

TIE-S:

LIST OF TABLES

Table 1. Variable RAMP gene expression in disease models ............................... 22
Table 2. Growth-mediators that influence R3 expression .................................... 24
Table 3. ERK signaling in specific cellular compartments ................................. 33

viii

LIST OF FIGURES

Figure 1. Amino acid sequences of RAMPs ...................................................................... 15
Figure 2. Recycling of the AM-lR .................................................................................... 19
Figure 3. Scansite analysis of putative human R3 protein interaction amino acid
sequences ........................................................................................................................... 49
Figure 4a. GST protein overlays illustrating R3 interaction with ERK ............................ 50
4b. GST protein overlays illustrating R3 interaction with activated human MEKSI
Figure 5. Potential Subcellular locations for R3:ERK interaction .................................... 53
Figure 6. Sequences of oligonucleotide primers used to generate ERKDD mutations in
human R3 ........................................................................................................................... 68
Figure 7. PDGFBB-Induced Proliferation of RMC Cells using MTS Assay .................... 69

Figure 8. ERK Inhibition of PDGFBB-Induced Proliferation of RMC Cells using MTS
Assay .................................................................................................................................. 70

Figure 9. Dose-response curve for NRK proliferation following FGF-2 treatment....... . .71

Figure 10. ERK Inhibition of F GF -2-Induced Proliferation of NRK Cells using 3 H

methylthymidine incorporation ......................................................................................... 72
Figure 11. Confocal Microscopy depicting expression levels of RAMPs ......................... 73
Figure 12. MTS Assay for RMC Proliferation following gene-specific knockdown ........ 74
Figure 13. Confocal Microscopy depicting Gene-Specific knockdown using siRNA ..... 75

Figure 14. MTS Assay for proliferation using Dharmacon gene-specific knockdown....76

Figure 15. Membrane-associated pERK following gene-specific R3 knockdown and
50ng/mL PDGFBB stimulation .......................................................................................... 77

Figure 16. The effect of R3 knockdown on maximal membrane-associated ERK
phosphorylation following PDGFBB treatment ................................................................. 78

Figure 17. 3 H Thymidine Incorporation Assay for proliferation of NRK following R3
transfection ......................................................................................................................... 79

ix

Figure 18. Effect of R3 on membrane-associated ERK phosphorylation following FGF-2

treatment ............................................................................................................................ 80
Figure 19. Membrane expression of R3 and R3 mutants .................................................. 81
Figure 20. Effect of R3 ERKD—Domain mutation on membrane-associated ERK

phosphorylation following FGF-2 treatment .................................................................... 82
Figure 21. The role of R3 ERK-DD in FGF-2-stimulated proliferation of NRK83
Figure 22. GST Overlay with R3 mutations ..................................................................... 84
Figure 23. Potential Subcellular locations for R3:ERK interaction ............................... 103
Figure 24. Signal peptide cleavage and processing ......................................................... 104

Figure 25. Comparison of PCDNAR3 and pCMVTag2R3(Flag-tag) transient transfection
on AMlR desensitization ................................................................................................. 105

Figure 26. Confocal Microscopy identifying subcellular location of Flag epitope

expression in HEK293 cells ................................................... . ......................................... 106
Figure 27. Optiprep Protocol for the subcellular fractionation of plasma membrane,
endosomes, golgi, and ER .............................................................................................. .107
Figure 28 Upper Panel. Western blot for PDGFBR expression in HEK293 cells... ......108
Figure 28 Lower Panel. Subcellular fractionation using Optiprep gradient following
PDGF-BB treatment ............................................................................... 108
Figure 29. Confocal Microscopy identifying co-localization of Flag epitope and pERK

in HEK293 cells following PDGF 4313 treatment .............................................. 109
Figure 30. Co-Immunoprecipitation of RAMP3 and ERK .................................. 110

Figure 31. Working model depicting N-terminal RAMP3szRK cellular location. . . ...1 11
Figure 32. RAMP membrane expression in RMC following FGF-2 treatment .......... 117

Figure 33. Membrane RAMP expression in RMC following Sng/mL treatment... ......1 18

KEY TO ABBREVIATIONS:

AM, adrenomedullin

AM 1 R, adrenomedullin-1 receptor (composed of CRLR and RAMP2)
AM2, intermedin ,
AM2R, adrenomedullin—2 receptor (composed of CRLR and RAMP3)
AMPAR, a-amino-3-hydroxy-5-methylisoxazolepropionate receptor
AngII, angiotensin-II

CaSR, calcimn-sensing receptor

CCl4, carbon tetrachloride

CGRP, calcitonin gene-related peptide

CRLR, calcitonin receptor-like receptor

CRT, calreticulum

CT, calcitonin

CTR, calcitonin receptor

DAG, diacylglycerol

d-siRNA, diced small interfering RNA.

ECM, extracellular matrix

EGF, epidermal growth factor

ER, endoplasmic reticulum

ERK1/2, extracellular signal-regulated kinase -1/2
ERKDD, extracellular signal-regulated kinase docking domain
ET-l, endothelin-l

FGF-2, basic fibroblast growth factor

GDP, guanosine diphosphate

GEF, guanine nucleotide exchange factor

GH, growth hormone

GPCR, G protein-coupled receptor

GST, glutathione S-transferase

HGF, hepatocyte grth factor

HSPGs, heparan sulfate proteoglycans

IL-l B, interleuken-l [3

ITSN, intersectin

JN K, c-Jun amino-terminal kinase

KSR, Kinase Suppressor of Ras

MAPK, mitogen-activated protein kinase

MC, mesangial cell

MC, mesangial cell

MEK, MAP kinase kinase

MERM, merlin/ezrin/radixin/myosin

MP-l, MEK-l Partner 1

NGF, nerve growth factor

NHERF -1 , Na+/I-I+ exchanger regulatory factor-1

NRK, normal rat kidney interstitial fibroblasts

xi

NSF, N-ethylmaleimide-sensitive factor

PDGF, platelet-derived growth factor

PDGF-01a, platelet-derived growth factor-alpha alpha
PDGF-0113, platelet-derived growth factor-alpha beta
PDGF 4313, platelet-derived grth factor-beta beta
PDZ, PSD-95/Discs-large/ZO-1 homology domain type-I
PH, pleckstin homology

PI, phosphatidylinositol

PI, phosphoinositide

PLCy, phospholipase Cy

PNS, post-nuclear supernatant

PNS, post-nuclear supernatant

PTB, phosphotyrosine binding

PTH—R, parathyroid hormone receptor

PTK, protein tyrosine kinase

RAMP, receptor activity-modifying protein

R2, RAMP2, receptor activity-modifying protein-2
R3, RAMP3, receptor activity-modifying protein-3
RKIP, Raf-l kinase inhibitor protein

RMC, rat mesangial cell

RTK, receptor tyrosine kinase

Sef, similar expression to F GF

8H2, Src homology 2

SOS, son of sevenless

SP, signal peptidase

SPP, signal peptide peptidase

SRP, sequence recognition particle

TGF-a, transforming growth factor type a

TNF-a, tumor necrosis factor type a

VEGF, vascular endothelial grth factor

VPACR, vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide
BZ—AR, BZ-adrenergic receptor

xii

1. INTRODUCTION:

The discovery of receptor activity modifying proteins (RAMPS) has broadened
our understanding of G protein-coupled receptor (GPCR) signaling. RAMPs 2 and 3 (R2
and R3) when expressed with the calcitonin receptor-like receptor (CRLR) generate
adrenomedullin (AM) receptors. In addition to their documented roles in the trafficking
and internalization of CRLR, RAMP3 have been shown to interact with other class B and
C GPCRs. In addition, studies from our laboratory have demonstrated that R3 interacts
with Na+/H+ exchange regulatory factor-1 (N HERF-l) and N-ethylmaleimide sensitive
factor (NSF) to mediate R3:CRLR internalization and recycling to the plasma membrane.
These protein interactions occur through a PSD-95/Discs Large/Zona occulens-l (PDZ-l)
domain located at the extreme C-terminus of R3. R3 mRNA and protein are up-regulated
in several proliferative diseases, and in several cell lines and animal models of growth.

In particular, R3 expression is increased in rat mesangial cells (RMC) following
PDGFBB treatment even though RMC express abundant basal levels of R3. In addition,
R3 harbors a putative extracellular regulated kinase docking domain (ERK-DD). ERK-
DD’s are important in regulating the specificity and efficiency of ERK activation in the
cell. The research within this thesis investigates the role of the R3 ERK-DD in ERK-
mediated proliferation utilizing RMC, normal rat kidney (NRK), and human embryonic
kidney (HEK)293 cells. It identifies the critical amino acid residues within the ERK-DD
that mediate R3:ERK interaction and subsequent ERK activation. It reveals the cellular
location of R3:ERK interaction following growth factor treatment.

This introductory chapter is followed by a comprehensive review of the literature

as it pertains to this thesis work. Subsequent chapters are organized according to the

proposed specific aims. Each of these chapters are comprised of an introduction,
materials and methods, experimental results, and a brief discussion of these results.
Chapter three identifies a putative ERK D-Domain within R3’s amino acid sequence, and
demonstrates R3:ERK interaction in vitro. Subsequent chapters investigate R3’s role in
ERK activation and proliferation following PDGFBB or basic fibroblast grth factor
(FGF-2) treatment, and the cellular location of R3:ERK interaction. Finally, it assesses
R3 protein expression following FGF-2 treatment of NRK. The concluding chapter
describes the major hypotheses tested and a summary of the results. It discusses the

positive outcomes, the limitations of the work, and future directions to be undertaken.

2. LITERATURE REVIEW:

2.1. Mesangial cells.
2.1.1. Mesangial cell functions

The glomerulus, a network of capillaries originating in the afferent arteriole, is the
filtration unit of the kidney. Mesangial cells are located primarily in the glomerular tuft.
The contractile properties of mesangial cells allow for an important role in the regulation
of glomerular hemodynamics. By contracting, mesangial cells can reduce the blood flow
to specific capillary loops, thus reducing glomerular filtration area. Unlike other renal
cell: cell interactions, mesangial cells are in direct contact with the endothelial lining of
the glomerular capillaries. There is no basement membrane separating them from the
capillaries. As a result, mesangial cells are in free contact with plasma and various
macromolecules such as lipids, immune complexes, grth factors and advanced
glycosylated end products [1, 2].

By regulating the amount and composition of the surrounding extracellular matrix
(ECM), mesangial cells provide the essential structural support for the glomerular
capillary tuft. The normal mesangial extracellular mat1ix consists of collagens IV, V, VI,
glycoproteins(1aminin and fibronectin), heparan sulfate (syndecan) and chondroitin
sulfate proteoglycans [3, 4]. Loss of coordinated regulation, associated with altered
composition and increased mesangial ECM deposition, ofien leads to progressive

glomerulosclerosis, loss of filtration function, and end-stage renal disease [5].

2.1.2. Mesangial cells in disease

The primary locus of renal dysfunction is the glomerulus, and the mesangial cell
(MC) appears to be a central site of the microscopically observable renal damage. By
regulating the amount and composition of the surrounding extracellular matrix (ECM),
mesangial cells provide the essential structural support for the glomerular capillary tuft.
MC proliferation is a general characteristic of a variety of progressive glomerular
diseases. Loss of coordinated regulation, associated with altered composition and
increased mesangial ECM deposition, often leads to progressive glomerulosclerosis, loss
of filtration function, and end-stage renal disease [5]. It follows, therefore, that an
understanding of the regulatory mechanisms for MC proliferation is important for our

understanding of glomerular disease and its eventual treatment.

The turnover rate of mesangial cells in the normal adult human kidney is low; the
renewal rate is less than 1% [6]. Under normal conditions, quiescent mesangial cells
either face few mitogenic signals, or are unable to respond to them. In several models of
progressive glomerular disease (immunoglobulin A nephropathy, lupus nephritis,
hemolytic uremic syndrome, and diabetic nephropathy), mesangial cell proliferation,
phenotypic change and increased grth factor expression precede up-regulation of
genes for ECM and mesangial expansion [7, 8]. Ultimately, mesangial cell
hypercellularity and deregulation of ECM production and are hallmarks of many

glomerular diseases.
2.1.3. Animal Models of Glomerulonephritis

Several animal models have been developed to investigate the effects of

mesangial proliferation in glomerulonephritis [9-13]. Of these models, the IgA

nephropathy and anti-Thy1.1 models have been the most extensively studied. In the rat
anti-Thy1.1 model, glomerulonephritis is induced by injection of an anti-mesangial cell
antibody (anti-Thy1.1) which leads to an acute phase of complement-dependent
mesangial cell lysis with disruption of the mesangial matrix, followed by a phase of
robust mesangial cell proliferation [14]. A similar pathology occurs, namely mesangial
cell degeneration (necrosis or lysis), and subsequent mesangial proliferation, in several
human renal diseases including diabetic nephropathy, IgA nephropathy, diffuse lupus

nephritis, renal transplant rejection, and acute glomerulonephritis [15].
2.2. PDGFBB.

2.2.1. PDGFBB Introduction

Platelet-derived growth factor (PDGF) is a potent simulator of growth and
motility of connective tissue cells, such as fibroblasts, smooth muscle cells, and cardiac
myocytes [16], but also acts on other cell types, including capillary endothelial cells and
neurons. PDGF has important roles during the embryonic development, as revealed by
recent studies on mice with PDGF or PDGF receptor genes ablated. Inactivation of the [3-
chain [17] or the [3 receptor [18] results in defective kidney development with a total
absence of mesangial cells in the glomeruli, as well as defective development of blood
vessels with an inability to attract pericytes to the vessel wa11[19]; these mice die around
the time of birth. In the adult, PDGF stimulates wound healing[20] and has an important
role in the maintenance of the interstitial fluid pressure[21]. ‘PDGF is implicated in the
development of certain disorders involving excess cell proliferation, including certain

malignancies, atherosclerosis and fibrotic conditions.

2.2.2. Interaction between PDGF isoforms and PDGF receptors

The level and types of PDGF receptors expressed by a target cell determines its
response to PDGF. PDGF is a dimeric molecule consisting of 30-kDa disulfide-bonded
A- and B-polypeptide chains. Homodimeric (PDGF-eta, PDGF-BB) as well as
heterodimeric (PDGF-(1B) isoforms exert their effects on target cells by binding with
different specificities to two structurally related protein tyrosine kinase receptors, denoted
a- and B receptors. The A- and B-chains of PDGF are both synthesized as precursor
molecules that undergo proteolytic processing. The mature A- and B-polypeptide
molecules, which consist of about 100 amino acid residues each and are about 60%
identical in their sequences, are encoded by separate genes located on chromosomes 7
and 22, respectively [22-24]. The three-dimensional structure of PDGF [25] is similar to
that of the related vascular endothelial grth factor (VEGF)[26], and show some distant
resemblance to those of nerve growth factor and transforming growth factor-[3 [27],
although the latter factors show no sequence similarities to PDGF. Since the PDGF
isofonns are dimeric molecules, they have two separate receptor binding epitopes each.
Thus, one PDGF molecule binds two receptor molecules simultaneously[28-30]. The a-
receptor binds both A- and B-chains. Thus, PDGF-a receptor binds all three isoforms,
whereas PDGF-[i receptors bind with high affinity to the BB homodimer [31-33]. The
a— and B-receptors for PDGF each contains five extracellular immunoglobulin domains

and an intracellular tyrosine kinase domain with a characteristic inserted sequence [34-

39]. PDGF thus forms a bridge between two receptor molecules. The ligand—receptor

complex is further stabilized by a direct interaction between domain 4 of the two
receptors [40-42].

Binding of PDGF dimmers to the extracellular portion of the receptor induces
receptor dimerization. Dimerization of the receptors is the key event in PDGF receptor
activation. It juxtaposes the intracellular parts of the receptors which then allows
phosphorylation in trans (autophosphorylation) between the two receptors in the complex
[43]. Autophosphorylation provides attachment sites for direct interaction between
substrate proteins of the different signaling pathways and the activated receptor [44].
Such interactions are exerted by specific domains, e.g. Src homology 2 (8H2) domains
and phosphotyrosine binding (PTB) domains which recognize phosphorylated tyrosine
residues in specific environments, SH3 domains which recognize proline-rich regions,
pleckstin homology (PH) domains which recognize membrane phospholipids and PDZ
domains which recognize a C-terminal valine residue and specific upstream sequences.

More than 10 different SH2-domain-containing molecules have been shown to bind to
different autophosphorylation sites in the PDGF a- and B-receptors.
2.2.3. PDGFBB and renal disease

Platelet-derived growth factor beta (PDGFBB) is a potent in vitro and in vivo
mesangial cell mitogen [14, 45, 46], and is involved in the regulation of mesangial cell
matrix synthesis [47]. The normal adult kidney expresses only low amounts of PDGF
protein and receptors, primarily by mesangial cells [44, 48]. In renal disease states,
infiltrating inflammatory cells (monocytes/macrophages) and platelets are a major source
of PDGF [49]. Exposure of mesangial cells in vitro to grth factors, such as epidermal

growth factor (EGF), growth hormone (GH), transforming growth factor type a (TGF-a),

basic fibroblast growth factor (FGF-2), and tumor necrosis factor type a (TNF-a) induce
expression of PDGF mRN As. EGF, TNF-a, and FGF-2 also stimulate MCs to secrete
PDGF [50] Inflammatory agents such as interleuken-l B (IL-1B) and IL-6, as well as
vasoactive mediators such as vasopressin, endothelin-1 (ET-1), and angiotensin-II
(AngII) [51]. Expression of PDGFBB and its receptor are also significantly increased in
mesangial cells of animal models of glomerulonephritis [52, 53] and in human
glomerulonephritis in which mesangial proliferation occurs [52, 54, 55]. The importance
of PDGFBB, in glomerulonephritis was first demonstrated experimentally by the
administration of anti-PDGFBB antibodies in the anti-Thy1.1 model. Anti- PDGFBB
antibody treatment did not affect the initial mesangiolysis that occurs, but did result in a
50% reduction in mesangial cell proliferation and a corresponding reduction in ECM
accumulation [53, 56]. More recent studies using the non-specific PDGFBB, inhibitors
trapidil [57], dipyridimole [58], and TNP-47O [59] as well as the selective PDGFBB
inhibitor STI 571 [60], have confirmed the importance of PDGFBB, in both animal models
and human glomerulonephritis.

2.3. FGF-2.
2.3.1. FGF-2 Introduction

The first fibroblast grth factor (FGF) was discovered as a mitogen for cultured
fibroblasts at least 30 distinct F GFs have been identified thus far. Basic fibroblast growth
factor (FGF-2) is a member that affects the growth, differentiation, migration and
survival of a wide variety of cell types [61]. F GF-2 and other members of the FGF
family bind to heparin and heparin sulfate. Binding of FGF-2 to heparin sulfate has been

demonstrated to reflect a complex biochemical regulatory mechanism for this growth

factor. FGF-2 is a proteolytic product of the primary 18 kDa form [61]. Larger forms of
FGF-2 (22, 22.5 and 24 kDa) have also been identified resulting from alternate CUG-
translation start sites. Sequence homology for FGF-2 is very high (>90%). FGF-2
contains four cysteine residues with no intramolecular disulfide bonds, a large number of
basic residues, two sites (Ser 64 and Thr 112) that can be phosphorylated by protein
kinases A and C, respectively[6l]. FGF-2 consists of 12 anti-parallel B-sheets organized
into a trigonal pyramidal structure. Heparan sulfate proteoglycans (HSPGs) are
membrane-bound (e.g., syndecans and glypicans) or extracellular matrix (e.g., perlecan)
macromolecules that consist of a core protein and one or more heparan/heparin sulfate
glycosarninoglycan chains. Heparin is only synthesized by connective tissue mast cells,
heparin sulfate is widely distributed throughout all mammalian tissues and organs
attached to core proteins as HSPGs [62]. Heparan sulfate proteoglycans are potent,
specific regulators of cell-matrix interaction, cell adhesion, migration, proliferation,
angiogenesis, and development. The binding of HSPGs facilitates the self-association of
FGF-2 molecules into dimer and higher-order oligomers [63]. HSPGs are found on
cell surfaces and in the extracellular matrix where they have been demonstrated to

interact with F GF-2 and modulate its distribution and function [64].

F GF-2 lacks a consensus signal sequence for secretion, however significant
amounts of the 18 kDa form of FGF-2 are found outside the cell, the higher molecular
weight forms are predominantly localized to the nucleus [61]. The mechanism of
secretion of the 18-kDa FGF-2 remains unclear. FGF-2 does not progress through the
endoplasmic reticulum and the Golgi via the regular secretory pathway. It has been

suggested that FGF-2 is released from cells as the result of cell damage, death and non-

 

 

 

 

lethal membrane disruptions [4]. Released FGF-2 is found stored in the extracellular
matrix and basement membranes bound to HSPG [65]. Extracellular FGF-2 binds to cell
surface receptors and HSP68 and is subject to internalization and lysosomal degradation.
However, a considerable amount of FGF-2 can translocate into the nuclear fraction of
various cell types [66]. Nuclear translocation is cycle dependent, occurring in the Gl-S
transition. This results in an overall decrease in FGF-2 degradation and correlates with
enhanced mitogenic activity [61, 66] . FGF-2 plays key roles in development,
remodeling and disease states in almost every organ system [61]. One of the best-
characterized activities of F GF -2 is its ability to regulate the growth and function of
vascular cells such as endothelial and smooth muscle cells. A role of HSPG in
modulating FGF-2 activity is its ability to modulate the binding of FGF-2 to its receptor.
Cells that do not express HSPG show reduced receptor binding affinity and reduced

biological response [63].
2.3.2. FGF-2 Signaling

F GF -2 interacts with specific cell surface receptor proteins derived from four
separate genes (FGF R1-4). Splice variants confer specificity in signaling in response to
the 23 identified FGF family members. FGF-2 has been proposed to have two separate
receptor binding sites which might allow a single F GF -2 to bind to two receptors or to
interact with a single receptor in two separate positions [67]. HSPGs can increase the
affinity of FGF-2 for its receptors [68] and act as a bridge to facilitate the dimerization of

receptors.

Like all receptor tyrosine kinases, the F GF R2 is composed of an extracellular

ligand-binding domain, a single transmembrane domain and a cytoplasmic domain. The

10

extracellular domain of FGF Rs is composed of three Ig-like domains (D1, D2, and D3) in
which D2 and D3 function as F GF-and heparin-binding regions. Formation of a ternary

F GF/heparin/F GFR complex results in FGF R dimerization and activation. The
cytoplasmic domain of FGF R contains in addition to the catalytic protein tyrosine kinase
(PTK) core, several regulatory sequences. The juxtarnembrane domains of FGFRs are
considerably longer than that of other receptor tyrosine kinases. There are seven tyrosine
residues in the cytoplasmic tail of FGFR-l that can be substrates for phosphorylation:
Tyr463, Tyr583, Tyr585, Tyr653, Tyr654, Tyr730 and Tyr766. Tyr766 is necessary for
FGFR activation of phospholipase Cy (PLCy) through SH2 domain binding [69].
Mutational analysis of Tyr766 has shown that the phosphorylation of this tyrosine residue
is essential for complex formation with and tyrosine phosphorylation of PLCy, resulting
in PLCy activation, stimulation of phosphatidylinositol (PI) hydrolysis and the generation
of the two second messengers, diacylglycerol (DAG) and lns (1,4,5) P3 [69]. This region
contains a highly conserved sequence that serves as a binding site for the PTB domains of
the two members of the F GF R signaling adaptor (FRSZ) docking proteins, FRS2a and
FRSZB [70, 71]. FGF-stimulation leads to tyrosine phosphorylation of FRSZa and
FRS2B, followed by recruitment of multiple Grb2/SOS complexes resulting in activation
of the RasfMAP kinase signaling pathway [72]. FRS2a contains four binding sites for
the adaptor protein Grb2 and two binding sites for the protein tyrosine phosphatase Shp2.
F GF -stimulation leads to tyrosine phosphorylation of Shp2 resulting in complex
formation with additional Grb2 molecules. Grb2/SOS complexes are thus recruited
directly and indirectly via Shp2 upon tyrosine phosphorylation of F RSZa in response to

FGF-stimulation [72]. In addition to enhancement of tyrosine phosphorylation, FGF-

11

stimulation induces MAPK-dependent phosphorylation of FRSZa on at least eight
threonine residues. Threonine phosphorylation of FRSZa is accompanied by reduced
tyrosine phosphorylation of FRSZa, decreased recruitment of Grb2 and attenuation of the

MAP kinase response [73].
2.3.3. FGF-2 in renal disease

F GF -2 is expressed by a variety of cells, including endothelial cells, smooth
muscle cells, macrophages, fibroblasts, and mesangial cells [74]. In addition to FGF-2
receptors, heparan sulfate proteoglycans, especially syndecan, are essential for the
binding of FGF-2 to its cellular receptor [75, 76]. Since mesangial cells express FGF-2
and its receptor components, its not surprising that FGF-2 is a potent mitogen for
mesangial cells [74]. However, FGF-2 lacks a signal peptide for secretion and is not
normally released into the circulation [77]. FGF-2 is released from adult mesangial cells
only following an injury and may lead to further mesangial cell injury by stimulating
nitric oxide production [78]. Nitric oxide can be toxic to cells by causing nitrosylation of
Fe-S groups on non-heme enzymes, resulting in respiratory inhibition. This feed-forward
progression of FGF-Z-induced mesangiolysis is an important feature of the early phase of
anti-Thy1.l nephritis. After the initial mesangiolysis, the marked proliferation of
mesangial cells that occurs in the anti-Thy] .1 model is associated with an increased
glomerulosynthesis of F GF-2 [74]. Further evidence of the importance of FGF-2 in this
model are the observations that injection of FGF-2 following anti-Thy] .1 antibody
administration resulted in increased mesangiolysis [74], while treatment with anti-FGF-Z
blocked the initial mesangiolysis, resulting in less mesangial proliferation and less ECM

deposition [79].

12

2.4. RAMPs and the AM signaling system.
2.4.1. The AM signaling system

AM belongs to a large regulatory peptide family that includes calcitonin gene-
related peptide (CGRP), amylin, calcitonin (CT), and a recently discovered member,
intermedin (AM2). These peptides bind to and act via selective receptors derived from
the calcitonin receptor-like receptor (CRLR). CRLR is a seven-transmembrane-spanning
G-protein coupled receptor (GPCR) belonging to the class B GPCR family (secretin
family of GPCRs). Human CRLR is comprised of 464 amino acids and is 54%
homologous to the human calcitonin receptor (CTR). CRLR acts as either a CGRP or an
AM receptor depending on its interaction with receptor activity modifying proteins
(RAMPS). RAMP] (R1), RAMP2 (R2), and RAMP3 (R3) are distinct gene products and
have been characterized as single-transmembrane domain proteins capable of direct
interaction with CRLR. An important study was published in 1998 by McLatchie et al.
that clarified the elusiveness of the AM and CGRP receptors, and provided a novel
paradigm of GPCR regulation. When CRLR was expressed with R1, a CGRP receptor
was produced. In HEK293 cells when CRLR was expressed with R2 or R3 virtually
identical (pharmacologically and biologically) AM receptors (AM-1R, AM2-R,
respectively) were produced [80], even though R2 and R3 share only approximately 30%
sequence identity.. RAMPs and their receptor partners form stable complexes that
originate in the endoplasmic reticulum (ER) and Golgi apparatus. These complexes are
maintained during the processes of translocation to the cell surface, agonist activation,

internalization and trafficking to lysosomes [81-83].

13

2.4.2. RAMP gene and protein structure

RAMPs are a family of three type I transmembrane proteins. R1 and R2 were
initially cloned from human neuroblastoma cell (SKN-MC) DNA library, whereas R3
was isolated from the human spleen. A scan of the human genome revealed no more
sequences similar to the RAMPs [84]. R1 and -3 share some similarities in their gene
composition, being comprised of three exons divided by large introns. The R1 and -3
genes are large in comparison to that of R2 (approximately 24 Kb vs. 5 Kb, respectively).
Despite the relatively low amino acid sequence identity between the RAMP isoforms
(approximately 30%), the hydrophobicity plot analysis suggests similar protein
structures. 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 R1, R2, and R3, respectively [85,
86]. RAMP proteins have a molecular weight of only approximately 14-17 kilo-Daltons.
R1 and R3 are 148 amino acid proteins, while R2 is composed of 175 amino acids. All
isoforms are made up of a large extracellular domain, an approximately 20 amino acid
transmembrane domain, and a 10 amino acid intracellular domain [87]. While some key
amino acid residues are conserved across all RAMP isoforms, their sequence
heterogeneity is the basis for their ligand binding, receptor trafficking, and cell signaling

diversity (Figure 1).

l4

 

 

  

 

RAM P2
RAM P3
Figure 1. Amino acid sequences of RAMPS.
0 R1, R2, and R3 share approximately 30%
N
c c sequence homology. The sequences are most
diverse within the long, extracellular N-
C C
N terminal regions of RAMPs. The N-terminal
N region of R1 and 3 are the most conserved
C C
E E among the isoforms, R2 is 26 amino acids
C longer. Conserved cysteine (C) residues and
N N
C C N-terminal glycosylation sites (N) are shown.
The transmembrane domain is highly
conserved between isoforms. All three
'- '- RAMPs harbor conserved serine (S) and
147 119
lysine (K) residues within their short,
‘5 b cytoplasmic tails. R3 contains a PDZ
V V
domain at the extreme C-terminus.
Fl R
167 s s 140
K K
D R
S T
E D
A I
176 2 L 143

 

 

 

Adapted from: TIBS 31(1 1): 631—38 ref [88]

2.4.3. Role of RAMP N—terminal and transmembrane domains

The N-terminal sequences of RAMPs are poorly conserved (less than 20% of
amino acid identity) but share in common a significant number of consensus sites for
cotranslational modifications including cysteine residues (potentially involved in
disulfide bond formation) and N-glycosylation consensus sites (Asn-X-Ser/Thr). The N-
terminus is comprised of amino acidsl-119 for R1 and 3, 1-147 for R2, and the
transmembrane domains are composed of 20 amino acids thereafter. The N-terminal
sequences have been reported to be important for ligand binding specificity, while the
transmembrane domain stabilizes the CRLR-RAMP heterodimer complex [80].

The structural domain(s) involved in selective AM binding have been studied using
various RAMP chimeras and deletion mutants. Co-expression of chimeric RAMPs and
CRLR in HEK293 cells revealed that residues 77-101, situated in the extracellular N-
terminal domain of human R2, were crucial for selective AM-evoked cAMP production.
In addition, deletion of hR2 residues 86-92 significantly attenuated high-affinity 125I-AM
binding and AM-evoked cAMP production despite cell surface expression of CRLR-R2.
Deletion of hR3 residues 59-65 had a similar effect.

Disulfide bonds are also critical for CRLR-RAMP activation. Four cysteine
residues are conserved in all RAMP isoforms, and R1 and 3 harbor two additional
conserved cysteine residues. While similar studies have not been completed using R1 ,
mutation of each of the six cysteines results in a significant loss of R3 cell-surface
expression and ligand binding [88]. Several consensus sites for N-glycosylation (Asn-X-

Ser/Thr) have also been identified on R2 and R3. Four N-glycosylation sites identified

16

on human, mouse and rat R3 are conserved in the mouse R2, R1 has no consensus sites
for N-glycosylation [89]. In a recent study, it was demonstrated that N-glycosylated R2
and R3 are expressed at the cell surface, and their transport to the plasma membrane
requires N-glycans. R1 is not N-glycosylated and is transported to the cell surface only
upon formation of heterodimers with CRLR [90].
2.4.4. Role of RAMP C-terminal domains

While many studies have investigated the functional roles of both RAMP
extracellular and transmembrane domains, recent work has implicated the importance of
short C-terminal domains in receptor function and cellular signaling. Deletion of the
cytoplasmic tail of either R1 or R3 does not alter trafficking of CRLR to the plasma
membrane, but its deletion from R2 inhibits CRLR transport to the cell surface.[91].
The C-termini of all three RAMP isoforms contain highly conserved serine and lysine
residues. While these residues do not effect internalization of either Rl-CRLR or R2-
CRLR, they negatively regulate internalization of R3-CRLR [92]. A PSD-95/Discs-
large/ZO-l homology domain type-I (PDZ) recognition motif (Asp-The-Leu-Leu) on the
extreme C-terminus of R3, but not R1 or R2, that is responsible for protein-protein
interactions important in the regulation of trafficking of the R3-CRLR complex [93, 94].
N-ethylmalemide-sensitive factor (NSF) is a hexameric ATPase that targets both [32-
adrenergic receptor ([32-AR) and the a-amino-3-hydroxy-5-methylisoxazolepropionate
receptor (AMPAR) for recycling by a PDZ-binding domain interaction [95, 96]. Recent
reports from our laboratory have also demonstrated the importance of a R3 C-terminal
PDZ-binding domain in R3-CRLR trafficking. R3 interaction with NSF results in the

recycling of the R3-CRLR complex following agonist stimulation, while the absence of

17

NSF, or deletion of the R3 PDZ domain, results in receptor degradation [97] (Figure 2).
In addition, the R3 PDZ-binding motif plays a role in CRLR internalization via novel R3
protein: protein interactions. NailH+ exchanger regulatory factor-1 (N HERF-l) tethers
membrane receptors to the cytoskeleton via PDZ interactions with the
merlin/ezrin/radixin/myosin (MERM) family of proteins [98]. Recent evidence has
revealed important associations between the NHERF -1 and several G protein-coupled
receptors such as the B2AR, the kappa-opioid receptor, and the PTHR, as well as growth
factor tyrosine kinase receptors such as the platelet-derived growth factor receptor and
the epidermal grth factor receptor [98-101]. In HEK293 cells, overexpression of
NHERF-l , together with CRLR-R3 (but not CRLR-R1 or CRLR-R2) abolishes agonist-
mediated internalization [102]. These results were confirmed in a primary human
proximal tubule cell line that expresses abundant amounts of NHERF-l. In these cells
AM treatment results in no internalization of CRLR, and knockdown of NHERF -1 by
RNA interference allows internalization to occur following AM treatment. Protein kinase
A and C phosphorylation consensus sites also are present on the C-terminal intracellular
domains of R1 and R3, but not R2 [85, 103]. The role of these phosphorylation sites has

not been fully elucidated.

18

Extracellular

 

 

Intracellular

 

Golgi

Endosome

 

-NSF \

Degradation
Adapted from: TIBS 31(11): 631-38 ref [104]

Figure 2. Recycling of the AM-lR. Co-expression of CLR and R3 in HEK293 cells
enables the receptor complex to form a stable dimer that originates at the ER and Golgi.
The dimer is maintained during translocation to the cell surface, receptor activation,
internalization and degradation. Activation of the receptor complex by AM results in
cAMP generation with subsequent desensitization (not shown). The receptor complex is
then internalized and targeted for degradation. Over-expression of NSF prevents the

CLR—R3 complex from undergoing degradation.

2.4.5. RAMP interaction with other class B and C receptors

In addition to CRLR, other members of the class B GPCR family include
receptors for secretin, glucagon, glucagons-like peptide, amylin, gastric inhibiting
peptide, vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide
(V PACR), growth-hormone-releasing-hormone, parathyroid hormone (PTHR), and CTR.
In a recent RAMP binding study, when co-expressed with RAMPS, 6 out of 10 class B
receptors demonstrated RAMP interaction [105]. VPAC2 receptor (V PAC2R),
glucagon-like peptide receptors, and growth-horrnone-releasing-hormone receptor did not
interact with any of the co-expressed RAMP isoforms. Three class B receptors, namely
VPAC, glucagon, and PTH, have been shown to associate with RAMPs. VPACl
receptor (V PAClR) interacts with all three RAMP isoforms, parathyroid hormone-1
receptor (PTHIR) and glucagon receptor with R2, and parathyroid hormone-2 receptor
(PTH2R) with R3. Unlike the interaction of RAMPs with CRLR, VPACIR is expressed
at the plasma membrane independently of RAMP presence. The classic coupling pathway
associated with VPAClR is a significant accumulation of cAMP with little
phosphoinositide (PI) hydrolysis. However, the VPACleR2 heterodimer exhibits
enhanced PI hydrolysis with no significant change in CAMP stimulation. These intriguing
results suggest RAMPs may modulate GPCR coupling efficiency through diverse cell
signaling pathways. Similar to the ability of R2 to alter VPACleRZ-mediated signaling,
it is conceivable that R3 may play a diverse role in cellular signaling

Several published reports have concentrated on the regulation of class B GPCRs

by RAMP interactions. However, a recent study has demonstrated that a class C GPCR, a

20

calcium-sensing receptor (CaSR), interacts with RAMPs [106]. In a paradigm similar to
CRLR, recombinant CaSR expressed in COS7 cells does not translocate to the plasma
membrane unless R1 or R3 (but not R2) is also co-expressed. In the same study, R3 was
shown to mediate both the glycosylation and trafficking of CaSR from the ER to Golgi.
In the absence of R3, CaSR was retained in an intracellular compartment that confocal
microscopy identified to be the ER. Further studies are needed to determine if and how
RAMPs interact/modulate other class C GPCR functions.
2.4.6. RAMP3 are a regulated component of the AM signaling system

RAMP mRN A distribution has been analyzed in several species including mouse,
rat, and human. R1 has shown predominant expression in human heart, brain, skeletal
muscle, pancreas, thymus, spleen, fat, and kidney; R2 expression is abundant in the heart,
aorta, kidney, spleen, fat, skeletal muscle, and lung. R3 is the most widely distributed; its
expression is greatest in the kidney, heart, brain, and lung [86, 87, 107-110]. Changes in
RAMP gene expression have been studied under many disease models, physiological
changes, and drug treatments. Variable RAMP gene expression has been reported in
several human/animal disease states including carbon tetrachloride (CCl4)—induced liver
cirrhosis [111], cancer[1 12-115], cardiac hypertrophy [116] , chronic heart failure [186]
pregnancy-induced hypertension [1 12-115] and hypoxia [reviewed in [117]] (Table 1).
In several of the disease models described above, RAMP expression did not correlate
with CRLR expression. These findings suggest specific, receptor-independent RAMP

cellular activities might contribute to disease prevention/progression.

21

 

Model Tissue R1 R2 R3 Ref.

 

LV Hypertrophy Rat L ventricle ND 11 11‘] [116]
Chronic Heart Failure Rat Atria T - [IT [118]
Rat Ventricles T] - T]
Hypertensive Rat Renal Cortex ND 1],] H [119]
DOCA Salt Loaded Renal Medulla ND 11 H [120]
Hypoxia Rat Lung TTT - T [121]
LPS Sepsis Mouse Lung/Spleen it 111 HT [107]
LPS Sepsis AM+/-Mouse Lung ND ill 111 [122]
Pregnancy Rat Mammary Gland ll - ii [112]
Liver Cirrhosis (CCl4) Rat Liver T] T TN [111]
Prostate Cancer Biopsy - - TT [123]

 

Adapted from: Semin Cell Dev Biol 15(3): 299-308 ref [117]

Table 1. Variable RAMP gene expression in disease models. Level of expression is
shown as increase (T), decrease (l), or no change (-). Single, double and triple arrows indicate

weak, moderate, and strong increase/decrease. ND: not determined.

22

2.4.7. Growth related mediators of R3 expression

Using both animal and cell culture models, several reports have indicated that
grth associated conditions can result in alterations of R3 gene expression. However the
mechanisms of R3 involvement in proliferation have not been thoroughly investigated.
Published reports from our lab demonstrate that R3, but not R2, is dose-dependently
upregulated in RMC following PDGF-BBjreatment [124]. The growth-stimulating
parathyroid hormone also increases R3 expression in primary mouse osteoblasts [125] in
a dose and time-dependent manner. R3 is expressed in the rapidly growing thymus of
newborn rats, but not in quiescent thymus of adult animals [126]. R3 is expressed in the
prostate cancer cell line, DU145 [123]; in prostate carcinoma biopsies the levels of R3
were significantly elevated (compared to prostate hyperplasia biopsies) while expression
levels of R2 were unchanged in these samples [123]. Estrogen and phytoestrogen are
important mediators of uterine grth and differentiation. In rats, administration of either
of these mitogens preferentially increases uterine R3 expression levels, and actually
significantly deceases CRLR levels [114, 127]. R3 is also exclusively upregulated in
cultured rat neonatal cardiac fibroblasts with IL-lbeta [128], and in a rat model volume
overload-induced cardiac hypertrophy R3 was significantly increased, while other AM
signaling components were only modestly or not increased at all [128, 129].
Angiotensin II induces hypertrophy of cultured rat neonatal cardiomyocytes. mRN A
levels of R1 and R3 are significantly elevated following 24-h treatment with Ang II
(without a change of those of R2 and CRLR), and the effects of Ang II on R1 and R3

expression were abolished by an Ang 11 type 1 (AT1) receptor antagonist [130]. It is

23

evident from the reports described above that R3 is differentially regulated compared to
other AM2R components, namely CRLR and R2. Further studies are warranted to assess

R3’s involvement in these conditions (Table 2).

 

 

Treatment Cell Line/ Animal Model R1 R2 R3 Ref.
*Ang 11 Rat Cardiomyocytesww m ____.- U TT -.____r_._ WZMHM .__ TT “[130] ._
PDGF-BB RMC ND - TT [124]
Estrogen Rat Uterus following injection - T TTT [114]
PTH Primary mouse osteoblasts - - TT [131]
Prostate Cancer DU 145 cells - - TT [123]
IL-IB Rat neonatal fibroblasts ND - TT [128]

 

Table 2. Growth-mediators that influence R3 expression. Level of expression is shown as
increase (T), decrease (l), or no change (-). Single, double and triple arrows indicate weak,

moderate, and strong increase/decrease. ND: not determined.

24

2.5. MAPK Cell Signaling.

2.5.1. MAPK Introduction

Cells recognize and respond to extracellular stimuli by engaging specific
intracellular programs, such as the signaling cascade that leads to activation of the
mitogen-activated protein kinases (MAPKs). All eukaryotic cells possess multiple
MAPK pathways, which coordinately regulate diverse cellular activities from gene
expression, mitosis, and metabolism to motility, survival and apoptosis, and
differentiation. Five distinct groups of MAPKs have been characterized in mammals:
ERKs 1 and 2 ERK1/2, c-Jun amino-terminal kinases (JNKs 1, 2, and 3), p38 isoforms
a, [3, Land 5, ERKs 3 and 4, and 5. MAPKs can be activated by a wide variety of
different stimuli, but in general, ERKl , and ERK2 are preferentially activated in response
to grth factors and phorbol esters, while the JN K and p38 kinases are more responsive
to stress stimuli ranging from osmotic shock and ionizing radiation to cytokine

stimulation.

Each family of MAPKs is composed of a set of three evolutionarily conserved,
sequentially acting kinases: a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase
(MAPKKK). The MAPKKKs, which are serine/threonine kinases, are often activated
through phosphorylation and/or as a result of their interaction with a small GTP-binding
protein of the Ras/Rho family in response to extracellular stimuli [36,98]. MAPKKK
activation leads to the phosphorylation and activation of a MAPKK, which then

stimulates MAPK activity through dual phosphorylation on threonine and tyrosine

25

residues located in the activation loop of kinase sub domain VIII. Once activated,
MAPKs phosphorylate target substrates on serine or threonine residues followed by a
proline; however, substrates selectivity is often conferred by specific interaction motifs
located on physiological substrates. MAPK cascade specificity is also mediated through
interaction with scaffolding proteins that organize pathways in specific modules through
simultaneous binding of several components. Since it is widely believed that the ERK
cascade is a primary mediator in many proliferative responses, inhibition of the ERK
signaling cascade has been targeted extensively in diseases such as cancer and vascular

remodeling [132, 133].
2.5.2. ERKl/2 Properties

The ERKl/2 module, also known as the classical mitogen kinase cascade, consists
of the MAPKKKS A-Raf, B-Raf, and Raf-1, the MAPKKs MEKl and MEK2, and the
MAPKs ERK] and ERK2. ERK1/2 are serine/threonine kinases that mediate
proliferation by transmitting signals from the cell surface to the nucleus to regulate the
activity of transcription factors. ERK] and ERK2 have 83% amino acid identity and are
expressed to various extents in all tissue. They are strongly activated by grth factors,
serum, and phorbol esters and to a lesser extent by ligands of the heterotrimeric G
protein-coupled receptbrs, cytokines, osmotic stress, and microtubule disorganization
[134]. Cell surface receptors such as tyrosine kinases, (RTK) and G protein-coupled
receptors transmit activation signals to the Raf/MEK/ERK cascade through different
isoforms (H-Ras, N-Ras, and K-Ras 4B/4A) of the small GTPase protein Ras [135, 136].
Activation of only 5% of Ras molecules is sufficient to induce full activation of ERK1/2

[137]. Ras proteins must associate with the cytosolic leaflet of cellular membranes to be

26

fully activated [138, 139]. Post-translational modifications of a conserved C-terrninal
CAAX sequence (C=cysteine, A=aliphatic amino acid, X=any amino acid) in Ras
mediates this membrane association [140]. Membrane-association of R33 is also
achieved through recruitment of SOS (son of sevenless), a Ras-activation guanine
nucleotide exchange factor (GEF). SOS stimulates Ras to change GDP to GTP, allowing
it to interact with a wide range of downstream effector proteins, including isoforms of the
serine/threonine kinase Raf [141]. Activated Raf binds to and phosphorylates the dual
specificity tyrosine/threonine kinases MEKl and -2, which in turn phosphorylate ERKl/2
within a conserved Thr-Glu-Tyr (TEY) motif in their activation loop. Phospho-Erk forms
dimers that are transported into the nucleus, where they phosphorylate the Ets family of
transcription factors, including Elk-l. Regulation of both Ras and Raf is crucial for the
proper maintenance of cell proliferation, as activating mutations in these genes lead to
oncogenesis [142]. Ras has been shown to be mutated in 30% of all human cancers,

while B-Rafis mutated in 60% of malignant melanomas [143, 144].

All signal transduction cascades transmit signals from the cell surface into the
cytoplasm and nucleus to elicit a cellular response. These cascades contain multiple
components, and classical cascades act by a sequential series of phosphorylation steps.
Since MAP kinases phosphorylate very similar motifs (consensus sequence Ser/Thr-Pro
(S/TP)), and several potential substrates contain this motif, it is important to provide
specificity to direct individual kinases towards the correct substrates. Multiple
mechanisms exist for generating signaling specificity within these cascades to ensure

specific cellular responses, including the association of the cascade components with

27

their activating kinases, inactivating phosphatases, and scaffold proteins that facilitate

distinct signaling complexes.

2.5.3. Scaffolding proteins are ERK-activity modulators

Scaffolding protein interaction is one means of ensuring efficiency and specificity
in MAPK signaling cascades [145]. Several proteins have been shown to interact with
members of the ERK cascade, including the scaffold proteins MEK-l Partner 1 (MP-1),
and Kinase Suppressor of Ras (KSR) and the modulators Connector Enhancer of KSR
(CNK) and Raf-l Kinase Inhibitor Protein (RKIP), resulting in stimulation or inhibition
of the ERK1/2 cascade [146]. MP1 was isolated in a yeast two-hybrid screen using
MEK-l as bait [147]. This small protein displays no sequence homology with known
proteins and does not contain any recognizable motif. MP1 selectively interacts with
ERK-1 and MEK-1 but fails to bind to ERK-2 and MEK-2. In vitro, MP1 enhances the
activation of MEK by Raf, and when overexpressed in cells, MP1 can selectively induce
the activation of ERK-l but not that of ERK-2, suggesting that MP1 could help to
discriminate between two closely related MAPKs in the same module. MP1 MAPK
scaffolding activity is localized to the cytoplasmic surface of late endosomes/lysosomes,
thereby combining catalytic scaffolding and subcellular compartrnentalization as means
to modulate MAPK signaling within a cell [148]. KSR is a protein kinase identified by
genetic screens in Caenorhabditis elegans and Drosophila. A database search revealed
that Raf family kinases are the closest relatives to KSR, however KSR lacks the
necessary Ras binding domain so it is unable to bind to activated Ras [149]. Biochemical

studies using mouse KSR-1 revealed that the kinase domain of KSR interacts strongly

28

with MEK-1 and MEK-2, ERK via the CA4 domain, and to Raf-l via the CA5 kinase
domain [150]. While the interaction with MEK appears constitutive, the ability of KSR
to interact with Raf-1 and ERK is induced by cell treatment with growth factors [151]. In
addition, KSR is cytosolic in resting cells but a fraction is translocated to the membrane
upon cell treatment with grth factors [152]. A physiological substrate for KSR has not
been identified therefore the exact mechanism by which this kinase regulates the
Raf/MEK/ERK pathway has not been elucidated. RKIP was isolated in a two-hybrid
screen using the kinase domain of Raf-l as a bait and belongs to the family of
phosphatidylethanol-binding proteins, which are widely expressed and highly conserved.
In vitro, RKIP was found to bind MEK, Raf-l , and ERK, but not to Ras. This interaction
is exclusive since the binding sites for Raf and MEK in RKIP overlap [153].
Consequently, overexpression of RKIP can disrupt the physical interaction between Raf-
l and MEK, thereby impairing MEK phosphorylation by Raf, without affecting that of
ERK by MEK. Cell treatment with growth factors provokes the release of RKIP from
Raf-l, allowing phosphorylation and activation of MEK. RKIP co-immunoprecipitates
with Raf-1 and MEK from cell lysates and colocalizes with Raf-l at the plasma
membrane when examined by confocal microscopy. RKIP represents a new class of
protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK module.

2.5.4. ERK Docking Domains are ERK-activity modulators

The first description of docking domain function arose from the observation that
the transcription factor c-Jun differs in several respects from its oncogenic counterpart v-
Jun. Namely, the 6-domain present in c-Jun is absent in v-Jun, suggesting an important

function for this motif. Mutational analysis revealed that the c-Jun 8-domain acts as a

29

binding site for the c-Jun N—terminal kinase (JNK) and this function is lost in v-Jun
[154-156]. The 8-domain was subsequently defined as a JNK MAP kinase-docking
domain. Binding of JN K to this docking site was shown to be important for efficient
phosphorylation of c-Jun by IN K and for the activity of its transcriptional activation
domain. The 6-domain has also been implicated in directing the site-specific
phosphorylation of c-Jun. In vitro, c-Jun is normally phosphorylated on Ser63 and
Ser73, but, upon deletion of the 6-domain, alternative phosphorylation sites in c-Jun
become preferentially phosphorylated decreasing the overall efficiency of c-Jun

activation [157].

Subsequently, other transcription factor—MAP kinase combinations were
studied, and an important theme that emerged was that many MAP kinase substrates
possess docking sites that serve to recruit MAP kinases and increase the efficiency of
their phosphorylation. In fact, each of the three major classes of MAP kinases (ERK,
JNK and p38) has been shown to bind to transcription factors, and, in each case, the

docking site serves to enhance substrate phosphorylation.

It is clear from the discussion above that one of the major functions of docking
sites is to promote the specificity, efficiency and accuracy of substrate phosphorylation
by MAP kinases. However, some scaffolding proteins contain docking domains; their
primary function is to bring several MAPK molecules in close proximity at a precise
subcellular location. For example, the scaffolding protein JIP-l binds specifically to JNK
MAP kinases via its docking domain and brings together several JNK MAP kinase

cascade molecules resulting in the cytoplasmic retention of JNK and its inhibition [158,

30

159]. Another example is JunB, which, like c-Jun, contains a docking site but lacks
phosphoacceptor motifs. In this case, JunB acts to recruit JNK MAP kinases, which can
then act in trans to phosphorylate other proteins such as its heterodimerization partner
JunD, which contains a phosphoacceptor site but lacks a functional docking site [157].
Thus, docking domains can serve to recruit MAP kinases either into signaling complexes
to enhance their regulation or onto promoters where they can phosphorylate other
transcription proteins. ERK, p38 and JN K all have conserved domains that are used for
docking to their specific activators (MAPKKs/MEKs), their inactivators (MKPs), their

substrates (MAPKAPKS), chaperones, and scaffolding molecules [160, 161].

Although there is no clear overall consensus, docking domains contain an LxL
motif located 3—5 amino acids downstream from a region containing several basic
residues. Differences in the spacing and composition of these motifs are apparent for
substrates that are recognized by different classes of MAP kinases. These specific
sequences within MAP kinase docking domains impose both the specificity and
efficiency of protein partner interaction. For example, the ERKDD of MEK allows it to
interact with ERK only, not other MAPK family members, such as JNK or p38.
Similarly, the docking sites of MEF2A and MEF2C only direct phosphorylation by a
subset of p38 isoforms [162]. However, some docking domains are recognized by two
different classes of MAP kinases, as observed with Elk-l, where the docking site directs
both ERK and JN K MAP kinases to this substrate [163], permitting responses to a wider

variety of stimuli.

31

The consensus sequences of MEK1/2 D-domains are located at their N-tenninal
region and consist of two residues of the basic amino acids arginine or lysine, followed
immediately by the helix breaking amino acid proline, followed two to six amino acids
later by the hydrophobic amino acids leucine or isoleucine [160, 164] (Consensus: -
(R/K)2-(P)-(X)2.6-(L/l-X-L/I)-). Experiments utilizing a blocking peptide consisting of
the MEK] ERK D-domain resulted in a complete, specific blockade of MEK] :ERKl
interaction suggesting that MEK D-domains are necessary to direct the specific
recognition, binding, and phosphorylation of ERK [160, 161, 165].

2.5.5. Plasma membrane and ERK signaling

Protein tyrosine kinase receptors (PTKR) located on the plasma membrane are
known to activate the Ras/MAPK signaling cascade. Regulation of ERK signaling can be
achieved through spatial regulation, utilizing scaffolding proteins that recruit ERK
cascade components to specific subcellular compartments. One such scaffolding protein
is kinase suppressor of Ras (KSR). Following EGF stimulation, KSRl colocalizes with
activated Ras and Raf-l at the plasma membrane, facilitating the phosphorylation
reactions required for the activation of MEK and MAPK [166]. Although the importance
of plasma membrane MAPK signaling has been studied extensively, a role for
endosomal, ER, and golgi targeting in MAPK mediated cellular events has recently been

suggested (Table3).

32

Cellular Locations of ERK Signaling

Plasma Membrane:
KSR-mediates raszEKzERK interaction/activation
CNK and SUR8-mediate raszraf interaction/activation

 

Endosomes: (HGF,EGF,PDGF,NGF)
MP1-facilitates MEK] :ERKl interaction/activation
[3_—;arrestin- targets ERK activity to cytoplasm

ER and Golgi:
RKTG-negative modulator of raf

RasGRPl-activates Ras on the Golgi
S_ef-recruits MEK and ERK and directs activated ERK

towards cytosolic targets instead of nuclear

Table 3. ERK signaling in specific cellular compartments.

33

2.5.6. Endosomes and ERK signaling

PTKRs are internalized via clathrin-dependent and independent pathways. This
ligand-induced endocytosis was originally thought to be a mechanism of receptor
inactivation. Recent studies, however, suggest that receptors remain active on the
cytoplasmic face of endosomes [167, 168] and that signaling can occur from these sites
[169, 170]. In EpH4 (murine mammary epithelial cells), Caco-2 cells, and HeLa cells,
MAPK scaffolding activity localizes to the cytoplasmic surface of late
endosomes/lysosomes, thereby combining scaffolding and subcellular
compartmentalization as means to modulate MAPK signaling within a cell [148].
Kerrnorgant et al. have recently demonstrated that traffic to endosomes is essential for
hepatocyte grth factor (HGF/c-Met) to trigger an ERK response [171]. In addition,
normal endocytic trafficking of EGFR is important for the full activation of MAPK.
Inhibition of clathrin-mediated endocytosis by dominant-negative dynamin in EGF-
activated cells revealed that internalization of EGFR is needed for maximal MAPK
activation [172]. Burke et al. have also reported that the EGF receptor (EGFR) activates
its targets Ras, Raf, and MAPKK (MEK) at the plasma membrane, but endocytosis had to
occur to activate ERK [167, 173]. Like EGFR, nerve growth factor (N GF ) activation of
TrkA receptors results in internalization of phosphorylated TrkA via endocytosis. Howe
et al. demonstrated that upon NGF stimulation, PC12 derived endosomes contained
phosphorylated TrkA, Shc, Raf, ERK, as well as activated Ras [174].

In similar fashion, following EGF stimulation, the scaffolding protein MEK]
partner 1 (MP1) is recruited to late endosomes where it binds MEKl, phosphorylates

ERK then releases the activated ERK to prolong its activation [175]. This ttu'nover could

34

provide an amplification mechanism that translates low MEK activity into sustained
ERK/MAPK activation, in which MP1 continues to supply MEKl with inactive ERK for
phosphorylation. MAPK organizer-1 (MORGl ), a recently isolated MP1-interaction
partner, also associates with Raf-1, B-Raf, MEK and ERK/MAPK on endosomal
vesicles. MORGl shows the typical properties of a scaffold, enhancing ERK/MAPK
activation at low concentrations and inhibiting it at higher levels. MORGl function
seems to be stimulus-specific — it affects ERK/MAPK activation by serum,
lysophosphatidic acid and phorbol esters, but not by EGF or PDGF [176]. B-arrestin also
localizes to endosomes and serves as a Raf-l/MEK/ERK scaffold downstream of GPCRs.
Originally characterized as a mediator of GPCR desensitization, B-arrestin is now
recognized as a multifunctional protein that regulates internalization of GPCRs into
clathrin-coated vesicles and serves as a scaffold for both the ERK and JN K MAPK
modules [177]. Several lines of evidence implicate B-arrestin functionally in the
transmission of signals from Raf-1 to MEK and ERK on endosomes. Raf-l
overexpression increased MEK and ERK binding to B-arrestin [177], and a dominant-
negative form of B-arrestin blocked ERK activation downstream of GPCR activation
[168]. Thus, endosomes serve as an organelle that supports both PTKR and GPCR
signaling to ERK, and each system uses its own scaffold.
2.5.7. Golgi membrane associated ERK signaling

As mentioned above, Ras proteins undergo post-translational modifications of
their CAAX motif that render them membrane-associated. One of these modifications,
namely farnesylation of the CAAX motif, targets Ras to ER and golgi membranes[l 78].

The motif is then processed by enzymes found exclusively in the ER and golgi [179,

35

180]. The CAAX motif alone targets Ras proteins to the endomembrane system where
they are methylated. Thus, rather than promoting nonspecific membrane association,
prenylation mediates specific Ras association with the ER and Golgi membranes, and
further processing there allows for its trafficking to the plasma membrane. Recently, it
was demonstrated that the information required for accurate membrane localization is
contained within the CAAX region [181], which also dictates how Ras proteins traffic to
their destinations. Whereas H- and N-Ras traffic to the PM along the secretory pathway
through the ER then Golgi complex, K-Ras4B is directly routed from the ER to the
plasma membrane [178, 182]. The presence of H-Ras in these intracellular locations
seems not to be a transient event associated with the transport and/or recycling of H-Ras
proteins to and from the PM; instead, a pool of H-Ras appears to permanently reside in
these organelles. These observations have led investigators to ask whether ER/golgi
associated H-Ras undergoes GTP-GDP exchange and subsequent MAPK pathway
signaling. Using live cell imaging utilizing a probe consisting of Raf-l ’s Ras binding
domain fused to green fluorescent protein (RBD-GFP), Chiu et al. demonstrated that
following EGF stimulation activated Ras is present at the plasma membrane, ER and the
Golgi [183]. Although the Golgi-associated Ras was initially conceived of as a pool that
does not traffic to the plasma membrane, recent studies have elucidated a plasma
membrane/Golgi cycle that functions as a consequence of
palmitoylation/depalmitoylation [184, 185]. Using transfected COS-1 cells, prenylated
but depalmitoylated Ras appears to traffic between membrane compartments though the
cytosol, probably bound to a chaperone. In this model, Ras could be activated at the

plasma membrane, and then traffic in a GTP-bound state to another compartment such as

36

the Golgi. Rocks et al. [184] investigated this palmitoylation/depalmitoylation cycle by
applying the palmitoyltransferase inhibitor 2-bromopalmitate and observed a decrease in
Ras activation on the Golgi, suggesting that the acylation/deacylation cycle is required
for Ras activation on this organelle. In addition, recent work in glutamate-stimulated
hippocarnpal neurons has revealed that GFP-tagged Ras dissociates from the plasma
membrane and associates with intracellular membranes (including Golgi) in a
calcium/calmodulin—dependent fashion [186].

Golgi membrane regulation of ERK signaling is also mediated through
proteinzprotein interactions. Sef (similar expression to FGF) is a transmembrane protein
that was identified as the product of an F GF-induced gene and feedback inhibitor of the
ERK/MAPK pathway in zebrafish [187]. The exact mechanism of inhibition is unclear,
but seems to involve interference at the level of phosphorylation of FGF receptor
substrates and at the level of MEK-mediated ERK/MAPK phosphorylation. A recent
study demonstrated that Sef captures activated MEK—ERK/MAPK complexes at the
Golgi apparatus [188]. Sef selectively bound to activated MEK and permitted
ERK/MAPK activation. However, Sef retained activated ERK/MAPK in the complex,
preventing its nuclear translocation and restricting ERK/MAPK signaling to cytosolic
substrates. Golgi-associated Ras proteins are activated by a pathway that is different from
the canonical one (through Grb2 and the GEF son-of-sevenless (808)) that is used by
RTKs to activate Ras at the cell membrane [189]. Localization at the Golgi could
therefore dedicate the ERK/MAPK module to selectively connecting a distinct set of

inputs with specific signaling outputs.

37

2.5.8. ERK signaling at the ER

In addition to evidence of signaling on the Golgi, recent evidence has been
presented for Ras signaling on the ER. When palmitoylation—deficient forms of Ras are
expressed in COS-1 cells, they accumulate on the ER and in the cytosol [178]. When
these palmitoylation-deficient forms of Ras are expressed along with the GFP-RBD Ras
activation reporter mentioned above, GTP-bound H-Ras accumulates on the ER. EGF
stimulation activates H-Ras at the ER in less than one minute [183], and this ER-
restricted H-Ras selectively engages the ERK pathway resulting in fibroblast
transformation. One interpretation for this result is that GEFs can rapidly activate ER-
associated Ras. This idea has been supported by Arozarena et al. who showed that
endogenous Ras GEFs are highly expressed in neurons and rapidly translocate to the ER
to activate H-Ras on this compartment [190]. Ras GEFs, both endogenous and over-
expressed, were present in the endoplasmic reticulum but not in the Golgi complex.
Utilizing H-Ras constructs specifically tethered to the plasma membrane, endoplasmic
reticulum, and Golgi complex, they demonstrated that RasGRFl and RasGRF 2 can
activate plasma membrane and reticular, but not Golgi-associated, H-Ras. Further
evidence for Ras signaling from the ER comes from the discovery of a Ras inhibitor that
is restricted to the ER compartment. Sobering et al. described a protein in Saccharomyces
cerevisiae designated ER-associated Ras inhibitor (Eril) that behaved genetically like an
inhibitor of Ras, bound preferentially to GTP-bound Ras via its effector domain, and was

localized to the ER [191].

38

2.5.9. Potential roles of compartment-specific ERK signaling

Compartmentalized signaling is a relatively new paradigm in signal transduction.
One hypothesis is that greater specificity in Ras/MAPK signaling might be achieved
through spatial regulation. In this fashion, compartmentalized signaling could increase
the signaling specificity by altering kinetic outputs down a single pathway and/or by
allowing for activation of distinct downstream pathways. This increase in complexity
may help explain how a single regulatory molecule such as Ras can control such a
plethora of cellular responses such as cell proliferation, differentiation, transformation,
and apoptosis. To test whether or not compartmentalized signaling mediates specific
signaling cascades, Chiu et al. designed experiments in which transmembrane tethers
were used to artificially and stringently target Ras proteins to various membrane
compartments [183]. When oncogenic Ras was targeted to the ER or Golgi with a
transmembrane tether, it retained full transforming activity, indicating that all the
signaling events required for the complex cellular phenotype of transformation can be set
into motion from internal membranes [183, 192]. This might suggest that Ras signaling
from internal membranes is no different than from the plasma membrane. However,
quantitative differences in signal output could be detected. In transfected COS-1 cells,
Golgi—associated Ras activated ERK and PI3K with potency equal to that of natively
targeted Ras, the JN K pathway was poorly activated. Conversely, ER-tethered Ras was a
potent activator of IN K but a relatively poor activator of ERK and PI3K. In addition, this
compartmentalization altered the signaling kinetics. Using the same COS-l system, Chiu
demonstrated that activated Ras accumulation at the plasma membrane was rapid

following EGF stimulation (1-3 minute onset), activation of Ras at the golgi was delayed

39

(10 minute onset) and sustained [183]. The compartment-associated effects of Ras
signaling seem to be cell specific. In antigen receptor stimulated Jurkat T cells, as well as
in primary T cells, Ras activation on the golgi is rapid, and no Ras activation is detected
at the plasma membrane [138]. Evidence for compartmentalized Ras signaling has also
emerged from the study of fission yeast. In Schizosaccharomyces pombe, Rasl controls
both the mating pathway via a MAPK cascade and elongated cellular morphology
through an exchange factor for Cdc42 [193]. ER-restricted Rasl can support morphology
but not mating and the converse is true for Rasl restricted to the plasma membrane.
ERK/MAPK activation at the ER could provide mechanisms for altering the quality,

quantity or kinetics of ERK/MAPK signaling.

40

3. In Vitro identification and characterization of the ERKDD amino
acid sequence in R3

3.1 Introduction

PDGF-BB and F GF-2 stimulate mesangial cell proliferation by transmitting signals
from the cell surface to the nucleus to regulate the activity of transcription factors. The
binding of grth factors to their tyrosine kinase receptors facilitates the exchange of
guanosine diphosphates (GDP) for guanosine triphopsphates (GTP). GTP bound Ras, a
small GTP binding protein, then initiates the activation of a linear cascade of protein
kinases defined sequentially as mitogen activated protein (MAP) kinase kinase kinase,
referred to as MEKK, and MAP kinase kinase (MEKl and MEK2), which in turn activate
MAP kinase (ERK1/2). Activated (phosphorylated) ERK then translocates to the nucleus
to exert effects on transcription. Since MAP kinases phosphorylate very similar motifs
(consensus sequence Ser/Thr-Pro (S/TP)), and several potential substrates contain this
motif, it is important to provide specificity to direct individual kinases towards the correct
substrates. Multiple mechanisms exist for generating signaling specificity within these
cascades to ensure specific cellular responses, including the association of the cascade
components with their activating kinases, inactivating phosphatases, and scaffold proteins

that facilitate distinct signaling complexes.

ERK-DDS are sites in which members of the ERK family bind to interacting protein
partners. These protein partners can be upstream ERK regulators such as the MAPKKs
MEK] and 2; which activate (phosphorylate) ERK following docking, ERK phosphatases
such as PTP-Sl, STEP, and MKPs; which inactivate (dephosphorylate) ERK following

docking, ERK chaperones; which translocate ERK to a specific subcellular location

41

following docking, or ERK scaffolding proteins such as KSR; which bring several ERK
signaling molecules together in close proximity [160]. Although docking domains can
sometimes be recognized by more than one class of MAPK, they are generally thought to
increase signaling specificity by directing the level of MAPK activation, the
phosphorylation of their substrates, and in some instances their subcellular localization.
The characterization of receptor activity modifying proteins (RAMPS) has enhanced
our understanding of mechanisms involved in the regulation of G protein-coupled
receptors (GPCRs). R1, R2, and R3 are distinct gene products and have been
characterized as single-transmembrane domain proteins capable of direct interaction with
the class B (secretin family) G-protein coupled receptors, calcitonin receptor (CTR) and
CR-like receptor (CRLR) [87, 105, 194]. When CRLR is expressed with R1 in HEK293
cells, a CGRP receptor is produced. Even though R2 and R3 share only approximately
30% sequence identity, when CRLR is expressed with R2 or R3 in HEK293 cells
virtually identical (pharmacologically and biologically) AM receptors (AM-1R, AM2-R,
respectively) are produced [80]. Upon activation, the CRLR-RAMP receptor complex
causes cyclic AMP activation in most systems, irrespective of whether the ligand is AM
or CGRP. For many years, RAMP interactions were thought to occur only between a few
select class B receptors. However, in a recently published RAMP binding study, when
co-expressed with RAMPS, 6 out of 10 class B receptors demonstrated select RAMP
interaction [105]. VPACl receptor (V PAClR) interacts with all three RAMP isoforms,
parathyroid hormone-1 receptor (PTHlR) and glucagon receptor with R2, and
parathyroid hormone-2 receptor (PTH2R) with R3. Another study has demonstrated that

a class C GPCR, a calcium-sensing receptor (CaSR), interacts with RAMPs [106]. In a

42

paradigm similar to CRLR, recombinant CaSR expressed in COS7 cells does not
translocate to the plasma membrane unless R1 or R3 (but not R2) is also co-expressed.
In the same study, R3 was shown to mediate both the glycosylation and trafficking of
CaSR from the ER to Golgi. In the absence of R3, CaSR was retained in an intracellular
compartment that confocal microscopy identified to be the ER. It is evident that diverse
amino acid sequences within the three RAMP isoforms allow them to play a much
broader role in GPCR regulation than what was initially proposed.

All three RAMP isoforms harbor short, intracellular C-terminal tails. RAMPs
contain a conserved Ser-Lys (S, K; brown) sequence in the intracellular domains. While
the Ser-Lys motif does not effect internalization of either Rl-CRLR or R2-CRLR, they
negatively regulate internalization of R3-CRLR [92]. The R3 cytoplasmic tail also
contains a conserved Leu-Leu motif. This motif is known to mediate internalization of
the human vasopressin V2 receptor through interactions with proteins involved in
endocytosis (clathrin, adaptins and arrestins) [195]. CRLR, R1 and arrestins do exist as
a ternary complex in HEK-293 cells, resulting in dynamin- and B-arrestin-dependent
internalization following CGPR exposure [196]. No such interaction has been identified
thus far for R2 or 3. All three RAMP isoforms also contain putative phosphorylation
sites (threonine in R1 and 3; serine in R2), however the potential roles of these residues
in GPCR intemalization/endocytosis have not been studied. A unique PSD-95/Discs-
large/ZO-l homology domain type-l (PDZ) recognition motif (Asp-The-Leu-Leu) on the
extreme C-terminus of R3 mediates protein-protein interactions involved in the regulation
and trafficking of the R3-CRLR complex. Recent reports from our laboratory have

demonstrated that in HEK293 cells, overexpression of the Na‘i/H+ exchanger regulatory

43

factor-1 adaptor protein (NHERF) together with CLR and R3 abolished agonist-induced
receptor internalization without affecting desensitization[102]. R3 PDZ motif interaction
with the N-ethylmaleimide-sensitive factor (NSF) also mediates the recycling of CRLR in
HEK293 cells [97]. In these experiments, the absence of NSF overexpression resulted in
CRLR degradation following agonist stimulation. In RMCs, CRLR is normally recycled
following agonist-induced desensitization [197]. R3 knockdown, as well as
pharmacological inhibition of NSF abolishes RMC CRLR recycling [97].

The N-tenninal sequences of RAMP3 share less than 20% of amino acid
homology but share in common a significant number of consensus sites for
cotranslational modifications including cysteine residues (potentially involved in
disulfide bond formation) and N-glycosylation consensus sites (Asn-X-Ser/Thr). The N-
terminal sequences have been reported to be important for ligand binding specificity and
efficient receptor transport to the membrane The structural domain(s) involved in
selective AM binding have been studied using various RAMP chimeras and deletion
mutants. Co-expression of chimeric RAMPs and CRLR in HEK293 cells revealed that
residues 77-101, situated in the extracellular N-terrninal domain of human R2, were
crucial for selective AM-evoked CAMP production. In addition, deletion of hR2 residues
86-92 significantly attenuated high-affinity ”SI-AM binding and AM-evoked cAMP
production despite cell surface expression of CRLR-R2. Deletion of hR3 residues 59-65
had a similar effect. Disulfide bonds are also critical for CRLR-RAMP activation. Four
cysteine residues are conserved in all RAMP isoforms, and R1 and 3 harbor two
additional conserved cysteine residues. While similar studies have not been completed

using R1, mutation of each of the six cysteines results in a significant loss of R3 cell-

44

surface expression and ligand binding [88]. Several consensus sites for N-glycosylation
(Asn-X-Ser/Thr) have also been identified on R2 and R3. Four N-glycosylation sites
identified on human, mouse and rat R3 are conserved in the mouse R2, R1 has no
consensus sites for N-glycosylation [89]. In a recent study, it was demonstrated that N-
glycosylated R2 and R3 are expressed at the cell surface, and their transport to the plasma
membrane requires N-glycans. R1 is not N-glycosylated and is transported to the cell
surface only upon formation of heterodimers with CRLR [90].

Published reports from our laboratory have demonstrated that RMCs
endogenously express both the AM1R(CRLR + R2) and the AM2R (CRLR + R3) [197].
Furthermore, Nowak, et al. demonstrated that R3 is differentially upregulated following
PDGFBB treatment in cultured RMCs [124]. The emphasis of this paper was to
investigate the mechanisms of R3 upregulation and the subsequent increases in AM-
mediated (antiproliferative) RMC responses. The possibility that R3 may play a role in
addition to (or instead of) its known contribution in CRLR activation was not addressed.
RAMP isoforms vary greatly in their amino acid sequences. Given the recent
characterizations of novel, R3 proteinzprotein interactions, it seems plausible that R3 may
independently mediate some aspect of growth. The major aim of this study was to
identify and characterize amino acid regions within R3’s sequence that could potentially
mediate PDGFBB mediated growth responses in RMC.

3.2 Materials and Methods
3.2.1. Materials
pGEXSX-Z vector was purchased from Amersharn (Piscataway, NJ). Activated

ERK2 and MEKl were obtained from Sigma Aldrich (St.Louis, MO) and inactive ERK2

45

from Calbiochem (San Diego, CA). R2, R3, and ERK2 antibodies were from Santa Cruz
(Santa Cruz, CA). Anti-MEKl/2 antibody was from Cell Signaling (Beverly, MA). BL
competent cells were from Stratagene (La J olla, CA). All other reagents were of the
highest quality available.

3.2.2. Scansite analysis of putative human R3 protein interaction amino acid
sequences

The human R3 (NP_005847) and human R2 (NP_005845) amino acid sequences
were entered into the database Scansite (Massachusetts Institute of Technology) to search
for putative protein interaction sequences [198]. This database compares the amino acid
sequences of more than 35,000 proteins. Images in this thesis/dissertation are presented in

color.

3.2.3. GST Protein Overlays

Full-length human R2 and R3 cDNA were sub cloned into a pGEX5X-2 vector
and induced as described previously [97, 102]. 10uG of R2 and R3 GST fusion proteins
were resolved on a 10%SDS-polyacrylamide gel and transferred to a PVDF membrane.
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 NaCl, 0.1% v/v Tween 20) for 1 hour at
room temperature then incubated overnight at 4 degrees in a solution containing either
25nM purified active or inactive ERK. The following day the blots were washed three
times with TTBS, probed with antibodies to ERK-2 (1:500), washed three times with
TTBS, and incubated with a horseradish peroxidase-conjugated secondary antibody to
detect ERK interaction. Protein bands were visualized by soaking the blots in

Supersignal West Pico chemiluminescent substrate (Pierce), and exposing to x-ray film.

46

The blots were then stripped using a solution of .5% sodium dodecyl sulfate and .1X
sodium chloride/sodium citrate (SSC) heated to 95 degrees Celsius and then re-probed as
described above with antibodies to either R2 or R3 (1:300) to confirm their presence on
the membrane. To test GSTR3zMEK interaction, lOuG GSTR3 and 5uG activated ERK
protein (positive control) were resolved on gels and probed with a solution containing
purified His-tagged MEK] protein. HRP-anti MEK1/2 antibody was used to detect MEK
interaction. Blots were then stripped as described above, and re-probed with R3 and

ERK-2 antibodies to confirm R3 and ERK presence on the blots.

3.3.Results

3.3.1. In Vitro identification and characterization of ERKDD amino acid sequences
in human R3

The Scansite database search revealed that the amino acid sequence of R3
contains a putative ERKDD motif at amino acids 6 through 18. In fact, the search
indicated a 100% match between R3 and the established ERK docking domains of MEK.
The consensus sequence of this domain consists of the basic amino acids arginine or
lysine, followed immediately by the helix breaking amino acid proline, followed two to
six amino acids later by the hydrophobic amino acids leucine or isoleucine [160, 164].
The N-terminal amino acid sequence of R2 harbors some similarity to an ERK D-
Domain, however, some critical residues were not present. (Figure 3).
3.3.2. GST Overlays to identify R3:ERK and R3:MEK interaction

GST overlay experiments demonstrated that R2 protein does not interact with

neither inactive nor active forms of ERK-2 protein. GSTR3, however, showed a robust

47

interaction with both inactive and active forms of the purified ERK-2 protein. No
detectable bands were observed when GST-R3 blots were incubated with purified,

activated MEK] protein and probed with anti-MEK1/2 antibody. (Figure 4A and B)

48

Docking Domain Sequences of human MAPKK (MEK)

Human MEK] 3KKK£TPIQLNPAPDGSIS
Human MEK2 4RRK£VLPALTINPTIAEG21
Human R3 SLRREQLLPLLLLLCGGCP“
Human R2 19VGR_I:AALRLLLL3’1

Consensus: -(R/K)2-(P_)-(X)2_6-(L/I-X-L/l)-

Figure 3. Scansite analysis of putative human R3 protein interaction amino acid
sequences. Human R3 (N P_005847) and R2 (NP_005845) amino acid sequences were
entered into the database Scansite (Massachusetts Institute of Technology) to search for
putative protein interaction sequences [198]. This database compares the amino acid
sequences of more than 35,000 proteins. The search revealed that the amino acid

sequence of R3 contains a putative ERKDD motif similar to that of human MEK] and 2.

49

A.R2R3 B.R2R3 C.R2R3 D.R2 R3

64 KDa

49 Kiln

Active ERK Inactive ERK

Figure 4A. GST protein overlays illustrating R3 interaction with ERK. lOuG of human
R2 and R3 GST fusion proteins were resolved on a 10%SDS-polyacrylamide gel and
transferred to a PVDF membrane. Filters were incubated with solutions containing either
25nM purified active or inactive human ERK. The following day the blots were probed
with antibodies to ERK-2 (A. and B.) then HRP-secondary antibody to detect ERK
interaction. Protein bands were visualized by soaking the blots in Supersignal West Pico
chemiluminescent substrate (Pierce), and exposing to x-ray film. The blots incubated with
active ERK were then stripped and then re-probed as described above with antibodies to

either R2 (C) or R3 (D) to confirm RAMP presence on the membrane. n=3.

50

A. ERK R3 B. ERK R3 C. ERK R3

64 KDa
°*" 49 KDa

Active MEK] R3 ERK-2
Figure 4B. GST protein overlays illustrating R3 interaction with activated human MEKI.
lOuG human R3 GST fusion protein and Sug purified active ERK protein were resolved
on a 10%SDS-polyacrylamide gel and transferred to a PVDF membrane. Filters were
incubated with a solution containing 50 units activated MEKl protein. The following day
the blots were probed with antibodies to MEK1/2 (A) then HRP-secondary antibody to
detect MEK] interaction. Protein bands were visualized by soaking the blots in
Supersignal West Pico chemiluminescent substrate (Pierce), and exposing to x-ray film.
The blots incubated with active MEKl were then stripped and then re-probed as
described above with antibodies to either R3 (C) or ERK (D) to confirm their presence on

the membrane. n=3.

51

3.4. Discussion

Although .RAMPs share only N30% sequence identity, the hydrophobicity plots
of the three RAMPs suggesting the typical structure of class I transmembrane proteins.
R2 is the least conserved, being 26 amino acids longer than R1 and R3 [87] The N-
terrninal extracellular domain of R3 is composed of amino acids 1-119. As expected, a
significant number of consensus sites for co-translational modifications including
cysteine residues (potentially involved in disulfide bond formation) and N-glycosylation
consensus sites (Asn-X-Ser/Thr) are located within this region. These sites are known to
mediate membrane expression and ligand-binding activity of the CRLR2R3 complex.
In addition to these established motifs, a Scansite database search of putative protein
interaction domains within the R3 N-terminal (aal-119) sequence revealed several
interaction domains within this region of R3 that could potentially be involved in growth
signaling. Using high-stringency parameters, Scansite predicted putative interaction
motifs for human SH2 and SH3 proteins, as well as an ERK D-Domain, similar to that of
human MEKl/2 within the extreme N-terminus of R3. Using the same parameters, no
such motifs were identified for R2. In addition, R3, but not R2, protein was capable of
human ERK2 interaction. R3 did not demonstrate interaction with purified activated
human MEK protein. From the preliminary results obtained thus far, we derived the
following “working hypothesis”:
R3 is a critical regulator of PDGF 36,6 -stimulated RMC proliferation through direct

interaction with the MAPK cascade within specific subcellular compartments. (Figure 6).

52

C
AMZR AM2R
Extracellular ]

Intracellular ! ‘1

    
 
 

Plasma Membrane

\ Il:ternalization
-(\/

Endosome
-NSF \

Degradation

   

Adapted from: TIBS 31(1 1): 631-38 ref [104]

Figure 5. Potential Subcellular locations for R3:ERK interaction. Potential R3:ERK

membrane interaction sites include the ER, golgi, caveolae, and endosomes.

53

4. The role of R3 in ERK phosphorylation and cellular proliferation
following growth factor stimulation

4.1. Introduction

The primary locus of renal dysfunction is the glomerulus, and the mesangial cell
(MC) appears to be a central site of the microscopically observable renal damage. By
regulating the amount and composition of the surrounding extracellular matrix (ECM),
mesangial cells provide the essential structural support for the glomerular capillary tuft. .
The turnover rate of mesangial cells in the normal adult human kidney is low; the
renewal rate is less than 1% [6]. Under normal conditions, quiescent mesangial cells
either face few mitogenic signals, or are unable to respond to them. Therefore, MC
proliferation is a general characteristic of a variety of progressive glomerular diseases.
Loss of coordinated regulation, associated with altered composition and increased
mesangial ECM deposition, often leads to progressive glomerulosclerosis, loss of
filtration function, and end-stage renal disease [5]. It follows, therefore, that an
understanding of the regulatory mechanisms for MC proliferation is important for our
understanding of glomerular disease and its eventual treatment.

Platelet-derived growth factor beta (PDGFBB), is a potent in vitro and in vivo
mesangial cell mitogen [14, 45, 46], and is involved in the regulation of mesangial cell
matrix synthesis [47]. The normal adult kidney expresses only low amounts of PDGF
protein and receptors, primarily by mesangial cells [44, 48]. In renal disease states,
infiltrating inflammatory cells (monocytes/macrophages) and platelets are a major source
of PDGF [49]. Exposure of mesangial cells in vitro to grth factors, such as epidermal

grth factor (EGF), growth hormone (GH), transforming growth factor type a (TGF-a),

54

basic fibroblast growth factor (F GF -2), and tumor necrosis factor type a ("INF-a ) induce
expression of PDGF mRNAs. EGF, TNF-a , and F GF-2 also stimulate MCs to secrete
PDGF [50] Inflammatory agents such as interleuken-l [3 (IL-10) and IL-6, as well as
vasoactive mediators such as vasopressin, endothelin-1 (ET-1), and angiotensin-II
(AngII) [51]. Expression of PDGFBB and its receptor are also significantly increased in
mesangial cells of animal models of glomerulonephritis [52, 53] and in human
glomerulonephritis in which mesangial proliferation occurs [52, 54, 55]. The importance
of PDGFBB, in glomerulonephritis was first demonstrated experimentally by the
administration of anti-PDGFBB antibodies in the anti-Thy1.1 model. Anti- PDGFBB
antibody treatment did not affect the initial mesangiolysis that occurs, but did result in a
50% reduction in mesangial cell proliferation and a corresponding reduction in ECM
accumulation [53, 56]. More recent studies using the non-specific PDGFBB, inhibitors
trapidil [57], dipyridimole [5 8], and TNP-470 [59] as well as the selective PDGFBB
inhibitor STI 571 [60], have confirmed the importance of PDGFBB, in both animal models
and human glomerulonephritis.

Basic fibroblast growth factor (FGF-2) is expressed by a variety of cells,
including endothelial cells, smooth muscle cells, macrophages, fibroblasts, and mesangial
cells [74]. In addition to FGF-2 receptors, heparan sulfate proteoglycans, especially
syndecan, are essential for the binding of F GF-2 to its cellular receptor [75, 76]. Since
mesangial cells express F GF-2 and its receptor components, its not surprising that F GF-2
is a potent mitogen for mesangial cells [74]. However, FGF-2 lacks a signal peptide for
secretion and is not normally released into the circulation [77]. FGF—2 is released from

adult mesangial cells only following an injury and may lead to further mesangial cell

55

injury by stimulating nitric oxide production [78]. This feed-forward progression of
FGF-2-induced mesangiolysis is an important feature of the early phase of anti-Thyl.1
nephritis. After the initial mesangiolysis, the marked proliferation of mesangial cells that
occurs in the anti-Thy1.l model is associated with an increased glomerular synthesis of
FGF-2 [74]. Further evidence of the importance of FGF-2 in this model are the
observations that injection of F GF -2 following anti-Thy1.1 antibody administration
resulted in increased mesangiolysis [74], while treatment with anti-FGF-Z blocked the
initial mesangiolysis, resulting in less mesangial proliferation and less ECM deposition
[79].

RAMP mRNA distribution has been analyzed in several species including mouse,
rat, and human. In fact, Ramps are expressed in some cell lines that do not express
CRLR receptors [87]. R3 is the most widely distributed RAMP isoforrn; its expression is
greatest in the kidney, heart, brain, and lung [86, 87, 107-110] Using both animal and
cell culture models, several reports have indicated that grth associated conditions can
result in alterations of R3 gene expression. However the mechanisms of R3 involvement
in proliferation have not been thoroughly investigated. R3, but not R2, is upregulated in
mesangial cells following PDGF-BBJreatment [124]. The growth-stirnulating
parathyroid hormone also increases R3 expression in primary mouse osteoblasts [125].
R3 is expressed in the rapidly growing thymus of newborn rats, but not in quiescent
thymus of adult animals [126]. R3 is expressed in the prostate cancer cell line, DU145
[123]; in prostate carcinoma biopsies the levels of R3 were significantly elevated
(compared to prostate hyperplasia biopsies) while expression levels of R2 were

unchanged in these samples [123]. R3 is also exclusively upregulated in cultured rat

56

neonatal cardiac fibroblasts with IL-lB [128], and in a rat model volume overload-
induced cardiac hypertrophy R3 was significantly increased, while other AM signaling
components were only modestly or not increased at all [128, 129]. Angiotensin II
induces hypertrophy of cultured rat neonatal cardiomyocytes. mRNA levels of R1 and
R3 are significantly elevated following 24-h treatment with Ang 11 (without a change of
those of R2 and CRLR), and the effects of Ang II on R1 and R3 expression were
abolished by an Ang 11 type 1 (AT1) receptor antagonist [130]. It is plausible from the
reports described above that R3 plays a role in growth-mediated events. The purpose of
this study is to identify the role of R3 in ERK phosphorylation and cell proliferation in
RMCs following PDGF-BBand FGF-2 stimulation
4.2. Materials and Methods
4.2.1. Materials

Rat mesangial cells (RMC) and Normal rat kidney interstitial fibroblasts (N RK-
49F) were obtained from ATCC. RPMIl640, DMEM, penicillin/streptomycin, fetal
bovine serum, non-essential amino acids, BLOCK-iT Dicer RNA interference kit, and
Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA). PDGF-BB was
obtained from Sigma Aldrich (St.Louis,MO) and FGF-2 from Oncogene (Cambridge,
MA). MTS assay kits were from Promega (Madison, WI). The MEK inhibitors,
PD98059 and U0126, were obtained from Calbiochem (San Diego, CA).
Gene-specific RNA oligonucleotides were purchased from Dharrnacon (Lafayette, CO)
and were transfected into RMC using Mirus TKO transfection reagent (Madison, WI).
Antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and

chemiluminescence reagents from Pierce (Rockford, IL). PfuTurbo DNA polymerase,

57

pCMVTag2 vector, and BL-21, DHSa competent cells were from Stratagene (La Jolla,
CA) and Fugene 6 transfection reagent was obtained from Roche (Nutley, NJ).
3 H methylthymidine was purchased from Perkin Elmer (Waltharn, MA). All other
reagents were of the highest quality available.
4.2.2. Cell Culture

NRK were maintained in Dulbecco's modified Eagle's medium supplemented with
4 mM L-glutamine, 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 5% bovine calf
serum, 5% Non-Essential Amino Acids, and 1% Penicillin/Strepomycin. RMC were
maintained in RMPI 1640 media containing 15% FBS and 1% penicillin-streptomycin.
4.2.3. MTS assay for RMC proliferation following PDGF-[3T3 treatment

RMC were plated at a concentration of 2000 cells/well in a 96-well tissue culture
plate. The following day, the cells were serum-starved overnight, then treated for 16
hours with 0, 1, 5, 10, 50, and 100ng/mL PDGFBB. An MTS assay was used to measure
the RMC proliferative response.
4.2.4. MTS assay for RMC proliferation following MEK inhibition

RMC were serum-starved overnight, pre-treated with MEK inhibitor, either lOuM
PD98059 or luM U0126 for fifteen minutes prior to 50mg/mLPDGFBB treatment. 16
hours later, proliferation of the cells was measured by MTS assay following
manufacturer’s directions.
4.2.5. MTS assay for RMC proliferation following gene-specific knockdown

Gene-specific knockdown was performed two ways. In the initial RMC
knockdown experiments, gene-specific d-siRNA for lacZ (control), R2, and R3 were

generated and purified using the BLOCK-iT Dicer RNA interference kit (Invitrogen).

58

RMC’s were transfected with d-siRNAs using Lipofectamine 2000 as per the
manufacturer's instructions (Invitrogen). 24 hours following transfection, cells were
plated on 96-well plates at a concentration of 200 cells/well. The following day the cells
were serum-starved overnight and treated with 50ng/mL PDGFBB. 16 hours later,
proliferation of the cells was measured by MTS assay as per manufacturer’s instructions.
To confirm the BLOCK-iT Dicer RNA interference results, gene-specific d-siRNA for
mismatched RNA (NS) and R3 (Dharrnacon) was transfected into RMC’s using Mirus
TKO transfection reagent as per manufacturer's instructions and proliferation was
measured by MTS assay.
4.2.6. Confocal Microscopy

5 days after gene-specific knockdown using d-siRNAs for lacZ (control), R2, and
R3 (Invitrogen), or mismatched (NS) and R3 (Dharmacon), RMC were plated on
coverslips. Cells were fixed with 4% paraformaldehyde then permeablized with 0.1% v/v
Triton X-100 in PBS. Coverslips were 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 (R2 or R3 at 1:200). Appropriate secondary antibodies were
applied for 1h at room temperature (Goat anti-rabbit Cy5 at 1:400). Coverslips were
mounted in Shandon mounting medium. Cells were visualized on a Zeiss 210 laser
confocal microscope. Images presented are representative single optical sections and are

representative of at least 20 fields imaged from at least three experiments.

4.2.7. Membrane-associated ERK phosphorylation following PDGF-BB treatment
and R3 knockdown in RMC

59

A PDGFBB-induced ERK phosphorylation time course was established by serum
starving RMC overnight, then treating the cells with 50ng/mL PDGFBB for 0 (control),5,
10, 15, 30, and 60 minutes. The media was removed and the plates were snap-frozen to
preserve the phosphorylation status of the cells. The cells were then scraped from the
plates in homogenization buffer (lOmMHepes, 0.15MnaCl, lmM EDTA, lmM PMSF,
0.2mM sodium orthovanadate, lOOnM NaF, 5ug/mL leupeptin, and lOug/mL aprotinin).
This lysate was dounce-homogenized for 20 strokes. The homogenates were centrifuged
at 1300xg for 20 minutes at 4°C to pellet nuclear and cell debris. The supernatant was
centrifuged at 100,000xg at 4°C for 1 hour to isolate the membrane (pellet) fraction.
Protein concentration was determined by the Bradford assay. For each time point, 40ug
membrane fraction protein was separated on a 10% SDS-polyacrylamide gel. Western
blotting was performed with anti-pERK antibody at 1: 500 to assess ERK
phosphorylation status. Bands were visualized using chemiluminescence (Pierce
Supersignal West Pico) on XO-Mat film. Blots were stripped by immersion in a 95°C
solution of.5% SDS/.1XSSC and then re-probed for ERK-2 at 1:500. The PDGFBB-
induced ERK phosphorylation time course described above was repeated in RMC
following gene-specific R3 knockdown using Dharmacon si-RNA as described above.
4.2.8. Membrane-associated ERK phosphorylation following FGF-2 treatment in
NRK An F GF mediated, membrane-associated ERK phosphorylation time course was
established in NRK by serum-starving NRK overnight, then treating the cells with
5ng/mL FGF-2 for 0, 5, 10, 15, 30, and 60 minutes. The media was removed and the

plates were snap-frozen to preserve the phosphorylation status of the cells. The cells

were then scraped from the plates in homogenization buffer (lOmMHepes, 0.15MnaCl,

60

lmM EDTA, lmM PMSF, 0.2mM sodium orthovanadate, 100nM NaF, 5ug/mL
leupeptin, and lOug/mL aprotinin). This lysate was dounce-homogenized for 20 strokes.
The homogenates were centrifuged at 1300xg for 20 minutes at 4°C to pellet nuclear and
cell debris. The supernatant was centrifuged at 100,000xg at 4°C for 1 hour to isolate the
membrane fraction. NRK cells were transfected with 5ug of pCMVTag2-R3 DNA per
100mm tissue culture dish using Fugene 6 transfection reagent (Roche) following
manufacturer’s instructions. Control samples were transfected with pCMV vector. 24
hours following transfection, cells were plated on 150mm tissue culture dishes, then
serum-starved overnight and the ERK phosphorylation time courses were repeated with
FGF-2. Protein concentration was determined by the Bradford assay. For each time
point, 40ug membrane fiaction protein was separated on a 10% SDS-polyacrylamide gel.
Western blotting was performed with anti-pERK antibody (Santa Cruz) to assess ERK
phosphorylation status. Bands were visualized using chemiluminescence (Pierce
Supersignal West Pico) on XO-Mat film. Blots were stripped by immersion in a 95°C
solution of.5% SDS/.1XSSC and then re-probed for ERK-2 (Santa Cruz).

4.2.9. R3 Cloning and Mutagenesis Procedure

Full-length cDNA of human R3 was cloned into a pCMV-Tag2 (Flag epitope tag
at N-terminus) vector using the restriction cut sites EcoRl/Xhol as described previously.
Site-directed mutagenesis was performed using a pair of complementary oligonucleotides
(Michigan State University Macromolecular Structure Facility) containing the appropriate
point mutations in the sequence of the R3 ERK DD (Figure 6). The PCR for the mutation
was introduced in a pCMV-Tag2 as follows: 94 °C for 2 min; 25 cycles of 94 °C for 30 s,

50 °C for 30 s, 68 °C for 4 min; final cycle of 68 °C for 7 min using PfuTurbo DNA

61

polymerase, then Dpnl digested for 2 hours. The mutated DNA sequences were
confirmed by automated sequencing (Michigan State University Genomic Technology
Support Facility). NRK cells were transfected with 3ug of pCMVTag2-R3, R3EE, or
R3EEG DNA using Fugene 6 transfection reagent following manufacturer’s instructions

Mutations were confirmed by automated sequencing (Michigan State University Genomic

Technology Support Facility).

4.2.10. 3H Thymidine Incorporation Assay for proliferation of NRKs

A dose-response curve for NRK proliferation following FGF-2 treatment was
established using 3 H methylthymidine incorporation . Briefly, cells were plated at a
concentration of 3000 cells/well on a 24-well plate. The following day the cells were
serum starved overnight, then treated for 16 hours with 0,1,5, and lOng/mL F GF -2.
0.50Ci 3 H methylthymidine was added to each well. Four hours later, cells were washed
with PBS, and 10% TCA was added to the wells to stop the incorporation. The TCA was
aspirated; the cells were lysed with0.2N NaOH. 3 H methylthymidine incorporation was
assessed by counting the samples on a B—counter. To measure the effect of MEK
inhibition on F GF-2-mediated NRK proliferation, cells were pre-treated withluM U0126
for fifteen minutes prior to 5mg/mL F GF -2 treatment and subsequent 3H
methylthymidine incorporation. For proliferation assessment following R3 introduction,
R3 was sub-cloned into the pCMV-Tag2 expression vector and 5 micrograms per 100mm
dish were transfected into the NRKs using Fugene 6 following the manufacturer’s
protocol. 24 hours after transfection, cells were plated at a concentration of 3000

cells/well on a 24-well plate. The following day the cells were serum starved overnight,

62

and proliferation following 5ng/mL FGF-2 was assessed using 3 H methylthymidine
incorporation.
4.2.11. GST Overlay with R3 Mutations

Site-directed mutagenesis was performed using a pair of complementary
oligonucleotides (Michigan State University Macromolecular Structure Facility)
containing the appropriate point mutations in the sequence of the R3 ERK DD. (Figure 7)
The mutation was introduced into a pGEX5X-2 vector using PCR as follows: 94 °C for 2
min; 25 cycles of 94 °C for 30 s, 50 °C for 30 s, 68 °C for 4 min; final cycle of 68 °C for
7 min using PfuTurbo DNA polymerase (Stratagene), then Dpnl digested for 2 hours.
The mutated DNA sequences were confirmed by automated sequencing (Michigan State
University Genomic Technology Support Facility). lOuG of R3, R3Q, R3EE, and
R3EEG GST fusion proteins were resolved on a 10%SDS-polyacrylamide gel and
transferred to a PVDF membrane. 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 NaCl, 0.1%
v/v Tween 20) for 1 hour at room temperature then incubated overnight at 4 degrees in a
solution containing either 25nM purified inactive or active ERK (Sigma and
Calbiochem). The following day the blots were washed three times with TTBS, probed
with antibodies to ERK-2 (1:500 Santa Cruz), washed three times with TTBS, and
incubated with a horseradish peroxidase-conj ugated secondary antibody to detect
R3:ERK interaction. Protein bands were visualized by soaking the blots in Supersignal

West Pico chemiluminescent substrate (Pierce), and exposing to x-ray film.

4.2.12. Statistics

63

Data are presented as mean :t S.E.M. Multiple group comparisons were made
using analysis of variance (ANOVA), followed by Bonferoni’s multiple comparison
between treatments. Two treatment comparisons were compared using Student’s t-test
method. Statistical significance was set at P<0.05.

4.3. Results

4.3.1. MTS assay for RMC proliferation following MEK inhibition

First, a PDGF-BB stimulated dose-response curve was established in RMC to
determine the PDGF 4313 concentration to be used for future experiments (Figure 7). A
dose of 50ng/mL PDGF-BB was chosen. The MEK inhibitor, U0126, significantly

decreased PDGF-BB- proliferation of RMC (25%i4.7 vs. 3.2%d: .7) (Figure8).

4.3.2. 3H Thymidine Incorporation Assay for proliferation of NRKs following MEK
inhibition

'An FGF-2 stimulated dose-response curve was established in NRK to determine
the FGF-2 concentration to be used for future experiments (Figure 9). A dose of 5ng/mL

F GF -2 was chosen. The MEK inhibitor, U0126, significantly decreased and FGF-2-

induced proliferation of NRK (4.80foldi0.7 vs. 1.2foldi0.3) (Figure10).
4.3.3. MTS assay for proliferation following gene-specific knockdown

Confocal microscopy was utilized to assess expression levels of RAMPs in RMC
5 days after gene-specific knockdown using BLOCK-iT Dicer RNA interference
(Figurel I). Dicer RNA R3 knockdown in RMC resulted in a significant decrease in
PDGF-BB -stimulated proliferation compared to non-specific (Lac-Z) knockdown
(Figure12). Confocal microscopy also illustrated R3 knockdown in RMC following

treatment with gene-specific d-siRNA (Figurel3). Using the Dharmacon si-RNA

64

technique, R3 knockdown significantly decreased PDGF-[3B -stimulated proliferation
(33.9"/oi6.6vs.3.1°/oi2.8) (Figure14).
4.3.4. Membrane-associated ERK phosphorylation following PDGF-BB treatment

and R3 knockdown

A membrane-associated ERK1/2 phosphorylation time course was established in
RMC by serum-starving them overnight, then treating the cells with 50ng/mL PDGF-BB
for 0 (basal), 5, 10, 15, 30 and 60 minutes. Phosphorylated ERK was normalized to ERK
protein levels to determine maximal ERK activation. Following PDFGF-BB stimulation,
ERK activation peaked at 10 minutes and returned to basal levels by 30 minutes. R3
knockdown, using gene-specific RNA interference technology, resulted in significantly
decreased maximal ERK activation at the 10 (91%:t5 vs. 50.7%:t6.9) and lS-minute time

points (56%i12.7 vs. 16.3%il .3) (Figures 15 and 16).
4.3.5. Proliferative effects of R3 introduction into NRK cells

3 H thymidine incorporation assays demonstrated that introduction of R3 into NRK
cells resulted in a significant increase of both basal (584cpmi43 vs. l483cpmd:1 10) and

5ng/mL FGF-2 stimulated (2403cpmi202 vs. 5461cpmzt437) proliferation (Figure 17).

4.3.6. Effect of R3 on membrane-associated ERK phosphorylation in NRKs

following FGF-2 treatment

A membrane-associated ERKl/2 phosphorylation time course was established in
NRK by serum-starving them overnight, then treating the cells with 5ng/mL FGF-2 for 0
(basal), 5, 10, 15, 30 and 60 minutes. Phosphorylated ERK was normalized to ERK
protein levels to determine maximal ERK activation. Following F GF-2 stimulation, ERK

activation peaked at 5 minutes and returned to basal levels by 10 minutes. R3

65

introduction resulted in significantly increased membrane-associated ERK activation at
the 10-minute time point compared to control (pCMV) (90.1%maximal.responsei0.5 vs.

45.2%:1:.08) (Figure 18).
4.3.7. Expression of R3 and R3 D-Domain mutants

Mutating the putative D-Domain of R3: R6R7 to E6E7 (AEE), R6R7P3 to
E6E7G3(AEEG), and Lu to Q1 .(AQ) did not effect the membrane fraction expression of

R3 (Figure 19).

4.3.8. Effect of R3 ERKD-Domain mutation on membrane-associated ERK
phosphorylation in NRKs following FGF-2 treatment

R3 significantly increased membrane-associated ERK activation levels at 5
(2.72i.3 fold) and 10 minutes (2.81.24 fold) following FGF-2 stimulation, compared to
respective basal (0 time point) levels of ERK activation. R3Q also significantly
increased membrane-associated ERK activation at these time points (3.4i.62 and 3.5:t.66

fold, respectively) (Figure 20).

4.3.9. The role of R3 ERK-DD in FGF-2-stimulated proliferation of NRK

Proliferation of NRKs following transfection of R3, R3AEE, R3AEEG, RAQ, and
subsequent FGF-2 treatment was assessed using 3 H Thymidine incorporation. R3
introduction in NRK resulted in a significantly increased proliferative responses
compared to control (pCMV) levels (3.98foldil .2 vs. 2.0d:.62). Introducing R3-AEE
and R3-AEEG mutations in NRK resulted in a significantly decreased the F GF-2
proliferative response compared to R3 (2.5fold 2t.7 and 1.6foldi.8 vs. 3.98foldi1.2) The

proliferation response of R3AEEG mutation introduction was also significantly different

66

...-.4,

L?EI:';L’-.‘¢I_‘.KL. A

.l

min"

 

from R3AQ (1.6foldi.8 vs. 3.31.6foldi.81.2). The proliferative responses following R3-
AEE, R3AEEG, and R3AQ introduction and FGF-2 stimulation were not significantly

different from control (pCMV) levels (Figure 21).
4.3.10. R3 Mutation:ERK interactions using GST Overlay

All of the GST-R3 ERK docking domain mutant proteins are capable of in vitro
ERK-interaction. However, using overlay experiments, R3AEE and R3AEEG mutants

demonstrated less ERK-2 binding than wild type R3 and the R3AQ mutant (Figure 22).

67

Putative ERK D-Domain Sequence:

5LRR_1:QLLPLLLLLCGGCP24

Primer Sequences Used to Generate ERKD-Domain Mutations:

AR7 to E:
5:GAG ACT GGA GCG CTG GAG CGC CCG CAA CTTf’
5 AAG TTG CGG GCG CTC CAG CGC TCC AGT CTC"

A’R7R3 to EE: ’
{GAG ACT GGA GCG CTG GAG GAG CCG CAA CTT CTC?
5 GAG AAG TTG CGG CTC CTC CAG CGC TCC AGT CTC3

412712822 to EEG: ,
{GAG ACT GGA GCG CTG GAG GAG GGG CAA CTT3
5 AAG TTG CCC CTC CTC CAG CGC TCC AGT CTC3
A142 to Q:

5:GGA GCG CTG CGG CGC CCG CAA CAG CTC CCG TTG CTG”,
5 CAG CAA CGG GAG CTG TTG CGG GCG CCG CAG CGC TCC3

Figure 6. Sequences of oligonucleotide primers used to generate ERKDD mutations in

human R3.

68

’0“, A. on ‘ 0"

—---- --—.
.Q Q. 1s .

sigma 14.; «Treat—y t.

 

4o.
35-
30-
25-
20-

1 0-
5.

% Change from Basal

 

 

1 5 10 so 100
PDGF-BB (ng/mL)

Figure 7. PDGFBB-Induced Proliferation of RMC Cells using MTS Assay. RMC were
plated at a concentration of 2000 cells/well on 96-well plates and serum-starved
overnight, and then treated with l, 5, 10, 50, or lOOmg/mL PDGFBB for 16 hours.
Proliferation of the cells was measured by MTS assay (Promega) following

manufacturer’s directions. * ps 0.05; n=3.

69

% Change from Basal

**

 

 

PDGFBB PDGFBB PDGFBB
+ PD98059 + U0126

Figure 8. ERK Inhibition of PDGFBB-Induced Proliferation of RMC Cells using MTS
Assay. RMC were plated at a concentration of 2000 cells/well on 96-well plates and
serum-starved overnight, pre-treated with either lOuM PD98059 or luM U0126
(Calbiochem) for fifieen minutes prior to 50mg/mL PDGFBB treatment. Proliferation of
the cells was measured by MTS assay (Promega) following manufacturer’s directions.

** p5 0.001 (U0126 and PD98059 vs. PDGF); n=3.

70

50007
40001

3000-t

pm

° 2000.

1000-

 

 

Basal lng/ml 5ng/mL10ng/mL

Figure 9. Dose-response curve for NRK proliferation following FGF-2 treatment.

NRK were plated at a concentration of 3000 cells/well on a 24-well plate. The following
day the cells were serum starved overnight then treated with F GF-2 for 16 hours. NRK
proliferation was assessed using 3H methylthymidine incorporation. *p50.05 (5, 10

ng/mL FGF-2 vs. Basal); n=4.

71

Fold Basal

 

 

FGF-2 FGF-2 + U0126

Figure 10. ERK Inhibition of FGF-2-Induced Proliferation of NRK Cells using 3H

methylthymidine incorporation as described above.**p$0.001 (U 0126 vs. FGF-2); n=3.

72

aim-mm. m ..- aL-qn .up‘d‘t‘F—t—fi

 

R3 LacZ (NS) R3 R2 R2
Control Knockdown Knockdown Control Knockdown

Figure 11. Confocal Microscopy depicting expression levels of RAMPs. 5 days after
gene-specific knockdown using d-siRNAs for lacZ, R2, and R3 (Invitrogen). RMC were
cultured on coverslips, fixed, and then permeablized with 0.1% v/v Triton X-100 in PBS.
Coverslips were 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 (R2 or R3 at 1:200). Appropriate secondary antibodies were applied for 1h
at room temperature (Goat anti-rabbit Cy5 at 1:400). Coverslips were mounted in
Shandon mounting medium. Cells were visualized on a Zeiss 210 laser confocal
microscope. Images presented are representative single optical sections and are

representative of at least 20 fields imaged from at least three experiments.

73

% Change from Basal

 

 

Figure 12. MTS Assay for RMC Proliferation following gene-specific knockdown. Gene-
specific d-siRNA for lacZ (NS), R2, and R3 were generated and purified using the
BLOCK-iT Dicer RNA interference kit. RMC’s were transfected with d-siRNAs using
Lipofectamine 2000 as per the manufacturer's instructions (Invitrogen). 48 h after
transfection, cells were serum-starved overnight and treated with 50ng/mL PDGFBB. 24
hours later, proliferation of the cells was measured by MTS assay. R3 knockdown
significantly inhibited PDGFBB-induced proliferation. **p§0.001 (vs. control and R2

knockdown); n=5.

74

YWETWT‘ —..-‘19'

 

NS Knockdown R3 Knockdown NS Knockdown R2 Knockdown

Figure 13. Confocal Microscopy depicting Gene-Specific R3 knockdown using
Dharmacon d-siRNA for mismatched RNA (NS) and R3. R3 knockdown was performed
as described in Methods section. Confocal microscopy was performed as in figure 12.
Cells were visualized on a Zeiss 210 laser confocal microscope. Images presented are
representative single optical sections from at least 20 fields imaged from at least three

experiments.

75

t FEW-rm jflfi— W‘F—‘E'

%Change from Basal

 

 

Figure 14. MTS Assay for RMC proliferation using Dharmacon gene-specific
knockdown. Gene-specific d-siRNA for mismatched RNA (NS control), R2, and R3
(Dharmacon) was transfected into RMC’s using Mirus TKO transfection reagent as per
manufacturer's instructions. 24 hours following transfection, cells were plated on 96-well
plates at a concentration of 2000 cells/well. The following day the cells were serum-
starved overnight and treated with 50ng/mL PDGFBB. 16 hours later, proliferation of the
cells was measured by MTS assay (Promega) as per manufacturer’s instructions.

**p50.001 (R3 vs. NS); n=5.

76

No R3 Knockdown R3 Knockdown

ERK
Essa...- ~----—----—- - p
-——~ - ...—.... R3

0min 5min 10min 15min 0min 5min 10min 15min

Figure 15. Membrane-associated pERK following gene-specific R3 knockdown and
50ng/mL PDGFBB stimulation. Gene-specific d-siRNA for mismatched RNA (NS) and
R3 (Dharmacon) was transfected into RMC’s using Mirus was transfected into RMC’s
using Mirus TKO transfection reagent as per manufacturer's instructions. Following
50ng/mL PDGFBB treatment, a membrane fiaction was prepared as described above.
Western blotting was preformed with anti-R3 antibody at 1:300 (Santa Cruz) and

visualized using chemiluminescence. Blot represents one of six experiments.

77

110-

 

 

5 5 100, +Control
-; “g 90- + R3 Knockdown
2 '5 80-
E; 7c»
E: a *
8 a
-= .§ 40‘
i: 5 3‘“
a: E 20‘ *
|.I.l g 1
0 1'0 30
Time (min)

Figure 16. The effect of R3 knockdown on maximal membrane-associated ERK
phosphorylation following PDGFBB treatment. Graph represents pERK/ERK ratios
observed following experimental conditions described in Figure 18. *pS0.05 (R3i Vs.

control at 10,15 min); n=6.

78

 

6000- **
-control

5000- =R3

2000‘ *

cool
10‘ I

Basal 5nglm L FGF-2

 

 

Figure 17. 3 H Thymidine Incorporation Assay for proliferation of NRK following R3
transfection. R3 was sub—cloned into the pCMV-Tag2A expression vector and 5
micrograms per 100mm dish were transfected into NRK using Fugene 6 (Roche)
following the manufacturer’s protocol. 24 hours after transfection, cells were plated at a
concentration of 3000 cells/well on a 24-well plate. The following day the cells were
serum starved overnight then treated with 5ng/mL FGF-2 for 16 hours. NRK proliferation
was assessed using 3 H methylthymidine incorporation. Control represents pCMV
transfected NRK. *p50.05 (R3 basal vs. control basal) "p50.001 (R3 FGF-2 treated vs.

control FGF-2 treated); n=5.

79

7651-. f i .2 (“:11‘7’ 1, s u ..._ ~— - PERK

0min 5min 10min 15min 30min 60min

1 .1-
1_o. * + Control
0.9- + R3

0.8-
0.7-
0.6-
0.5-
0.4-
0.3-
0.2-
0.1-
0.3

pERK/ERK
% Maximal Stimulation

 

 

od
""1
d
O
A
m4
to
O
O
0

Figure 18. Effect of R3 on membrane-associated ERK phosphorylation in NRK following
FGF-2 treatment. An FGF-2-induced, membrane-associated ERK phosphorylation time
course was established in NRK as in Methods section for control (pCMV) and
pCMVTag2(Flag)-R3 transfected cells. Protein concentration was determined by the
Bradford assay. For each time point, 40ug membrane fraction protein was separated on a
10% SDS-polyacrylamide gel. Western blotting was performed with anti-pERK antibody
(Santa Cruz) to assess ERK phosphorylation status. Bands were visualized using
chemiluminescence (Pierce Supersignal West Pico) on XO-Mat film. Blots were stripped

by immersion in a 95°C solution of.5% SDS/.1XSSC and then re-probed for ERK-2

(Santa Cruz). Graph represents pERK/ERK ratios. *pS0.05; n=5

80

ALLA A-_-..Af“
‘ ‘ A;_‘.__-_Lt ——A A A m ‘n i
‘7‘ --.___. ....I -. ..‘1 r' " .M . SJ R3
O 41th -..__;.._ Z; A .. - 1-2.4:» db- _"5-"“"

Control R3 R3AEE R3AEEG R3AQ

Figure 19. Membrane expression of R3 and R3 mutants. NRK cells were transfected
with 5ug of pCMV vector (control), pCMVTag2-R3, R3AEE, R3AEEG, or R3AQ DNA.
Membrane protein fractions were obtained as described in Methods section. Protein
concentration was determined by the Bradford assay. 40ug membrane fraction protein
was separated on a 10% SDS-polyacrylamide gel. Western blotting was performed with
anti-R3 antibody (1:300 Santa Cruz). Bands were visualized using chemiluminescence
(Pierce Supersignal West Pico) on XO-Mat film. Blot is representative of four

experiments.

81

 

4.5-

 

 

4-0‘ -0 min
3.5-1 -5 min
E a 3,0. - - C310 min
0)
$3 2.5- -15 min
a: E 2-0‘ -30 min
ll] 0 151
QI-I- ' y _ r y ' 560 m1"
tot . t = . l t 5
u I = u I i i v E i t E I
o.-5:;g:55;;5::s !
0.0. L 4 I n A 2 L a = a I = l

:1
u

R3AQ R3AEE R3AEEG

 

Figure 20. Effect of R3 ERKD-Domain mutation on membrane-associated ERK
phosphorylation following FGF-2 treatment. NRK cells were transfected with 5ug of
pCMVTag2-R3, R3AEE, R3AEEG, or R3AQ DNA. Control samples were transfected
with pCMV vector. 24 hours following transfection, cells were serum-starved overnight
and ERK phosphorylation time courses were performed by treating the cells with
5ng/mLFGF-2 for 0 (basal), 5, 10, 15, 30 and 60 minutes. Membrane fractions were
prepared as described previously. Protein concentration was determined by the Bradford
assay. For each time point, 40ug membrane fraction protein was separated on a 10%
SDS-polyacrylarnide gel. Western blotting was performed with anti-pERK antibody
(Santa Cruz) to assess ERK phosphorylation status. Bands were visualized using
chemiluminescence (Pierce Supersignal West Pico) on XO-Mat film. Blots were stripped
by immersion in a 95°C solution of.5% SDS/.1XSSC and then re-probed for ERK-2
(Santa Cruz). Graph represents pERK/ERK ratios, **p£0.001 (R3, R3AQ 5 and 10 min

time points vs. their respective 0 min-basal time points); n=5.

82

4.5- a
4.0-
3.5-1
3.0-
2.51
2.0--1
1.5-
1.0-
0.5-
0.0-

Fold Basal

 

 

Con RAMP3 AEE AEEG AQ

Figure 21. The role of R3 ERK-DD in FGF-2-stimulated proliferation of NRK. Site-
directed mutagenesis was performed using a pair of complementary oligonucleotides
(Michigan State University Macromolecular Structure Facility) containing the appropriate
point mutations in the sequence of the R3 ERK DD. The mutated DNA sequences were
confirmed by automated sequencing (Michigan State University Genomic Technology
Support Facility). NRK cells were transfected with 5ug of pCMVTag2-R3, R3AEE,
R3AEEG, or R3AQ DNA Control samples were transfected with pCMV vector. 24 hours
later, the cells were plated on 24-well tissue culture plates, and the following day the cells

were serum-starved overnight. NRKs were then treated for 16 hours with 5ug/mL FGF-2.

NRK proliferation was assessed using 3 H thymidine incorporation. apSO.05 (R3 and

R3AQ vs. Control), bp50.05 (R3 vs. R3AEE and R3AEEG); n 2 4.

83

1mm mm

R3 R3AQ R3AEE R3AEEG

 

ERK-2

, ... -w - .

Figure 22. GST Overlay with R3 mutations. Site-directed mutagenesis was performed to
produce the appropriate point mutations in the sequence of the R3 ERK DD. The mutated
DNA sequences were confirmed by automated sequencing (Michigan State University
Genomic Technology Support Facility). lOuG of R3, R3AQ, R3AEE, and R3AEEG GST
fusion proteins were resolved on a 10%SDS-polyacrylamide gel and transferred to a
PVDF membrane. Filters were blocked then incubated overnight at 4 degrees in a
solution containing either 25nM purified inactive or active ERK. The following day the
blots were washed and probed with antibodies to ERK-2, washed, and incubated with a
horseradish peroxidase-conjugated secondary antibody to detect R3:ERK interaction.
Blots were stripped and re-probed for R3. Blot is representative of 4 overlay

experiments.

84

 

4.4 Discussion

PTKR activation of MAPK is probably the most extensively investigated
mechanism of mesangial cell proliferation. However, both FGF-2 [199-202] and PDGF-
BB [203-205] can induce a proliferative response in certain cell types by activating
phospholipase C-y, resulting in increased intracellular Ca”. The specific MEK
inhibitors, U0126 and PD98059, were utilized to assess the contribution of ERK in l
RMCs. U0126 is a chemically synthesized organic compound that inhibits both active |

and inactive MEK1/2, while PD098059 inhibits activation of inactive MEK 1/2 by

binding to inactive MEK to prevent its activation. In RMC, MEK inhibition almost

 

totally inhibited PDGF-BB induced proliferation, and it significantly decreased the FGF-2
induced proliferation of NRK. In NRK, approximately 12% of the proliferative response

was not blocked by MEK inhibition. This small amount may be due to a non—ERK

mediated response, such as activation of PLC-y.

RMCs endogenously express both R2 and R3, however; only R3 expression is
upregulated following PDGF-BB treatment. A previous study from our laboratory
focused on the consequences of R3 upregulation on AM-mediated (anti-proliferative)
responses [124]. However, the non-AM associated effects of increased RMC R3
expression were not addressed. Given the recent reports identifying novel RAMP
receptor and protein interaction partners [97, 102, 105, 106], it seemed plausible that R3
might play an alternative role in PDGF- BB mediated cellular responses. Studies were

undertaken to identify and characterize such roles.

85

PDGF-BB is a potent mitogen for RMC, and deregulated PDGF-BB-induced RMC
grth known to play a role in a variety of glomerular diseases. PDGF-BB treatment of
cultured RMC results in a dose-dependent increase in proliferation, with 50ng/mL PDGF-

BB treatment resulting in a maximal proliferative response [124]. Initial experiments
employed to identify R3’s role in PDGF-BB-induced proliferation employed Invitrogen’s
“dicer” technology. In these experiments, R3 knockdown completely inhibited the
proliferative effects of 50ng/mL PDGF-BB treatment. Dicer generates short, double-
stranded interfering RNA for an entire gene of interest. This technology has been utilized

successfully for specific gene knockdown with certain short gene products, and the

3 ”.-.-eons ...)Jmiflbfi :nflim—T-‘fl

inhibition of RMC proliferation was not observed with R2 or lac-Z knockdown.
However, dicer can result in some non-specific effects. Specific double-stranded RNA

oligonucleotides knockdown confirmed the R3 effects on proliferation.

Since RAMPs are class I transmembrane proteins, crude membrane preparations
were utilized to assess the role of R3 in PDGF-BB mediated actions. PDGF- BB treatment
resulted in quick (5 minute) peak activation of membrane-associated ERK that steadily
decreased to control levels in 20 minutes. Consistent with the RMC proliferation data,
gene-specific knockdown of R3, but not R2, resulted in a decrease of membrane-
associated ERK activation. While the peak (5minute) ERK activation trended towards,
but did not reach statistical significance, maximal membrane-associated ERK activation

was decreased in both the 10 and lS-rninute time points.

To further validate the effects of R3 knockdown on proliferation, R3 DNA was
transfected into NRK cells. Rat interstitial fibroblasts (N RK) express CRLR and R2, but

not R3 [206]. AM is anti-proliferative in NRK, however, as in the case of RMC, non-

86

AM mediated RAMP effects have not been studied. Since FGF-2 is a well-characterized
fibroblast mitogen, a F GF-2 dose-response curve to assess proliferation was performed in
NRK. Maximal proliferation was observed following 5ng/mL FGF-2 treatment.
Consistent with the RMC data suggesting a role for R3 in PDGF-BB induced
proliferation, the introduction of R3 in NRK resulted in a significant increase in both

basal and 5ng/mL FGF-2 treatment compared to vector-transfected cells. R3’s effect on

 

it:
serurn-starved basal proliferation may be a result of culturing the newly transfected cells
with serum for one day after plating them on 24-well plates prior to serum-starving them
the following evening. These cells had ample exposure to numerous grth factors :
within the serum that, together with R3, may have affected their rate of proliferation. It i

may also be possible that the introduction of R3 into these cells independently results in
an increased cell-turnover rate. Although the mechanism is unclear, introducing R3 into a
cell line that does not normally express it resulted in a robust basal and F GF-2 stimulated

proliferation.

To characterize R3 involvement in NRK MAPK activation, membrane-associated
ERK activation was assessed. First, a time ecurse for NRK membrane-associated ERK
activation following FGF-2 treatment was established. Like the quick (5 minute peak and
decrease back to basal at 15 minutes) membrane-associated ERK activation observed in
RMC following PDGF-BB treatment, the F GF -2 response in NRK peaked at 5 minutes
and returned back to basal levels by 10 minutes. Although the introduction of R3 into
NRK did not effect the level of ERK activation at 5 minutes it did result in a significant
increase in membrane associated ERK activation at the 10-minute time point compared to

vector transfected control. This membrane-associated ERK activation in the R3

87

transfected cells was sustained, but not statistically significant at the 15 and 30-minute
time points. By utilizing both R3 knockdown and introduction it is clear that R3

influences membrane-associated ERK activation in RMC and NRK.

The N-terminal amino acid sequence of R3 harbors a putative ERK D-Domain.
Mutating one or both of the basic arginine residues of the putative ERKDD to acidic
glutarnic acids did not affect in vitro binding of GST fusion proteins to either inactive or

active (phosphorylated) ERK protein. The sequential addition of a hydrophobic, helix-

. . '— —,i‘-'~’m

breaking praline to an uncharged, polar glycine residue mutation resulted in a slight, but

significant decrease in ERK:R3 binding. Mutating the downstream hydrophobic amino

 

acid leucine to glutamine also had no effect on ERK:R3 interaction.

In NRK a significant amount of FGF-2 induced proliferation is mediated through
ERK cascade activation. R3-GST fusion protein overlays demonstrated that R3 could
physically interact with both active and inactive ERK protein. In addition, R3 expression
resulted in an increased ERK activation and proliferation of NRK. It seems plausible that

the D-Domain of R3 might mediate membrane-associated ERK activity.

Although R3 membrane expression was assessed in NRK, mutating the R3 D-
Domain may result in a decreased membrane expression of the corresponding mutated
proteins. R3 ERK D-Domain mutant expression in the NRK membrane fraction was
verified, and the effects of mutating key amino acid 3 within the ERK D-Domain were
measured. Transfecting NRK with R3 resulted in increased membrane-associated ERK
activity, mutating the extreme N-terminal D-Domain residues from basic, positively
charged, hydrophilic arginines to acidic, negative charged, hydrophilic glutamates

(R3EE) resulted in decreased ERK activation levels that were similar to control (pCMV)

88

levels. Mutating the adjacent neutral, helix breaking amino acid proline to a neutral
glycine (R3 EEG) residue did not significantly decrease ERK activation from control
levels. Mutating the downstream hydrophobic amino acid leucine to glutamine did not
significantly decrease ERK activity levels from R3 transfected levels.

While R3 introduction into a cell line (N RK) that does not endogenously express
it resulted in a robust proliferative response to FGF-2, but the biological responses
following specific ERK D-Domain mutation would provide a much better assessment of
R3’s role in ERK-cascade activation. To this end, proliferation of NRK following mutant
R3 transfection and subsequent FGF-2 treatment correlated well with the membrane-
associated ERK activity. Transfecting NRK with the R3EE mutant did not significantly
alter FGF-2 stimulated proliferation from control (vector) transfected levels, but it was
significantly lower than the R3 response. Introducing a R3EEG mutant resulted in a
small, but insignificant decrease from control, and a significant decrease in proliferation
compared to the R3Q mutation. Proliferation following R3Q mutant introduction was not

statistically different from R3.

The findings reported thus far suggest that the ERK D-Domain of R3 might play
an important role as a mediator of ERK cascade activation and the subsequent
proliferative response following PDGF-BB treatment in RMC and FGF-2 treatment in
NRK. This D-Domain is located in the extreme N-terrninus of the class I transmembrane
protein, R3. How then, is this integral membrane protein able to interact and mediate the

activity of a cytosolic protein such as ERK?

89

 

5. Identification of the subcellular site(s) of R3:ERK interaction

5.1 Introduction

All signal transduction cascades transmit signals from the cell surface into the
cytoplasm and nucleus to elicit a cellular response. These cascades contain multiple
components, and classical cascades act by a sequential series of phosphorylation steps.
Since MAP kinases phosphorylate very similar motifs (consensus sequence Ser/Thr—Pro
(S/TP)), and several potential substrates contain this motif, it is important to provide

specificity to direct individual kinases towards the correct substrates. Multiple

' Panamanian IFJRWiu-T‘r'r'

mechanisms exist for generating signaling specificity within these cascades to ensure
specific cellular responses. One important mechanism for ensuring signaling specificity
involves targeting MAPKs to their substrates within distinct cellular compartments.
Compartmentalized signaling is a relatively new paradigm in signal transduction. Most
work thus far has focused on the roles of scaffolding proteins that chaperone and tether

MAPK components together in close proximity in specific cellular locales.

Kermorgant et al. have recently demonstrated that traffic to endosomes is
essential for hepatocyte growth factor (HGF/c-Met) to trigger an ERK response [171]. In
addition, normal endocytic trafficking of EGFR is important for the full activation of
MAPK. Inhibition of clathrin-mediated endocytosis by dominant-negative dynamin in
EGF- activated cells revealed that internalization of EGF R is needed for maximal MAPK
activation [172]. Burke et al. have also reported that the EGF receptor (EGFR) activates
its targets Ras, Raf, and MAPKK (MEK) at the plasma membrane, but endocytosis had to

occur to activate ERK [167, 173]. Like EGF, nerve grth factor (N GF) activation of

90

TrkA receptors results in internalization of phosphorylated TrkA via endocytosis. Howe
et al. demonstrated that upon NGF stimulation, PC 1 2 derived endosomes contained
phosphorylated TrkA, Shc, Raf, ERK, as well as activated Ras [174]. Since it is believed
that the R3:CRLR dimer is maintained during translocation to the cell surface, receptor
activation, internalization and degradation, it is plausible that R3:ERK interaction also

occurs at the endosomal membrane.

Cell surface receptors such as tyrosine kinases, (RTK) and G protein-coupled

receptors transmit activation signals to the Raf/MEK/ERK cascade through different

 

isoforms (H-Ras, N-Ras, and K-Ras 4B/4A) of the small GTPase protein Ras [135, 136].

Far-f .

Activation of only 5% of Ras molecules is sufficient to induce full activation of ERK1/2
[137]. Compartmentalized signaling may also help explain how a single regulatory
molecule such as Ras can control such a plethora of cellular responses such as cell
proliferation, differentiation, transformation, and apoptosis. Ras proteins must associate
with the cytosolic leaflet of cellular membranes to be fully activated [138, 139].
Recently, it was demonstrated that the information required for accurate membrane
localization is contained within the CAAX region [181], which also dictates how Ras
proteins traffic to their destinations. Whereas H- and N-Ras traffic to the PM along the
secretory pathway through the ER then Golgi complex, K-Ras4B is directly routed from
the ER to the plasma membrane [178, 182]. The presence of H-Ras in ER/golgi
subcellular locations seems not to be a transient event associated with the transport and/or
recycling of H-Ras proteins to and from the PM; instead, a pool of H-Ras appears to
permanently reside in these organelles. These observations have led investigators to ask

whether ER/golgi associated H-Ras undergoes GTP-GDP exchange and subsequent

91

MAPK pathway signaling. Using live cell imaging utilizing a probe consisting of Raf-1 ’s
Ras binding domain fused to green fluorescent protein (RBD-GF P), Chiu et al.
demonstrated that following EGF stimulation activated Ras is present at the plasma
membrane, ER and the Golgi [183]. The studies describing the kinetics/mechanisms of
Ras activation within these subcellular compartments have expanded our understanding
regarding the complexities of RaszERK activation. They allow us to consider how other
membrane-associated proteins, such as R3, could influence ERK signaling in locales

other than the PM. (Figure 23)

 

Most secreted, and many membrane proteins contain cleavable N-terrninal signal l.
sequences that coordinate their targeting to and translation across the ER [207, 208].
Signal sequences are usually located at the extreme N-terminus of these proteins and are
typically 20-30 amino acids in length. Not surprisingly, all three RAMP isoforms contain
signal peptide sequences at their N-termini. R3’s putative ERK D-Domain is located

within its signal peptide region.

Traditionally, signal sequences were thought to be simple, interchangeable
domains that played a somewhat passive role in protein processing. Although all signal
sequences share a recognizable three-domain structure consisting of a basic “N” domain,
followed by a 7-13 residue hydrophobic “H” domain, terminating with a slightly polar
“C” domain [209], overall, prokaryotic, as well as eukaryotic signal sequences lack any
significant homology.[207]. Recently, several broad-ranging studies, assessing a variety
of functions and experimental systems, have disputed the “passive” signal sequence

paradigm. Indeed, the diversity of signal sequences within membrane and secretory

92

proteins can effect their targeting pathways, translocon interaction, cleavage, and post-

cleavage processing.

The sequence is first recognized by the signal sequence recognition particle
(SRP). SRP interacts with the ribosome-nascent chain in the ribosome, slows translation,
and targets the chain to the SRP receptor located on the ER membrane. Following GTP
hydrolysis, the ribosome engages with the translocon. SRP is then released from the
complex, and translation resumes. The amino acid residues within the hydrophobic
region of the signal sequence have been shown to orchestrate the interaction with Sec61
and the efficiency, and thus kinetics, of the translocation and translation process [210].
Signal sequences are usually released from the precursor protein by signal peptidase (SP)
during passage of the growing polypeptide chain through the ER membrane, but the
details/kinetics of this step are not well characterized. For example, while the signal
peptide sequences of most precursor proteins are cleaved rapidly (ass soon as the C-
region enters the ER lumen), the signal sequences of HIV-1 and feline immunodeficiency
virus are cleaved very slowly (very late in their protein maturation process) [211, 212].
Signal peptides are thought to span the ER membrane at their hydrophobic region, with
the N terminus facing the cytosol and the C terminus exposed toward the ER lumen [213-
216]. Following liberation by SP, many, but not all, of these peptides are processed
further by signal peptide peptidase (SPP) within the ER membrane, resulting in fragments
that are released into the cytosol [217]. Cleavage by SPP is mediated by helix-breaking
residues, such as proline(s) located in the membrane spanning region of the peptide

[216].(Figure 24)

93

 

Conventionally, it was assumed that signal peptides were degraded rapidly
following cleavage by SPP, and therefore had no other cellular function. However, SPP is
found in the genomes of plants and animals, but not in fungi or bacteria, both of which
process proteins with signal sequence, suggesting the possibility of additional roles.
Recent studies have also demonstrated that liberated peptides can have additional cellular
functions. One such example is SPP cleavage of Fas ligand (F asL). F asL binding to Fas f"
receptor triggers the apoptosis that plays a pivotal role in the maintenance of immune
system homeostasis. SPP cleavage liberates a small, unstable peptide fragment. This

fragment translocates to the nucleus and is capable of inhibiting gene transcription [218].

 

Another example indicating a post-targeting function of SPP- liberated peptides is
observed in the generation of human lymphocyte antigen (HLA)-E epitopes in humans.
HLA-E epitopes are derived from major histocompatability complex (MHC) class I
molecules. During biosynthesis of MHC class I molecules, the signal sequence is first
cleaved by SP, then SPP, and a nine-residue N-terminal fragment is released into the
cytosol, loaded onto the HLA-E molecule, and transported to the cell surface for
presentation to natural killer cells. HLA-E expression on the cell surface of presenting
cells results in a decreased natural killer response [215] In addition, liberated signal
peptides have been shown to interact with cytosolic signal transduction molecules. Site-
specific photo-crosslinking studies have followed the fate of the signal sequence of
preprolactin in a cell-free system. The preprolactin SP cleaved signal sequence is
processed by SPP and an N-terminal fragment is released into the cytosol. This signal
peptide fragment was found to interact efficiently with Ca+2/calmodulin [219, 220].

Similar to preprolactin, a signal peptide fragment of the HIV-1 envelope protein p-gpl60

94

is cleaved by SPP, released into the cytosol, and subsequently binds to calmodulin
through interactions within its signal peptide [221]. A cytosolic form of the ER
chaperone calreticulin was found to arise by an aborted translocation mechanism
dependent on its signal sequence. In this study, in addition to its well-established ER
functions, a portion of calreticulin influenced glucocorticoid receptor-mediated gene
activation in the cytosol.[222]. Taken together, these studies suggest bioactive roles for
liberated signal peptides in transcriptional regulation, cell surface presentation, and
interaction with various cytosolic cell-signaling mediators.

Although the signal peptide of R3 contains a putative SP cleavage site at amino
acid 23, no study to date has determined if it is indeed cleaved. We hypothesized that if
the signal peptide is cleaved, R3:ERK membrane interaction occurs at the ER. If, it is not
cleaved during protein processing, R3:ERK membrane interaction may occur in another
subcellular compartment. The purpose of this study is to determine if the signal sequence
of R3 is cleaved at the ER, and to identify the subcellular location of R3;ERK interaction.

5.2. Materials and Methods
5.2.1. Materials

NRK and HEK 293T cells were obtained from ATCC. DMEM,
penicillin/streptomycin, and fetal bovine serum were purchased from Invitrogen
(Carlsbad, CA). Adrenomedullin was purchased from Bachem Bioscience, Inc. (King of
Prussia, PA). CAMP level was measured using the Biotrak cAMP Enzyme Immunoassay
System from Amersham Biosciences (Piscataway, NJ). Fugene 6 transfection reagent was
obtained from Roche (N utley,NJ). Anti EEA, Calnexin, and gm230 antibodies were from

BD Biosciences (San Jose, CA). Anti-R3 and anti-pERK antibodies were from Santa

95

 

Cruz Biotechnology (Santa Cruz, CA). Chemiluminescence reagents were from Pierce
(Rockford, IL). pCMVTag2 vector and DHSa competent cells were from Stratagene (La
Jolla, CA). Anti-Flag antibody and optiprep reagent was purchased from Sigma Aldrich I
(St.Louis,MO). All other reagents were of the highest quality available.

5.2.2. Cell Culture

HEK 293 cells were maintained in Dulbecco's modified Eagle's medium low F.
glucose medium containing 10% fetal bovine serum, and 1% penicillin-streptomycin.
NRK were cultured using Dulbecco's modified Eagle's medium supplemented with 4 mM

L-glutamine, 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 5% bovine calf serum, 5% :-

 

Non-Essential Amino Acids, and 1% Penicillin/Strepomycin.
5.2.3. R3 Cloning and Expression

Full length cDNA of human R3 was cloned into a pCMV-Tag2(flag) vector using the
restriction cut sites EcoRl/Xhol as described previously. 5ug pCMV-Tag2R3 was
transfected into HEK 293 and NRK cells using F ugene 6 as per manufacturer’s
directions.

5.2.4. Desensitization Assays

HEK 293 cells were transiently transfeCted with CRLR and either PCDNAR3 or
Tag2(flag)R3 DNA using F ugene 6 as per manufacturer’s directions. 48 h after
transfection, HEK293 were seeded on a 24-well plate until reaching 75-80% confluency
and then incubated in serum-free medium overnight before the experiment." Cells were
pretreated with or without 10 nM AM in Dulbecco's modified Eagle's medium containing
0.2% bovine serum albumin for one hour. After agonist exposure, cells were washed three

times with Dulbecco's phosphate—buffered saline. Cells were then re-challenged with AM

96

 

(100 nM) for 10 min at 37 °C. and frozen at -80°C for cAMP accumulation assays.
Determination of cAMP level was measured using the Biotrak cAMP enzyme
immunoassay system according to the manufacturer's instructions. cAMP levels in RMCs
were calculated using a standard curve ranging from 10 to 104 frnol of cAMP. Each
experiment was done in triplicate and repeated at least three times. Data is expressed as

the percentage of maximal response, percentage of forskolin.

5.2.5. Optiprep Protocol for the subcellular fractionation of plasma membrane,
endosomes, golgi, and ER

 

HEK 293 cells were transfected with 5ug pCMVTag2R3 using Fugene 6 as per l
manufacturer’s directions. 24 hours later, the cells were plated on 150mm tissue culture l
plates. The following day, the cells were serum-starved overnight. Cells were treated for
10 minutes with 50ng/mL PDGF-BB then snap frozen at -80°C. The Optiprep protocol
was adapted from Puglielli [223]. Briefly, cells were scraped in a small volume of buffer
containing .25M sucrose, l40mM NaCl, lmM EDTA, 20mM Tris-HCI, lmM PMSF,
0.2mM sodium orthovaadate, 100nM NaF, 5ug/mL leupeptin, and lOug/mL aprotinin.
and homogenized using 20 strokes in a Dounce homogenizer. The homogenates were
then spun at 800g for 5 minutes at 4°C to pellet debris and the nuclear pellet. The PNS
was used for subsequent centrifugation. A continuous 8-34% iodixanol gradient was
prepared in 13 mL tubes using a gradient mixer. 1mL of the PNS was carefully loaded
onto the gradient and samples were spun at 4°C for 18 hours at 100,000g in a Sorvall
ultracentrifuge. .3 5mL fractions were carefully removed from the meniscus, precipitated
with cold 10% trichloroacetic acid, spun, and washed with cold acetone. Fractions were

resuspended in 4X protein loading dye. Proteins were separated by electrophoresis

97

through a 4-20% SDS- polyacrylamide gel. Western blotting was performed with anti-
pERK, anti-R3, anti-Flag, anti-EBA, anti gm-230 and anti-calnexin antibodies. Bands
were visualized using chemiluminescence (Pierce Supersignal West Pico) on XO-Mat

film.

5.2.6. Confocal Microscopy

HEK 293 cells were cultured on coverslips then permeablized 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 (mouse anti EEA, Calnexin, or
gm230 at 1:200), or rabbit/mouse anti-Flag (at 1:300), or rabbit anti pERK (at 1:300) in
blocking buffer for 2h at room temperature. Appropriate secondary antibodies were
applied for 1h at room temperature (anti-rabbit Cy3 at 1:400, anti mouse Alexa 488 at
1:500). Coverslips were mounted in Shandon mounting medium. Cells were visualized
on an Olympus laser confocal microscope. Images presented are representative single
optical sections from at least 20 fields imaged from at least three experiments.
5.2.7. Co-Immunoprecipitation experiments
HEK293 were transfected with pCMVTag2R3 (or vector control) as described above and
serum-starved overnight. The following day, cells were treated for 15 minutes with
50ng/mL PDGF-BB and snap-frozen. Cells were lysed in a buffer containing 50mM
HEPES, 150mMNaCI, 1.5mM MgClz, lmM EGTA, 10% glycerol, and 1% Nonidet P-40
and spun for 15 minutes at 4°C at 1200xG. Supematants were then spun at 4°C for 1 hour
at 100,000xG to obtain cytosolic and membrane fractions. Cytosolic fractions were
concentrated by spinning through a 5,000MWCO filter to a volume of 600ul. Pellets were

re-suspended in a volume of 600u1 lysis buffer. Both cytosolic and membrane fractions

98

 

were pre-cleared by the addition of washed protein A sepharose beads and subsequent
rocking at 4°C for 1 hour. The protein A sepharose was pelleted by centrifugation at
14,000xG for 1 minute. Supematants were divided in half; and either lug polyclonal anti-
F lag antibody or polyclonal anti-ERK antibody was added to the tubes. These samples
were rocked for 1.5 hours at 4°C, and then 40ul washed protein A sephaorse was added

to each tube. The cell extract/antibody/protein A sepharose samples were rocked at 4°C

fl...
for 2 hours, spun, and washed three times in lysis buffer. Samples were eluted in SDS l
loading buffer and resolved on a 10% SDS-polyacrylamide gel and transferred to a

nitrocellulose membrane. Blots were probed with monoclonal anti-pERK or rabbit 2
polyclonal R3 antibodies. _L.

 

5.2.8. Statistics

Data are presented as mean :1: S.E.M. Multiple group comparisons were made
using analysis of variance (ANOVA), followed by Bonferoni’s multiple comparison
between treatments. Two treatment comparisons were compared using Student’s t-test
method. Statistical significance was set at P<0.05.
5.3. Results

5.3.1. Desensitization Assays following N-terminal Flag addition

In order to establish whether or not the putative signal peptide sequence of R3 is
indeed cleaved during protein processing, full-length human R3 cDNA was cloned into a
pCMVTag2 vector containing a flag tag at the N-terminus of the multiple cloning site.
Studies were undertaken to access any alterations in AM signaling that may arise due to
addition of the N-terminal flag tag by comparing the normal desensitization process in

PCDNAR3/PCDNACRLR and pCMVTag2R3/PCDNACRLR transfected HEK 293

99

cells. For these assays, transfected cells are exposed to lOnM AM (pretreat) for one hour,
washed, then re-challenged with 100nM AM for 15 minutes. cAMP levels were
measured to assess desensitization of the AM-l R. No significant difference was
observed between PCDNAR3 and Tag2R3 transfected HEK 293 cells, indicating that the
receptor is able to desensitize normally following AM exposure (Figure 25).

5.3.2. Confocal Microscopy to detect Co-localization of R3 in pCMVTag2R3

 

transfected HEK293 In
For these experiments, EEA, gm230, and calnexin were used to identify ;

endosomal, golgi, and ER compartments, respectively. Confocal microscopy experiments

in pCMVTag2R3-transfected HEK293 cells demonstrated the colocalization of Flag with :

calnexin at the ER, but not golgi (gm230), or endosomal (EEA) compartments
(Figure 26).
5.3.3. Subcellular fractionation to identify R3 signal peptide cleavage

For these studies, the N-terminal flag epitope tag of pCMVTag2R3 was utilized to
monitor the signal peptide fate of R3. Anti-flag antibody, as well as an antibody that
recognizes the C-terminal region of R3, were utilized to identify the cellular location of
N- and C-termini. A continuous 8-34% iodixanol gradient was used to separate plasma
membrane/endosomes, golgi, and ER membranes. Antibodies to EEA, gm230, and
calnexin were used to identify endosomal, golgi, and ER membrane compartments. The
N-terminal flag tag was observed only in ER membrane fractions, while an antibody
detecting the C-terminus of R3 demonstrated that it localized to ER, golgi, and plasma
membrane/endosomal compartments. In serum-starved conditions, pERK was not

detected in any membrane compartment (Figure 27).

100

 

5.3.4. Subcellular fractionation to identify pERK locale following PDGF-BB
stimulation in HEK293

First, the presence of PDGF-BR in HEK293 cells was confirmed (Figure 28 upper
panel). HEK 293 cells were transfected with pCMVTag2R3 to identify the cellular
locations of N- and C-terminal R3 and pERK, and lysate were fractionated using a
continuous 8-34% iodixanol gradient following PDGF-BB stimulation. pERK localized

to ER and plasma membrane/endosomal fractions. R3 was detected in ER, golgi, and

“in?
1.

plasma membrane/endosomal fractions under PDGF-BB—stimulated conditions, and the
Flag epitope localized in ER membrane fractions (Figure 28 lower panel).

5.3.5. Confocal Microscopy identifying colocalization of Flag epitope and pERK in _
HEK293 cells following PDGF-BBtreatment ....

 

For these experiments, a rabbit anti-F lag antibody and a mouse anti-calnexin
antibody were used to identify the presence or absence of R3 at the ER compartment.
Confocal microscopy demonstrated the colocalization of Flag with calnexin at the ER
following PDGF-BB treatment (Figure 29).

5.3.6. Co-Immunoprecipitation of R3 and pERK

Antibody irnmunoprecipitation of both membrane (100,000Gpellet) and cytosolic
(100,000G supernatant) fractions of pCMVTag2R3 transfected HEK 293 cells using
polyclonal anti-Flag antibody demonstrated co-immunoprecipitation of activated ERK
under serum-starved and PDGF-BB treated conditions. The Flag antibody co-
immunoprecipitation of activated ERK following PDGF-BB treatment was significantly
increased in both cytosolic and membrane fractions. Co-immunoprecipitation of R3 with
the Flag antibody occurred only in the membrane fractions of both serum-starved and

PDGF-BB treated samples. Polyclonal ERK-2 antibody co-immunoprecipitation of

101

activated ERK occurred in both membrane and cytosolic fractions, but was significantly
increased in cytosolic fractions following PDGF-BB treatment. Ployclonal ERK antibody
co-immunoprecipitation of R3 occurred only in the membrane fraction following PDGF-

BB treatment (Figure30).

‘ ......”

 

102

Extracellular

  

Intracellular "iii Plasma Membrane

 

Degradation

Adapted from: TIBS 31(11): 631-38 ref [104]

Figure 23. Potential Subcellular locations for R3:ERK interaction. Potential R3:ERK

membrane interaction sites include the ER, golgi, PM, and endosomes.

103

 

 

Signal Peptidase

Adapted from Biochemical Society Transactions 31(6): p1243-6 ref [224]
Figure 24. Signal peptide cleavage and processing. A newly synthesized protein
containing a signal peptide is targeted to the ER membrane by the ribosome and i N’
translocated into the ER lumen. For most secretory and transmembrane proteins, signal
peptidase then cleaves the signal peptide from the precursor protein. The amino acid
residues within the signal sequence determine the exact timing and efficiency of signal
peptidase activity. The signal peptide spans the ER membrane with the N-terminus facing
the cytosol and the C-terminus within the ER lumen. For some, but not all proteins,
signal peptide peptidase then cleaves he signal peptide within its hydrophobic region
(shown as a barrel shape) to release the signal peptide into the cytoplasm. The liberated
peptide can then undergo degradation, or participate in other cellular functions. These
functions include cytosolic Can/calmodulin binding and glucocorticoid receptor-

mediated gene activation, as well as cytosolic/nuclear shuttling.

104

 

DPCDNA R3+CRLR
- Tag2R3(Flag tag)+CRLR

‘e‘

0)
O
l

cAMP (mmollmg protein)
[°/o Forskolln]
A N
s e
at

 

 

 

 

No Pretreat AM Pretreat

Figure 25. Comparison of PCDNAR3 and pCMVTag2R3(Flag-tag) transient transfection
on AMlR desensitization. HEK 293 cells were transiently transfected with CRLR and
either PCDNAR3 or Tag2(flag)R3 DNA using Fugene 6 as per manufacturer’s directions.
Cells were plated on 24-well plates, serum starved overnight, then treated for one hour
with 10nM AM (pretreat). Cells were then washed repeatedly and assayed for cAMP
accumulation to assess receptor desensitization by being re-challenged with 100nM AM
for 15 minutes then snap frozen. cAMP levels were measured using the Biotrak cAMP
enzyme immunoassay system (Amersham Biosciences) according to manufacturer’s
instructions. Cellular cAMP levels in the transfected HEK293 cells were calculated from
a standard curve ranging from 10 to 104 fmol of cAMP. Each cAMP measurement was
done in triplicate. Data is expressed as percent maximal response, % forskolin. *p50.05;
n=3. No significant difference was observed between PCDNAR3 and Tag2(Flag)R3

transfected HEK 293 cells.

105

 

rFlag mEEA Overlay

 

Overlay

 

rFlag mCalnexin Overlay

Figure 26. Confocal Microscopy identifying subcellular location of Flag epitope
expression in HEK293 cells. Tag2(flag)R3 DNA transfection and confocal microscopy
were performed as described in Methods. Images shown are representative of at least 20

fields imaged from at least three experiments. Bar scales on all images represent 20pm.

106

 

 

. “‘-" f .- _‘ -‘.”‘"- .
twlguun-o an -
" D... n. t '—_-I-'——" .I .~ I- + rmw‘mfim; trumr— I'm‘ ‘ w-‘I ‘ V ‘- .

 

g... _,>_, , _ . ,. Till“; - "\.""v'n w“. “A _ uw‘ 5ka ._. . R
an r 3 "' ‘ ,- . . . _ __. gm230

 

 

i """ “‘7'“? Cal
3" - _-.. "Le-11"?!” Flag

1 28 Fraction #

Figure 27. Optiprep Protocol for the subcellular fractionation of plasma membrane,
endosomes, golgi, and ER. HEK 293 cells were transfected with 5ug pCMVTag2R3
using Fugene 6 as per manufacturer’s directions. 24 hours later, the cells were plated on
150mm tissue culture plates. The following day, the cells were serum-starved ovemight.
The Optiprep protocol was adapted from Puglielli [223]. The PNS was used for Optiprep
centrifugation. A continuous 8-34% iodixanol gradient was prepared in 13 mL tubes
using a gradient mixer. 1mL of the PNS was carefully loaded onto the gradient and
samples were spun at 4°C for 18 hours at 100,000xG. .3 5mL fractions were carefully
removed from the meniscus, precipitated with trichloroacetic acid and washed with
acetone. Proteins were separated by electrophoresis through a polyacrylamide gel.
Western blotting was performed with anti-pERK, anti-R3, anti-Flag, anti-EEA, anti gm-
230 and anti-calnexin antibodies. EEA antibody was used as an endosomal marker, gm
230 as a golgi marker, and Calnexin as an ER marker. Blot is representative of 4

experiments.

107

 

293 RMC
f5 «uh-‘6 -- ' ' «- EEA
E. ’ L 3’ - - "It-o"... 5.3-al.? Cal
WT " ”“ _ i " 7 gm230
:5 .- m’r "' ": ...- ...-a .... .. pERK F2.
5 i.
W- 5:. Flag :
Le.‘ - .o'-'~ :21? ..- -.. ._ 5353M“: R3
1 28 Fraction#
Figure 28 Upper Panel. Western blot for PDGF BR expression in HEK293 cells.

 

l!‘

Lower Panel. Subcellular fractionation using Optiprep gradient following PDGF-

BB treatment. HEK 293 cells were transfected with 5ug pCMVTag2(Flag)R3 using
Fugene 6 as per manufacturer’s directions. 24 hours later, the cells were plated on
150mm tissue culture plates. The following day, the cells were serum-starved overnight.
Cells were treated for 10 minutes with 50ng/mL PDGF—BB then snap frozen at -80°C. The
Optiprep protocol was adapted from Puglielli [223] as described above. Western blotting
was performed with anti-pERK, anti-R3, anti-Flag, anti-EEA, anti gin-230 and anti-

calnexin antibodies. Blot is representative of 4 experiments.

108

 

rFlag mpERK Overlay

_ I
II. No PDGF-B's

 

       

 

rFla mCalnexin Overla

rpERK mCalnexin ' Ovrlay
PDGF-BB

rFlag mpERK Overlay
PDGF-BB

 

Figure 29. Confocal Microscopy identifying colocalization of Flag epitope and pERK in
HEK293 cells following PDGF-BB treatment, Confocal microscopy was performed as
described in Methods. Images shown are representative of at least 20 fields imaged from

at least three experiments. Bar scales on all images represent 50pm.

109

Transfection pCMVTag2R3 Vector

PDGF-BB + + — — — —

   

Fraction M C M C M C 7 IP: poly Flag
.... WB: pERK
E 5.-.: "" WB:R3
Transfection pCMVTag2R3 Vector
PDGF-BB + + — — — -
Fraction M C M C M C 7 lP:poly ERK
:1: l’ __ =23 . ‘ WB: pERK
"" WB: R3

7

Figure 30. Co-immunoprecipitation of R3 and pERK. Co-immunoprecipitation
experiments were performed as described in Materials and Methods. P=pellet from
100,000G spin, S=supematant from 100,000G spin. Vector=pCMV transfected,
Lane7=IgG + Protein A Sepharaose negative control. Blots are one representative of

three experiments.

110

 

Working Model for R3:ERK in RMC

 

Nucleus

Figure 31. Working model depicting N-terminal R3szRK cellular location. Following
PDGF-BB stimulation results in autophosphorylation of the PDGF-BR, and the
recruitment of Grb/SOS. Ras switches to it’s GTP-bound state, resulting in raf activation.
Rafthen phosphorylates MEK, which in turn phosphorylates ERK. A portion of the
activated ERK then travels to the ER where it binds to the ERKD-Domain of R3, which
is located within its signal peptide. This signal peptide is cleaved by SP, then SPP, and
the peptide is released into the cytoplasm. The activated ERK may be released fi'om the
signal peptide (or remain attached), and translocate to the nucleus, or the cytoplasm to

phosphorylate specific nuclear/cytosolic substrates.

111

5.4. Discussion

In mammals, approximately one third of all proteins reside in, or transition
through the secretory pathway. The ER chaperone calreticulum (CRT) was first
identified as a cytosolic integrin binding protein [225, 226], a nuclear export factor [227],
as well a p21 translation regulator [228]. In each of these studies, functional and physical
interactions have been demonstrated in the cytosol. Another ER resident protein,
glucosidase II [3 subunit, was originally described as a cytosolic protein kinase C target
[229] that modulates calcium homeostasis by binding to the cytosolic tail of the TRPS
channel [230]. Recently, it has been shown to interact with Grb3 [231], serving a shuttle
between the cytosol and nucleus. Some class I transmembrane signal peptides display
novel subcellular localizations. They include transforming growth factor-[3 [232],
cytochrome p450 [233], and Slit3 [234] (all mitochondrial), as well as IL-15 [235] and
cathepsin L [236] (both cytoplasmic). Many studies have incidentally observed
unexpected cellular locations for signal sequence-containing proteins presumably
intended for the secretory pathway. Levine, et al. suggested that for many secretory
pathway proteins, a biologically significant portion of the total protein can have access to
the cytosol [23 7]. Clearly, many signal peptide-containing proteins, or portions of them,
exhibit physiological functions other than their documented ER roles. In each instance,
the diversity within the signal peptide sequences of these proteins dictated the efficiency
of SP/SPP cleavage, as well as the final fate of the peptide and/or protein.

To that end, the results of this study demonstrate that the signal peptide is indeed
cleaved from mature R3 at the ER, as indicated by confocal microscopy identifying

colocalization of the N-terminal Flag epitope and the C-terminal portion of R3 at the ER,

112

 

but not at other subcellular compartments. Data from the subcellular fractionation studies
suggest that under serum-arrested conditions, a portion of R3’s N-terminus remains in an
ER fraction, as indicated by Flag epitope detection in this compartment. In addition,
while no membrane-associated pERK was detected under serum-arrested conditions, a
small portion of pERK was detected at the ER membrane following PDGF-

BB treatment. Confocal microscopy indicated that the Flag epitope colocalized with

 

R
pERK at the ER. Co-immunoprecipitation experiments identified interaction between R3 1
and pERK in a supernatant fraction from a 100,000G spin, which probably represents a :
cytosolic cellular fraction. This interaction was increased following PDGF-BB treatment.
R3 was detected only in the 100,000G pellet fraction (representing membrane) when co- :

immunoprecipitated with the N-terminal Flag epitope. Further experiments are warranted
to determine the fate of R3’s signal peptide. Taken together, these results suggest a novel

role for R3 in ERK-mediated proliferation in NRK and RMC (Figure 31).

113

6. The effects of FGF-2 treatment on membrane R3 expression in RMC

6.1. Introduction

 

Several laboratories have published RAMP gene expression profiles for many cell
lines and tissues. Many studies have reported variable RAMP gene expression in diverse
disease models, physiological states, and following certain drug treatments. R3
expression was specifically upregulated in several growth-associated cell culture and F
disease models. This upregulation did not correlate with an increase in AMlR 4‘
components, namely CRLR and R2.

RMC express all components of the AM signaling system; R1, R2, R3, and

 

CRLR. Published reports from our laboratory have demonstrated specific membrane-
associated R3 upregulation in RMC following PDGF-BB treatment. The physiological
consequences of R3 upregulation were described in the context of the AM signaling
system. The AM-independent cellular effects of increased R3 expression were not
investigated. The focus of the present studies has focused on R3-mediated cellular
actions outside the usual paradigm of the AM system.

PDGF-BB and FGF-2 are important regulators of mesangial cell proliferation and
matrix deposition following glomerular injury. Experiments within this document have
demonstrated that the FGF-2-induced proliferation observed in RMC is primarily ERK-
mediated. Furthermore, R3 introduction into the R3-deficient cell line, NRK, increased
membrane-associated ERK activation and subsequently also increased proliferation.
Does FGF-2 treatment of RMC also preferentially increase R3 expression? Studies were
undertaken to assess the effect of FGF-2 treatment on membrane-associated R3

expression in RMC.

114

6.2. Materials and Methods
6.2.]. Materials

RMC were obtained from ATCC. RPMIl 640, penicillin/streptomycin, and fetal
bovine serum were purchased from Invitrogen (Carlsbad, CA). F GF -2 was obtained from
Oncogene (Cambridge, MA). R2 and R3 antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA) and chemiluminescence reagents from Pierce
(Rockford, IL). SDS-polyacrylamide gels were from Bio-Rad Laboratories (Hercules,
CA). All other reagents were of the highest quality available.

6.2.2. Cell Culture

 

RMC were maintained in RMPI 1640 media containing 15% FBS and 1% .1—
penicillin-streptomycin.
6.2.3. Membrane expression of R3 following FGF-2 treatment in RMC

RMC were plated on 150mm tissue culture dishes, then serum-starved overnight.
The following day the cells were treated with 0, 1, 5, and lOng/mL FGF-2 for 24 hours.
The media was removed and the plates were snap-frozen. The cells were then scraped
from the plates in homogenization buffer (lOmMHepes, 0.15MnaCl, 1mM EDTA, 1mM
PMSF, 0.2mM sodium orthovanadate, 100nM NaF, 5ug/mL leupeptin, and lOug/mL
aprotinin). This lysate was dounce-homogenized for 20 strokes. The homogenates were
centrifuged at 1300xg for 20 minutes at 4°C to pellet nuclear and cell debris. The
supernatant was centrifuged at 100,000xg at 4°C for 1 hour to isolate the membrane
fraction. Protein concentration was determined by the Bradford assay. For each time
point, 40ug membrane fraction protein was separated on a 10% SDS-polyacrylamide gel.

Western blotting was performed with anti-R2 or R3 antibody (Santa Cruz) to assess

115

RAMP protein levels. Bands were visualized using chemiluminescence (Pierce
Supersignal West Pico) on XO-Mat film. Blots were stripped and re-probed for actin for
protein normalization.
6.2.4. Statistics

Data are presented as mean i S.E.M. Multiple group comparisons were made
using analysis of variance (AN OVA), followed by Bonferoni’s multiple comparison
between treatments. Two treatment comparisons were compared using Student’s t-test
method. Statistical significance was set at P<0.05.
6.3. Results
6.3.1. Membrane expression of R3 following FGF-2 treatment in RMC

24-hour FGF-2 treatment of RMC resulted in a concentration-dependent increase
in membrane-associated R3 expression (Figure 32). Membrane-associated R3 expression
increased (2.8: 33 fold basal) following 24-hour 5ug/mL FGF-2 treatment (Figure 33).
Membrane-associated R2 expression was not statistically different from basal levels

following 24-hour 5ng/mL FGF-2 treatment.

116

 

 

“...—......- ‘M W W” ' R3

Figure 32. RAMP membrane expression in RMC following FGF-2 treatment.

RMC were plated on 150mm tissue culture dishes, serum-starved, and treated with 0,1,5,
and 10ng/mL FGF-2 for 24 hours. Membrane fi'actions were prepared as described in
Methods. Western blotting was performed with anti R2 antibody. Blots were stripped

and re-probed for R3 and actin. Blots are representative of four experiments.

117

Fold Basal

 

 

Basal R2 R3

Figure 33. Membrane RAMP expression in RMC following 5ng/mLFGF-2 treatment.

Graph represents RAMP/Actin ratios of the experiments described in Figure XX

following 5ng/mL FGF-2 treatment. *pS0.05; n=4.

118

6.4. Discussion

Like PDGF-BB, F GF -2 treatment of RMC resulted in a specific up-regulation of
R3 in a cellular membrane component. It is intriguing that two potent mesangial cell
mitogens significantly increased R3, but not R2, expression. Clearly, ample evidence
exists for differential RAMP expression in cell lines, and in animal disease models. Since
AM exerts anti-proliferative responses in many cells, most of these studies, especially
those investigating renal pathologies, have associated these RAMP increases with AM-
mediated, anti-proliferative responses. The results observed herein suggest that the
specific upregulation of R3 may play a noteworthy role in non-AM mediated cellular

responses.

119

 

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 AM-independent R3
actions in ERK-mediated proliferation. It was hypothesized that R3 is a critical regulator
of renal mesangial cell proliferation following stimulation by PDGFBB, by direct
interaction with the MAPK cascade. Hence, as summarized below, the following specific

aims were addressed and hypotheses relating to these aims were tested:

Specific Aim #1: In RMC, PDGFBB-induced activation of the ERK signal transduction
cascade and subsequent proliferation requires R3.

Hypothesis 1: A significant portion of the PDGFBB-induced proliferative response in
RMC occurs through activation of ERK.

Hypothesis 2: The amino acid sequence of R3 contains a punitive ERK-Docking domain
that may allow for direct R3:ERK physical interaction.

Hypothesis 3: R3 is involved in ERK-mediated RMC proliferation following PDGF BB
treatment.

Hypothesis 4: R3 mediates PDGFBB-induced RMC proliferation by modulating ERK

activation.

Results:

a. The PDGF-BB-induced proliferation of RMC is almost exclusively ERK-mediated.
Similar findings were observed for F GF-2-induced proliferation of NRK.

b. A putative ERK D-Domain was identified using Scansite database software.

c. GST-tagged R2 protein does not interact with neither inactive nor active forms of
ERK-2 protein in vitro. GST-R3 showed a robust interaction with both inactive and

active forms of purified ERK-2 protein.

120

 

 

 

(1. Using two separate approaches, R3 knockdown completely abolished PDGF-BB-
induced proliferation in RMC.

e. Introducing R3 into a cell line that normally does not express it, NRK, resulted in a
significantly increased basal and FGF-2-induced proliferative response.

f. R3 knockdown resulted in a decreased membrane-associated ERK activation in

RMC. Introducing R3 into NRK significantly increased membrane-associated ERK

activation following FGF-2 treatment.

g. R3 does not mediate cytosolic ERK activation in RMC.

‘Pflh'JT— . _

Specific Aim #2: To identify the subcellular location of R3:ERK interaction.

Hypothesis 1: R3 interacts with ERK at a cellular membrane component.
Hypothesis 2: R3:ERK interaction occurs at an ERK D-Domain located within R3’s N-
terrninal signal sequence. This sequence is cleaved at the ER during R3 synthesis,

suggesting that R3:ERK interaction occurs in this subcellular compartment.

Results:
a. R3 and pERK colocalized at the ER in HEK293 cells following PDGF-
BB.treatment.

b. R3’s N—terminal signal sequence is cleaved at the ER.

Specific Aim #3: To determine the specific amino acid requirement of the R3 ERKDD in

PDGFBB-induced ERK activation and subsequent proliferation.

121

Hypothesis 1: Mutating specific amino acid residues within the R3 ERK D-Domain will

result in decreased R3:ERK protein binding in vitro.

Hypothesis 2: Mutating specific amino acid residues within the R3 ERK D-Domain will

decrease membrane-associated ERK activation following FGF-2 treatment in NRK:

Hypothesis 3: The ERK D-Domain mutations that result in decreased membrane-
associated ERK activation will also result in a decreased FGF-2 proliferative response in

NRK following FGF-2 treatment.

Results:

a. The extreme N-terminal arginine (positions7 and 8) residues, as well as the
adjacent praline (position 9) residue, of R3’s D-Domain are necessary for
R3:ERK protein interaction in vitro.

b. The7/8 arginines, and the adjacent proline residue of the ERK D-Domain mediate
ERK membrane-associated activation in NRK following F GF -2 treatment. These
residues also mediate the subsequent FGF-Z-induced proliferative response in
NRK.

c. The downstream D-Domain leucine (at position 11) does not mediate in vitro
R3:ERK protein interaction, membrane-associated ERK activation, or NRK

proliferation following FGF-2 treatment.

122

 

 

 

Specific Aim #4: To assess the effects of FGF-2 treatment on R3 expression.

Specific Hypothesis: F GF-2 treatment of RMC will result in an increased expression of

R3.

Results:

a. FGF-2 treatment of RMC results in a dose-dependent increase in R3 expression in
a membrane fraction.

...- .. ...... -m‘m; (Isis?!

b. 5ng/mL FGF-2 treatment of RMC increases basal R3 expression 2.8 fold..

r..- Inf-J

 

7.2. Limitations of the study

1

1. The primary limitation of this study was the use of cell culture systems
exclusively to demonstrate the role of R3 in ERK-mediated proliferation. Clearly,
cell culture systems were warranted for investigating detailed molecular
mechanisms. However, mesangial cells in vivo are in a dynamic environment, in
which endocrine/paracrine factors, as well as cell:cell interactions within the
glomerulus, ultimately contribute to their cellular responses.

2. All of the experiments in this study were performed using rat mesangial and
kidney fibroblasts, or human embryonic kidney cells. Although these cells have
been used extensively in the study of renal disease, the results obtained may not
necessarily reflect the responses and regulatory mechanisms in human cells. The
cell lines were chosen because the AM signaling system has been well
characterized in these cells, they are easily cultured and manipulated, resulting in

highly reproducible responses.

123

ERK activation, and the resulting proliferative response were elicited by the
exogenous administration of PDGF-BB and FGF-2. While concentrations used
were clearly within previously published ranges, it is difficult to assess the
concentrations that mesangial cells and renal fibroblasts are exposed to in vivo.
Although this preliminary study clearly demonstrates a role for R3 in ERK-
mediated proliferation, more work is warranted to unravel the mechanisms
involved in activated ERK translocation to the nucleus following its interaction
with R3 at the ER. To date, most studies describing signal peptide release into the
cytoplasm utilize complex, cell-free in vitro reticulocyte methodologies. Future
studies employing these systems could potentially provide additional mechanistic

information regardingR3zERK interaction at the ER as well as signal peptide fate.

7.3. Positive outcomes of the study

AM, the ligand for R3:CRLR, is known to exhibit potent anti-proliferative and

anti-migratory effects in RMC. However, prior to these studies, the extent of non-AM

mediated RAMP involvement in the anti-proliferative process has not been thoroughly

investigated. R3 expression has been shown to be upregulated in several growth-related

animal and cell culture models, even when CRLR (and R2) levels remain unchanged, or

even decrease. For example, in RMC, PDGF-B treatment results in an increased R3 (but

not R2) protein expression, and subsequent increases AM-mediated adenylate cyclase

activity result in an anti-proliferative response. But, in all published results, AM-

independent R3-mediated effects on proliferation were not investigated. Are all R3

ef‘fects on proliferation AM-mediated?

124

 

R2 and R3 exhibit great diversity within their amino acid sequences (they are only
30% homologous), yet both elicit an identical cellular response following agonist
exposure. Recent studies from our lab have demonstrated the importance of R3’s unique
C-terminal PDZ domain in post-endocytic trafficking of the R3:CRLR complex. In
addition, several recent studies have shown promiscuity in RAMP interactions. For
example, even though in a recent RAMP binding study, when co-expressed with
RAMPS, 6 out of 10 class B receptors demonstrated RAMP interaction [105] Recently
published results suggest RAMPs may also modulate GPCR coupling efficiency through
diverse cell signaling pathways. Unlike the interaction of RAMPs with CRLR, VPACIR
is expressed at the plasma membrane independently of RAMP presence. The classic
coupling pathway associated with VPAClR is a significant accumulation of cAMP with
little PI hydrolysis. However, the VPACleRZ heterodimer exhibits enhanced
phosphoinositide (PI) hydrolysis with no significant change in cAMP stimulation. So,
although R2 was not necessary to shuttle the VPAClR to the plasma membrane, it’s
interaction with this receptor altered the conventional signaling response. The scope of
RAMP-mediated GPCR regulation was further expanded by studies identifying a class C
calcium-sensing receptor, (CaSR), interaction with RAMP3 [106]. In a paradigm similar
to CRLR, recombinant CaSR expressed in COS7 cells does not translocate to the plasma
membrane unless R1 or R3 (but not R2) is also co-expressed. In the same study, R3 was
Shown to mediate both the glycosylation and trafficking of CaSR from the ER to Golgi.
In the absence of R3, CaSR was retained in an intracellular compartment that confocal

microscopy identified to be the ER. Clearly, all of these studies suggest that RAMPs

125

 

participate in many diverse cellular functions, well beyond their classical AM-associated
roles.

This study was the first to describe a novel, AM-independent role for R3 in ERK-
mediated proliferation. While ERK involvement in the proliferation of many cell types
has been extensively studied, other cell signaling cascades can contribute significantly to
this process. This study demonstrated that both PDGF-BB (in RMC) and F GF-2 (in NRK)
exert mitogenic actions primarily through ERK activation.

The extreme N-terminus of R3, but not R2, contains a putative ERK D-Domain that
is very similar to the ERKD-Domains of MEK] and 2. We hypothesized that R3 had the
potential to mediate ERK activity by interacting with ERK through its D-Domain. By utilizing
both R3 overexpression and knockdown methodologies, this study demonstrated that R3 is a
critical mediator of membrane-associated ERK activity. Specifically, using RMC, we employed
two separate technologies to knockdown endogenous R3 prior to PDGF-BB stimulation. Both of
these approaches resulted in significantly decreased PDGF-BB stimulated growth. To confirm
these results, R3 was introduced into a cell line that does not endogenously express it (N RK). R3
introduction into these cells resulted in an increased proliferation in both basal and FGF-2-
stimulated conditions.

Published reports have indicated the importance of basic arginine residues in
MEKzERK interaction. These residues are located at the extreme N-terminus of the ERK
D-Domain. These residues were also found to be important in R3 modulation of

membrane-associated ERK activity and proliferation following FGF-2 treatment of NRK.

A helix-breaking proline residue located just downstream of the arginines has also been

reported to be necessary for MEKzERK interaction. This was not observed for R3:ERK

126

 

interaction, membrane-associated ERK activation, or proliferation in NRK following
F GF -2 treatment.

As with almost all class I transmembrane proteins, the amino acid sequence of R3
contains a signal peptide that targets them ER and orchestrates their protein processing
there. Many, but not all, signal peptides are cleaved by SP at the ER. They can then be
processed ftu'ther within the ER membrane by SPP, resulting in the release of the peptide
into the cytosol. The actual mechanisms and kinetics involved in protein processing have
been thoroughly investigated for only a few transmembrane proteins. Even less is
understood regarding signal peptide release/fate in the cytoplasm. Initially, it was
assumed that signal peptides were degraded rapidly following cleavage by SPP, and
therefore had no other cellular function. However, recent studies have demonstrated that
liberated peptides can have additional cellular functions. One notable study
demonstrating novel signal peptide functions includes MHC class I molecules [215],
whose signal peptide interacts with the HLA-E molecule and transports it to the cell
surface. Other signal peptides, including those of preprolactin and HIV-lp-gpl60, have
been reported to modulate cytosolic Cawcalmodulin signaling [219-221, 224]. In
addition, it has been demonstrated that a small percentage of calreticulin’s signal peptide
pool influences glucocorticoid receptor-mediated gene activation in the cytosol.[222].

Thus, bioactive roles for liberated signal peptides have been demonstrated for
transcriptional regulation, cell surface presentation, and interaction with various cytosolic
Cell-signaling mediators.

Both confocal microscopy and differential centrifugation were employed to

establish the subcellular localization of R3:ERK interaction. First, confocal microscopy

127

 

revealed that the N-terminus of R3 is present only at the ER, while an antibody
recognizing the C-terminal region of R3 is observed at both the ER and plasma
membrane. This data suggests that the signal peptide of R3 is indeed cleaved from
mature R3 protein during synthesis. Secondly, differential centrifugation demonstrated
that the N-terminal region of R3, as well as activated ERK, were both present in an ER
membrane fraction following PDGF-BB treatment of pCMVTag2-R3 transfected HEK
293 cells. Confocal microscopy indicated their colocalization at the ER, but not golgi or
endosomes.

This thesis has identified a novel role for R3 in mediating ERK activity and

proliferation at a unique subcellular membrane compartment. Given the fact that, in

 

several models of progressive glomerular disease, loss of coordinated mesangial cell
proliferation, phenotypic changes and increased growth factor expression precede up-
regulation of genes for ECM and mesangial expansion. A better understanding of the
mechanisms involved in R3:ERK mediated mesangial proliferation may allow us to
develop novel anti-proliferative strategies for the treatment of glomerular disease.
7.4. Future studies
1. As discussed previously in this thesis, the use of animal models, such as R3
knockout mice, would greatly advance or understanding of the physiological role
R3 plays in proliferation. Caron, et al. recently generated R3 knockout mice. They
reported that mice lacking R3 exhibited a normal phenotype, other than decreased
body weight [238]. It would be interesting to induce glomerulonephritis (by
injection of the anti-mesangial cell antibody Thyl .1) in these animals. In control

mice, an acute phase of complement-dependent mesangial cell lysis, followed by

128

a phase of deregulated mesangial cell proliferation and altered matrix deposition
would ensue [14]. Would similar mesangial cell effects be observed in R3
knockout mice?

2. AM is known exhibit anti-proliferative effects in several cell types, including
mesangial cells, fibroblasts, and vascular smooth muscle cells. Conversely, in
keratinocytes, epithelial cells, and endothelial cells, AM exhibits robust
proliferative effects. Interestingly, in bone AM promotes osteoblast growth both
in vitro and in viva [239, 240]. It is intriguing that, although the physiologic
consequences were not investigated, PTH treatment of primary mouse osteoblast
cells results in the specific upregulation of R3 [131]. Using both cell clines and
animal models, AM has been demonstrated to be proliferative in several types of
carcinomas including breast, endometrial ovarian, melanoma, prostate, and
glioblastomas. Interestingly, administration of the uterine mitogens estrogen or
phytoestrogen to rats preferentially increased uterine R3 expression levels, and
actually significantly deceased CRLR levels [114, 127]. Hewitt et al [241]
recently found that R3, with a 43-fold increase, was one of the most potently
induced genes in the uterus of wild-type mice treated with estrogen. Other studies
have shown that regulatory region of the R3 gene contains an estrogen response
element [127].

Although RAMP expression, (and the physiological consequences of altered
RAMP expression) have not been extensively studied in cancer, some noteworthy
findings have recently been published. Dysregulation of the Wnt/ beta-catenin

signal transduction pathway has been extensively implicated in the pathogenesis

129

 

of mammary gland and colon tumors. Gene expression microarray analysis of
mouse mammary epithelial cells inducibly expressing a constitutively active
mutant of beta-catenin identified R3 as one of a handful of target genes of this
pathway [242]. In addition, R3 is expressed in the prostate cancer cell line,
DU145 [123], and in prostate carcinoma biopsies the levels of R3 were
significantly elevated (compared to prostate hyperplasia biopsies) while
expression levels of R2 were unchanged in these samples [123]. R3 knockdown
experiments (unpublished data) from our laboratory have suggested that R3
mediates EGF-stimulated proliferation of DU145 cells. Is it plausible that in
growth-associated pathologies, altered cellular levels of R3 can influence certain
cell types to preferentially proliferate, while not affecting others? Clearly, RAMP
expression profiles, with characterization of the ensuing physiological

consequences of altered expression, are needed to address these questions.

130

10.

ll.

12.

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