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dissertation entitled
A1 Adenosine Receptor Gene:
Characterization and Analysis of its Transcription
Start Site Heterogeneity
presented by

Samita Bhattacharya

has been accepted towards fulfillment
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Ph.D. Physiology

 

 

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A1 ADENOSINE RECEPTOR GENE :
CHARACTERIZATION AND ANALYSIS OF ITS TRANSCRIPTION START

SITE HETEROGENEITY

By

Samita Bhattacharya

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Physiology

1993

ABSTRACT
A1 ADENOSINE RECEPTOR GENE: CHARACTERIZATION AND
ANALYSIS OF ITS TRANSCRIPTION START SITE HETEROGENEITY
By

Samita Bhattacharya

The primary objective of the research presented in this thesis was to characterize the A1
adenosine receptor gene thereby expanding our understanding of the adenosine receptor-
signaling system. Adenosine receptors (AR) are broadly divided into two classes, A1 and
A2, based on their differential binding selectivity for a series of adenosine analogs and
on their ability to inhibit (AlAR) or stimulate (A2AR) the adenylyl cyclase system.
While the A2AR is only known to be coupled to the adenylyl cyclase system, the AlAR
has been reported to interact with multiple effector systems. Studies using the AlAR
cDNA and the gene will help in understanding the mechanism by which this complicated
system is activated, and its ability to interact with multiple effector systems. Recently
both Al and A2 receptors have been cloned and expressed from dog thyroid, rat brain,
bovine brain and human brain cDNA libraries. Using a radiolabelled oligonucleotide
probe designed from the dog thyroid AlAR clone (RDC7), a partial (2kb) cDNA clone
was isolated from a rabbit kidney cDNA library. This clone, designated SB4, lacks the
5’-untranslated region and nucleotides corresponding to those coding for the first 74
amino acids of RDC7. 834 has 91% identity at the nucleotide level with RDC7 and
codes for a protein with 94% identity to the canine AlAR. To obtain the remaining 5’-

end of the cDNA, a rabbit genomic library was screened using an 860 base probe, an

EcoRI/Smal fragment derived from the 5’-end of SB4 and an AlAR fragment generated
by PCR from rat brain mRNA (gift of Dr. M. Lohse). One positive clone was isolated
containing a 3.2 kb XhoI fragment, which included 2 kb of 5’-flanking region, an exon
of 341 nucleotides coding for the first 113 amino acids of the AlAR, followed by an
intron. The exon sequence of this clone was 100% identical to SB4. Together the cDNA
and the genomic clones provided the entire open reading frame for the rabbit AlAR
(RbAl). The cDNA for the rabbit AlAR was further used to isolate and characterize
rabbit genomic DNA clones containing the entire coding sequence for the AlAR. Results
of restriction analysis and sequencing of genomic clones were consistent with the
existence of a single AlAR receptor gene. The complete receptor coding sequence is
contained in 2 exons separated by an intron Of greater than or equal to 34 kb. Primer
extension analysis indicated that transcription of the gene begins at multiple initiation
sites, ~78, -106, -268 and -322 nucleotides 5’ to the ATG translation initiation codon.
Neither TATA nor CAAT boxes were found to be present in the 1000 nucleotides that
has been sequenced immediately upstream of the translational start site. Examination of
the 5’-end sequences of the gene revealed sequences with homology to several
transcriptional regulatory sequences. Knowledge of the structure of the receptor gene
should facilitate future studies on the structural determinants of the receptor function, the
regulation of the receptor expression in various physiological and pathophysiological

conditions and lastly the molecular basis of multiple AlAR functions.

To my parents
and

to the memory of my sister Santa.

iv

ACKNOWLEDGMENTS

During the years of my graduate life I have received enormous support from many
a people, which resulted in the completion of this work. First and foremost among these
is my mentor Dr. William S. Spielman. His guidance allowed me to think independently,
develop self-confidence and to acquire scientific insight.

I would like to express my deep appreciation to Dr. David L. Dewitt for
introducing me to the field of molecular biology with unsurpassed endurance and care,
and for assisting me in developing a critical eye for science. I hope I can some day be
as critical a thinker and as compassionate a person as him.

I would like to acknowledge my other committee members, Dr. Seth R. Hootman,
Dr. Donald B. Jump and Dr. William L. Smith for their suggestions leading to the
improvement of my thesis. I would specially like to thank Dr. William L. Smith for
giving me the opportunity to use his laboratory facilities.

Thanks to my friends and colleagues, Dr. Lois J. Arend and Dr. David L. Levier
for all their support. My special thanks for Dr. Maria Burnatowska—Hledin for all her
intellectual and technical support. Thanks to Linda Shi, Stacey Kramer, Odette Laneuville,
Marc Lccomte, Beth Meade, Jim Otto, and Marty Regier for all their help and more so
for making me feel a member of their lab. Special thanks for Stacey Kramer for her

stimulating discussions and her help with computer programs. Thanks to John Freeman

for all the encouragement. John will always be in my thoughts.

My deep appreciation to Susan Harkema, a good friend in need, for being a pillar
of strength and support which enabled me to ride over the hard times of graduate life.

I would like to thank the members in the Department of Physiology, specially
Sharon Shaft for her excellent secretarial assistance.

In the end I would like to convey my deepest gratitude to my family, especially
my parents, my sisters Tanushree, Jayashree and Santa, my uncle Ganesh Maitra and my
aunt Savitri Goswami. Their constant love and encouragement were the foundation of my

work.

vi

TABLE OF CONTENTS

List of Tables

List of Figures

1.

II.

III.

IV.

Vi.

Introduction

Literature Review
A. Adenosine metabolism
B. Adenosine receptors
C. Physiological effects of
adenosine receptor activation

Molecular Cloning of A1 Adenosine
Receptor cDNA From Rabbit

A. Introduction

B. Materials and Methods

C. Results

D. Discussion

Characterization of the A1 Adenosine Receptor Gene
and Analysis of Transcription Start Site Heterogeneity
A. Introduction
B. Materials and Methods
C. Results
D. Discussion

Summary and Conclusions

Literature Cited

vii

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19

26
26
27
33
54

56
56
58
62
89

94

97

Table I.

Table II.

Table III.

LIST OF TABLES

Responses coupled to A1 adenosine receptor

Functional effects of adenosine receptor activation

Southern blot analysis of the genomic clones

viii

flgu_rc
Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

LIST OF FIGURES

Adenosine metabolism

Model for adenosine receptors and their
interaction with adenylyl cyclase system

The alignment and comparisons of the predicted
amino acid sequences of the adenosine receptors
isolated from different species

Flow diagram depicting the strategy involved
in screening the cDNA library

Northern blot analysis of AlAR mRNA

Comparison of amino acid sequence of RDC7,
the canine AlAR with SB4

Schematics of the A—EMBLB cloning site of the
genomic library vector and the probes used to
screen the library

Southern blot analysis of the genomic clone-1

Sequence obtained from the 3.2 kb Xhol
fragment and its comparison with SB4

Schematic representation of the strategy involved in
generating a full length clone encoding the rabbit AlAR

Nucleotide and the deduced amino acid
sequence of the rabbit AlAR

Comparison of the deduced amino acid sequences
of the canine, rat, bovine, rabbit and human AlAR

Analysis of the rabbit AlAR gene by
Southern blot hybridization

ix

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Risers

Figure 14.

Figure 15.
Figure 16.
Figure 17.

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Figure 22.

LIST OF FIGURES, CONT"D

The different probes used for characterization
of the genomic clones

Southern blot analysis of the eight genomic clones
Southern blot analysis of the eight genomic clones
Schematic representation of the rabbit AlAR gene and cDNA

Southern blot hybridization analysis to determine
the size of intron-l of AlAR gene

The nucleotide and the amino acid sequence
of the rabbit AlAR gene

Primer extension analysis of the transcription
start site for the rabbit AlAR gene

Nucleotide sequence of the 5’-flanking
region of the rabbit AlAR gene

The locations of the exonfrntron splice sites of
G-protein linked receptors are compared

68

70

75

77

8O

83

86

I. INTRODUCTION

Adenosine is an ubiquitous purine nucleoside and a byproduct of both extracellular
and intracellular metabolism (Collison et al. 1987; Olsson et a1. 1990). Due to its short
half life (Conway and Cooke 1939), it is likely that adenosine acts in a paracrine or
autocrine manner and produces a wide array of physiological effects, including
hemodynamic changes, neurotransmitter release, platelet aggregation, lipolysis and renin
release and solute transport by the kidneys (McCoy et al. 1993; Lang et al. 1985;
Ramkumar et al. 1988; Linden 1991). The actions of adenosine are mediated through G-
protein coupled plasma membrane receptors.

Adenosine receptors (AR) are broadly classified as A1 or A2AR, based on their
affinities for a variety of adenosine analogs (Daly et al. 1987) and on their ability to
inhibit (AlAR) or stimulate (A2AR) the adenylyl cyclase system (Londos et al. 1980).
The second messenger systems coupled to AlAR, however, are remarkably diverse, and
include adenylyl cyclase, phospholipase C and various ion channels (Linden et al. 1991).
This diversity of responses generated by AlAR activation has been interpreted to indicate
that subtypes of AlAR may exist, either as products of differential splicing of a
multiexon gene or as products of entirely different adenosine receptor genes. A possibility

of a single receptor capable of interacting with multiple effector systems, also exists.

2

Characterization of the AlAR gene or genes is a necessary first step in distinguishing
between these possibilities.

The research presented in this dissertation describes the molecular cloning of the
cDNA and the characterization of the gene encoding the AlAR from rabbit. The first
chapter, the literature review is composed of three sections, which discuss the metabolism
of adenosine, the adenosine receptor system and lastly the physiological functions
involved with adenosine receptor activation. The projects comprising the thesis are
represented as two separate chapters, each comprising of an introduction, methods, results
and discussions relevant to each project. The final chapter summarizes the significance

of the research.

II. LITERATURE REVIEW

A . Adenosine Metabolism Adenosine, the ubiquitous purine nucleoside, is
produced intracellularly as well as extracellularly by two distinct metabolic pathways,
involving hydrolases. The first pathway involves the enzymatic hydrolysis by 5’-
nucleotidase of adenine nucleotides, (Baer et al. 1966 and Pearson et al. 1980) and the
second pathway involves the hydrolysis of S-adenosyl homocysteine (SAH) by SAH
hydrolase (Schrader 1981). Since the SAH hydroxylase is inhibited by its products,
adenosine and inosine, it is postulated to be a minor source of adenosine (Eloranta 1977;
Lloyd and Schrader 1987; Schatz et al. 1977). The major source of adenosine is therefore
believed to arise from the metabolism of adenine nucleotides (Figure 1).

Adenosine consists of an adenine purine ring and a ribose moiety and is a
precursor to, and a metabolite of, adenosine 5’-monophosphate (AMP), adenosine 5’-
diphosphate (ADP) and adenosine 5’-t1iphosphate (ATP). Adenosine is also a component
of S-adenosyl homocysteine.

Adenosine produced extracellularly or intracellularly, is also degraded by two
mechanisms: 1) deamination by adenosine deaminase to inosine or 2) phosphorylation
by adenosine kinase to AMP (Plagemann and Wohlhueter 1980). Adenosine deaminase
is present both intracellularly as well as in the interstitium (Conway and Cooke 1939;
Trams and Lauter 1974), whereas adenosine kinase is an intracellular enzyme. The
concentrations Of intracellular and extracellular adenosine are maintained by a balance

between its rate of production and degradation.

Figure 1 . Adenosine Metabolism : Flow diagram of adenosine sources and sinks.

 

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The movement of adenosine into and out of the cells is governed by the
mechanism of facilitated diffusion, a concentration dependent process. Dipyridamole and
related compounds act as inhibitors of this process via the nucleoside transporter. This
transporter has been well characterized in red blood cells (Kolassa and Pfleger 1975;
Kubler and Bretschneider 1963), aortic endothelial and smooth muscle cells (Pearson et
al. 1978), platelets (Sixma et 211.1976), and in hepatoma cells (Plagemann 1971;
Plagemann and Wohlhueter 1980; Plagemann and Richey 1974). The nucleoside
transporter is closely associated with the membrane bound 5’-nucleotidase, thereby
suggesting that the production and translocation of adenosine is a single step process (Fox

and Kelly 1978).

B . Adenosine receptors

Adenosine consists of a purine ring and a ribose moiety, and is a precursor to,
and a metabolite of adenine nucleotides. Under conditions of hypoxia, resulting in a state
of decreased cellular energy, the intracellular production of adenosine increases and
adenosine is released from the cell, by facilitated diffusion (Olsson and Pearson 1990).
Adenosine thus released acts, as described by Newby as "a retaliatory metabolite"
(Newby 1987), binding to its plasma membrane receptors and triggering intracellular

signaling.

B . l . Receptor subtyg

Adenosine receptors are members of the purine receptor family, as originally

classified by Bumstock (1978), as P1 or P2 purinergic receptors, depending on their

7

preference for adenosine or adenine nucleotides, respectively. The xanthine sensitive
adenosine receptors, a subclass of P1 purinergic receptors, are further divided into two
subtypes, A1 and A2 adenosine receptors. The major criterias used to distinguish between
the two adenosine receptors are the binding affinity of the receptors for adenosine and
its analogs and their ability to interact with the adenylyl cyclase system. AlAR is a high
affinity receptor activated by nanomolar concentrations of agonist which on activation
produces inhibition of adenylyl cyclase systems. In comparison A2AR is a low affinity
receptor activated by micromolar agonist concentrations and on activation produces
increased activity of the adenylyl cyclase. The most commonly used adenosine analogs
are, N°-cyclohexyl adenosine (CHA), R( -)N‘- (2-phenylisopropyl) adenosine (R-PIA), and
5’-N-ethylcarboxamido adenosine (NECA). Agonist binding at the AlAR have a rank
order of potency in CHA>R-PIA>NECA, while the potency series for A2AR mediated
responses is NECA>R-PIA>CHA. Analogs selective for AlAR typically are substituted
at the N‘5 positions, whereas analogs substituted at 5’ or 2—positions are selective for the
A2AR (Daly and Padgett 1992).

Other than A1 and A2AR, there is another intracellular adenosine receptor located
on the catalytic subunit of adenylyl cyclase. This site termed the P-site, is responsible for
inhibition of adenylyl cyclase. The P-site is activated by millimolor concentrations of
adenosine and is not antagonized by methylxanthine, a nonselective antagonist for
adenosine receptors. The physiological relevance of the P-site is still in question, since
adenosine kinase and adenosine deaminase normally maintain an intracellular
concentration of adenosine below the micromolar range. A model for adenosine receptors

and their interaction with adenylyl cyclase is presented in Figure 2.

Figure 2 . Model for adenosine receptors and their interaction with
adenyl cyclase system. View text for details.

 

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Pharmacological studies suggest that subtypes of AlAR may exist. The order of

potency for AlAR in rat adipose tissue is NECA >>> R-PIA, whereas binding
characteristics in rat heart and brain indicate similar affinities for NECA and R—PIA
(Burns et al. 1987 ; Linden et al. 1985). In various nerves it has also been observed that
the potency of NECA to inhibit neurotransmitter release equals R-PIA (Fredholm et al.
1988). The order of AlAR antagonist potency also supports this hypothesis. Gustafsson
et al. (1990) have shown that in brain the AlAR specific antagonist, 1,3-dipropyl-8-
cyclopentylxanthine (DPCPX) is much greater in potency in comparison to the xanthine
amino cogener (XAC), which has only moderate AlAR selectivity, whereas at the
neuromuscular junction XAC is as potent as DPCPX. The functional diversity observed
with AlAR stimulation also indicates that subtypes of AlAR may exist. In spite of all
these evidence, however the presence of subtypes of AlAR is still not universally
accepted. This is due to the fact that other factors such as, 1) differences in the
distribution of the generally hydrophobic compounds used to characterize the receptors
or 2) differences in the receptor-effector coupling could contribute to these discrepancies.
Cloning and expression of the hypothetical subtypes is essential for confirming the
existence of AlAR subtypes.

Recently Zhou et al. 1992, reported the molecular cloning and characterization of
an A3AR (R220) from rat brain. R220 encodes for a protein of 320 amino acids with
seven transmembrane domains which possess 50% identity in the amino acid sequences,
when compared to the canine A1 and A2AR. R220 was expressed in CHO or COS-7
cells and was observed to bind to the non-selective adenosine agonist [3H]-NECA and to

the A1 selective agonist [‘“IJ-APNEA (N‘-2-(4-aminophenyl)ethyl adenosine), but not

11

to the A1 selective antagonist [3H]-DPCPX or to the A2 selective agonist ligand
[3H]CC821000. Competitive binding studies using [mm-APNEA demonstrated that the
binding could be inhibited by adenosine ligands with a potency order of R-PIA = NECA
> S-PIA but not by the AlAR specific antagonist DPCPX. Although functional studies
indicated that R220 inhibited forskolin stimulated cAMP accumulation through a pertussis
toxin sensitive G-protein, a phenomenon linked to AlAR activation, pharmacological

studies suggest that R220 could be an A3 receptor.

B . 2 . Receptor structure

Photoaffinity crosslinking of [‘”I]APNEA into rat cerebral cortex
membranes, followed by isolation of cross-linked proteins by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (Koltz et a1. 1985; Lohse et al. 1986; Stiles et al.
1985) identified a protein of molecular weight 36,000 which displayed all of the
pharmacological properties of AlAR. Purification of AlAR through chromatographic
methods also supported this interpretation. Further studies of AlAR structure indicated
that the protein is glycosylated. Deglycosylation of AlAR from rat brain and adipose
cells results in a protein of molecular weight of 32,000 (Klotz and Lohse 1986; Stiles
1986). The functional role for this glycosylation remains to be determined. Similar
photoaffinity labelling of A2AR by [”51] PAPA-APEC, a selective A2AR agonist,
demonstrated that the molecular weight of A2AR is 45,000.

The structures of the adenosine receptors has been further established by
molecular cloning. Recently a number of A1 and A2 adenosine receptors have been

cloned, sequenced, and expressed from different species (Maenhaut et al. 1990; Libert et

12
al. 1991; Mahan et al. 1991; Olah et al. 1992 and Bhattacharya et al. 1993). Both the A1

and the A2 receptors show a high degree of homology between species (91-92%). These
receptors are small relative to other receptors of the G protein-coupled receptor
superfamily, the AlAR being 36,000 and the A2AR being 45000 in molecular weight,
as indicated by photoaffinity labelling.

Like other G-protein linked receptors, the A1 and A2AR have seven
transmembrane domains (Mahan et al. 1991). Comparing the AR with several other G-
protein coupled receptors however reveal some unique characteristics, which include: 1)
a relatively low molecular weight; 2) one or two consensus glycosylation sites on the
second extracellular loop rather than on the amino terminus; and 3) the presence of a
relatively short third intracellular loop (34 amino acids) in the AlAR, compared with

other receptors that inhibit adenylyl cyclase.

B . 3 . Molecu_l_ar cloning of adenosine ream

Although much is understood regarding adenosine and its effects on a variety of
physiological systems, only recently have the primary structures of the adenosine
receptors been determined by molecular cloning. Libert et al. (1989) were the first to
clone the adenosine receptors, and used oligonucleotides designed on the basis of
conserved regions of other genes encoding the superfamily of G-protein linked receptor
proteins. Sets of degenerate oligonucleotides from putative transmembrane regions (TM3
& TM6), were used as primers in the polymerase chain reaction (PCR) to amplify cDNA
fragments of human thyroid tissue. These PCR products were subsequently used as

probes to screen a dog thyroid cDNA library to obtain full length clones. Five clones

13

were isolated and identified as receptors : l) RDCl, identical to a recently cloned human
vasoactive intestinal peptide receptor (Shreedharan et al. 1991); 2) RDC4, thought to be
a 5-HT receptor (Libert et a1. 1989); 3) RDC5, the dog analog of the or-adrenergic
receptor, and two orphan clones, namely 4) RDC7 and 5) RDC8. Based on ligand
binding, tissue distribution and second messenger coupling RDC8 was subsequently
identified as A2AR (Maenhaut et al. 1990). Tissue distribution has been an useful criteria
for identifying orphan receptors, a classical example of which is the characterization of
rat brain cannabinoid receptor (Matsuda et al. 1990). The 51% identity of RDC7 to RDC8
in its nucleotide sequence made RDC7 a possible candidate for AlAR cDNA clone.
Subsequently ligand binding and second messenger coupling experiments confirmed that
RDC7 codes for AlAR (Libert et a1. 1991).

cDNA clones encoding the AlAR from rat brain (Mahan et al. 1991; Reppert et
al. 1991), bovine brain (Olah et al. 1992), human brain (Libert et al. 1992) and from
rabbit kidney (Bhattacharya et al. 1993) were subsequently isolated using probes derived
from RDC7. The amino acid sequence of adenosine receptors are aligned and compared
in Figure 3. The two adenosine A2 receptors cloned from rat and canine cDNA libraries
not only share 97% identity in their amino acid sequences, but the rat A2 receptor also
shares 65%, 64% and 63% amino acid identities with those of rat A1, canine A1 and
bovine A1 receptors. Thus not only are A2 and A1 receptors highly homologous within
species, but there is also a high degree of homology between the two receptors in their
amino acid sequences. The maximum homologies between the A2 and the A1 receptors
are in the transmembrane domains, which in G-protcin coupled receptors is the region

thought to be involved in ligand binding; the major differences in the A1 and A2

Figure 3.

14

The alignment and comparisons of the predicted amino
acid sequences of the adenosine receptors isolated from
different species. The figure shows the comparison of the
A2 and A1 adenosine receptors isolated from rat, canine
and bovine cDNA libraries. Amino acids identical to the rat
A2 are boxed. The putative seven transmembrane domains
are overlined.

15

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sequences third intracellular loop and in the C-terminal, which are thought to be involved
in G-protein coupling.

As mentioned before, Zhou et al. (1992), have reported the molecular cloning and
characterization of an adenosine receptor from rat brain (R220) which possesses an
unique binding characteristics and which they therefore refer to as A3AR. R220 encodes
for a protein of 320 amino acids, with seven transmembrane domains. When compared
with RDC7 and RDC8, R220 is only 50% identical. R220 when expressed in CBC and
COS-7 cells, was observed to bind to the nonselective adenosine agonist NECA and the
A1 selective agonist APNEA, but not to the A1 selective antagonist DPCPX or to the A2
selective agonist ligand CC821000. Competitive binding studies using [‘”I]-APNEA
demonstrated that the binding could be inhibited by adenosine ligands with a rank order
of potency of R-PIA = NECA > S-PIA, but not by the antagonist DPCPX. Functional
studies indicated that the protein R220 inhibited the forskolin stimulated CAMP
accumulation through a pertussis toxin sensitive G-prorein, a phenomenon observed with

AlAR activation.

B . 4 . Receptor - effector com

Adenosine receptors belong to the class of hormone receptors that are coupled to
the intracellular effector systems via guanine nucleotide binding proteins. The low affinity
A2AR is coupled to activation of adenylyl cyclase, whereas the high affinity A1 receptor
inhibits its activity. Both Al and A2 receptors are coupled to adenylyl cyclase through
the guanine nucleotide binding protein Gs and Gi, respectively (Spielrnan and Arend

1991). The second messenger systems coupled to AlAR, however are remarkably diverse

17

(Table I), and includes adenylyl cyclase, phospholipase C and various ion channels
(Linden 1991). This diversity of responses generated by AlAR activation has been
interpreted to indicate that subtypes of AlAR may exist, either as products of differential
splicing of a multiexon gene or as products of entirely different adenosine receptor genes.
A third possibility is that a single receptor exists which is capable of interacting with

multiple effector systems.

B . S . Adenosine receptor regulation :

Regulation of receptor function protects the cell from either over or under-
activation of its effector systems. It is generally accepted that prolonged exposure to an
agonist leads to desensitization of the receptor which may be receptor specific
(homologous) or generalized (heterologous) for receptors coupled to similar second
messenger system. The alternate situation, exposure to antagonists, often results in
enhancement of subsequent responses to agonists. Although desensitization of the
adenosine receptor has been observed in various systems, the molecular mechanisms
responsible for desensitization are yet to be determined. Generally it is believed that
desensitization can result from reduction of the size of the receptor population by
sequestration, by regulation of receptor transcription and or translation rates, from
alteration in expression of G~proteins, or from covalent modification such as
phosphorylation leading to reduced function of the receptor (Hausdroff et al. 1990).

Homologous desensitization of the A2AR has been observed in rat brain striatum
(Porter et al. 1988), rat vascular smooth muscle cells (Anand-Shrivastava et al. 1989),

hamster DDT1-MF-2 cells, a cell line derived from the vas deferens smooth muscle

18

 

 

8.3.0 .28. {2.0
2.8 «8.50..

2.8 5.3.... 0:0
5:00 a... 005:0

2.8 20...... 0.0:...
unto“.

0:00 (own—.00:

0:050: E00000 00005

E00008. 02.020 00000.03

8:88... 92.80.... .28... .o 8:32...

E02030... 0.2.002... 3:00... 00000.00.

0.05.2.0 100 .0 00:02.00...

 

 

 

0...< 0.00:2.0 :03. .0. :0..0>..0<
88.3
05.2...
:8:
. 2.8 3300.. 8......» 33.8.. .o 8:32...
00:00.» 0020000..

 

 

 

0.0.0000. 0:.00..00< p< 0 ... 00.0000 00000000.. . . 0.00...

 

19
(Ramkumar et al. 1991), rat kidney fibroblasts (Newman and Levitzki 1983) and in

LLCPKI cells, a cell line developed from porcine kidney (Levier and Spielman, 1992).
The desensitization of A2AR in all these systems, appears to be rapid, within a period
of 24 hrs.

Desensitization studies of AlAR have been carried out using adipocytes (Hoffman
et al. 1986; Parsons and Stiles 1987; Green 1987; Longabaugh et al. 1989; Green et a1.
1990; Green et al. 1992), DDTl-MF-Z cells (Ramkumar et al. 1991), rabbit kidney (Arend
and Spielman 1992) and embryonic chicken heart (Shryock et al. 1989). Desensitization
in all these systems, unlike the A2AR was observed to have a slower time of onset. In
DDTrMF-Z cells, it was observed that prolonged exposure to the A1 agonist resulted in
decreased numbers of AlAR. Phosphorylation, sequestration, and uncoupling of the
AlAR appeared to account only in part for the decrease in the AlAR number. These
results suggest that the receptor may be regulated at the transcriptional and or
translational level. A1 adenosine receptor numbers are also observed to increase in
response to stimulation by glucocorticoids in DDT,-MF-2 cells (Gerwins et al.1991) and
thyroid hormones (Rapiejko et a1 1987). With the molecular cloning of adenosine receptor
cDNA and the gene, it is now possible to elucidate the molecular mechanisms underlying

regulation of the adenosine receptors.

C . Physiolpgical effects of a_denos£re receptor amufion:
The physiological role of adenosine has been recognized for more than 60 years.
Drury and Szent-Gyorgi, in 1929 first demonstrated a role for adenosine in the regulation

of the cardiovascular system and vascular muscle tone. The effects of adenosine,

20

however, are not confined solely to the cardiovascular system, but are manifested in the
nervous system, renal system, pulmonary system, gastrointestinal and immune systems
(Table II). The physiological actions of adenosine are initiated by binding of adenosine
to its plasma membrane G-protein coupled receptors and the subsequent change in
intracellular signaling mechanisms. The actions of adenosine on different systems are

summarized below :

1. Cardiovascular system : Adenosine has negative chronotropic,
dromotropic and inotropic effects (Olsson and Pearson et a1. 1990) in the heart. Drury and
Szent Gyorgi (1929) were the first to report, a transient decrease in heart rate following
adenosine infusion. Further investigations revealed that adenosine acts directly on the SA
node, AV nodes and Bundle of His in experimental animals (Belardinelli et al. 1980).
The effects of adenosine are blocked by methylxanthines and increased by inhibitors of
adenosine uptake such as dipyridamole (Belardinelli et al. 1981; Bume et al. 1963). The
negative inotropic effect of adenosine may be mediated by the activation of K-channels
through AlAR (Kurachi et al. 1986; Belardinelli et al. 1987). In ventricular tissues it
appears that adenosine acts as an inhibitor of adrenergic stimulation (Dobson et al. 1987).

In vascular beds, adenosine acts as a potent vasodilator (Li and Fredholm 1983)
except in the renal vascular system (Macias et al. 1983) and in the placenta (Olsson et
al. 1990). The vasodilatory effect of adenosine is probably mediated via activation of
A2AR on endothelial cells and releasing the endothelium derived relaxing factor (Olsson

and Bunger 1987).

21

Table II. Functional Eliects Oi Adenosine Receptor Activation :

 

 

EFFECT

 

 

 

 

 

 

 

 

 

TISSUE RECEPTOR
Nervous System
peripheral V transmitter release A 1
central V neuronal firing A1
Heart anti-adrenefgic A 1
Smooth Muscle
vascular relaxation A 2
trachea relaxation A 2
taenia coll relaxation A 2
Platelets antl-aggregatory A 2
Fats cells anti-lipoiytic A 1
Masts cells degranulation ?
Lymphocytes immunosuppression 7
Kidneys
afferent art. contraction A 1
efferent art. relaxation A 2
JGA, renin inhibition A 1
release stimulation A 2
erythropolstin inhibition A 1
stimulation A 2
adren. trans. presynaptic inhibition A 1
collecting cAMP ( Aliza perm. 7) A 2
tubule cAMP ( V H20 perm. 7) A 1
thick limb CAMP ( Ll Me) A 2
cAMP (' .1 Na) A 1

 

 

 

 

 

22

2. Nervous system : The role of adenosine as a modulator of neurologic
function was first suggested by Drury and Szentz-Gyorgi in 1929. Subsequently
adenosine was reported to inhibit the release of neurotransmitters, including
norepinephrine, dopamine, serotonin, acetylcholine, GABA, aspartate, and glutamate
(Stone 1981; Spignoli et al. 1984; Ebstein et al. 1982). This inhibition appears to be
independent of calcium entry into neurons.

In 1954 Feldberg and Sherwood were the first to demonstrate the cessation of
spontaneous motor activity in cats upon intraventricular injection of adenosine. This led
to further investigation and now adenosine and its analogs have been found to produce
sedative actions. Adenosine in high doses also has been shown to protect against seizure

(Dragunow ct a1. 1985).

3. Renal system : The diverse effects of adenosine are best illustrated
in the renal system. In the kidney, adenosine has been reported to produce a variety Of
responses, including hemodynamic changes, renin release and solute transport (Spielman
and Arend 1991; Churchill and Churchill 1988; Osswald 1983; Spielman and Thompson
1982; Yagil et al. 1990; Dillingham and Anderson 1985). In contrast to peripheral
vascular beds, adenosine infusion into the renal artery results in vasoconstriction
(Osswald et al. 1978) which in turn decreases glomerular filtration rate (GFR). The
mechanism underlying the adenosine induced decrease in GFR, appears to be due to a
decrease in glomerular hydrostatic pressure resulting from preglomerular AlAR-mediated
vasoconstriction and a more slowly developing postglomerular A2AR-mediated

vasodilation (Rossi et al. 1988; Rossi et al. 1987). Since an increase in distal nephron

23

perfusion rate produces an afferent arteriolar vasoconstriction and a resultant decrease in
single nephron GFR, it can be interpreted to serve as a mechanism to limit large increases
in fluid and solute delivery and consequent alterations in tubule and excretory functions
(Briggs and Schnermann 1990). This whole phenomenon has been termed tubulo—
glomerular feedback (TFG). Studies have shown that the specific AlAR antagonist
DPCPX inhibits TGF when administered either into the tubule lumen or into the
peritubular capillary circulation (Schnennann et al. 1990).

In addition to hemodynamic changes, adenosine infusion also produces a biphasic
renin release. At low adenosine concentrations A1 receptors are activated inhibiting renin
release, whereas at high adenosine concentrations which activates A2 receptors, renin
release is augmented (Osswald et a1 1978).

Adenosine also has been Shown to regulate erythropoietin production (Ueno et al.
1988) and inhibition of neurotransmitter release (Hedqvist et al. 1976) in the kidney. In
exhypoxic polycythemic mice, activation of A2AR produces stimulation of radioiron
incorporation into red blood cells and AlAR activation is shown to inhibit this process.
Moreover, stimulation of A2AR enhances erythropoietin production in renal carcinoma
cells whereas AlAR stimulation inhibits erythropoietin production (Ueno et al. 1987).

In the kidney, adenosine acts at prejunctional AlAR to inhibit the release of
norepinephrine from sympathetic neurons. However, it has also been reported that
adenosine increases the sensitivity of the kidney to norepinephrine through a post-
junctional mechanism (Hedquist et a1. 1976).

Decrease in urine flow and solute excretion also has been reported to result from

elevated levels of intrarenal adenosine (Y agil et al. 1990; Miyamoto et al. 1988). A direct

24

tubular action of adenosine has been reported in rabbit cortical collecting tubule
(Dillingham and Anderson 1985;), canine medullary thick ascending limb and cortical

thick ascending limb (Anand-Shrivastava et al. 1986).

4. Pulmonary system : Adenosine causes bronchoconstriction in
asthmatic lung which is antagonized by the adenosine receptor antagonist, theophylline
(Cushley et al. 1984). Theophylline also has been demonstrated to inhibit the action of
adenosine to potentiate the antigen—induced histamine release from guinea pig lung

(Welton et al. 1976).

5. Gastrointestinal system : In rats, the adenosine receptor antagonist,
theophylline, augments the production of stress induced gastric ulcers (Geiger and Glavin
1985; Watt et al. 1987). Adenosine analogs have been shown to induce gastric lesions
(Ushijina et al. 1985). It has also been shown that in pancreas, caffiene induces pancreatic
secretion during fasting and theophylline induces the release of amylase from isolated

pancreatic acinar cells (Korman et a]. 1980).

6. Immune system : Inherited deficiency of adenosine deaminase, the
enzyme that catalyzes the breakdown of adenosine to inosine, which leads to elevated
levels of adenosine, has long been known to have detrimental effects on the development
of the immune system. Among the major functions of adenosine in the immune system,
are lectin stimulated proliferation of lymphocytes, inhibition of interleukin-2 production

by T-lymphocytes (Averill et al. 1985), modification of T-lymphocyte effector functions,

25

inhibition or facilitation of cytokine production, and suppression of natural killer activity

of neutrophils (Cronstein et al. 1985).

7. Platelet : The anti-aggregatory effect of adenosine on platelets has
long been recognized (Born 1964). This effect is blocked by the methylxanthines and

enhanced by dipyridamole, the inhibitor of adenosine uptake.

III. MOLECULAR CLONING OF Al ADENOSINE RECEPTOR cDNA FROM

RABBIT

A . INTRODUCTION

Adenosine receptors are members of the purine receptor family, broadly classified
as P1 (adenosine) or P2 (ATP, ADP, or AMP) purinergic receptors (Brunstock 1978). The
P1 receptors have been further subdivided into A1 and A2 subtypes based on their
affinities for a variety of adenosine analogs (Daly et al. 1987) and on their ability to
inhibit (Al) or stimulate (A2) adenylyl cyclase (Londos et al. 1980). The second
messenger systems coupled to the A1 adenosine receptor, however, are remarkably
diverse, and include adenylyl cyclase, phospholipase C and various ion channels (Linden,
1991). This diversity of responses generated by AlAR activation has been interpreted to
indicate that subtypes of the AlAR may exist, either as products of differential splicing
of a multiexon gene or as products of entirely different adenosine receptor genes.
However, the possibility of a single receptor capable of interacting with multiple effector
systems also exists.

Our laboratory has long been interested in adenosine receptors, its structure,
function and signalling in the rabbit kidney. In RCCT-28A cells, a cell culture line
developed from the rabbit cortical collecting tubule, adenosine receptors were shown to
be coupled to adenylyl cyclase via guanine nucleotide binding proteins (Arend et al.

1987). The A2AR, which is coupled to the stimulatory guanine nucleotide protein,

26

27

activates the cyclase system, whereas the AlAR, which is coupled to the inhibitory
guanine nucleotide binding protein (0,), inhibits the cyclase system. It also has been
observed that stimulation of the AlAR in RCCT-28A cells not only produces an
inhibition of the cyclase but also a stimulation of the phospholipase C activity (Arend et
al. 1989). It is not clear whether there is one AlAR which is capable of interacting with
more than one effector system or whether there are subtypes of AlAR which have the
same ligand binding domains but different effector coupling domains. Molecular cloning
and expression of the AlAR will help to resolve this controversial area.

This report describes the cloning of an AlAR from rabbit. The deduced amino
acid sequence of this receptor and its comparison with other cloned AlAR is presented
in this chapter. This data show that the rabbit AlAR displays a high degree of similarity
with the dog thyroid, rat brain, bovine brain and the human brain AlARs (Libert et al.,

1991; Mahan et al., 1991; Reppert et al., 1991; Olah et al., 1992; Libert et al., 1992).

B . MATERIALS AND METHODS

MATERIALS: [y-3ZP]ATP (specific activity of 6000 Cmeol), [35S]ATP
(specific activity of 1200 Ci/mmol) and [or-”PMCTP (specific activity of 800 Ci/mmol)
were obtained from DuPont-New England Nuclear; restriction enzymes, T4 DNA ligase
and Random primer labelling kit were obtained from Boehringer-Manheim or BRL; 7.-
ZAPII cDNA library was from Stratagene; A—EMBLB SP6/T7 rabbit genomic library
containing Sau3AI partial digest of rabbit kidney DNA was from Clontech; nitrocellulose

filters (pore size 0.45 pm) were from Schleicher and Schuell; agarose (type I) was

28

obtained fiom Sigma; oligonucleotides used for library screening and for sequencing were
synthesized by the Macromolecular Structure, Synthesis, and Sequencing Facility
Laboratory at Michigan State University and the Sequencing Laboratory at SmithKline
Beecham Pharmaceuticals; DNA sequencing kits were obtained from United States

Biochemicals.

METHODS:

Desiming an oligonucleotide probe: The nucleotide sequence of the canine
AlAR protein RDC7 (Libert et al. 1991 was deduced with the help of a computer
program "Backtranslate" distributed by the Genetics Computer Group, Wisconsin. Each
amino acid is coded by a set of three nucleotides called a codon. However most of the
amino acids can be coded by more than one codon, which is referred to as degeneracy
of codon. It has been observed that in case of degenerate codons, each species has a
preference for a particular codon over others. The Backtranslate program statistically
compares a group of different messages from a particular species, and generates a
nucleotide sequence for a protein containing the best fit codon for that species. The
nucleotide sequence of RDC7 thus obtained from this Backtranslate program was used
to generate a 60-mer oligonucleotide probe, termed WSSl, in order to screen the rabbit
kidney cDNA library.

The 60 mer oligonucleotide designated, WSSl (5’-GAAGAAG'I'I‘GAAGTAIA-
CCATGTACTCCATGGAGATIACCITCI‘CAAACTCACACI'I‘GAT-3’) was synthesized

based on the region comprising a part of the second extracellular loop and fifth

29
transmembrane domain (166-186 amino acid) of RDC7, the dog thyroid AlAR (Libert

et al. 1991). WSSl was end labeled with [7-32P]ATP and T4 polynucleotide kinase and

the labelled probe was first used for Northern blotting.

Northern Blot : Poly-(A)* RNA was isolated by a two step process from kidney
and brain tissues of white male New Zealand rabbit and also from RCCT-28A cells, a cell
culture line developed from rabbit cortical collecting tubule (Arend et al. 1989). Total
cellular RNA was first isolated by extraction with guanidinium isothyocyanate and cesium
chloride separation, followed by poly-(A)+ mRNA isolation using oligod(T) cellulose
column (Sambrook et al. 1989). The mRNA isolated (10 jig/lane) was electrophoresed on
a 1% agarose-fonnaldehyde gel and blotted overnight onto nitrocellulose, immobilized by
baking at 80°C for 1 hour and hybridized to labelled WSSl. Hybridization conditions
were 20% fonnamide, 5X SSPE (0.75M NaCl, 0.05M NaHzP04, 0.005M EDTA, pH 7.4),
5X Denhardt’s solution (a stock solution of 50X Denhardt’s containing 5 gm Ficoll Type
400 (Pharrnacia), 5 gm Polyvinylpyrrolidone and 5 gm Bovine Serum Albumin Fraction
V was used to prepare the working solution), 0.1% sodium dodecyl sulphate, 0.25 mg/ml
of salmon sperm DNA, with 2X10° cpm/ml y-“P labelled probe, for overnight at 42°C.
The filters were washed with 6X SSC (0.9M NaCl, 0.09M Sodium Citrate, pH 7.0)
containing 0.1% SDS for 15 min. at room temperature, followed by 15 min wash at 55°C
and exposed for autoradiography. WSSI was shown to hybridize to a message length of

2.4 kb, the reported size for canine AlAR.

30
QNA libraiy screening: End labelled WSSl was then used to screen a rabbit

kidney cDNA library (l-ZAPII cDNA library cloned in the EcoRI cloning site). Duplicate
nitrocellulose filters which were used for phage lifts were hybridized with 20%
formamide, 5X SSPE, 5X Denhardt’s solution, 0.1% sodium dodecyl sulphate, 0.25 mg/ml
of salmon sperm DNA, with 2X10° cpm/ml of the 7—32P labelled oligonucleotide, WSSl,
for overnight at 42°C. The filters were washed with 6X SSC/0.1% SDS for 15 nrin at
room temperature, followed by a 15 min wash at 55°C and exposed for autoradiography.

One positive clone designated SB4, was isolated, purified and sequenced.

DNA seflencing; The insert isolated from the positive clone was ligated in
Ml3mp19 in both orientations and sequencing was performed on both strands using
[”S]dATP, as per the manufacture’s instruction, using Sequenase ver. 2.0 kit (U. S.
Biochemical). Sequencing data indicated that SB4 was the rabbit analog of RDC7 but was
missing the first 74 amino acid of RDC7. In order to get a full length clone I further

screened a rabbit genomic library.

Genom_ic library screening : A rabbit genomic library (a A-EMBL3 library
cloned in the BamHI restriction site) was purchased from Clontech. The library was
screened with an 870 nucleotide fragment from the 5’-end of SB4 (an EcoRI and a SmaI
fragment; SB4E/S), as per the cDNA library screening protocol, with the exception that
the 1) probe was labeled with [or-”PMCI‘P using a random primer labeling kit
(Boehringer-Manheim); 2) the hybridization solution contained 50% forrnarnide and 3)

the washes following hybridization were more stringent (2X SSC containing 0.1% SDS

31

for 15 min at room temperature followed by 0.2X SSC containing 0.1% SDS for 15 min
at 65°C). Eight positive clones were isolated and plaque purified. Duplicate nitrocellulose
filters used for plaque lifts from the eight clones were hybridized to two sets of probes:
a) the 5’-end EcoRI/Smal fragment of SB4 (SB4E/S) and b) a PCR fragment generated
from rat brain mRNA, using oligonucleotides (21 mer) designed from RDC7, which
included the first 300 nucleotide of the 5’-end starting from the translation start site
(kindly provided by Dr. Lohse, Genzentram/MPI for Biochemistry, Martinsried, FRG).
This PCR fragment therefore contained the nucleotides that were missing in SB4. One
genomic clone, named clone-1, gave a positive signal with both the probes and was

therefore further characterized.

Southern blot : Phage DNA isolated from Clone-1, was digested with XhoI and
electrophoretically separated on a 1% agarose gel containing 0.5 pg/ml of cthidium
bromide, blotted overnight onto nitrocellulose and immobilized by baking at 80°C for 1
hour. Southern blot analysis was performed using labelled SB4E/S fragment and the PCR
fragment as probes. A 3.2 kb Xhol fragment of clone-1 was found to hybridize to both
SB4E/S and the PCR probe. Sequencing data indicated that this 3.2 kb XhoI fragment
contained approximately 2 kb of 5’-flanking region, an exon of 341 nucleotides followed

by an intron.

PCR and ligation of genomic clone-l and SB4 to generate a full lengtlr cDNA

 

encoding the AlAR from rabbit: The genomic clone-1 and SB4 were ligated using the

polymerase chain reaction (PCR) to generate a full length cDNA encoding the AlAR

32

from rabbit (Figure 10). A pair of reverse and complementary primers were synthesized

from the exon-l and intron-l of genomic clone-l as follows :

PRIMER SEQUENCE PRODUCT SIZE
7033 (intron- 1) 5'GGCACTGCCAGGCTCGTG'ITCCT’' 571 nucleotide

7 198 (exon-1) 5'CCIGAA'I'I‘CTGCTGATGTGCCCAGCI‘G3'

Primer 7198, contains a restriction site for EcoRI, as underlined and is 30 bp
upstream of the translational start site. For PCR, a mixture including 5 pmol primers, 2
U Taq DNA polymerase (Perkin Elmer), 1.25 mM each of dATP, dCTP, dGTP and d'l'I'P
was used. The total volume was brought to 100 pl with 1 X PCR buffer (80 mM Tris-
HCl, pH 8.9; 20 mM (NIL)ZSO,,; 5 mM MgC12) and 50 ng of the 3.2 kb Xhol fragment
as template. The PCR cycle included denaturation at 95°C for 1 min, annealing at 50 °C
for 1 min and extension at 72 °C for 1 min, for 25 cycles. After PCR, one-tenth of the
sample was electTOphoresed on a 1% agarose gel to verify the size of the products. The
PCR product was digested with EcoRI and Sacl to obtain a product of 310 nucleotides,
starting 30 bp upstream of the translational start site and subcloned into pUC19
(pUC19+5’ORF) for sequencing and further verification. SB4 and pUC19+5’ORF were
digested with SacI and PstI and religated to generate the full open reading frame for the

rabbit AlAR.

33
C. RESULTS

(a) Isolation of all AlAR-encoding cDNA

An oligonucleotide probe, WSSl was synthesized based on the nucleotide
sequence of RDC7, the canine AlAR (amino acid 166-186; Figure 4). On Northern blot
analysis of poly(A)+ mRN A isolated from rabbit cortical collecting tubule cells (RCCT-
28A), rabbit kidney and rabbit brain, end labeled WSSl hybridized to a 2.4 kb mRNA,
the reported size of mRNA for canine AlAR (Figure 5). Therefore WSSl was used as
a probe to screen a rabbit kidney A-ZAPII cDNA library, cloned in the EcoRI cloning
site. A single positive clone containing a 3 kb insert was isolated. The cDNA insert of
the clone was comprised of two EcoRI fragments 2 kb and 1 kb in length. To verify
whether both of the fragments originated from the same mRN A, a Northern blot analysis
of rabbit kidney mRNA was performed with the two individual EcoRI fragments. The 2
kb and the 1 kb fragments hybridized to two different sized mRNAs indicating that they
were probably derived from different messages (data not shown). This may be due to an
artifact in the cDNA library two different messages were ligated bluntly resulting in a
single insert. Since the 2 kb fragment (designated SB4) hybridized to a 2.4 kb mRNA,
the reported size of mRNAs for canine AlAR, we sequenced SB4 in both orientations.
Comparison of amino acid sequence obtained from the largest open reading frame to that
of the canine receptor protein sequence (RDC7) indicated 94% identity. SB4 was
however, found to lack 5 ’—UTR sequences and nucleotides corresponding to the fast 74
amino acids of RDC7 (Figure 6). Moreover, no poly (A) tail was observed at the 3’-end

of the clone, although oligo dT primers were used to construct this library.

Figure 4 .

34

Flow diagram depicting the strategy involved in
screening the cDNA library. RDC7, the canine AlAR,
with its putative seven transmembrane domains is shown to
the right. The shaded amino acids indicate the region from
which WSSl was designed.

RDC7 Amino acid sequence

    
       

0 go o 0
° 080808
000

l O
)3’60 0

 

Backtranslate $

0
C
O
O
O
O
O
O
O

00

°o

0
060900000

name

0 C0% 0°
009600000600

-m It memes

35

sun-summers”
O ”summer-name

—>

 

0
0
C
0
3
8
in
0
E
.8.
0
3
Z
r.
O
O
I

l

 

Oligonucleotide probe

Northern blot

l

 

l

Screen rabbit kidney cDNA library

Figure 5 .

36

Northern blot analysis of AlAR mRNA. Poly(A)*
mRNA (10 jig/lane) was run on an agarose/formaldehyde
gel, transferred to nitrocellulose and probed with 32P-
labelled oligonucleotide, WSSl. Tissues analyzed were
rabbit kidney and brain and RCCT-28A cells, a cell culture
line developed from rabbit cortical collecting tubule.
Position of the RNA molecular weight markers are shown
to the right.

 

37

 

Figure 6 .

38

Comparison of the amino acid sequence of RDC7, the
canine AlAR, with SB4. Vertical lines indicate arrrino
acids that are identical and dots indicate the differences in
nucleotide numbers in a set of codon for a particular amino
acid.

omN
NNm
com
vhw
own
vNN
ooa
whn

on

vNH

va 02m!
. ...
mNm D=m<

mmm4Dmowaw¢m<m10¢hrozzdehh>¢mxzH¢h<fi>umZZszwzfiam
... .... H....................u..................
mmmmamn>..mmbmdothGZZH84hh>¢h¥OH¢h4N>HmzxdmZOZBAh

Heex>00meadowmomae>uzaH=000300400000000maeuxamxoaa
......................._......_..........._......_
HeszqHmmzmummumqenozqHzaqumammamqHaeqmzequmxoxw

xzmQOmmemezxmqommHaxm>mawH0>xqaqmmq>z>mmzmx>zmemH
....................._?_.............._......_....
200000m¢0>xxoqommnaxm>quHq>zqaqmoa>3>mezex>2>020H

>xmmmoxn>0mo>002<¢z<¢o>mquzzomzmeao>>000H:OO<H<><
....._._....._........._ ......u..._._........._..
>xmmmox~>00000024<3¢¢o<moqzzzomamaao>>mmaH300<H<><

<0¢ae>>¢xxmamux>¢axzo><~<aa¢a~000e000>00<>xqoa=mue
...................._...._........................
<m¢0e>>exxxame>maxmn>¢H<00<aH000a0H0>moe>zaoe=mae

HmN
mmm
HON
th
and
own
new
and
an

mNH

mh

vmm

FUGm

. 0mm

roam
can
hoax
vmm
Foam
vmm
Fund
vmm

bond

40
(b) Isolation of genomic AlAR clones

To obtain a clone encoding the N-terminal sequence of the rabbit AlAR we
screened a rabbit genomic library (Clontech) with an EcoRI/Smal fragment derived from
the 5’-end of SB4. A schematic of the A-EMBL3 vector of the genomic library with its
cloning site and the probes used to screen the library is depicted in Figure 7. Eight
positive clones were identified and rescreened with a PCR fragment from the 5’-end of
the rat brain AlAR generated using oligonucleotide primers designed from RDC7. This
PCR fragment included the first 300 nucleotide of the 5’-end of the rat brain AlAR
begining with the translation initiation codon (kindly provided by Dr. Martin Lohse,
Genzentram/MPI for Biochemistry, Martinsried, FRG). Only one of the eight clones,
designated clone-1, hybridized to this PCR fragment. Clone-1 was rescreened, purified
and its DNA was extracted (Sambrook et al. 1989). Southern blot analysis of the PCR
positive clone revealed a 3.2 kb Xhol fragment which hybridized to both the SB4 probe
and the PCR fragment (Figure 8). Sequence analysis demonstrated that the 3.2 kb XhoI
fragment contained the 5’—UTR, an exon of 341 nucleotides followed by an intron with
a consensus exon/Intron splice site. The exon sequence was 100% identical to SB4 at the
region of overlap (Figure 9) and the deduced amino acid sequence was 98% identical to
that of the canine AlAR (RDC7). Together, the cDNA and the genomic clone provided

the entire open reading frame for the rabbit AlAR.

(c) The amino acid sgquence of rgbbit AlAR and its comparison to other AlARs

A part of the 5’-open reading frame was amplified from the 3.2 kb XhoI fragment

using PCR with primers made from exon-l with an EcoRI site at the 5’-end, 30 bp

41

Figure 7 . Schematics of the l-EMBL3 cloning site of the genomic
library vector and the probes used to screen the library.

42

mom

 

 

 

 

 

 

.V///////////A
0 mm
_ . _ _
.m. 00m . «Em ... 00m
E0 :55. .0059... .33....» 8.0 :0.
n... a A... 3 0.. pm
I'VE .0om .... Eon .2: .3x =.. Son .08 .....m

ml! a0 .05 .... 8.“. ... com .2... .. AH

Figure 8 .

43

Southern blot analysis of the genomic clone-1. DNA
isolated from genomic clone-1 was digested with the
restriction enzyme XhoI, run on agarose gel, transferred to
nitrocellulose and probed with random labelled SB4(E/S)
and the PCR fragment. Positions of the k-HindIII digest,
molecular weight markers are shown to the left.

EcoRI 5mg

3'

 

sac/s 9m!

5' .\\\\\\\\‘ 3'

PCR SB4E/S

23K -->

9.4K —>
6.5K —>

4.3K —>

2.3K —>
2.0K —>

   

Figure 9 .

45

Sequence obtained from the 3.2 kb XhoI fragment and its comparison
with SB4. The top figure illustrates the sequencing map of the 3.2 kb
X hoI fragment from genomic clone-1. Arrows indicate the length and
direction of sequence obtained from each primers. The middle figure
shows the nucleotide sequence obtained from the 3.2 kb XhoI fragment,
including the fast exon and the exon/rntron border. The 5’ UTR and the
intron sequence is shown in small letters. The figure below shows the
nucleotide sequence compared to SB4.

61
121
181
241
301
361

46

 

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47

upstream from the translation start site and a reverse and complementary primer from
intron-1 (Figure 10). The 571 nucleotide product thus obtained, after size verification on
an agarose gel, was digested with EcoRI and Sacl to obtain a fragment of 310 bp in
length. This EcoRI and Sacl fragment from PCR was ligated into pUC19 (pUC+5’ORF),
and sequenced. Both genomic clone-1 and SB4 possess an unique 5061 site at the 5’-end
and therefore SB4 was digested with SacI and PstI, generating a 1487 bp long fragment
containing the nucleotides coding for a part of the open reading frame and a part of the
3’-UTR. This fragment was then ligated to the SacI/Pstl site into the vector containing
the PCR product (pUC+5’ORF; Figure 10). The vector containing the entire open reading
frame of the rabbit AlAR (pUC+RBA1) was sequenced from both ends by the forward
and reverse primers made from pUCl9 for further verification. The nucleotide and the
amino acid sequence obtained from the rabbit AlAR is shown in Figure 11.

The rabbit AlAR was compared to other AlARs as shown in Figure 12. This
comparison demonstrated that: (1) there is a high degree of interspecies homology among
the AlARs (2) each contain seven putative transmembrane spanning domains
characteristic of G-protein linked receptors, (3) the rabbit AlAR contains two additional
amino acid residues, (4) each receptor contains serine residues in the third intracellular
loop, which may be a potential site for phosphorylation involved in regulation of receptor
function and (5) all the AlAR cloned so far with the exception of canine AlAR contain
two consensus glycosylation sites. Canine AlAR has only one consensus glycosylation
site. The five AlARs cloned to date are 92% identical in their transmembrane region I,
II and V, and in the first intracellular loop. Histidine 278 and 251 in bovine AlAR has

been identified as an important residue in both agonist and antagonist binding (Olah et

Figure 10 . Schematic representation of the strategy involved in
generating a full length clone encoding the rabbit
AlAR.

49

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Figure 11 .

The nucleotide sequence of the rabbit AlAR clones and
its deduced amino acid sequence. The oligonucleotide
probe WSSl derived from the canine AlAR (Libert et a1.
1991) was used to isolate a 2-kb rabbit AlAR cDNA (SB4)
comprised of nucleotide 260-2215. The 5’-end of SB4 and
a PCR fragment derived from the 5’-end of the rat AlAR
were used to isolate a genomic clone (clone-1), which
contained the 5’-UTR, an exon of 341 nucleotide and a part
of the first intron of the rabbit AlAR gene. The region of
overlap between exon-1 of genomic clone-1 and SB4 is
shown in bold (nucleotide 260-378). The putative mRNA
splice site is underlined. The 5’-end and the 3’-end UTRs
are shown in lower case whereas capital letters indicate the
open reading frame. The stop codon is shown as an

asterisk. The sequence is registered with the GeneBank

Database under accession No. L01700.

61
121
2’
101

241

2161

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8 A P
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A I L
CSCCTOICC!
P V L

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V A I

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r c x

ICACCTCCC?
S H L

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P S I

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120
26

100
40

240
60

300
00

160
100

420
120

400
140

540
160

600
100

660
200

720
220

700
240

040
260

900
290

960
300

1020
120

1000
340

1140
1200
1260
1320
1100
1440
1300
1560
1620
1600
1740
1000
1060
1920
1900
2040
2100
2160
2215

Figure 12 .

52

Comparison of the deduced amino acid sequences of
canine, rat, bovine, rabbit and human AlAR. Identical
amino acids are represented by dashes and the dissimilar
amino acids are in italics. Solid lines represent putative
transmembrane domains.

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54
al. 1992). Mutation of His—278 to Len-278 decreased both agonist and antagonist binding

by 90%. When His-251 was mutated to Len-251 antagonist affinity and the number of
receptors recognized by the antagonist decreased, however the binding affinity of agonist
remained the same although the number of receptors detected by an agonist was reduced.
These His residues are conserved among all of the AlAR isolated so far. The major
differences between the various AlARs are in the C-termini, in the transmembrane

domain IV and VII, and in the extracellular loop II and HI.

D. DISCUSSION

A partial cDNA encoding an AlAR which lacks nucleotides corresponding to
those coding for the first 74 amino acids of the canine AlAR was isolated from a rabbit
kidney cDNA library. The remaining 5’-end was obtained from a rabbit genomic library
clone which contained the 5’-flanking region, the first exon, and part of the first intron.
Together, the cDNA and the genomic clone provided the entire open reading frame
encoding the rabbit AlAR. This conclusion was based on a comparison of the open
reading frame obtained from the rabbit clone to other cloned AlARs.

The deduced amino acid sequence of the rabbit AlAR was shown to possess a
significant homology to AlARs from canine, rat, bovine and human. The encoded
proteins are all identical in molecular mass with 326 amino acids except for the rabbit
AlAR which has 328 arrrino acids. The five AlARs cloned to date are 92% identical in
their overall amino acid structure. The rabbit AlAR differs from the canine, rat, bovine

and human receptors by 26, 26, 33, and 23 amino acid substitutions, respectively. It is

55

generally believed that the transmembrane domains of the G-protein coupled receptors are
involved in ligand binding (Strader et al. 1989; O’Dowd et al. 1989) and therefore are
more conserved within species. Comparisons of the putative transmembrane domains of
the AlARs isolated from different species revealed the presence of only 6, 4, 11 and 4
amino acid differences between the canine, rat, bovine and human AlARs. There are
100% identity in the transmembrane domains I and 11 while the most diverse are domains
° IV and VII.

The recent cloning and purification of the AlAR and A2AR and the development
of selective radioligands will help to understand the structure, function and regulation of
the adenosine receptor systems. Although the physiological effects of adenosine have been
well characterized for more than 60 years, only recently have the receptors been cloned
which will help us to understand the system at the biochemical and molecular biological

levels.

IV. CHARACTERIZATION OF THE Al ADENOSINE RECEPTOR GENE

AND ANALYSIS OF TRANSCRIPTION START SITE HETEROGENEITY

A . INTRODUCTION

Although a physiological role of adenosine have been observed since the last 70
years, (Drury and Szent-Gyorgi 1929; and Bume 1963) it is only recently that the cDNA
for the adenosine receptors have been cloned thereby allowing for investigations at the
molecular level. Recently, we and others have reported the cloning, sequence analysis and
expression of the cDNA encoding both the A2 and the A1 adenosine receptors (Libert
et al.1989; Maenhaut et al. 1990; Reppert et al. 1991; Libert et al. 1992; Olah et al. 1992;
Bhattacharya et al. 1993). Analysis of both A1 and A2 receptors amino acid sequence and
function indicated the proteins belong to the superfamily of the G-protein coupled
receptors. This superfamily consists of a diverse group of receptors in terms of ligand
binding and functional characteristics, yet they all appear to share common structural
features ( i.e. seven transmembrane domains) and mechanisms of signal transduction (i.e.
activation of specific G-proteins). In addition to the adenosine receptors, this receptor
superfamily includes the opsins (Nathans and Hogness et al. 1983), the adrenergic
receptors (Lefkowitz and Caron 1988), the muscarinic receptors (Kubo et al. 1986; Peralta
et al. 1987; Bonner et al.1987; Bonner et al 1988; Shapiro et al. 1988), dopamine

receptors (Grandy et al. 1989; Sokoloff et al. 1990; Van Tol et al. 1991), thyroid

56

57

stimulating hormone receptors (Parmentier et al. 1989), leutinizing hormone receptors
(Loosfelt et al. 1989; McFarland et al. 1989), endothelin receptor (Hosoda et al. 1992),
tachykinin receptors (Hershey et al. 1991; Takahashi et al. 1992), the secretin receptor
(Ishihara et al. 1991), the cannabinoid receptor (Gerard et al. 1991), the serotonin 1c
receptor (Julius et al. 1988), thromboxane A2 receptor (Hirata et al. 1991), the
antidiuretic hormone receptor (Birnbaumer et al. 1992; Alain et al. 1992; Lolait et al.
1992), the oxytoxin receptor (Kimura et al. 1992), the product of the oncogene mas
(Young et al. 1986; Jackson et al. 1988), the yeast mating factor receptors (Burkholder
et al. 1985; Nakayama et a1. 1985), and a dictyostelium CAMP receptor (Klien et al.
1988).

A distinguishing feature that all G-protein coupled receptors have in common is
their genomic structure. Most of the receptor genes that belong to this superfamily have
been found to be intronless, although there are some exceptions including the dopamine
D2, D3 and D4 receptors (Grandy et al. 1988; Sokoloff et al. 1990; Van Tol et al. 1991),
the opsins (Nathans and Hogness et al. 1984), the tachykinins (Hershey et al. 1991;
Takahashi et al. 1992), the leutinizing hormone receptors (Tsai-Morris et al. 1993) and
the endothelin receptor (Hosoda et al. 1992). In the present work I have cloned and
characterized the gene encoding the AlAR which belongs to the intron containing G-
protein linked receptor gene family.

Adenosine receptors are found in a wide variety of cell types and tissues and are
known to be regulated by agonist-induced desensitization (Parsons et al. 1987;
Longabaugh et al. 1989), antagonist-induced sensitization (Ramkumar et al. 1988),

thyroid hormone (Rapiejko and Malbon 1987) and by glucocorticoids (Gerwins and

58

Fredholm 1991). Little is known about the biochemical mechanisms underlying the
regulation of this receptor system. To begin to investigate the regulation of the receptors,
1 have cloned and characterized the 5’-flanking region of the gene encoding the rabbit

AlAR using the AlAR cDNA from rabbit as a probe (Bhattacharya et al. 1993).

B . MATERIALS AND METHODS

MATERIALS :

Reagents were obtained as follows : [y-32P]ATP (specific activity, 6000 Ci/mmol),
[a-32P]dCTP (specific activity, 800 Ci/mmol), and [”S]dATP (specific activity, 1200
Ci/mmol) were from DuPont-NewEngland Nuclear, restriction endonucleases, T4
polynucleotide kinase, T4 DNA ligase, and a random primer DNA labelling kit, were
from Boehringer-Manheim; RNAse inhibitor was from Promega; an avian myeloblastosis
virus reverse transcriptase was from GIBCO; A—EMBL3 SP6/I‘7 rabbit genomic library
containing Sau3AI partial digest of rabbit kidney DNA was from Clontech; nitrocellulose
filters (pore size 0.45 pm) were from Schleicher & Schuell; agarose (type I) was from
Sigma; Bluescript KS(+) was from Stratagene; a Taq dye primer cycle sequencing kit was
from Applied Biosystems; DNA sequencing Kit was from United States Biochemicals;
oligonucleotides were synthesized by the Macromolecular Structure, Synthesis, and
Sequencing Facility Laboratory at Michigan State University and by the Sequencing

Laboratory at SmithKline Beecham Pharmaceuticals, Philadelphia.

59
METHODS :

Analysis of the gene copy number : Rabbit kidney genomic DNA (Clontech),
was digested with restriction enzymes BamHI and Pstl, electrophoresed on 1% agarose
gels, transferred to nitrocellulose and immobilized by baking for 1 hour at 80°C. A 3’-
HaeIl fragment of 658 bp derived from 3’-untranslated region of SB4 was labelled with
[a-32P]dC1‘P (800 Ci/mmol) by random priming and hybridized to genomic DNA blots
in 20% formamide, 6X SSPE (0.75 M NaCl, 0.05 M NaH2P04, 0.005 M EDTA, pH 7.4),
5X Denhardt’s solution, 0.1 % Sodium Dodecyl Sulphate, 0.2 mg/ml Salmon Sperm DNA
at 42°C for overnight. DNA blots were washed in 2X SSC (0.3 M NaCl, 0.03 M Sodium
Citrate, pH7.0) containing 0.1% SDS for 15 min at room temperature followed by a 15

min wash with 0.2X SSC containing 0.1% SDS at 55°C.

Selection and analysis of genomic clones : As mentioned in chapter 111 a rabbit
genomic library was screened with a gel—purified EcoRI-Smal fragment containing the
coding sequence of the previously cloned cDNA encoding the rabbit AlAR, SB4
(Bhattacharya et al. 1993). The cDNA probe was randomly labelled with [or-”PMCI'P
(800 Ci/mmol) by random primer synthesis and approximately 2X 10°recombinants were
screened at moderate stringency (20% fonnamidc at 42°C for overnight). Eight genomic
clones were isolated from the library which were further purified and amplified. As
mentioned in chapter 111, one genomic clone named as clone-1, contained the 5’—UTR,
exon-l and a part of intron-l. In order to isolate the rest of the gene, phage DNA from

the remaining 7 genomic clones were isolated and characterized by restriction

60
endonuclease mapping and Southern Blot analysis, using the cDNA encoding the rabbit

AlAR, the primers originating from different regions of the AlAR cDNA isolated from
rabbit and also the cDNA for the canine A2AR as probes. The A2AR was used as a
probe to distinguish and eliminate clones that would produce stronger signals with A2AR
probes in comparison to AlAR probes. The pattern of hybridization of the restriction
digests of these genomic clones to the different AlAR cDNA fragments, primers
originating from the cDNA, and the canine A2AR cDNA, facilitated in identifying three
overlapping genomic clones namely, clone-4, 5 and 7, containing a 2.3 kb Xhol
restriction fragment which contained the remaining part of the gene encoding the AlAR
cDNA. Clone-7 was used as a representative of the three clones for characterization.

The DNA restriction fragments of clone-7, containing exons and flanking regions
of the gene were subcloned into the M13mp19 or pUC19 vectors and sequenced.
Sequencing was performed by the dideoxy method (Sanger et a1. 1977) with Sequanase
version 2.0 (United States Biochemical) using either double stranded pUC 19 or single
stranded M13mpl9. Sequencing data indicated that genomic clone-7 contained a part of
intron-1 and exon-2. Since a polyadenylation signal was not obtained from the 2.3 kb
fragment of genomic clone-7, direct sequencing of the phage DNA from clone-7 was
performed using the TanyedeoxyTM Terminator Cycle Sequencing Kit (Applied
Biosystems) until a polyadenylation signal was obtained.

A restriction map of the AlAR gene was obtained from Southern blot analysis
using the T7 and SP6 primers as well as primers that were synthesized corresponding to
the 5’ and 3’-ends of the AlAR cDNA. These oligonucleotide primers were end labeled

with [y—SZPMTP (6000 Ci/mmol) and T4 polynucleotide kinase and were used as probes

61

for Southern hybridizations of restriction digests of genomic clones.

The size of intron-1 was determined by restriction digest and Southern blotting
of the genomic DNA isolated from rabbit (Clontech) using fragments from two ends of
intron-1. Digestion of genomic DNA was carried out for 24 hours with 10 pg of DNA
and 100 U of the restriction enzyme EcoRI, blotted onto nitrocellulose, baked at 80°C and
hybridized for 24 hrs using cDNA and genomic probes labeled randomly with [Ot-

32P]dC'I'P (800 Ci/mmol).

Primer Extension Analysis : RNA extracted from rabbit brain (Sambrook et al.
1989) was used to isolate poly(A)* RNA by chromatography on oligo(dT) cellulose
(Pharmacia) and used for primer extension analysis. Two 30 mer oligonucleotides,
WSSZ3, 5 ’-CI‘CGATGCCAATGTAGGCGGCCTGGAAGGC-3 ’ complementary to base
pairs 19 to 48 of the rabbit AlAR gene; and WSS30 5’-AGCCCITCCI‘CI‘G-
CCFAGGTCCCCI‘CCC'I'I‘~3’, complementary to the base pairs -206 to -l77 of the
AlAR gene, starting from the translation start site, were used in primer extension. These
oligonucleotides were end labelled at the 5’-ends with T4 polynucleotide kinase and [y-
32P]ATP (6000 Ci/mmol) to a specific activity of 2X 108 cpm/pg. Labeled primer (2X
10" cpm) was combined with 10 pg of poly(A)+ mRNA in 30 pl of 40 mM Pipes pH 6.4,
containing 1 mM EDTA pH 8.0, 0.4 M NaCl, and 80 % formamide. Samples were
denatured for 10 rrrin at 85°C and then hybridized for overnight at 30°C. The resultant
oligonucleofideszNA hybrids were precipitated with ethanol and resuspended in 20 pl
of 25 mM Tris-Cl, pH 8.3, containing 10 mM dithiothreitol, 37.5 mM KCl, 1.5 mM

MgC12, 40 U RNasein, and 1 mM each of dATP, dCTP, dGTP and dTTP. This was

62

subjected to reverse transcription by addition of M-MLV reverse transcriptase (200 U)
and incubating at 50°C for 60 min following extraction with phenolzchloroform (1:1) and
ethanol precipitation. The pellet was resuspended in 80% formamide sample buffer. The
products were denatured by heating to 95°C and then loaded onto a 5% polyacrylamide
gel containing 7 M urea along with sequencing reactions using the 3.2 kb XhoI fragment

as the template.

C . RESULTS

Gene copy number determination: To determine the organization and the
structure of the rabbit AlAR gene, Southern blot analysis of the chromosomal DNA
isolated from rabbit kidney was performed. As shown in Figure 13, a single hybridization
band was obtained on using a probe from the 3’-end of the rabbit AlAR cDNA, SB4
(HaeII fragment, 658 bp; Figure 17). Digestion of the genomic DNA with BamHI or Pstl
gave rise to a single positive band (2.1 kb for BamHI and 1.5 kb for PstI) that correlated
with the genomic restriction map (Figure 17). A smear of radioactivity was however,
observed at the upper portion of the blot, which may indicate the presence of other
undigested restriction fragment or may be due to the presence of multiple copies of the
gene. Therefore this preliminary observation may indicate the presence of a single copy
of the AlAR gene and the absence of pseudogenes in the rabbit genome, but for further
verification of this data rabbit genomic DNA needs to be digested with other restriction

enzymes and probed with different regions of the cDNA or the gene.

Figure 13 .

63

Analysis of the rabbit AlAR gene by Southern blot
hybridization. High molecular weight rabbit DNA (10
pg/lane) digested with PstI and BamHI restriction enzymes
(lane 1 and 2 respectively), size fractionated by
electrophoresis on 1% agarose gel, transferred to
nitrocellulose, immobilized and probed with rabbit AlAR
cDNA (658 bp HaeII fragment from 3’-UTR), as described
under "Materials and Methods". Sizes of the molecular
weight markers are indicated on the left.

23

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1.0 it"

 

 

 

 

65

Isolation and characterization of genomic clones encoding rabbit AlAR: A
rabbit cDNA clone encoding the AlAR (SB4) has previously been isolated and
characterized (Bhattacharya et al. 1993). With an 860 bp EcoRI/Smal fragment of SB4
we screened a rabbit genomic library, a A-EMBL3 library (Figure 7) and isolated eight
positive clones: clone-l, clone-2, clone-4, clone-5, clone-6, clone-7, clone-2.1 and clone-
2.2. As mentioned before clone-1 was found to contain the 5’ flanking region, exon-1 and
part of intron-1. So in order to investigate which of the other seven clones contained the
rest of the gene encoding the AlAR, we decided to characterize the remaining genomic
clones. The phage DNA isolated from the genomic clones were digested with XhoI and
Southern blot analysis was carried out using the probes depicted in Figure 14. The A2AR
has not been cloned from rabbit, but data suggests that A2AR cloned so far from
different species show a high degree of identity in their amino acid sequences (Figure 3)
within species and also to the AlAR sequence cloned from different species. We
therefore used RDC8, the canine A2AR, as a probe to isolate and eliminate the clones
that would give stronger signals with RDC8 in comparison to SB4.

Results of Southern blot analysis are shown in Figure 15 and 16 and is
summarized in Table III. The results indicated as follows: 1) clone-2 and 6 hybridized
strongly to the canine A2AR cDNA probe as opposed to the rabbit AlAR cDNA, and
therefore may contain the gene encoding the A2AR; 2) clone-2.1 and 2.2 are identical
clones that hybridize more strongly to the rabbit AlAR cDNA probe but did not
hybridize to all the other primers made from the cDNA, and hybridized less strongly to
A2AR cDNA. A 2.2 SacI restriction fragment from clone 2.1 which hybridized to most

of the cDNA probes was sequenced. When this sequence was compared to the SB4

Figure 14 .

The different probes used for characterization of the
genomic clones. 1) EcoRI and SmaI fragment from the 5’-
end and EcoRI and HaeII fragment from the 3’-end of the
rabbit cDNA, SB4; the primers designed from different
regions of SB4, namely, WSS6, WSSl, WSS4, WSSS and
WSSB; and the canine A2AR cDNA, RDC8. The arrow
denotes the position of the intron-1 with respect to the
cDNA sequence.

67

 

 

fit I
o 0
0 0‘3” .1, g!
a -m ‘0 O
- -_ : :0
5°Wl l T‘
l l l J l 3
t 1 1 i
U! U, (n 0‘! m
m to m or a,
as d A 0' u
IZSOI
kb
884

. SB4, 5'-end Eco Ri/Smsl iragmsnt
. SB4, 3'-end Eco Rl/Hae ll tragment
. WSSG, 17 mer, position 80

. WS81, 60 mer, position 286

. W884, 17 mar, position 642

. W885, 17 mar, position 1124
.WSSS, 17 mar, position 1485

. RDCB, the canine A2 adenosine receptor cDNA.

Figure 15 .

68

Southern blot analysis of eight genomic clones. The
phage DNA isolated from each clone was digested with
XhoI, size fractionated on 1% agarose gel, transferred to
nitrocellulose, immobilized and hybridized with different
probes. Blots A-D are from the same gel, each containing
XhoI digest of the eight genomic clones. The clones are
designated on the top of each lanes. The radiolabelled
probes used for high stringency hybridization were the
following region of the cDNA SB4: blot (A) 5’-end EcoRI
and SmaI fragment; (B) 3’-end EcoRI and HaeII fragment
(C) W881 and (D) WSS3. Shown to the left are the sizes
in kb of the molecular weight markers.

69

1 2 4 5 6 7 2.12.2 1 2 4 5 C 7 2.12.2
| .q .
a .- 0.. I-

   

2.22.1765421 1245672.1

23.0—
$.4—
6.0—

4.4—'

 

 

—__—_ -_—— -——.—-———-—--
r— — —

Figure 16 .

70

Southern blot analysis of the genomic clones. The
genomic clones are designated on top of each lanes. The
radiolabelled probes used for each high suingency
hybridization were (A) WSS4, (B) W885 and (C) RDC8,
the canine A2AR. Shown to the left are the sizes in kb of
the high molecular weight markers.

23.0—
9.4—
0.5—

4.4—

4 5

8 7 2.1 2.2

 

Table IH

72

Southern blot analysis of the genomic clones. The
genomic clones were digested with restriction enzyme X hoI,
size fractionated on an 1% agarose gel, transferred to
niuocellulose and probed with different regions of the
AlAR cDNA (SB4) and the canine A2AR cDNA (RDC8)
(depicted on the top pannel). The arrow depicts the position
of intron-1 with respect to the cDNA. The table shows the
probe used for hybridization and the results obtained. (+)
denotes positive signal, (-) denotes negative signals and
(++) denotes very strong positive signal.

73

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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74

sequence, it matched 100% to a part of the third intracellular loop after which it
completely diverged (data not shown). The genomic library was made with a partial
digest of Sau3AI restriction enzyme and a Sau3AI restriction site was observed at the
point of divergence. The data therefore indicated that they could encode for different
adenosine receptor clone or may result due to an artifact of the genomic library; 3) a 2.3
kb Xhol fragment from three genomic clones, namely clone-4, 5, and 7 hybridized very
strongly to both the 5’-end (EcoRI/Smal) and 3’-end (EcoRI/HaeII) fragments and to all
the sequencing primers except for W886, the primer upstream of intron-1, suggesting that
clone-4, 5, and 7 are overlapping clones containing a major portion of the AlAR gene
starting from a part of the first intron. Clone-7 was chosen arbitrarily as a representative
for all three clones for characterization of the AlAR gene. The 2.3 kb XhoI fragment
from clone-7 was subcloned and sequenced. Sequencing data indicated that genomic
clone-1 and 7 contained the entire coding region of the gene in two exons, exon-l present
in clone-1 and exon-2 in clone-7.

Structure of the rabbit AlAR gene: Southern blot analysis and comparison of
the sequence of the rabbit AlAR gene with that of the cDNA (Bhattacharya et al. 1993)
established the restriction map of the AlAR gene (Figure 17). The size of intron-1 was
deduced from Southern blot analysis. Genomic DNA from rabbit was digested with
EcoRI and hybridized to the 3’-end of genomic clone-1 (the 3.2 kb Xhol fragment) and
a 658 bp HaeII fragment from the 3’-untranslated region of the gene, present in genomic
clone-7. The restriction enzyme EcoRI was selected based on the restriction map which
indicated the absence of a restriction site for EcoRI in the intron segment present in the

two genomic clones, clone-l and 7. (Figure 17). As shown in Figure 18 both the probes

Figure 17 .

75

Schematic representation of the rabbit AlAR gene and
cDNA. (A), the restriction map of the two genomic clones,
clone-l and 7. There is no overlap between the two
genomic clones; B, BamHI; E, EcoRI; H, HaeH; P, Bid; 8,
SfiI; X, XhoI. (B), the schematic of the AlAR gene. The
closed boxes represent the exons. The restriction sites
described in A are represented by vertical lines. (C), the
structure of the rabbit AlAR cDNA. The region encoding
the membrane spanning domain are represented by closed
boxes and are numbered. The region encoding the non-
membrane spanning domain are represented by open boxes.
The translation start site (ATG) and the termination point

' (GAC)are indicated.

76

3 Chi

 

 

 

 

 

 

 

(200 C<p< .0
2.3 .22 .m
J _ d
h 2.20
p 2.0.0
— _ _ d _ E—
u u a 3:8... ...—0.0
= fl — _ 0.5—0:00 .<
3 I e e

Figure 18 .

77

Southern blot hybridization analysis to determine the
size of intron 1 of AlAR gene. 10 pg of rabbit genomic
DNA digested with EcoRI were probed with the 5’-end of
intron-1 (lane2) and the 3’-end of exon-2 (lane 1). Sizes in
kb of the molecular weight markers are indicated on the
left.

78

23K->

9.4K->

6.5K->

4.3K,

2.3K'>

2.0K ">

79
hybridized to fragments of different size. The 3.2 kb XhoI fragment hybridized to a 14

kb fragment and the 658 HaeII fragment hybridized to a 25 kb EcoRI fragment.
Therefore by comparing to the restriction map (Figure 17) and by simple addition and
substraction of the intron size obtained from the two genomic clones, namely clone-l and
7, the size of intron-l was calculated to be either equal to or greater than 34 kb. For
better approximation of the intron size, the rabbit genomic DNA needs to be digested
with restriction enzymes that recognizes eight bases, as NotI or SfiI and probed with
restriction fragments originating from 5’ and 3’-ends of the intron-l, in order to obtain
a single hybridization band with both the probes. Other G-protein linked receptor genes
also have been known to contain introns of great length whose exact sizes are not
determined. Some examples includes the human endothelin-A receptor (intron-2, >16 kb;
intron-3 >11 kb;; Hosoda et al. 1992) and the Substance P receptor (intron-1 > 15 kb;
intron-2 >23 kb; Hershey et al. 1991). As shown in Figure 19, the AlAR gene is
composed of two exons and the splice site agrees with consensus donar/acceptor (GT/AG)

splice site sequence (Breathnach and Chambon 1981).

Analysis of the 3’-flanking sequence of the rabbit AlAR gene: A potential
polyadenylation sequence in the 3’—end of the gene was observed on sequencing the
genomic clone-7. This feature has been reported to represent conserved areas for
transcription termination and 3’-end processing (Birnstiel et al. 1985). However, on
comparing the sequence obtained from the 3’-end of the gene to the cDNA revealed that
the 3’-end of the gene does not match with the cDNA sequence. This region is shown

in bold in Figure 19. Since polyadenylation signal was obtained at the 3’-end of the gene,

Figure 19 .

80

The nucleotide and the amino acid sequence of the
rabbit AlAR gene. Exon sequences are indicated in
uppercase. The 3’-flanking region that do not match with
the cDNA are indicated in bold. The polyadenylation signal
is underlined.

70
15

140
38

210
61

280
85

350
108

379
117

449
141

519
164

589
187

659
211

729
234

799
257

869
281

939
304

1009

327
1079
1149
1219
1289
1359
1429
1499
1569
1639
1709
1779
1849
1919
1989
2170

81

TTCTGCTGAT GTGCCCAGCC TGTGCTCGCC ATGCCGCCCT CCATCTCGGC
M P P S I S A

GCATCGAGGT GCTCATCGCG CTGGTCTCGG TGCCAGGGAA CGTGCTGGTG
I E V L I A L V S V P G N V L V
CCAGGCACTG CGGGACGCCA CCTTCTGCTT CATCGTGTCG CTGGCAGTGG
Q A L R D A T F C F I V S L A V A

CTGGTCATCC CGCTGGCCAT CCTCATCAAC ATCGGCCCCG AGACCTACTT
L V I P L A I L I N I G P E T Y F

CCTGTCCTGT CCTCATCCTC ACCCAGAGCT CCATCCTGGC CCTGCTGGCC
C P V L I L T Q S S I L A L L A

CCGCGTCAAG ATTCCTCTCC thgagtcca ----------
R V K I P L R

(INTRO. 1)
GCAGTGGTGA CGCCGCGCAG GGCGGCGGTA GCCATCGCCG GCTGCTGGAT
A V V T P R R A A V A I A G C W I

TGACGCCCAT GTTCGGCTGG AACAACCTGC GGGAGGTGCA GCGGGCCTGG
T P M F G W N N L R E V O R A W

ATGGAGTACA
M E Y M

GGAGCCGGTG ATCAAGTGCG AGTTCGAGAA GGTCATCAGC
E P V I K C E F E K V I S

ACCTGGAGGT
L E V

GTGTGGGTGC TGCCCCCGCT
V W V L P P L

ACTGCTCATG GTCCTCATCT
L L M V L I Y

CAAGTACTAC
K Y Y

AGCTCAGCAA GAAGGCGTCG GCCTCCTCCG GAGACCCGCA
L S K K A S A S S G D P H

TGGCTGCCTC
W L P L

CAAGTCGCTG
K S L

GCCCTCATCC TCTTCCTATT CGCCCTCAGC
A L I L F L F A L S

TCTACACCGC
Y T A

ACCCTCTTCT
T L F C P S C

GCCCATCCTG CCAGAAGCCC AGCATCCTCG
O K P S I L V

CAAGTTCCGG
K F R

GTCTACGCCT TCCGCATCCA
V Y A F R I H

ACTCGGCCAT GAACCCCATC
S A M N P I

GGCGACGAGG
G D E D

CGCCGGCGAC
A G D

GAACGACCAC
N D H

TTCCGCTGCC GGCCCGCACC
F R C R P A P
AACGACTAGG CGCTGCCCTC TGCTCTTCCA GCCCAGCCGG TCCTCCCCTC
N D ‘

CTTCCCAAAG
GGAGCGTCTT
TGGGCCCCGG
GGCAGGGGTC
CACGTGGGGA
CAGGACTCAA
GACTCAGGAT
CTGCACCAGC
AGCACTGAGC
ATACTGCCCA
GAGGGGAAGG
GGICTGGCCG
TGGGACGCTC
CICIGGGCCT
IGICCGGACC

GGGCTCGGAG
CCACCCCAGG
GGCCGTGTGG
ATGCAGAAGA
TTCTCTGCGC
CAAGGACTCT
AGAGTGGGTC
TGGAGTGGTG
CTGGGCGGCG
TGCCAACATG
TGCTCACCGG
TCCACTGGCC
ATTIAGGGA!
CTTCTGTTGG

TGGGGAGGGA
AGTGAGCAGG
GCGGATGAGG
CAGGATGCTC
AGCAGAAGGG
GCCCAGGGCC
CAAAAATCAT
GGTGCTGGCC
CCTGGCTGTG
CTGCCTGCCA
GGCACCTGCG
CTTLGATGCC
CTGGGAIIGG
IGGCGGGTGT

GAGTGCACCC
GAGCCCACCC
GGAGGCTGGG
TGCTTGTTGG
GGGGGTGTCT
GGAGCCTCCA
CGCACTTTGG
CCTTGGACTG
CCTCCTTGCC
ATGGAGGGGG
GTCCCTGCAG
GAGACCGCTC
TTGACAGIIG
GCTGGILGGG
CTGGCGGAGA

CTGCACCCCG
ACCTGCCTGA
CGTGCAGAGG
GGTGCTGGTG
TGGCCTTGCT
TCCCCACCTC
AGAGGCAGAA
TGGGCAGGGG
CTGCACCTCG
AACCCAACCA
GCTTGGTCGG
TCGAGGAAGG
GICTCCIGGC
GTTCIIGICC
1C1!

CTTCCAGGCC
F O A

ATCTGGGCCG

I W A V

CTGACGTGGC
D V A

CCACACCTGC
H T C

ATCGCCGTGG

I A V D

-—--gccccg

CCTCTCGCTC
L S L

GCGGCCAACG

A A N G

TGGTGTACTT
V Y F

CTTCTACCTG
F Y L

GGCAAGGAGC

G K E L

TGCACATCCT
H I L

CATCTTCCTC
I F L

GTCACCTTCC

V T F L

ACCTCCCGGA
L P E

CCCTCCGCCC

ACCCGCGCCC
GCCCCCGTTC
CTGCCTGGAC
TCCGGGAGGG
GCTCAGAAGC
GTGCCAGGTG
TTCTGACCTG
TCACACAGCC
CCAGCCGGAT
GCCCCCCCCA
GGGTGGGAAG
CAAAGCTCAG
CCTCGCCTGC
GIGGOCATGI

GCCTACATTG
A Y I G

TGAAGGTGAA
K V N

CGTGGGCGCC
V G A

CTCATGGTGG
L M V A

ACCGCTACCT
R Y L

cagGTACAAG
Y K

GTGGTGGGCC
V V G L

GCAGCGTCGG
S V G

CAACTTCTTC
N F F

ATCCGCCGGC
I R R 0

TGAAGATCGC
K I A

GAACTGTGTC
N C V

ACGCACGGCA
T H G N

TCAAGATCTG
K I W

AGAGAAGCCC
E K P

CAAGGGGCCC

CAGGGCGCGA
AAGGGGTGGG
TTCTGCGCCA
AGGGGAGCAG
AGCTAAGGGA
CCAGGCGGCC
GGCTTTTATT
ACCAGGCGGC
GGCACCGTGT
GGAAGACGAA
TGGCGGGCAI
GCTGGGGCTT
IGCCTCTGTG
GGCIALIIIA

82

it is highly unlikely that there is any artifact in the genomic clone. On the other hand, as
reported before no polyadenylation tail was observed at the 3’-end of the cDNA although
the library from which the cDN A clone was isolated was designed by reverse transcribing

the mRNA using oligo dT primers. Therefore it is likely to be an artifact of the cDNA

library.

Analysis of the transcription start site of the rabbit AlAR gene: The
transcription initiation site of the AlAR gene was mapped by primer extensions using
two primers WSS-23 (PE-1, 19 to 48) and WSS-BO (PE-2, -206 to -117) (Figure 21). In
primer extension with the rabbit brain poly(A)+ RNA, WSS-23 revealed four signals at
-78, -106, -268 and -322 and WSS-3O revealed two strong signals at -268 and -322
(Figure 20). No bands were observed in the tRNA control lanes. WSS-23 produced a
single large extended product and a smaller product (Figure 20). Since the primer WSS-
30 produced a single extended product, this probably reflects an incomplete extension by
primer WSS-23. RNase protection assay was used to confirm the location of the
transcriptional start site determined by primer extension using a cRNA produced by in
vitro transcription of a plasmid constructed by subcloning the region containing the 5’-
end of the AlAR gene from the genomic phage clone-l into the plasmid Bluescript
KS(+) using T7 RNA polymerase. Poly(A)*RNA from rabbit brain was used to hybridize
to the cRNA, tRNA and yeast RNA. Yeast RNA and tRNA were used as negative
controls. RNase protection assay with the cRNA produced protected fragments that were
not only different from the extended products but were also present in the negative

control lanes. This reflects that either the cRNA had secondary structure and therefore

Figure 20 .

83

Primer extension analysis of the transcription start site
for the rabbit AlAR. Primer extension using
oligonucleotides WS823 (PE-l, 19 to 48) and WSS30 (PE-
2, -206 to -177). The end labelled primers were hybridized
to 10 pg of poly(A)+ RNA from the rabbit brain and
extended with reverse transcriptase (lane 2). 10 pg of tRNA
was used as negative control (lane 1). The primer extended
products were analyzed on a 5% polyacrylamide denaturing
gel and their sizes determined by comparison with the
dideoxy-termination products from sequencing reactions run
in adjacent lanes, of the rabbit AlAR gene using the same
primer.

84

mccrccom Trrchcrcx_
P1 «04 . 0 u.
.14.. .

stifle? n ....

 

AC(}T

  

 

 

 

 

 

 

 

. 4
_CCCTMGGG_ATTGMGTCA_ —ACCCGMGGGG_AGGGTGGG
__ _ . _ _
2
“I. ' . ' 9 sill-....

AC(3T

 

W
1

h:

85

was protected or the polymerase was transcribing nonspecifically from the opposite
strand.

When the transcription start site for other G-protein linked receptors were looked
for, it was observed that transcription from most G-protein receptor genes originated from
a specific point. Some of the examples include the human endothelin-A receptor, the
transcription start site of which determined by primer extensions is 502 bp upstream of
the translation start site (Hosoda et al. 1992); the bovine rodopsin has a transcription start
site at 96 bp upstream of the methionine initiation codon (Nathans and Hogness 1983);
and the transcription initiation site of the substance P receptor as identified by solution
hybridization-nuclease protection assay, is at 576 bp upstream from the translation start
methionine (Hershey et al. 1991). For LH receptor, however, primer extension studies
indicate the presence of multiple transcriptional initiation sites (Tsai-Morris et al. 1991).
AlAR transcripts as determined by primer extension appears to be heterologous and four

sites were identified.

Characterization of the 5’-end of the rabbit AlAR gene: The sequence of
approximately 1000 bp of the 5’-flanking region upstream of the translation start site was
determined and is presented in Figure 21. As shown in the figure, the 5’-flanking region
of the rabbit AlAR gene was observed to have a high G+C content (67.5%). A TATA
or a CAAT box sequence, which determines the specificity of mRNA synthesis initiation
by RNA polymerase II, was not found in the 5’-flanking region of the AlAR gene
(Breathnach and chambon 1981; McKnight and Tijan 1986). Therefore the heterologous

transcription initiation observed on primer extension analysis may have resulted from a

Figure 21 .

86

Nucleotide sequence of the 5’-flanking region of the
rabbit AlAR gene. The position of the oligonucleotide
used for primer extensions are underlined. The transcription
start site as indicated by the primer WSS23 and WSS30
(underlined) are marked by an astericks. The putative
regulatory sequences are marked. Numbering begins with
the translation initiation codon.

87

-1097 arrorcacrc xocroccoco ccooooocoa.cooaoococo oococccoao
-1047 rcoaorcccc occoocoacc accrcccroo.acccaoccoo cccroorrrc
-997 rccaaocaao.aococaocoa.ocoocroccc aaoocorcoa.oocorroooo
GEE/Rev

- 947 acccr-ooraoccoo oxococcoco oocrrrooro
-397 accrrooo'ro no'rcromcc rccorroocc ccooooacumzoo
-a47 reocxooxoc CGGC‘I‘GCAGG nococc'roo nrroccccc'r roooocaooc
-797 oarooocnoo oorcocrocc aoroc'rooao cooo'rccooo GATGGCTAAG
-747 aomoor'r onoccmorc: roaoorooo'r ooococoooo caoocoroco
-597 ococacocn ccoooroooo corccacooc croocc'ru'r cccoo'rcoca
- 6 47 oc'rccocaoa cccacocc'ro ccncoccooc ccoaoo'roco monooooc
-597 ocrnocrcr'r runocorc cooocaoaoc crc'roccocn common—n
-547 ocrronnoo cooooc'rooo aooocroc-Smaoccro oaoooccaro
-497 macro-choc occcoonocc caocoo'rocc roraocaocc coooaocrcc
-447 oooc'rccooo roc'rcomo r-r'roooccco oocrcroooc cccrc'rocro
-397 occnocaooc aooarooroo roaocrzocc'r ocaroo'roc'r c'ro'rooococ
~347 ooomocao noccxoocro ooooacccc'r; oroooo'ro'ro rororororo
- 2 97 romoonc rorcrcwormcx caoreao'roc aooaoaoaor
-247 oc'r'oc'ram rmo'r'rocro AATGGAACCT C‘I‘GGGGATGA ow
- 197 Woomococ caocaocc'ro oofaoocacr
-147 oocao'roccrt aoroooocxo ccaoruccca oaocao'rooo crocccccrc
-97 cmcoaoocc ooccrcccwc croaccacao o'rocccoccr
-47 corococcrc coroccorro rocroaroro cccaoccror GCCCGCCatg

ccoccctcce tetcoWWmococt

88
lack of a well defined TATA or a CAAT box motif. However, there are a few G + C-rich

regions that contains a GC box homologous sequence, GGGCGG in its core (5 out of 6
base pair matches with the consensus sequence). The GC box sequence which binds to
the transcription factor SP1, when found in multiple copies can direct transcription
initiation from a TATA less promoter (Smale et al. 1990; Miwa et al. 1987). In human
endothelin-A receptor the 5’-flanking region lacks a typical TATA box but contain a
potential SP-l binding site which is thought to be responsible for initiation of
transcription (Hosoda et al. 1992).

The 5’-flanking region of the gene also contained several sequences with
homology to other known transcriptional regulatory elements. Consensus, potential cis-
acring elements for MyoD (TCI‘CACACCI‘GAGTAA; Lassar et a1. 1989) is present at
positions -285 to -270. Sequences with homology to the AP-2 consensus regulatory
binding site [(T/C)C(G/C)CC(C/A)(G/C)(G/C)(G/C)] are also located at the positions ~78
to -70 and -859 to -850 (Imagawa et a1. 1987). A consensus sequence for binding of the
transcription factor SP1 (GGGCGG) is located at position -519 to -514 (Li et al. 1991).
There are however presence of reverse SP—l sequences at positions -1040 to -1035 and
at -54 to -49. When the 5’-flanking region was searched for hormone responsive element
consensus sequences, a sequence in reverse orientation to the glucocorticoid responsive
element (GGGGCAGAACAGCI‘CC; Lerner et al. 1980) was found between position

-942 to -927.

89
D . DISCUSSION

In the present study the organization, structure and the copy number of the rabbit
AlAR gene have been clarified. Genomic clones containing a rabbit AlAR gene have
been isolated using cDNA as a probe. The present study also demonstrates that the rabbit
genome may contain a single copy of the AlAR gene, and that the gene contains only
one intron in the coding region (Figure 17).

The genes for most of the G-protein coupled receptors lack introns within their
coding regions (Kobilka et al. 1987), which has greatly facilitated isolation of many
receptor genes (Bonner et al. 1987). Subsequent studies have however revealed the
presence of introns in the coding region of certain G-protein coupled receptor genes. This
include the genes for the dopamine D2, D3 and D4 receptors (Grandy et al. 1988;
Sokoloff et al. 1990; Van Tol et al. 1991), the tachykinins (Hershey et al. 1991;
Takahashi et al. 1992), the opsins (Nathans and Hogness et al. 1984), the leutinizing
hormone receptors (Tsai-Morris et al. 1993) and the endothelin receptor (Hosoda et al.
1992). The AlAR gene elucidated in the present study belongs to the family of intron
containing G—protein linked receptor genes. The exon/intron splice sites of the G-protein
coupled receptor genes are compared in Figure 22. Comparison data shows that AlAR
gene is unique from the rest of the G-protein linked receptor gene, in its exon/intron
arrangement, since it has only one intron that separates the coding region. The genes for
the ET-AR, SKR, SPR, NKR, D2R, and D4R all have an intron at the same location
immediately after the third membrane spanning domain. This finding may suggest that

the genes for these receptors may have originated from the same ancestral intron-

Figure 22 .

90

The locations of the exon/intron splice sites of G
protein-linked receptors are compared. The exon/intron
splice site are compared between the cDNAs for the AlAR,
dopamine D2 receptor (D2R), endothelin receptor (ET-AR),
substance K receptor (SKR), substance P receptor (SPR),
neuromedin K (NKR), opsins, and luteinizing hormone
receptor (LHR). The seven transmembrane spanning AlAR
is shown schematically in the first line. The closed boxes
represent the membrane spanning domains. The linear lines
represents the cDNAs for G protein-linked receptors. The
vertical lines indicates the exon/intron splice sites and are

, numbered.

91

 

 

 

 

 

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92

containing gene whereas the gene encoding the AlAR may belong to a different gene
family.

Multiple initiation sites for rabbit AlAR transcription were observed on primer
extension analysis. Sequence analysis of the 5’-end demonstrated the absence of a
classical TATA box, which plays a role in directing the start of transcription to a specific
site. The absence of well defined TATA or CAAT boxes is consistent with the
heterogeneity of the transcription initiation sites. It would be interesting to determine
whether or not any stimulation could induce transcription predominantly from one of the
four initiation sites.

The 5’-flanking region of the rabbit AlAR gene was sequenced and all sequence
compilation, alignment and searches for the putative regulatory elements were performed
using the Wisconsin Genetics Computer Group Programs. A sequence reverse in
orientation to the glucocorticoid responsive element was found in the 5’-flanking region.
Glucocorticoids have been shown to upregulate the AlAR (Gerwins and Fredholm 1991).
Glucocorticoids affect gene expression via activation of the glucocorticoid receptor, which
in turn, interacts with glucocorticoid responsive sequences to increase or decrease the
transcription of the hormone responsive gene. It is therefore tempting to speculate that
glucocorticoids might be involved in the modulation of AlAR gene expression. To
determine if this mechanism is physiologically relevant, transfection assays with plasmids
containing the appropriate flanking sequences and reporter genes will be required.

Other possible transcription factor binding site consensus sequences include the
SP1 binding site, an AP2 binding site and MyoD binding site. Particularly interesting is

the presence of the alternating purine and the pyrimidine nucleotides which are reported

93
to have the potential to form Z-DNA (Nordheim and Rich 1983). It is noted that variation

in the length of the repeating units among individuals causes genetic polymorphism
(Weber and May 1989). Therefore the CA repeat turned out to be an abundant source of
genetic markers. The sequence (GT),8 in the AlAR gene could be used as a polymorphic
DNA markers to determine whether or not the AlAR gene is linked to any genetic
diseases.

In, summary, several putative regulatory sequences have been identified in a rabbit
AlAR gene. To determine the significance of these sequences will require further in viva

analysis of their enhancer activities.

V . SUMMARY AND CONCLUSIONS

The research presented in this dissertation provided the following results:

1. The deduced amino acid sequence of rabbit AlAR shows significant homology to

AlARs cloned from canine, rat, bovine and human.

2. Rabbit AlAR belongs to a family of intron-containing G-protein linked receptor
genes.

3. Preliminary studies indicate that the rabbit genome may contains a single copy of
AlAR gene.

4. The transcription start site for the rabbit AlAR gene as analyzed by primer

extension, appears to be heterogenous.
5. Several putative regulatory elements are present in the 5’-flanking region of the

gene.

The deduced amino acid sequence of rabbit AlAR was shown to possess a
significant homology to AlARs from canine, rat, bovine and human. The encoded
proteins are all identical in molecular mass with 326 amino acid except for rabbit AlAR
which has 328 amino acid. The five AlARs cloned so far are 92% identical in their
overall amino acid structure. The identity is more pronounced in the transmembrane
domains, which in G-protein coupled receptors are known to be involved in ligand
binding.

The cloned AlAR from rabbit, will help to evaluate the structure, function and

94

95

regulation of the A1 adenosine receptor system at the biochemical and molecular
biological levels. The expression of the AlAR clone in a cell system, will help to resolve
the controversial issue of whether or not a single receptor is capable of interacting with
multiple effector systems.

The gene encoding for the rabbit AlAR elucidated in the present study belongs
to the family of intron-containing G-protein linked receptor genes. Comparison of AlAR
gene structure to other intron-containing G-protein linked receptor gene revealed that the
exon/intron arrangement of the A lAR gene is unique from the rest, since it possesses only
one intron that separates the coding region. The genes for the endothelin receptor,
substance K receptor, substance P receptor, and dopamine receptor have multiple introns
and they all have an intron at the same location immediately after the third membrane
spanning domain. This may indicate that the genes for these receptors may have
originated from the same ancestral intron-containing gene, whereas the gene encoding the
AlAR may belong to a different family.

Introns are intervening sequences that are spliced out when heteronuclear RNA is
processed to mRNA. Occasionally errors arises due to splicing which result in splice
variants which may be similar in structure but different in function. One such example
is the dopamine D2 receptor. The two splice variants of the D2 dopamine receptor varies
in 81 nucleotides, and they have the same pharmacological character. It is however not
known if they have any differences in function. The gene structure for AlAR will help
to classify if subtypes of AlAR may arise due to differential splicing of the same gene.

The present study also indicated that the rabbit genome contains a single copy of

the AlAR gene which is a necessary first step for studies concerning AlAR gene

96

regulation and expression.

Analysis of the transcription start point by primer extension has indicated the
heterogeneity of its transcription. Sequence analysis of the 5’-end demonstrated the
absence of a classical TATA box, which has the role of directing the start of transcription
to a specific site. The absence of well defined TATA box is consistent with the
heterogeneity of the transcription initiation sites. It would be interesting to determine
whether or not any stimulation could induce predominant transcription from one of the
four initiation sites.

The 5’-flanking region of the rabbit AlAR gene was sequenced and all sequence
compilation, alignment and searches for the putative regulatory elements were performed
using the Wisconsin Genetics Computer Group Programs. A sequence reverse to the
glucocorticoid responsive element was found in the 5’-flanking region. Other possible
transcription factor binding site consensus sequences include, SP1 binding site, AP2
binding site and MyoD binding site. In summary, several putative regulatory sequences
have been identified in a rabbit AlAR gene. To detenrrine the significance of these
sequences will require further in viva analysis of their enhancer activity.

Cloning and characterization of the AlAR gene is therefore the first necessary step

in evaluating the AlAR function and regulation at the biochemical and molecular level.

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