CHARACTERIZATION OF NOVEL G PROTEIN-COUPLED

RECEPTOR GENES AND THE NOVEL LIGAND APELIN

by

Dennis K. Lee

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Pharmacology

University of Toronto

O Copyright by Dennis K. Lee 1999

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Characterization of Novel G Protein-Coupleci Receptor Genes and the Novel Ligand Apelin

Dennis K. Lee, MeSc. 1999 Department of Pharmacology

University of Toronto

G protein-coupled receptors (GPCRs) make up the large family of integral

membrane bound proteins which mediate the signaling of extracellular stimuli to

intracelMar responses via activation of heterotrimeric G proteins and their subsequent

interaction with effector proteins. GPCRs have been implicated in a number of

physiologicd Functions including behaviour. homeostasis, cognition, appetite, and drug

addiction. This thesis describes the molecular characterization of the human and rat

genes encoding apelin, the cognate ligand for the APJ receptor. Human and rat DNA

sequences encoding apelin were retrieved by a search of the GenbanP databases, their

full length sequences cloned and used as probes for expression analyses. Comparative

sequence and tissue distribution analyses revealed both apelin and the APJ receptor to

resemble the angiotensin II peptide and the angiotensin Il receptors, suggesting roles in

similar physiological systems. In addition, a degenerate PCR strategy, database

searching and the patent literature revealed DNA sequences encoding novel GPCRs,

namely the thyrotropin-releasing hormone receptor TRH-R2, orphan GPCRs GPR54,

GPR57, GPR58, GPR6 1, GPR62 and a pseudogene, vGPR57. With the initiai discovery

of each of these sequences, full length GPCR-encoding sequences were detennined and

used for mRNA distribution analyses by northern blot and in situ hybridization. In

addition, novel GPCR-encoding genes were localized to chromosomes by fluorescence

in situ h ybridization (FISH) and expressed for pharmacological characterization.

ACKNOWLEDGEMENTS

1 would Iike to thank my supervisors, Dr. Brian F. O'Dowd and Dr. Susan R.

George, for their support and guidance and for the opportunity to pursue my degree under

their direction. 1 would also like to express my gratitude to Tuan Nguyen for his work on

cloning rat apelin cDNA, TRH-R2, GPR54 and yGPR57, Regina Cheng for her work on

the northern blot and in situ hybridization anaIyses, and Yang Liu for her work on

isolating and sequencing the GPCR search clones- I would also thank Dr- Adriano

Marchese, Marek Sawzdargo, Ziedong Xie, Teresa Fan and the members of the O'Dowd

and George lab, both past and present, for their generous and amiable counsel, technical

support and our collaborative efforts at work and play. In addition, 1 wish to thank Brett

Clayton and acknowledge his excellent work on the three-dimensional schematic of the

GPR54 receptor. Finally, 1 would Iike to thank my family and friends for their love and

support, in hopes that 1 have returned both in kind-

PUBLICATIONS

This thesis contains work that has been published in or submitted to the scientific literature:

A ~ e h and APJ Lee, D.K., Cheng, Ra, Nguyen, T., Fan, T., Kariyawasam, A.P., Liu, Y., Osmond, D.H., George, S.R., and O'Dowd, B.F. "Charactenzation of apelin, the ligand for the APJ receptor", J. Neurochem. In press.

TRH-R2 O'Dowd, B.F., Lee, D.K., Huang, W., Nguyen, T.. Cheng, R., Liu, Y., Wang, B., Gershengorn, M C , and George, S.R. "TRH-R2 exhibits simiiztr binding and acute signaling but distinct replation and anatomic distribution compared to TRH-RI". Mol. Endocrin. In press.

GPR54 Lee, D.K.. Nguyen, T., O'Neill, G.P., Cheng, R., Liu, Y., Howard, A. D., Coulombe, N., Tan, C.P., Tang-Nguyen, A.-T., George, S.R., and O'Dowd, B. F. "Discovery of a receptor related to the galanin receptors". FEBS Lett. (1999) 446, 103- 107.

vGPR57. GPR57. GPR58 Lee, D.K., Lynch, KR., Nguyen, T., Xie, Z., Cheng, R., Saldivia, V.R., Liu, Y., Liu. I.S.C.. Heng, H.H.Q., Seeman, P., George, S.R., O'Dowd, B.F. and Marchese. A. "Cloning and characterization of additional members of the G protein-coupled receptor farnily". Submitted.

OTHER PUBLICATIONS

Lee, D.K., Nguyen, T., Porter, C.A., Cheng, R., George, S.R., and O'Dowd, B.F. 'TWO related G protein-coupled recepton: The distribution of GPR7 in rat brain and the absence of GPR8 in redents". Brain Res. Mol. Brain Res- (1999) 71,96403.

Sawzdargo, M., Nguyen, T., Lee, D.K., Lynch, KR., Cheng, R.. Heng, H.H.Q., George, S.R.. and O'Dowd, B.F. "Identification and cloning of three novel human G protein- coupled receptor genes GPR52, yGPR53 and GPR55: GPR55 is extensively expressed in human brain". Brain Res. Mol. Brain Res. (1999) 64, 193-198.

TABLE OF CONTENTS

TITLE PAGE

ABSTRACT

ACKNOWLEDGEMENTS

PUBLICATIONS

TABLE OF CONTENTS

LIST OF ABBREVIATIONS

LIST OF TABLES

LIST OF FIGURES

PAGE

I

II

III

IV

v

IX

X

XI

1.0 INTRODUCTION

Overview of Introduction 1

The GPCR family 2

Agonists for GPCRs 5

Discovery of GPCR Genes by Molecular Cloning 6

Discovery of GPCR Genes by Database Searches 13

Reverse Pharmacology: Assigning Ligands to Novel GPCRs 16

Researc h Objectives 22

2.0 MATERIALS AND METHODS

2.1 Materials

2.1.1 Chernical Reagents

2.1.2 Enzymes

2.1.3 Isotopes and Ligands

2.1.4 Oiigonucieotides

2.1.5 Kits

2.1.6 CeU lines, Plamnids and DNA Libraries

2.2 Methods

Computationd DNA and Protein Sequence Andysis

PCR: Polymerase Chain Reaction

GenbankTM Database Searches

Subcloning of PCR Products

DNA Minipreparation, Restriction Digestion and

Electrophoresis

DNA Sequencing

DNA Probe Extraction and Radiolabelhg by Nick

Translation

Genomic and cDNA Library Screening

Bacteriophage DNA Preparation

2.2.10 Southern Blotting

2.2.11 Northern Blotting

2.2.12 In Situ Hybridization

2.2.13 Chromosomal localization

2.2.14 Creation of an Intronless GPR58 Receptor Gene

Expression Construct

2.2.15 Maxi DNA Preparation

2.2.16 Calcium Phosphate TC811Sfection

2.2.17 Membrane Preparation and Binding Studies

3.0 RESULTS

Determination of the Human Apelin Genomic Structure

and Cloning of Rat Apeiin cDNA

mRNA Tissue Distribution of Apelin and the APJ Receptor

Discovery and Cloning of the TRH-R2 Receptor Gene

mRNA Tissue Distribution of the TRH-R2 Receptor

Discovery and Cloning of the GPR54 Receptor

mRNA Tissue Distribution of the GPR54 Receptor

Discovery and Cloning of the GPR57 and GPR58 Receptor

Genes and a Pseudogene F R 5 7

GPR57 and GPRSS Expression

Attempted Pharmacological Characterization of the

GPR58 Receptor

Chromosomal Locaîization of the GPR58 Receptor Genes and 74

Pseudogene yGPR57

Discovery and Cloning of the GPR61 and GPR62 Receptor 74

Genes

mRNA Tissue Distribution of the GPR61 Receptor 79

4.0 DISCUSSION

4.1 Apelin: Characterization of the Endogenous Peptide 80

Ligand for the APJ Receptor

4.2 TRH-R2: Discovery and Characterization of a Second 82

GPCR for Thyrotropin-Releasing Hormone

4.3 GPR54: Discovery and Characterization of a NoveI GPCR 85

related to the Gala& Receptors

4.4 GPR57 and GPR58: Discovery of a Novel Subfamily of 86

GPCRs

4.5 GPR61 and GPR62: Discovery of a Novel S u b f d y of 88

GPCRs

4.6 Conclusions 89

5.0 REFERENCES 91

LIST OF ABBREVIATIONS

ATP BLAST bp cDNA CHO DAPI DNA EST FISH GPCR(s) G protein G U ( s ) G S S hr HTGS MAGE IPTG kb LB min mRNA NCB 1 nr oGPCR ORFW PCR PKA PKC RNA P m SDS sec STS TM T m UTR(s ) x-gal

adenosine triphosphate basic local aiignment search tool base pairs complementary DNA chinese hamster ovary 4' '6'-diamidino-2-pheny lindole deoxyribonucleic acid expressed sequence tag fluorescence in situ hybridization G protein-coupled receptor(s) guanine nucleotide regulatory protein G protein-coupled receptor kinase genomic survey sequences hours high throughput genomic sequence Integrated Molecular Analysis of Gene Expression isopropylthio-B-D-galactosidase kilobase pairs Luria-Bertani media minutes messenger RNA Nationai Center for Biotechnology Information non-redundant orphan G protein-coupled receptor open reading frame(s) polymerase chain reaction CAMP-dependent protein kinase A protein kinase C ribonucleic acid revolutions per minute sodium dodecyl sulfate seconds sequence tagged sites transmembrane domain thyrotropin-releasing hormone untranslated region(s) 5-bromo-4-chloro-3-indolyl-B-D-galactosidase

LIST OF TABLES

PAGE

Table 1 Rhodopsin family of GPCRs with known endogenous ligands 7

Table 2 Orphan GPCRs 18

LIST OF FIGURES

FIGURE PAGE

The G protein-coupled receptor: Structure and Conserved Residues

Alignment of oGPCRs used for design of degenerate oligonucleotides

ApeIin amino acid sequence alignrnents and the human apelin genomic structure

Northem blot analyses of human and rat preproapelin mRNA

In situ hybridization analyses of rat preproapelin mRNA

In situ hybridization analyses of rat A H M A

Amino acid sequence aiignment between rat TRH-R 1 and TRH-W

Northem blot analyses of rat TRH-R2 mRNA

In situ hybridization analyses of rat TRH-R2 mRNA

Schematic representation of the GPR54 receptor

Nonhern blot anaiyses of rat GPR54 rnRNA

In situ hybridization of rat GPR54 mRNA

Sequence of the vGPR57 pseudogene

Amino acid sequence aiignment between GPR57, GPR58 and related receptors

FISH analyses for iyGPR57 and GPR58

Amino acid sequence alignment between GPR6 1, GPR62 and related receptors

Northem blot analyses of human and rat GPR6 1

1.0 INTRODUCTION

1.1 Overview of Introduction

G protein-coupled receptors (GPCRs) represent the largest farnily of integral

plasma membrane proteins mediating the signd transduction of external stimuli to the

interna1 environment of cells. As a result of its size and diversity, the GPCR family is

collectivel y the largest group of targets for today ' s pharmaceutical reagents and research

(Stade1 et ai., 1997). Over the last two decades, molecular cloning of genes encoding

GPCRs has greatly facilitated the understanding of these signal transduction systems by

permitting in vitro expression and investigation of GPCRs. In addition, moIecular

cloning has resulted in the discovery of most of the GPCRs known today, which has

provided an abundance of novel pharrnaceutical targets for exarnination and an impetus

for the identification of many as yet undiscovered transmitter systems in the brain and

periphery .

This thesis describes the discovery and characterization of six novel genes

encoding GPCRs and a GPCR pseudogene, as well as the characterization of apelin, a

novel peptide agonist for the GPCR, APJ. An introduction to the GPCR family is first

presented followed by a brief exarnination of the diversity of GPCR ligands. This is

followed by an account of successful strategies, both past and present, in cloning novel

GPCRs and the current efforts in assigning ligands to novel GPCRs. The introduction

will conclude with a final word on research objectives.

1.2 The GPCR family

The GPCR farnily contains a diverse group of cell-surface mediators of signal

transduction, its size exceeding other cell-surface protein receptors including the tyrosine

kinase receptors, guanylyl cyclase receptors and ligand-gated ion channels. The GPCR

family has nearly 300 unique members (not including the odorant GPCRs) (Marchese et

al., 1999), a membership that is expected to grow to over 400 by the completion of the

human genomic project (Stade1 et al., 1997). GPCR-encoding genes have been isolated

from a variety of species, from mamrnalian species to fish, birds, and even extending to

various plants, fungi and single ce11 organisms.

GPCRs act through G proteins, to which they are bound during their basal or

inactive state. Upon activation, GPCRs undergo a conformational change, which releases

the G protein as active a and By subunits into the cytosol. These subunits regulate the

activity of downstrearn effectors, such as adenylyl cyclase and phospholipase C, which in

turn regulates second messenger levels and subsequent intracelIular cascade reactions

resulting in a cellular response.

GPCRs are characterized by their serpentine-like structure of seven hydrophobic

a-helical transrnembrane domains (TM) connected by alternating intracellular and

extracellular loops and flanked by an extracellular amino-terminus and intracellular

carboxy-terminus (Fig. 1).

TM regions are 20 to 27 amino acids in length and contain a number of conserved

amino acid residues and motifs found throughout the GPCR family. TM 1 usually

contains a conserved "GN motif while TM2 is often characterized by a "(N/S)LAXAD"

motif (Fig. 1). Perhaps the best known GPCR motif is the "DRY" sequence found at the

Fig. 1. The G protein-couplod receptor: Structure and Conserved Residues. S h o w are conserved residues, motifs and generai structure of a G protein-coupled receptor. The rectangle represents the outer cellular membrane spanned seven times by a single polypeptide protein with an extracellular amino terminus (indicated by "NH2"), three extracellular and intracellular loops, and the intracellular carboxy terminus (indicated by "COOH). A disulphide bridge joining two cysteines is shown as a bent bar, and a palmitoylated cysteine is shown, creating a fourth intracellular loop.

TM3 and second intracellular loop junction (Fig. l), which is thought to play a role in

receptor activation (Gether and Kobilka, 1998). While conserved substitutions are found

within this motif, the central arginine is almost invariably conserved. This arginine is

hypothesized to be confined within a hydrophilic pocket composed of conserved polar

residues in TM 1, TM2 and TM7, and only exposed to the cytoplasrnic interior of the ceIl

during receptor activation (Gether and Kobilka, 1998). TM'S 2,4,5, 6 and 7 contain well

conserved aromatic residues and prolines (Fig. 1) which are thought to conuibute to

ligand poçket stabilization and receptor activation (Sealfon et al., 1995; Wess et al.,

1993). Another weil conserved motif among GPCRs is the "(N/D)PXXY1 motif found in

TM7 (Fit. l), which has been implicated to play a role in receptor endocytosis (Ferguson

and Washbum, 1998).

While the extracellular and intracellular portions of GPCRs exhibit a wide variety

of lengths and poor sequence conservation between different GPCRs, a number of

conserved residues and motifs still exist within these regions which are responsible for

post-translat ional modifications essential for proper receptor func tion. "NX(S/T)"

consensus sequences for asparagine-linked glycosylation are found in the arnino terminus

and extracellular loops (Fig. 1). Cysteines are often conserved in the first and second

extracellular loops (Fig. 1) which are believed to form disulphide bridges with each other

to stabilize the GPCR (Probst et al., 1992)- In the third intracellular loop and carboxy

terminus, serine and threonine residues (usually accompanied by nearby acidic residues)

are potential sites for phosphorylation by GPCR kinases (GRK), a mechanism which

ini tializes agonist-dependent desensitization (Krupnick and Benovic, 1998). In addition

to GRK sites, the intracellular loops and the carboxy terminus often contain CAMP-

dependent protein kinase (PKA) and protein kinase C (PKC) consensus sites for

phosphorylation essential for regulating GPCR signailing in both agonist-dependent and

independent desensitization (Ferguson et al., 1996). Finally, palmitoylation of cysteines

found in the carboxy terminus near to TM7 have been identified and are thought to

anchor the cysteines to the ce11 membrane, in turn creating a fourth intracellular loop

(Bouvier et al., 1995). The precise function of this fourth intracellular loop is still being

in vestigated.

1.3 Agonisa for GPCRs

While different GPCRs al1 share similar structures and conserved residues, the

external stimuli or ligands they bind are a diverse group, ranging from light to various

small chernical compounds to large complex molecules and even enzymes. For example.

the first GPCR cloned was rhodopsin, which is activated upon exposure to photons of

light. Ligands known to stimulate GPCRs include small biogenic amines (eg.

acetylcholine, epinephrine, norepinephrine, dopamine, and histamine), nucleosides and

nucleotides (eg. adenosine, adenine and uridine), phospholipids (eg. sphingosine 1-

phosphate and lysophosphatidic acid) and peptide neurotransmitters and hormones (eg.

opioids, somatostatin, fomyl peptide, thyrotropin- releasing hormone, angiotensin II.

apelin and orexins). A GPCR can also act as its own ligand. In the case of thrombin and

other protease-activated receptors, cleavage of a portion of the amino terminus leaves a

truncated amino terminus, which in turn acts as a tethered ligand for the receptor (Ji et al.,

1998).

1.4 Discovery of GPCR Genes by Molecular Cloning

Currently, the favoured methods of novel GPCR discovery rely heavily on the

significant levels of sequence identity or homology found amongst GPCRs, particularly

within conserved regions of the TM domains. Homology cloning (eg. low-stringency

library screening, PCR with degenerate oligonucleotide primers and database searching)

has proven to be successful, and constitutes the major method of GPCR discovery, as

seen in Table 1, which lists current GPCRs of the Rhodopsin farnily with known ligands.

However, it was not until the first few GPCRs were cloned and sequences compared that

receptor similarities were known to exist. Initial ventures to clone GPCRs by protein

purification relied on receptor pharmacology (ie. known ligands were used to isolate their

receptors), known tissue expression and required large amounts of the receptors

(Marchese et al.. l998a). While time consuming and technical ly demanding. this

technique allowed the isolation of receptors without any prior knowledge of their DNA

sequences. Purified GPCRs were cleaved into fragments and sequenced. These

sequences were used to design oligonucleotides, which in mm were used to screen cDNA

libraries from tissues known to express these receptors. The full open reading frames

(OWs) of the receptors were deduced from the positive cDNAs retrieved by screening

(Marchese et al., 1998a). Protein purification yielded the first cloned genes encoding

GPCRs, which included the P2-adrenergic (Dixon et al., 1986), Ml acetylcholine (Kubo

et al., 1986), M2 acetylcholine (Peralta et al., 1987), a2A-adrenergic (Kobilka et al..

l987a), and the a 1 B-adrenergic (Cotecchia et al., 1988) receptors.

An alternative GPCR discovery method was expression cioning, which involved

mRNA extraction from tissues known to express the GPCR in question. This mRNA was

Table 1. Rhodopsin famiiy of GPCRs with known endogenous ligands.

[ Receptors Cloning Strategy Species Genbank Acc. # 1

Adenosine Al Adenosine Au Adenosine Aze Adenosine A3

Adrenergic al* Adrenergic ale

Adrenergic al0

Adrenergic au Adrenergic Adrenergic azc Adrenergic Pl Adrenergic P2 Adrenergic B3

Anaphylatoxin C3a Anaphylatoxin CSa

Angiotensin ATlA Angiotensin ATlB Angiotensin AT2

Apelin

Bombesin 66, Bombesin BB2 Bombesin BB3

Bradykinin B1 Bradykinin B2

Cannabinoid (brain) CB1 Cannabinoid (periphery) CB2

Chemokine CCR1 Chemokine CCR2 Chemokine CCR3 Chemokine CCR4 Chemokine CCRS Chemokine CCRG Chemokine CCR? Chemokine CCR8 Chemokine CCR9

Chemokine CXCR1 Chemokine CXCR2 Chemokine CXCR3 Chemokine CXCR4 Chemokine CXCRS

Chemokine CX,CR1

PCR PCR PCR PCR

low strhgency protein purification low stringency protein purification low stringency Iow stringency low stringency protein purification low m-ngency

low stringency low stringency

expression cloning PCR expression cloning

PCR

Iow stringency protein purification low stringency

expression cloning expression cloning

Iow stnngency ?CR

PCR PCR low stringency PCR PCR PCR PCR PCR PCR

expression cloning Iciw stringency PCR PCR PCR

PCR

human human rat rat

human hamster rat human rat human human hamster human

human human

rat rat rat

human

rat mouse guine pig

human rat

rat human

human human human hurnan human human human human mouse

human human human COW

human

human

Table 1. (Continueci)

1 Receptor Cloning Stmtagy Specks Geribank Acc. # 1 Cholecystokinin CCKA Cholecystokinin CCKa

protein purification expression cloning

rat dog

human rat rat human human

cow rat

rat

human human

human human human

mouse

COW

dog human

human human

rat

mouse

human human rat mouse human

Xenopus human

human

pig pig rat rat human

Dopamine 01 Dopamine 02 Dopamine 03 Dopamine 04 Dopamine 05

PCR low stringency low stringency low stringency low strigency

Endothelin ETA Endothelin E '

expression cloning expression cloning

Follicle-stimulating hormone (FSH) low stringency

expression cloning low stringency

Galanin type-1 Galanin type-2 Galanin type-3

expression cloning PCR PCR

Gonadotropin-releasing hormone (GnRH)

PCR

Histamine Hl Histamine Hz Histamine H3

expression cloning PCR database

Leukotriene LTB4 Leukotriene CysLT1

low stringency database

Lutropin-choriogonadotropin hormone (LH-CG)

protein purification

Lysophosphatidic acid PCR

Melanocortin MC1 Melanocortin MC2 Melanocortin MC3 Melanocortin MC4 Melanocortin MC5

PCR PCR low stringency PCR PCR

Melatonin MLlA Melatonin MLIB

expression cloning PCR

Motilin MTL-RI low stringency

Muscarinic Acetlycholine M l Muscarinic Acetlycholine M2 Muscarinic Acetlycholine M3 Muscarinic Acetlycholine M4 Muscarinic Acetlycholine MS

protein purification protein purification low stringency low stringency low stringency

Table 1. (Continued)

Receptor Cloning Stratagy Species Genbank Acc. # 1 Neurokinin NK1 (substance P) Neurokinin NK2 (substance K) Neurokinin NK3 (neuromedin K) Neurokinin NK4

Neuropeptide Y Y1 Neuropeptide Y YI-like

Neuropeptide Y Y2 Neuropeptide Y Y4 Neuropeptide Y Y5

Neurotensin NTRl Neurotensin NTR2

Opioid 6 Opioid K Opioid p Orphanin

Oxytocin

Platelet-activating Factor

Prolactin-releasing peptide

Prostanoid EP, Prostanoid €Pz Prostanoid EP3 Prostanoid €PI Prostanoid OP Prostanoid FP Prostanoid IP Prostanoid TP

Protease-activated 1 Protease+activated 2 Protease-activatcd 3 Protease-activated 4

Purinoceptor P2yT Purinoceptor P2y2 Purinoceptor P2ys Purinoceptor PZy4 Purinoceptor PZy6 Purinoceptor P2yI

low stringency expression cloning low stringency expression cloning

low stringency low stringency

expression cloning low stringency expression cloning

expression cloning low stnngency

expression cloning PCR PCR ?CR

low stringency low strïngency protein purification

database database

expression cloning

expression cloning

PCR

low stringency low stringency low çtn'ngency low stringency PCR PCR low stringency protein purification

expression cloning low stringency PCR database

PCR expression cloning low sbingency PCR low sûingency PCR

rat COW

rat human

rat mouse

human human rat

rat rat

mouse mouse rat human

human human human

human human

human

guinea pig

human

mouse human mouse mouse mouse bovine human human

human mouse rat human

guinea pig mouse chicken human rat Xenopus

Table 1. (Continued)

[ Receptor Cloning Stratsgy Species Genbank Act. # 1 Purinoceptor P2Yll

Serotonin 5-HT,, Serotonin 5-Hf,, Serotonin 5-Hf,, Serotonin 5-Hf,, Serotonin 5-HT,, Serotonin 5-Hf,

Serotonin 5-HT,, Serotonin 5-HT2,

Serotonin 5-HT, Serotonin 5-HT,,

Serotonin 5-HT,, Serotonin 5-HT,

Serotonin 5-HT,

Somatostatin sstl Sornatostatin sstZ Somatostatin ssb Somatostatin sst, Sornatostatin sstS

Sphingosine 1-phosphate

Thyrotropin-releasing hormone C T W

Thyrotropin-stimulating hormone (TSW

Vasopressin VIA Vasopressin VIB Vasopressin V2

low stringency

low stringency

PCR PCR PCR low sûingency low stringency

low stringency expression cfoning

PCR PCR PCR

PCR PCR

PCR PCR low stringency low stringency PCR

differential hyb.

expression cloning

PCR

expression cloning PCR PCR

human

human human

human mon key

mouse

rat rat

rat

rat mouse mouse

rat rat

human human mouse rat rat

human

mouse

human

rat human rat

used to construct cDNA libraries, which were divided into pools and the transcribed

mRNA from each pool injected into Xenopus laevis oocytes or mammalian cells and

assayed using the ligands under investigation. Positive ce11 lines were further subdivided

and the above procedure repeated until a single cDNA was isolated, which was

sequenced and used to predict the ORF encoding the GPCR (Marchese et al., 1998a).

Like the protein purification technique, expression cloning was technically demanding

and did not require previous knowledge of the GPCR structure or sequence. However, if

successful, expression cloning would retrieve specific DNA encoding the desired

receptor. The first cDNAs encoding novel GPCRs isolated by this technique included the

neurokinin NK2 (Masu et al., 1987) and serotonin 5-HT2C (Julius et al., 1988) receptors.

With the discovery and sequencing of the fint cloned genes and cDNAs encoding

GPCRs, it became evident that GPCRs shared common seven TM structures, and more

importantly, significant levels of sequence identity, particularly within the TM dornains.

With each novel GPCR-encoding gene or cDNA cloned, highest levels of sequence

identity were observed between GPCRs which bound the same ligand. To a lesser

degree, strong sequence identities were also observed between GPCRs which bound

different but stmcturally similar ligands. New strategies employed to isolate novel

GPCR genes were based on shared sequence identity. Low-stringency hybridization was

the first of these homology cloning techniques, and involved screening of cDNA and

genomic libraries with radiolabeled fragments of DNA encoding previously cloned

GPCRs. Each search required optimizing the stringency of probe binding, either by

varying wash temperature or salt-concentration conditions, to retrieve novel related genes

(or paralogues) without significant levels of non-GPCR hybndization signals. The low-

stringency hybridization method to discovering novel GPCR-encoding DNA proved to be

a faster and less arduous a task than its predecessors, and was used to successfully clone

genes and cDNAs encoding the muscarinic M3 and M4 (Bonner et ai., 1987), dopamine

D3 (Sokoloff et al., 1990), D4 (OIMalley et al., 1992), D5 (Sunahara et al., 1991) and

serotonin 5-HT 1 A (Fargin et ai., 1988; Kobilka et al.. 1987b) receptors.

Finally, what has proven to be the fastest and most powefil molecular method of

discovering novel GPCR-encoding genes, is the polymerase chain reaction (PCR)

utilizing degenerate oligonucleotides. First devised as a means to amplify quickly and

selectively any known fragment of DNA (Mullis and Faloona, 1987), PCR has become

invaluable in novel GPCR discovery by exploiting the conservation of shon sequences or

motifs found in the GPCR family. PCR utilizing a set of two primers (sense and

antisense) based on conserved sequences found in a wide variety of different GPCRs

increases the odds of amplifying an even wider assortment of GPCR-encoding DNA

fragments, regardless of the difference of sequences found encoded between the two

prirners. By cornparison, the low-stringency screening technique is limited in the variety

of positive GPCR signals by the use of probes encoding large stretches of a particular

GPCR. The TM3 "DRY" and TM7 "(N/D)PXXY motifs, in particular, have proven to

be valuable sequences upon which to design PCR primers for GPCR gene discovery. In

our laboratory, 10 novel GPCRs, named GPRl through GPRIO, were found using the

same set of degenerate oligonucleotide primers designed frorn TM3 and TM7 (Heiber et

al., 1995; Marchese et al., 1994; Marchese et al., 1995; OIDowd et al., 1995). The

strategy of using degenerate oligonucleotides (which are composed of a mixture of

primers with substituted nucleotides at specific locations) further diversifies the mixture

of PCR-amplified GPCR-encoding DNA by the degenerate nature found wi thin the

genetic code. Among the first GPCRs to be discovered in this manner were four novel

GPCRs with unknown ligands (Libert et al., 1989), three of which were later

characterized to be the adenosine Al (Libert et al., 1991), A2A (Furlong et al., 1992), and

serotonin 5-HTlD (Maenhaut et al., 199 1) receptors. Other novel GPCRs quickly

followed, inciuding the dopamine Dl (Sunahara et al., 1990), the long f o m of D2

(O'Dowd et aI., 1990), histamine H 2 (Gantz et al-, 1991), serotonin 5-HTlB (Jin et al.,

1992) and melanocortin MC 1 and MC2 (Mountjoy et al., 1992) receptors.

1.5 Discovery of GPCR Genes by Database Searches

Over the past decade, gene discovery has been greatly enhanced by the

introduction of nucleotide and protein sequence databases. Of particuiar importance are

the GenbankTM databases maintained and made accessible to the public by the National

Center for Biotechnology Information (NCBI), a division of the National Library of

Medicine. Such databases include the non-redundant (or "nr", a comprehensi ve

collection of known gene, cDNA, protein and genomic contig sequences), the expressed

sequence tag (EST), the sequence tagged sites (STS), genomic survey sequences (GSS)

and high throughput genomic sequences (HTGS) databases. These databases are updated

daily, with data exchanged with other collaborating databases such as the DNA Databank

of Japan and the EMBL Data Library. Each Genbankm sequence entries are given

unique accession numbers, are annotated with bibliographic and biological data, and are

accessible by either word searches (eg. accession number, author, sequence name, etc.) or

may be retrieved by BLAST (Basic Local Alignment Search Tool), a computer sequence

similarity search program executed via the internet (http://www.ncbi.nlm.nih.pv)

(Altschul et al., 1997). As an extension of the traditional molecular homology cloning

techniques already in place, database BLAST searching utilizing sequences of known

GPCRs has retrieved many novel GPCR sequences (Table l), taking full advantage of the

high-throughput, daily growth of these databases. Examples of GPCR discovered by

database searching include GPR19 (O'Dowd et al., 1996) and GPR24 (Kolakowski et al.,

1996) receptors.

One particularly noteworthy database is the EST database, which has been

responsible for the majority of novel GPCR sequences found by database searching. The

EST database is made up of short, partial sequences of cDNA (usuaily 150 to 400 bp in

length), sampled from various cDNA libraries representing an enormous variety of

animal species and tissue types. The advantages of searching the EST database stem

from the sheer size of the database (which accounts for approximately 70% of

GenbankTM entries), the diversity of its entries (by species and tissues sarnpled) and the

database's representation of only expressed sequences, which excludes intron and other

non-coding sequences. However, the EST database does have problems with sequence

redundancy (which are inherit in any high-throughput system) as well as

underrepresentation of less abundant genes and genes expressed only in discrete tissues.

A typical search of the EST database with any GPCR sequence usuaIly retrieves

thousands of sequences, of which only the first few hundred are shown. With the rapid

expansion of gene discovery by database searching over the past decade, the vast yield of

any current database search will represent known sequences. However, careful sequence

analyses of these searches still reveal ESTs with novel GPCR-like sequences. Such ESTs

can often be ordered from the IMAGE (Integrated Molecular Analysis of Gene

Expression) consortium, who in a collaboration with the Washington University Genome

Sequencing Center contribute the vast majority of ESTs submitted to Genbankmf, and

used as probes to determine full-length GPCR-encoding sequences,

Two other databases of note are the HTGS and n r databases, which respectively

house in-progress and completed genomic contig sequences. The submission of these

sequences continue as part of the world-wide effort to sequence the genomes of various

species, including the human genome. The process is generally broken down into two

steps: mapping of yeast artifical chromosomes and radiation hybrids and sequencing

individual artificial chromosomes localized to mapped regions. High throughput

sequencing is made possible by cutting the artificial chromosomes into smaller

overlapping fragments which can be subcloned into the pUC plasmid or M l 3 phage

vectors. These fragments are sequenced and deposited as unordered sequences or

unfinished sequences with gaps into the HTGS database. Further gap-determining

sequencing and determination of order by sequence analysis of overlapping fragments

yield final sequences, usually well over 100 kb in length, which are deposited in the nr

database. In terms of novel gene discovery, the genomic sequence databases contain

underrepresented genes not found in the EST databases. Disadvantages of genomic

database searching include the inclusion of intronic sequences and size of the databases,

which currently pale in cornparison to the EST databases. However, the sequencing of

the human genome, which is scheduled for completion within three years (Venter et al.,

1998), holds the promise of the discovery of every human (and subsequently,

mammalian) gene, including the entire family of GPCRs.

1.6 Reverse Pharmacology: Assigning Ligands to Novel GPCRs

As stated earlier, the first GPCR-encoding genes were discovered using their

endogenous (or cognate) ligands as probes. These GPCRs were then expressed and

translated into functional proteins, ready for the investigation of their signalling

pathways, mechanisms of regulation, etc. However, the method of discovering GPCR-

encoding genes and cDNAs by homology cloning has Ieft investigators with the major

obstacie of assigning each novel GPCR to an endogenous ligand, a process which has

been dubbed "reverse pharmacology" (Stade1 et al., 1997). When a GPCR is first

discovered by homology cloning it is termed an "orphan" GPCR (oGPCR), requiring a

cognate ligand for purposes of classification and further physiological study. Early

oGPCRs found their cognate ligand by their high level of sequence identity (usually 40%

or greater) with other GPCRs. For example, the serotonin 5-HT1A receptor was

originally an oGPCR known as G21, yet strong sequence identity to the 5-HT2C receptor

led to its elucidation as another serotonin receptor (Fargin et al., 1988). In other cases,

GPCRs with less than 40% sequence identity have been found to share the same cognate

ligand. In such instances, the tissue distributions of oGPCRs can play a major role in

determining the cognate ligand. For example, the cannabinoid CB 1 receptor was

originally an oGPCR with little sequence identity with other known GPCR sequences, yet

expression analyses of CB 1 mRNA revealed strong overlap with the expression patterns

of cannabinoid receptors (Matsuda et al., 1990)-

However it is generally diffxcutt to assign ligands to oGPCRs by sequence identity

and expression analyses, and it is evident that a large body of unknown cognate Iigands

exist for the GPCR family. As a result, the list of oGPCRs has grown to number close to

100, constituting nearly a third of the GPCRs currently cloned (Table 2). The major

problem of assigning ligands to the current list of oGPCRs stems from the general

equation of proper GPCR characterization, which requires knowledge of an agonist, the

GPCR itself, and the determination of G protein (of which there are many types) and

effector systems involved. Identification of an agonist and its GPCR can elucidate which

G protein and effector system are involved. In return, knowledge of the GPCR and its

effector systems aIIows for assays to determine a cognate ligand. However, the exclusion

of both ligand and effector system identities makes the characterization of current

oGPCRs a daunting task, requiring new and inventive strategies for reverse

pharrnacology.

Recent successes in assigning ligands to oGPCRs have involved various screening

strategies to isolate peptide ligands. While such screening techniques have varied

sornewhat in their use of particular assays (eg. calcium mobilization, adenylyl cyclase

activity, change in the rate of extracellular pH), they are in essence the sarne. Briefly,

oGPCRs are expressed in various ce11 lines with established GPCR and G protein

expression and downstream effector system machinery and assayed with tissue extract

fractions from regions known to express the oGPCR (Wilson et al., 1998). Fractions

exhibiting specific dose-responses may contain the cognate ligand, and undergo further

fractionation until the ligand is purified. The first successfully isolated ligand by this

method was orphanin FQhociceptin (Meunier et al., 1995; Reinscheid et al., 1995), a

neuropeptide found to be the endogenous ligand for the oGPCR, ORLI (Bunzow et al.,

1994; Mollereau et al., 1994), utilizing brain extracts which inhibited adenylyl cyclase

Tobie 2. Orphan GPCR. ( ~orno loqy Imm8 ~~ 1%- I A e d o n No- I R e m u a f OPlOtD AND SOMATOSTATiN GPW RECEPTOR-UKE

CHEMOKlNE RECEPTOR-UKE GPF12

CKRX

GPR28

STRL33

PPRI

glod

ROC1

TWSFl

CLRl

Dez

FPRK

FPR2

GPRl

GPR30

human

human

human

rat

rat

t%Uman

hUITm

mwse

mouse

human

hufnan

bovine

rat

human

human

chicâen

human

human

human

human

human

h u m

mous8

629b GPR8 4036sst5

û2?6 GPRï (5%ss15

33% ssQ 32% ssts

29% popioid 28% -4

37% W R 2 35% GALRl

41% CXCR3 4096 CCR7

53% €01 43% CCRI

53% CKRX 36% CCRI

62% CCR 1 50% CCFW

43% CCR7 38% CCR6

37% CCR7 37% CCR6

39?6 CCR7 37% GPm8

33% F I E 1 30% CCR9

33% gl Od 30% CXCR2

22% GPRS 14% CCR6

51% BLR-1 36% CXCRl

37% GPRl 35% FPR2

72% FPR2 56% FPRl

72% FPRL2 69% FPRl

37% Dez 34% FPR2

3Z6 FPRL2 32% FPR2

39% FPRl 35% FPRL2

36% GPR32 36% Dez

O'üowd et al.. 1995

O'DoHlid et a.. 1995

Kolakowski et al.. 1996

Marchese et al.. 1995

Lee et al.. 1999

Marches et aL. 1944

unpublished

unpu büshed

Gao and Murphy. 1995

unpubiiihed

Liao et al.. 1997

Matsuaka et al.. 1993

Hanison et al.. 1993

tiben et al.. 1989

Spangenberg et al.. 1998

Gupta et ai.. 1998

Methner et al.. 1997

Bao et al.. 1992

Bao et al.. 1992

Marchese el al.. 1994

O'Dawd et al.. 1998

Marchese et aL. 1998

Marctiese el al.. 1998

Table 2. ( C o n t i n w ~~ornoloqy h n 8 bod.0 1% ldmtm IAcauion No, IRohmlcm 1 CHEMOAlïRACTANT RECEPTOR-LIKE (con't)

GPR44 human

mus oncoggne h u m

MRG

#TA

GPR53p

ANGIOTENSM RECEPTOR-UKE GPRlS

GPR25

CANNABlNOfD RECEPTOR-LIKE GPR3

GPRG

GPRl2

€DG4

GPR4 RECEPTOR-LIKE OGRl

GPR4

TDAGB

G2A

AMINE RECEPTOR-LIKE

NEUROPEPTIDE Y RECEPTOR-LIKE GIR

GPR19

GPR22

PNR

GPR26

GPR27

AGRS

human

rat

human

huITW'I

human

human

human

rat

human

human

human

human

mouse

r n o w

human

human

human

human

mou=

rat

34% MRG 26% CSoR

34% mas OllOOQene 34% CSeR

32% mas oncogene 33% MRG

35% MRG 28% mas oncogene

34% GPR25 3t% APJ

34% GPRIS 32% APJ

57% GPW 56% GPR6

46% EDGB 44% EDG-1

34% GPR4 31% OGRl

27% GalRl 26% NPY Y2

26% NPY Y6 24% CCKA

Maniiese et al.. 1999

Young et al.. 1986

Monmt et al.. 1991

Ross et al.. 1990

Çawzdargo et al.. 1999

Heiber et al.. 1996

Jung et al.. 1997

Marchese et al.. 1994

Heiber et al.. 1995

ldne et al.. 1991

Gmîer et al.. 1998

Xu and Casey. 1996

Heiber et al.. 1995

Kyaw et al.. 1998

Weng et ai.. 1998

Ham.gan et al.. 1991

O'Dowd et al.. 1996

O'Dowd et al.. 1997

Zeng et al.. 1998

unpublished

O'Dowd et al.. 1998

lshuaka et ai.. 1994

Table 2 (Con<lnii.d) [nornoiopy Ininr la--'-- I%#mirv l . r ? i No. l m 1

P2 RECEPTOR-CIKE

AMINE RECEPTOR-UKE (-3) GPR21

PSW4

GPR45

A-2

GPR52

RE2

GPA57

WU58

GPR61

GPR62

GPR23

RBintron

GPR35

'P2Y 1 O-

GPR17

GPRl8

HM74

GPR31

RSC338

€81 2

Hg63

GPR41

GPR42

GPR40

27% Om 24%

26% -4 23% &AR

70% PSP24 21% NK2

21% ÇHTIF 19% 5-HTlE

71% GPR21 27% H2

25% &.AR 2 s o 4 c A R

5996 GPR58 37% PNR

599: GPRn 42% PNR

2% GPRM 3û% ÇkK6

27% GPR61 28% ÇHT6

53% RBintmn 33% 'P2Y10"

53% GPFi23 38% P2Y4

32% GPR23 30% HM74

34% RBintmn 339i GPR23

35% P2Y2 34% MY4

30% RBintron 29% GPRl7

36% GPR3l 29% P2Yi

36% H m 4 29% P2Y t

33% Hg63 28% lp2y

33% RBintron 30% CCRl

33% RSC338 28% PAFR

98% GPR42 41% GPR43

98% GPR41 28% GPR23

31% GPR43 26% CXCRI

U66580

u92642

AF118266

U47928

Am96784

Am91890

NIA

WA

NIA

N'A

U66578

L11910

AM27957

AF000545

U33447

L42324

010923

U654ûî

D 1 3626

D l 7 7

AFOM986

-4688

AM24689

AF024ôô7

O'Dowd et al.. 1997

W l p r i M i

M a r c h e s e et al.. 1999

Ansan-Lan et ai.. 1946

Samdargo et al.. 1999

ungwished

WJw-

unpublished

unp-

u n p u b l m

Jung et al.. 1997

Tagududa et al.. 1993

O'Dawd et ai.. 1998

UnpuMished

Rapott et ai.. 1 9 9 6

Gantz et al.. 1997

Nomura et al.. 1993

Zingani et ai.. 1997

unpublished

Birkenùach et al.. 1993

Jacobs et al.. 1997

Samdargo et al.. 1997

Samtiaqo et ai.. 1997

Samdargo et al.. 1997

PZ RECEPTOR-UKE (eon't) GPR43

G P W

GP W

GPRSS

NEUROTENSIN RECEPTOR-LIKE GHS-R

GPR38

GPR39

ûPR66

MELATONIN RECEPTOR-LIKE

EN DOTHELIN RECEPTOR-LIKE

GLYCOPROTEIN HORMONE RECEPTOR-LIKE

OPSlN RECEPTOR-UKE

LGRS

Encephalopsin

RGR

human

human

human

human

human

human

human

human

human

human

human

human

human

human

67% GHSR 34% MR2

38% GPR38 34% GHS-R

26% FSH-R 25% LH-R

Samdargo et al.. 1997

O'Dawd et al, 1997

Marchese el al.. 1999

Sawdargo et ai.. 1999

Howard et al.. 1996

McKee et al.. 1997

McKee et al.. 1997

Tan et al.. 1998

f eng et al.. 1ç97

Valdenaire et al.. 1998

McOOMI~ el al.. 1998

Blackshaw et al.. 1999

Shen et ai.. 1994

stimulation in ceIl lines expressing ORLl. More recent discovenes utilizing these

screening methods include the hypocretin/orexin peptides for the hypocretin/orexin

receptors 0x1 and 0x2 (Sakurai et al., 1998), the prolactin-releasing peptide (Hinuma et

al., 1998) for the GPR 1 O receptor (Marchese et al., 1993, the apelin peptide (Tatemoto et

al., 1998) for the APJ receptor (O'Dowd et al., 1993) and the melanin-concentrating

hormone (Chambers et al., 1999; Saito et al., 1999) for the GPR24 receptor (Kolakowski

et al.. 1996).

1.7 Research Objectives

In this research, 1 proposed to isolate and characterize novel genes encoding

GPCRs to provide additionai receptors for use in the reverse pharmacology method of

GPCR ligand discovery. The identification of such novel transmitter systems creates a

basis for the discovery of novel therapeutic agents and study of GPCR-linked disease and

disorders. As an extension of characterizing GPCRs, 1 also proposed to characterize

apelin, a novel ligand discovered by reverse pharrnacoIogy for the oGPCR, APJ. Based

upon the sequence and structural similarities between apelin and a

hormone/neurotransrnitter angiotensin II as well as between the APJ and angiotensin AT1

recepton, 1 hypothesized a significant degree of correspondence between the expression

patterns and physiological roles of apelin and angiotensin II. To further the

understanding of the apelin system, apelin and APJ mRNA distribution patterns and

apelin's physiological functions were investigated and compared to the angiotensin II

system.

2.0 METHODOLOGY

2.1 Materials

2.1.1 Chemical Reagents

Bacto@ aga., BactoO tryptone, BactoB yeast extract were purchased from

Difco. 1 kb DNA ladder was purchased from Life Technologies. Acrylamide and

dex tran sulfate were purchased from Caledon. B isacrylamide, ammonium persul fate,

agarose, low-melt agarose and urea were purchased from ICN- Salmon sperm

deoxyribonucleic acid was purchased from Sigma; glycine and AG@ 50 1 -X8 resin from

Bio-Rad; ethidium bromide, bromophenoi blue and xylene cyan01 FF from International

Biotechnologies hc . ; phenol from Toronto Research Chemicals; chloroform frorn JT

Baker; ethanol and methanol from Consolidated AIcohols Ltd.; and sodium chloride,

ammonium acetate, potassium phosphate and magnesium chloride from Mallinchrodt.

Sodium citrate and potassium acetate were from J. T. Baker, sodium hydroxide from

Anachemia, sodium acetate from BDH. Calcium chloride was from Fisher Scientific.

Ammonium acetate, calcium chloride, potassium chloride, potassium hydroxide and

magnesium sulphate were from BDH. Ampicillin was from B I ; maltose from Toronto

Research Chemicals; glucose from BDH; IPTG (isopropyl- 1 -thio-p-D-galactoside) and

Xgal (5-bromo-4-chloro-3-indoly1-~-D-galactoside) from BRL; and tris base from ICN.

Boric acid, EDTA (ethylenediaminetetracetic acid) and glacial acetic acid were

purchased from BDH. Hydrochloric acid, SDS (sodium dodecyl sulfate) from Bio-Rad:

glycerol and isopropanol from BDH; adenosine 5'-triphosphate, dATP, dCTP, dGTP, and

dTTP from Pharmacia.

2.1.2. Enzymes

Restriction enzymes and modifying enzymes (Klenow fragment, alkaline

phosphatase, T4 DNA ligase and T4 polynucleotide kinase) were purchased from

Pharmacia. Taq DNA polymerase was from Life Technologies. Pfum DNA polymerase

was from Stratagene. Advantagem Taq was from Clontech. Proteinase K was from

United States Biochemical.

2.1.3. Isotopes and Ligands

a-)'P-dCTP was from ICN. ~ - " s - ~ A T P was from Amersham. The ligands

methiothepin, mianserin and SDZ-205,557 HCl were purchased from RBI.

2.1.4 Oligonucleotides

Oligonucleotides used for PCR and sequencing were obtained from either

Biotechnology Service Centre, University of Toronto or the ACGT Corporation,

2.1.5 Kits

T7 SequencingTM kits was from Pharmacia Biochemicals. Maxi prep kit and Gel

Extraction kit were from Qiagen. TOPO TA- cloning kit was from Invitrogen. Nick

translation ket was from Amersham. NACS Prepacm columns were from Bethesda

Research Laboratories. Nylon membranes were either from Amersharn or Mill ipore.

The Calcium Phosphate Transfection kit was from Life Technologies.

2.1.6 CeU lines, Plasmids and DNA Libraries

E. coli bacterial strain DlOB was from Stratagene. PBluescript SK(-) plasmid

was from Stratagene and pcDNA3 plasmid was from Invitrogen. Marathon-readym

whole-brain cDNA for human and rat. E M B L 3 ' I 7 f S p 6 human genomic library, human

hypothalamus library, and rat 5' stretch brain cDNA Iibrary in hgtl1 were from Clontech.

2.2 Methods

2.2.1 Computational DNA and Protein Sequence Analysis

DNA Strider v.2.1 sequence analysis software was used for storage, manipulation

and analysis of nucleotide and protein sequences. Nucleotide sequences were translated

into 6 potential reading frames and analyzed manually for ORFs. Hydropathy plots

(KyIe-Doolittle index) were utilized for analysis of arnino acid sequences.

2.2.2 PCR: Polymerase Chain Reaction

PCR reaction mixtures contained 1 pg DNA, 200 ng of each primer, 5 pl of 10X

PCR buffer (100 rnM Tris-HC1 (pH 8.3), 500 rnM K I ) , 3 mM MgCl?, 0.25 mM each of

dATP, dCTP, dGTP, and dïTP, and 2.5 U Taq, Pfu, or Advantage Taq polymerase. The

PCR amplifications were done using a Perkin-Elmer Cetus thermal cycler under the

foIIowing conditions: 94 "C for 30 sec, followed by 30 cycles of 94 "C for 30 sec,

annealing at 45 "C, 50 OC, 55°C or 68 OC for 30 sec, and extension at 68 'C or 72 'C for 1

min to 3.5 min, and final extension at 68 "C or 72 "C for 7 min.

For human apelin, primers used to perforrn M C E (Frohman et al., 1988) on

Marathon-readyTh' human whole brain cDNA included the Marathon-linker primers

supplied by the manufacturer and gene-specific primers as follows: for the 3' end, 5' -

CGATGGGAATGGGCTGGAAGACGG-3' with a nested primer 5'-GCAATGTCCGC-

CACCTGGTGCAGC-3', for the 5' end, 5'-CCGCTGGCGGCGGAATTTCCTCC-3'

wi th a nested primer 5'-CTGCCAGGGCCCTGGCCCATTCC-3'. Primers flanking the

vector cloning site used to amplify rat apelin cDNA using a library-screened isolated

plaque as a template were are follows: P l (5'-GGTGGCGACGACTCCTGGAGC-3')

and P2 (5' -GACACCAGACCAACTGGTAAT-3*)-

Degenerate oligonucleotide primers used to isolate DNA fragments encoding

TRH-R2 and GPR54 were designed from conserved sequences encoding TM3 and TM7

of GPR l through GPR IO, GPRL4, GPR 15, GPRl9 through GPR25, GPR27, GPR30 and

GPR31 (Fig. 2). The primer sequences for TM3 and TM7 were: Degl (5'-

CTGACCGGCATGA(C/G)(C/T)(NG/T)T(C/GR)GA(C)CG(C)TA-3 ) and Deg2

(5'-GAAGGCGTAGA(C/GTT)(C/G)A(A/C/GTT)(A/CIG)GG(A/G)TT-3'). These

primers were used to amplify a rat brain 5' suetch cDNA library. The TM7 or antisense

primer was also paired with two primers specific for the 5' and 3' regions flanking the

cDNA library inserts named PI and P2 (shown above). The full length ORF encoding

GPR54 was obtained from the rat 5' stretch cDNA library by PCR amplification with the

fol low ing primers: P3 (5'-ATGGCCGCAGAGGCGACG-3') and P4 ( 5 ' -

TCAGAGTGGGGCAGTGTG-3').

Human genomic DNA was amplified by PCR using oligonucleotide primers

based upon sequences encoding GPR57 (P5: 5'-CTCATCCTCCTGGAAAGA-3'; P6: 5'-

TAACAATCTCATTTGCAA-3') and GPR58 (PT ST-TGCTCAGTG(G/T)C(A/C/G/T)-

AT(A/CIT)GA(C/T)(A/C)G-3'; P8: 5' -ACCATATATTAACGGATT-3').

GPRl C T G GPFP C T G WfU C T ~ GPRQ C T G GPRS C T G GPFB C T G

GPFPB C T A GPR9 C T G GPRIO C TE ffiPR14 C T 9

GPR3

G C C T G C A T C A G C T T T G A C C G C T A A C C A C C A T C G C ~ G ~ G G A ~ C ~ C T A J ILL l.3

C T C A C T T G C A T

Ic T I c ' & l T ~ c A T

Transrnembrane 3 L L A 1 T V D R Y

G A G

Transmembrane 7 N P 1 1 Y A F

G A G OMO

T G T C A T C G T C A T T T T T G T C A T C G T C G T C T T T T T -

G G A C C G C T A T G A T A G A T A G G A C A G A T A 1 G G A T c GOTE T G A C C G C T A G G A C C G C T A CFJCCGCTA C G A C C G C T A G G A C C G ~ T A

A A C C C C A T C C T W T A ~ A A T C C C G T ~ C T C T A C G C C T T C

A A C C C C T T C C T C T A C G C C T T S A A C C C C T T C C T C T A C G C C T T T A A ~ ~ ~ G ~ T G ~ T ~ T A ~ G ~ ~ T ~ A A C C C C T T C A T C T A C G C C T G G A A T C C C T T G C T & T A C ~ T C T ~

C A T HG T

C T G A C C G G C A T G A C C A T C G A C C G A T A A A C C C C A T C A T C T A C G C C T T C G T G G T C T G C G C

T T T G G T

primer Deg-1 primer 089-2 (antl-parallel)

Fig. 2. Alignment of oGPCRs used for design of degeneiate oligonucleotides. Transmernbrane regions used for design indicated at top with a sample arnino acid sequence from GPR3. Conserved nucleotides are boxed and shaded. Degenerate primer Deg-1 and Deg-2 are shown at bottom. For convenient comparison to the transmembrane 7 sequences, the anti-parallel sequence for Deg-2 is shown.

For GPR61, primers designed from a rabbit patent encoding TM3 (PIS: 5'-

ATCAATGTGGAGCGCTAC-3') and TM6 (P 16: 5'-GAAGTAGGGCAACCAACA-3')

were used to amplify human and rat genomic DNA. To obtain the ORF for human

GPR61, RACE (Frohman et al., 1988) was performed on Marathon-readyTM human

whole brain cDNA including the Marathon-linker primers supplied by the manufacturer

and gene-specific primers as follows: for the 3' end, 5'-ACGTAGTCCACCCCATGCG-

CTACG-3' with a nested primer 5'-TGCTGGTGGGTGTGTGGGTGAAGG-3', for the

5' end, 5'-CCTTCACCCACACACCCACCAGC-3' with a nested primer 5 ' -

GCGCACCTCGTAGCGCATGGGGTGGACTACG-3'. The GPR62 ORF was

amplified using primers P 17 (5'-ATGGCCAACTCCACAGGGCTG3') and P 18 ( 5 ' -

TCAGGAGAGAGAACTCTCAGG-3').

2.2.3 Genbankng Database Searches

The EST, HTGS and nr sequence databases maintained by NCI were queried with

amino acid sequences of human preproapeIin and selected GPCRs using the sequence

similarity program TBLASTN (Altschul et al., 1997). For GPCR searches, retrieved

sequences were examined for recognizable GPCR TM motifs and novelty of sequence by

comparison with sequences of known GPCRs contained within the nr database.

2.2.4 Subcloning of PCR Products

5 pl of PCR products were separated by electrophoresis on regular agarose as

described in section 2.2.5 to confirm expected sizes of amplified products. The

remaining PCR sample was subcloned by the TOPO TA cloning kit following the

protocol supplied by the manufacturer or by the following protocol: the PCR sarnple was

phenol/chlorofonn extracted, precipitated with sodium acetate (3 M, pH 5.2) and 100%

ethanol. The pellet was resuspended in 24 pl TE buffer (IO mM Tris-HCl (pH 8.0). 1

mM EDTA), 2 pl one-phor-al1 buffer (Pharmacia), 2 p l 1 rnM ATP, 1 pl T4

polynucleotide kinase (5 U) and incubated at 37 OC for 15 min. pBluescript or pcDNA3

plasmid vector was digested with EcoRV endonuclease and dephosphorylated by

treatrnent with alkaline phosphatase (5 U) and incubated at 37 OC for 1 hr. The altered

PCR product and vector were electrophoresed on a low-melt agarose gel stained with

ethidium bromide, bands of the appropriate size excised from the gel, melted together at

70 "C for 5 min, cooled at 37 "C for 10 min, T4 ligase (5 U) and ATP (ImM) added, and

incubated at roorn temperature ovemight. The mixture was then melted and transfomed

into competent bacteria, plated ont0 Li3 plates containing arnpiciliin (100 pg/ml), IPTG

and X-gal, and incubated ovemight at 37 OC.

2.2.5 DNA Minipreparation, Restriction Digestion and Electrophoresis

Colonies were picked from LB plates and transferred to 5 ml of LB media and

incubated for 16 hr at 37 'C in an orbital shaker. The culture was then centrifuged at

6000 rpm for 2 min at 4 OC, the pellet resuspended in 100 pl of solution I (50 mM

gIucose, 10 mM EDTA and 25 m M Tris-HCI (pH 8.0)). lysed for 2 min in 200 pl of

solution II (0.2 N NaOH and 1% SDS) and incubated on ice for 3 min after addition of

170 pl of solution III (5 M sodium acetate (ph 5.2)). The mixture was centrifuged at

11000 rpm for 2 min and supernatent transfered to a new microcentrifuge tube. The

plasmid DNA was phenol/chlorofonn extracted, ethanol precipitated and washed with

70% ethanol and dned before resuspension in LOO pl TE buffer (10 rnM Tris-HCI (pH

8.0) and 1 mM EDTA (pH 8.0)).

5 pl of DNA were incubated for restriction digestion in a solution with 1 pl of

RNase and 1 X enzyme buffer (1 mM Tris-acetate (pH 7 . 3 , 1 mM magnesium acetate, 5

mM potassium acetate), 10 U of restriction enzyme to a total volume of 20 pl. Sarnples

were loaded with 1 kb ladder into separate wells in an agarose gel stained with ethidium

bromide, electrophoresed in 1X TAE buffer and analyzed under UV light and

photographed.

2.2.6 DNA Sequencing

40 pl of miniprep plasmid DNA was denatured in 5 pl alkali solution (7mM

EDTA and 2 M NaOH) for 5 min, neutralized in 25 pl of neutralizing solution (3 M

sodium acetate, pH 5.2), precipitated with 100% ethanol, washed in 70% ethanol and the

pellet resuspended in 100 pl double-distilled, filtered water.

5 pl of this denatured DNA was sequenced by the Sanger dideoxy method using

the T7 SequencingTM kit and following a protocol supplied by the manufacturer.

Sequencing samples were denatured by incubation at 85 OC for 3 min prior to loading on

a polyacrylamide gel (100 ml of gel soiution containing 8 g acrylamide, 20 ml 5X TBE,

0.69 g N, N'bisacrylamide, 41 g urea, 0.54 ml of 10% ammonium persulfate and 50 pl

TEMED). The gel was electrophoresed in 1X TBE running buffer (one liter of 5X TBE

containing 54 g Tris-base, 27.5 g boric acid, 20 ml of 0.5 M EDTA) at 2000 V for 3-4

hours. Gels were fixed with a mixture of 10% methanol and 10% glacial acetic acid,

transferred to filter paper and vacuum dried at 80 "C for I5 min. The gels were then

exposed to Kodak XAR film overnight.

2.2.7 DNA Probe Extraction and Radiolabelihg by Nick Translation

Probes consisted of DNA fragment cut from vectors by restriction digestion (see

section 22.5) and electrophoresed on a low-melt agarose gel stained with ethidium

bromide. excised from the gel. and extracted and radiolabeled with a-3'~-dCTP using the

Gel Extraction kit and nick translation kit respectively. following the protocol supptied

by the manufacturer. The probes were purified using NACS PrepacTM mini columns

according to the protocol supplied by the manufacturer (Gibco BRL).

2.2.8 Genomic and cDNA Library Screening

A human hEMBLSP6/T7 genomic library, human hypothalamus library and rat

brain 5' stretch cDNA library were screened with radiolabeled probes obtained by nick

translation. The libraries were titered to yield approximately 50000 plaques per plate and

plates lifted with replica nylon filters as per manufacturers instructions. The filters were

prehybridized with 50% formamide, 2X SSC ( 0.3 M NaCl and 0.030 M sodium citrate

(pH 7) ) , 10X Denhardt's solution (0.2% polyvinylpyrrolidone, 0.2% Ficoll and 0.2%

B S A), O . 1 % SDS, O. 1 % sodium pyrophosphate, 20% dextran sulphate and 50 m g h l

sheared salmon sperrn DNA. Probe was added and hybridization occurred overnight at

42 "C and terminated with two washes in solution (0.2X SSC and 1% SDS) at 55 O C . The

fiIters were exposed to Kodak XAR film with an intensifying screen at -70 OC for 3 days.

2.2.9 Bacteriophage DNA Preparation

To purify bacteriophage DNA from library screening, - 10 pl of bacteriophage

particles was added to a bacterial culture of E.coli LE392 grown to exponential phase in

LB containing 0.1% glucose and 10 rnM MgCl, and incubated overnight at 37 "C under

continuous agitation. The culture was centrifuged at 6000 rpm at 4 "C for 2 min, the

supernatent transferred to a ultracentrifuge tube, and the supernatent centrifuged for 30

min at 4 "C at 30000 rpm using a SW41 rotor in a Beckman Optima L-80 ultracentrifuge-

The pellet was resuspended in 400 pl SM ( 1 liter SM contains 5.8 g NaCI, 5 ml 2%

gelatin, 50 ml LM Tris-HCI (pH 7 3 , 2 g MgSO,) with proteinase K ( 1 mg/ml) and

incubated for 2 hr at 37 "C. DNA was phenol/chloroform extracted, ammonium acetate

(7.5 LM, pH 5.2) and ethanol precipitated, and resuspended in 100 pl TE buffer.

2.2.10 Southem Blotting

Phage DNA from genomic library screening was digested with various restriction

endonucleases (see section 2.2.5), electrophoresed alongs ide 1 kb ladder on an agarose

gel stained with ethidium bromide and photographed together with a fluorescent d e r .

The gel was transferred to a HybondfM-N nylon membrane using a vacuum apparatus

(Tyler Research Instruments) as per manufacturers instructions. Briefly, the gel was first

placed in denaturing solution (0.5 M NaOH and 1.5 M NaCI) followed by neutrdizing

solution (0.5 M Tris-HC1 (pH 7.4) and 1.5 M NaCI) and finally in 800 ml 1OX SSC for 2

hr. The nylon and transferred DNA were UV cross-linked with a StratalinkerB UV

crosslinker (Stratagene). Blots were hybridized and washed as described in section 2.2.8.

2.2.11 Northem Blotting

Both human and rat tissues were probed with respective hurnan and rat DNA

probes for northern blot analyses. Total RNA was extracted from tissues by the method

described previously (Chomczynski and Sacchi, 1987). Tissues were frozen and

homogenized by polytron in solution (SM guanidium thiocyanate (pH7.2) and 8% P-

mercaptoethanol), p henoVchloroform extracted, RNA precipitated by acetic acid and

ethanol, pellet resuspended in DEPC-treated sterÏle water and poly(~)* RNA isolated

using oligo-dT spin columns (mRNA isolation kit, Pharmacia) as per manufacturers

instructions. The poly(~)' RNA was heat denatured and size fractionated by

electrophoresis on a 1 % formaldehyde agarose gel. The quality of the poly(A)' RNA

was inspected by ethidium bromide visualization under UV light. The RNA was

transferred ont0 a nylon membrane and cross-linked (see section 2.2.10). The blot was

then hybridized with a nick-translated a-"P-dCTP labeled probe (see section 2-27),

washed in 2X SSPE (3 M NaCl, 0.2 M sodium hypophosphate and 0.2 M EDTA (pH

7.4)) and 0.1 % SDS at 50 OC for 2 min, washed again with O. 1X SSPE at 50 "C for 2 hr.

The blot was exposed to Kodak XAR film with an intensifying screen at -70 "C for at

least one week.

2.2.12 In Situ Hybridization

Rat brain sections were probed with rat DNA probes. Male rats (Charles River)

were killed by decapitation and their brains removed within 30 sec and frozen quickly in

dry ice. The brains were sectioned at 14 pm thickness using a Reichert-Jung cryostat at

-10 "C and thaw-mounted ont0 microscope slides. These sections were fixed in 4%

paraformaldehyde in 0.02% DEPC sterile water for 20 min at 4 OC, washed for 5 min in

cold phosphate buffered saline (pH 7.4), dehydrated through graded alcohols, air-dned

and stored at -70 OC. DNA probes were labeled as in section 2.2.7 but with a - " s - d C ~ ~ .

Brain sections were incubated for 2 hr at 42 "C in prehybridization solution (containing

50% deionized formamide, 0.6 NaC1, 10 mM Tris-HCI (pH 7.5). 10% dextran sulfate. 1

ri, polyvinylpyrrolidone, 2% SDS. 100 rnM DTT, and 200 pg/ml hemng sperrn DNA).

The labeled probe was added (106 cpm/slice) and allowed to hybridize for 16 hr.

Sections were washed greater increments of annealing stringency (ie* increasing

temperature and descreasing salt concentration) dehydrated in a graded alcohol series,

exposed to Dupont MRF-34 x-ray film for 4-6 weeks at -70 "C and developed. As a

control, adjacent brain sections were treated with RNase and hybridized with the

radiolabeled probe to confirm specificity of the hybridization.

2.2.13 Chromosomal localization

Fluoresescence in situ hybridization (FISH) was performed as previously

described (Heng et al., 1992; Heng and Tsui, 1993) to localize genes to regions on human

chromosomes. Human lymphocytes were cultured in an a-minimal essential medium (a-

MEM) supplemented with 10% fetal calf serum and phytohemagglutinin (PHA) at 37 "C

for - 3 days. The cultures were ueated with BrdU (0.18 rnghl, Sigma) for another 16 hr

to synchronize the ce11 population, washed three times with semmfree medium and

incubated at 37 "C for 6 hr in a-MEM with thymidine (2.5 pg/mI, Sigma). Cells were

harvested and slides made using hypotonic treatment, fixation and air drying. Slides

were aged for a few days, heated at 55 'C for 1 hr, treated with RNase A and denatured in

solution (70% formamide and 2X SSC) for 1 min at 70 'C followed by dehydration by

ethanol. Purified bacteriophage DNA probes containing the gene of interest (see section

2.3.9) were biotinylated with dATP using a Bionick labeling kit (BRL), denatured at 75

'C for 5 min in solution (containing 50% formamide and 10% dextran sulphate) and

loaded ont0 slides. Following overnight hybridization, detection and amplification, the

FISH signals and diamidino phenylindolote (DAPI) banding pattern were visualized by

fluorescence microscopy and photographed on Kodak slide film ASA 800/1600.

In addition to the FISH technique, chromosomal localization mapping was also

performed using a Southem blot analysis (see section 2.2.10) of a human

rnonochromosoma~ somatic ce11 hybrid panel (BIOS laboratones) as per manufacturers

instructions. Finally, human contigs revealing novel GPCR-encoding genomic sequences

(see section 2.2.3) were annotated with chromosomal locatizations.

2.2.14 Creation of an Intronless GPR58 Receptor Gene Expression Constmct

To construct the full-length ORF of GPR58, two DNA fragments encoding the

two exons of GPR58 were amplified and joined from human genomic DNA by PCR.

Fragment 1 was amplified using oligonucleotide primers based upon the GPR58 5'

untranslated region (UTR) (P9: 5'-TGACAAAA'M'CTATCTGTTCTTG-3') and the 3 '

end of exon 1 (P 10: 5'-CATACTATATGGCATGATGG-3'). Fragment 2 was arnplified

using primers based upon the 5' end of exon 2 (Pl 1: 5'-ATCAGATCGGT-

GGAGAACTGC-3') and the sequence surrounding the stop codon (P12: 5 ' -

TGCAGAAAAAGCCTACTCACTTTC-3'). PCR conditions were as shown in section

2.2.2. The PCR products were subcloned into the pCR 2.1 -TOPO vector (Invitrogen)

with the TOPO TA Cloning Kit and sequenced. Fragments 1 and 2 were joined and

amplified to form the full GPR58 ORF by two further rounds of PCR using primers

whose sequence spanned both fragments (Pi3: Y-ATAGTATGATCAGA-

TCGGTGGAGA-3' ; P 14: 5'-GATCTGATCATACTATATGGCAT-G-3 '). A second

round of PCR amplified an aliquot of the first round with primers P9 and P12. The PCR

products were subcloned into pcDNA3 and sequenced to verify correct orientation for

expression.

2.2.15 Maxi DNA Preparation

The GPR58/pcDNA3 construct was prepared for transfection using the Maxi prep

kit as per manufacturers instructions (Qiagen). Bnefly, bacterial cultures of E. coli

transforrned with GPRSWpcDNA3 constructs were grown overnight in LB media

containing ampicillin (IO0 pg/ml) at 37 OC under constant agitation. Cultures were

centrifuged to produce pellets, resuspended in Buffer 1, lysed in Buffer P2 and bacterial

debris precipitated with chilled Buffer P3 for 10 min at 4 T and centrifuged at 12000

rpm for 30 min at 4 OC. The supernated was gravity-eluted through an equilibrated

Qiagen-Tip, washed, the DNA precipitated with isopropanol, washed with 70% ethanol,

and resuspended in TE buffer.

2.2.16 Calcium Phosphate Transfection

Cells were transfected using the Calcium Phosphate Transfection System as per

manufacturers instructions. Briefly, COS-7 cells were cultured in a-MEM containing

IO% fetal bovine semm at 37 "C in 5% CO2 and plated in a LOO mm tissue culture dish ( 1

x 106 cellsllO0 mm dishl10 ml complete medium) 24 h r pnor to transfection. Cells were

supplied with fresh complete medium 3 hr pt-ior to transfection, with each dish containing

100 pl IOX HBS buffer (HEPES, NaCl), 15 pl 1 N NaOH, 70 pl calcium phosphate, 10

pl phosphate solution, 1 pg carrier DNA, 10 pg plasmid and brought up to 1 ml with

sterile water. Cells were treated with cdciurn-phosphate DNA precipitate for 16 hr in

complete medium at 37 "C in 5% CO2, replaced with fresh medium and incubated a

further 24 hr.

2.2.17 Membrane Preparation and Binding S tudies

Cells were harvested in a binding buffer (containing 50 m . Tris-HC1 (pH 7.8), 5

rnM MgC12, 1 mM EGTA, leupeptin (5 p a l ) , soybean trypsin (10 pg/pl) and

benzamidine (10 p a l ) ) - Cells were homogenized twice using a Polytron (Brinkmann)

for 20 sec and collected by centrifuging at 800 rpm for 10 min at 4 OC. Pellets were

resuspended in binding buffer and used imrnediately for radioligand binding studies. Ce11

membranes (25 pg to 80 pg protein) were incubated with radioligands for 1 hr at room

temperature in the presence or absence of competing agents. Nonspecific binding was

defined as the radioactivity remaining bound in the presence of 10 p M of cornpetitor.

Binding actions were stopped by adding chilled 50 mM Tris-HC1, pH 7.4 and rapid

filtration over Whatman FGIC g l a s fiber filters. Filters were washed twice with 5 ml

chilled solution (containing 50 mM Tris-HC1, pH 7.4) and the bound radioactivity

detennined by using a liquid scintillation counter. The data were analyzed by nonlinear

leas t square regression utilizing the computer program GraphPad.

3.0 RESULTS

3.1 Determination of the Human Apelin Genomic Structure and Cloning of Rat

Apelin cDNA

The apelin peptide was discovered by a reverse pharmacology method using the

APJ receptor expressed in Chinese Hamster Ovary (CHO) cells (Tatemoto et al., 1998).

WhiIe the apelin protein sequence was reported , no DNA sequences or detailed tissue

distributions were reported. Ln order to investigate the physiological hnctions of apelin

and APJ, 1 first endeavored to determine the DNA sequence of the apelin gene and

mRNA distributions of apelin and APJ.

A search of the Genbankm database with the human preproapelin peptide

sequence retrieved a human genomic DNA sequence mapped to chromosome Xq25-26.1

(Clone-ID PAC 454M7, Genbankm Acc. #AL022 162). This gene sequence revealed the

presence of an intron -6 kb in length with recognized introdexon boundaries interrupting

the OEW at the position encoding ~ l y ~ (Fig. 3). Using oligonucleotide pimers encoding

portions of the second deduced exon, 1 performed 5' and 3' RACE of Marathon-readymf

hurnan whole brain cDNA (RACE primers in section 2.2.2). 3' RACE amplified a DNA

fragment -1.4 kb in size and analysis revealed a sequence identical to PAC 454M7

intempted by an intron -800 bp in length 3' of the stop codon (Fig. 3c). 5' RACE

amplified several fragments, the largest -250 bp in length and identical to PAC 454M7

sequence, but was truncated upstream of the sequence encoding the start methionine.

The 3' RACE fragment was used to screen a human hypothalamus Iibrary, resulting in

the purification of four cDNA phage. Sequence analysis of one phage

40

Bovine

Rat Hurnan Bovine

Y L V K P R T S R T H L V Q P R G S R N Y L V Q P R G P R S "ni

Apelin Angiotensin I I

PAC 454M7 (

M e t (98072)

Stop (92070)

Fig. 3. Apelin amino acid sequence aiignments and the human apelin genomic structure. (A) An alignment of amino acid sequences of rat preproapelin with human and bovine preproapelin. Conserved amino acids are shown boxed. The mature apelin peptide are shaded. Numeric amino acid positions are indicated on the right. (B) An alignment of amino acid sequences of human apelin and angiotensin II. Consewed amino acids are shown boxed. (C) The genomic structure of the human preproapelin gene as found in the human PAC 454M7 clone (GenBank Accession # AL022162). Nucleotide positions of PAC 454M7 defining preproapeiin gene exons (boxes) are shown at the top. The ORF is shown in black, with the nucleotide positions of the start and stop codons shown at bottom.

revealed additional 5' UTR sequence found in PAC 454M7 (Fig. 3c). Sequence analysis

of the 13 amino acid mature peptide revealed identity, albeit lirnited, to angiotensin U

(Fig. 3b). The proposed start methionine did not conform to a Kozak consensus sequence

due to the presence of an adenosine immediately following the start codon (Kozak,

1996). The peptide sequence following the start methionine included a signal secretory

peptide hydrophobic region followed by a stretch of polar amino acids.

1 conducted a seatch of the EST database with the hurnan preproapelin peptide

sequence and retrieved a rat EST (GenbankTM Accession #AA94283 1) which partially

encoded the 5' portion of rat preproapelin. Primers based on this sequence were designed

and arnplified a 425 bp fragment from a rat brain 5' stretch cDNA library. This fragment

was used to screen the same library arnplified, resulting in the purification of three phage.

These phage were arnplified using prîmers designed upon regions flanking the cDNA

library inserts (Primers P l and P2 in section 2.2.2). Sequence analysis revealed a phage

(-1.5 kb in size) which encoded a protein with identity to the reported human

preproapelin. The rat preproapelin cDNA encoded a peptide of 77 amino acids (identical

in size to human and bovine), with identities of 82% and 77% with human and bovine

preproapelin respectively (100% identity within the mature peptide) (Fig. 3a). Similar to

the human preproapelin cDNA, the reported start codon for rat preproapelin did not

appear to conform to the Kozak consensus sequence, again by the presence of an

adenosine irnrnediately following the start codon.

3.2 mRNA Tissue Distribution of Apelin and the APJ Receptor

Northem blot analysis with a 3' RACE derived human cDNA probe reveaied

preproapelin mRNA detected as two bands migrating at 3.5 and 3.0 kb in extracts of

several human brain regions including the caudate nucleus, thalamus, hypothalamus,

hippocampus, midbrain, basal forebrain and frontal cortex (Fig. 4a). Northern analysis

utilizing the rat preproapelin-encoding cDNA as a probe revealed preproapelin mRNA

detected as two bands at 3.7 and 3.1 kb in several rat brain and peripheral tissue extracts

including the frontal cortex, cortex, striatum, midbrain, hippocampus, medulla pons,

cerebellum, pituitary, olfactory tubercle, septum, adrenal, vas deferens, testis, intestine,

kidney, and in the fetus (Fig. 4b and c). Higher levels of expression were indicated by

strong signals in the heart and faint signals were also detected in the spleen and liver (Fig.

42).

In situ hybridization anaiysis of rat brain utilizing the same probe used in the rat

northern analysis revealed preproapelin mRNA was abundantly expressed in cerebral

cortex in the frontal, parietal, and more caudal striate regions encompassing the

somatosensory areas, such as the primary visual, auditory and olfactory cortices (Fig. 5) .

There was also dense expression in the claustmm, anterior and posterior cingulate,

retrosplenial area, the subiculum and the olfactory tubercle. The medial and lateral septal

nuclei, nucleus of the diagonal band and the caudate-putamen had moderate labeling.

Several thalarnic nuclei were very densely labeled, such as the anterodorsal, mediodorsal,

ventroposterior, ventromedial, centrolateral and the media1 habenular nuclei, whereas

others such as the anteromedial thalamic nucleus had a much lower expression level.

Overall in hypothalamus, preproapelin rnRNA was not widely distributed, with dense

Fig. 4. Northern blot analyses of human and rat preproapelin mRNA. The distribution of preproapelin rnRNA in human (A) and rat (B and C) tissues. Each lane contained 10 pg of poly(A)+ RNA isolated from various tissues.

Fig. 5. In situ hybridization analyses of rat preproapelin mRNA. Darkfield autoradiograms of coronal sections of rat brain showing the localization of preproapelin mRNA, showing in distance from bregma according to the stereotactic coordinates (Paxinos and Watson, 1982). Shown are representitive sections at Ievels relative to the bregma at -0.3 mm (A), -0.5 mm (B), -1.8 mm (C), -2.8 mm (D), -3.9 to -4.2 mm (E) and -5.8 mm (F). 3v, third ventricle; 4v, fourth ventricle; 7, facial nucleus; AC, anterior cingulate cortex; AD, anterodorsai thalamic nucleus; AM, anterornedial thalamic nucleus; APT, anterior pretectat area; AUD, auditory area; CA, field of Ammon's horn; Ch. choroid plexus; CL, centrolateral thalamic nucleus; CP, caudate putamen; DG, dentate gyrus; DK, nucleus of Darkschewitsch; DM, dorsomedial hypothalamic nucleus: FP, C

frontoparietal cortex; IC, inferior colliculus; IPN, interpeduncular nucleus; LG, lateral geniculate complex; LS, lateral septal nucleus; MA, magnocellular preoptic nucleus: MD, mediodorsal thalamic nucleus; MG, medial geniculate nucleus; MEA, media1 amygdaloid nucleus; MEPO, median preoptic nucleus; MH, media1 habenular nucleus; MPO, medial preoptic area; OB, olfactory bulb; OT, olfactory tubercle; PAG, petiaqueductal gray; PB, parabrachial nucleus; PE, periventricular hypothalamic nucleus; Pi, pineal gland; PO, primary olfactory cortex; PT, parietal region; PVN, paraventricular hypothalamic nucleus; RS, retrosplenial area; S5, sensory root of the trigeminal nerve; SC, superior colliculus; SF, septofimbrial nucleus; SH, septohypothalamic nucleus ; SO, supraoptic hypothalamic nucleus; SS, primary somatosensory area; STISTH, subthalamic nucleus; SUB, dorsal subiculum; TS, tnangular septal nucleus; V, vestibular nucleus; VAL, ventral anterior-lateral complex thalamus; VIS, primary visud area; VM, ventromedial thalamic nucleus; VP, ventroposterior thalamic nucleus; ZI, zona incerta.

Fig. 5. (Continued)

Fig. 5. (Continued)

Fig. 5. (Continued)

labeling visualized only in the magnocellular neurons, with sparse signal present in the

media1 preoptic area, periventricular, ventromedial and dorsornedial nuclei. In

hippocampus, preproapelin mRNA was present in the CA regions of Ammon's horn and

the dentate gyrus. In midbrain, preproapelin mRNA expression was detected in the

medial and lateral geniculate complex, and very dense signal was observed in the zona

incerta and the subthalamic nucleus. Prominent expression was also detected in the

anterior pretectal nucleus, interpeduncular nucleus. inferior colliculus and the nucleus of

Darkschewitsch, with somewhat lesser amounts in the superior colliculus and the

periaqueductal gray. Preproapelin rnRNA was also present in the pineal gland,

parabrachial nucleus and the nucleus of the trigeminal nerve.

Previously. the APJ receptor gene was cloned in Our laboratory (O'Dowd et al.,

1993). DNA encoding the rat APJ receptor was used as a probe for in situ hybridization

of rat brain, which revealed the distribution of mRNA encoding the APJ receptor was

detennined in comparable sections as mapped for preproapelin (Fig. 6). APJ mRNA was

rnuch more discretely expressed, with extremely dense expression in the choroid plexus

lining the cerebroventricular system. There was abundant expression detected in the

olfactory bulb and the pineal gland. APJ mRNA was highly concentrated in the

paraventricula. nucleus and supraoptic nucleus of the hypothalamus, and was also present

in the Islands of Calleja, olfactory tubercle, dentate gyrus and the pontine gray. Sparse

labeling of cortex was evident, in the frontoparietal and pnmary olfactory regions.

Fig. 6. In situ hybridization analyses of rat APJ mRNA. Darkfield autoradiograrns of sagittal and coronal sections of rat brain showing the localization of APJ receptor mRNA (A through D). (A) shows a lateral representative section at 0.4 mm. Also shown are representitive sections at levels relative to the bregma at 1.2 mm (B), -1.8 mm (C) and - 4.8 mm (D). See Fig. 5 for abbreviation definitions.

Fig. 6. (Continued)

O

3.3 Discovery and Cloning of the TRE-R2 Receptor Cene

With the previous success of homology cloning by the degenerate PCR method, 1

proceeded to design new degenerate primers in the highly conserved TM3 and TM7

regions, yet based over a wider spectrum of GPCRs in order to diversify the products

arnplified and therefore increase my chances of arnplifying novel GPCR-encoding DNA.

To isolate novel GPCR genes, 1 aligned the TM3 and TM7 regions of various GPCRs

cloned in our Iaboratory inciuding GPRl through GPRlO, GPR14, GPRIS, GPR19

through GPR25, GPR27, GPR30 and GPR3 1 and designed two new degenerate prirners,

Deg 1 and Deg2 (Fig. 2). A rat brain 5' stretch cDNA Iibrary was amplified by PCR with

De@ paired with two prirners specific for the 5' (primer Pl) and 3' (primer P2) regions

flanking the cDNA library inserts to further increase the degeneracy of the search. One

PCR product (-400 bp) was found to encode a novel GPCR from TM4 to TM7, sharing

the greatest translated sequence identity of 4 4 8 to the thyrotropin releasing hormone

receptor TRH-Ri. This cDNA was labeled with [ 3 2 ~ ] d ~ T P - a and used to probe the

same rat brain cDNA library. which resulted in the isolation of two cDNA clones. These

clones were amplified by PCR using primers Pl and P2 and the products subcloned into

the pcDNA3 vector. Both cDNAs reveded identical sequences encoding the full-length

receptor, which 1 named TRH-R2. TRH-R2 encoded a protein of 352 amino acids which

shared the greatest sequence identity of 68% in the TM domains with TRH-RI (Fig. 7).

TRH-R1 had many conserved residues and motifs typical of the GPCR family, including

an asparagine in TM 1, an aspartate in TM2. prolines in TM'S 5 through 7, one consensus

sequence for N-Iinked glycosylation in the amino terminus. cysteines in

A a < A > O < 2 > C

O t a n a a a x W ,

t A O u r 0 > 2 z W ur U Y a

w a c n UI r 2 W r

N 1 - Y ) 1 CI

- x W

a Y < C

2

V)

t a > C

O UY UY

a a z Y

UI

ur < d

O C

O a rr V)

> Y C

t UY > t t O t

> C - O O a x W Y t W

f A t rra a u O u r

gj - < > a

if

Fig. 8. Northern blot analyses of rat TRH-R2 mRNA. Each lane contains 10 pg of

p o l y ( ~ ) + RNA isolated from various rat tissues. The molecular size is indicated on the right.

the first and second extracellular loops, a PKA/PKC consensus sequence in the third

intracellular loop and two possible palmitoylation cysteine sites in the carboxy tail.

In a collaboration with Dr. Marvin C. Gershengorn of the Department of

Medicine, Weill Medical College of Corne11 University, TRH-R2 was expressed in COS

cells and found to have similar affinities for thyrotropin-releasing hormone (thyroliberin,

TRH) with the mouse and rat TRH-RI receptors in affinity and cornpetition binding

experiments (O'Dowd et al., in press). in addition. binding experiments for mouse and

rat TRH-RI and the rat TRH-R2 receptors with TRH analogs also revealed binding with

similar affinities (O' Dowd et al., in press). Together, these binding data experiments

revealed TRH-R2 to be a second thyrotropin-releasing hormone receptor.

3.4 mRNA Tissue Distribution of the TRH-R2 Receptor

Tissue distribution of TRH-R2 mRNA transcripts was obtained by northern blot

analysis using a cDNA fragment encoding T M - R 2 from TM4 to TM7 and po ly (~)+

RNA isolated from various rat tissues. In the brain, a major transcript of 9.4 kb length

(and a faint band of 3.8 kb length) was seen in the pons, hypothalamus and midbrain (Fig.

8). Faint bands of 9.4 kb length were also found in the striatum and pituitary (data not

shown).

TRH-R2 receptor mRNA distribution visualized, by in situ hybridization

histochemistry, revealed abundant expression in very discrete nuclei and regions of rat

brain (Fig. 9). There was extremely dense expression in frontoparietal cortex,

particularly in the primary somatosensory and motor areas, and also in the pnmary visual

area and primary olfactory cortex. Strong signais were also present in other areas of

Fig. 9. In situ hybridization analyses of rat TRH-R2 mRNA. Darkfield autoradiograms of coronal sections of rat brain showing the localization of preproapelin mRNA, showing in distance from bregma according to the stereotactic coordinates (Paxinos and Watson, 1982). Shown are representitive sections at levels relative to the bregma at -0.7 mm (A), -0.8 mm (B), -1.3 mm (C), -3.3 mm (D), -3.9 mm (E), -5.3 mm (F), -6.8 mm (G), and -7.9 mm (H). (I) Sagittal section of rat brain 2.4 mm from the midline. (J) Section through pituitary gland. ACg, anterior cingulate area; AHi, amygdalohippocarnpal area; M y , anterior hypothalamic nucleus; AP, antenor lobe of pituitary; AV, anteroventral nucleus; BST, bed nucleus of the stria terminalis; CM, central media1 nucleus of thalamus; CR, centrai nucleus raphe; DGgr, dentate gyrus crest; En, endopiriform nucleus; FrP, frontoparietal cortex; FrPm, primary motor area; FrPss, primary somatosensory area; HDB, nucleus of the diagonal band; IC, inferior colliculus: [P. interpeduncular nudeus; LI), laterodorsal nucleus of thalamus; LGd, lateral geniculate nuclear complex, dorsal part; LH, lateral hypothalamic area: LP, lateral posterior nucleus of thalamus; MeA, media1 nucleus of amygdala; MGd, dorsal mediai geniculate nuclear complex; MGv, ventral media1 geniculate nuclear complex; MHb, media1 habenular nucleus; MPO, medial preoptic area of hypothalamus; MR, mesencephalic reticular nucleus; MRN, mesencephalic reticular nucleus; NIL, neurointermediate lobe of pituitary; OT, olfactory tubercle; PAG, periaqueductal gray; PB, parabrachial nucleus; PCg, posterior cingulate area; PH, posterior hypothalamic nucleus; PMd, dorsal premammillary nucleus; PMv, ventral premarnmillary nucleus; PN, pontine nucleus; PO, primary olfactory cortex; PRN, pontine reticular nucleus; PRt, pontine reticular nucleus; PVH, paraventricular nucleus of hypothalamus; PVT, paraventricular nucleus of thalamus; Rem, nucleus reuniens, media1 part; RL, rostral linear raphe nucleus; RSp, retrospenial area; S, subiculum; SAG, nucleus sagulum; SC, superior colliculus; Sd, dorsal subiculum; Sp, pyramidal layer of the subiculum; STh, subthalarnic nucleus; Str, striate areas of primary visual cortex; Sv, ventral subiculum: VL, ventrolateral nucleus of thalamus; VM, ventromedial nucleus of thalamus; VP, venteroposterior nucleus of thalamus; VTA, ventral tegmental area; 23, zona incerta.

Fig. 9. (Continued)

Fig. 9. (Continued)

Fig. 9. (Continued)

Fig. 9. (Continued)

Fig. 9. (Continued)

cortex, such as the anterior cingulate area, concentrated in the deeper rather than in the

superficial layers of cortex. Further caudally, TRH-R2 mRNA expresssion was

moderately dense also in the posterior cingulate area, retrosplenium, striate areas and

throughout the subiculum in both dorsal and ventral portions.

Several thalamic nuclei displayed extremely dense labeling, such as the

paraventricular, centromedial, anteroventral, and ventroposterior thalamic nuciei and the

media1 habenular nucleus. TRH-R2 mRNA was present less abundantly in other

thalamic nuclei, such as the laterodorsal, lateroposterior and ventromedial nuclei. and the

media1 reuniens nucleus. In hypothalamus, TRH-R2 mRNA was most abundant in the

anterior hypothalamic area, and also present in the medial preoptic and lateral

hypothalamic areas, the paraventricular nucleus and some of the mammillary nuclei.

Moderate labeling was also seen in the bed nucleus of the stria terminalis, the nucleus of

the diagonal band, some of the arnygdaloid nuclei and in the subthalarnic nucleus. The

geniculate nuclear complex contained very dense expression in the medid geniculate, in

both the dorsal and ventral divisions of the nucleus, whereas in the Iateral geniculate,

moderate expression was observed largely in the dorsal division.

In midbrain, a punctate pattern of TRH-R2 mRNA expression was observed in the

superior colliculus, penaqueductal gray and the mesencephalic reticular nucleus. Small

amounts of mRNA were present in the ventral tegmental area. The pontine gray

expressed TRH-R2 mRNA very abundantly, and the central and rostral Iinear raphe

nuclei showed moderately dense labeling as well. Lesser amounts were evident in the

in ferior colliculus, the nucleus sagulum, the pontine reticular nucleus and the

parabrachial nucleus. TRH-R2 receptor mRNA was detected in pituitary gland, as

sections revealed a very small amount of labeling in the anterior lobe, whereas the

neurointermediate lobe was devoid of any signal.

3.5 Discovery and Cloning of the GPR54 Receptor

In addition to TRH-R2, the degenerate PCR approach dso retrieved DNA

panially encoding the novel GPCR, GPR54. The degenerate primers Degl and Deg2

were used to arnplify the 5' stretch rat brain cDNA Iibrary. One of the resulting rat

clones appeared to partially encode a galanin-like receptor. The partial cDNA was

radiolabeled and used to screen the cDNA library employed in the degenerate PCR. Two

positive plaques were purified and their inserts amplified by PCR with the Pl and P2

primers flanking the cloning site of the hgtl 1 vector. Sequence analysis revealed that

each plaque encoded a region of a putative GPCR from TM3 to the carboxy terminus

identical to each other and the originai probe. A second round of screening of 1 x IO6

plaques freshly plated from the same library y ielded an additional three positive plaques.

PCR amplification of these positive plaques with P l and P2, each paired with an interna1

primer, revealed that only one of these positive plaques contained the entire ORF. This

plaque was purified, the insert subcloned into pBluescript and was confirmed to contain

the 5' end of the full-length ORF. Finally, two specific primers from the 5' and 3' ends

of the ORF were used to arnplify the full length rat cDNA 1.2 Kb clone, named GPR54.

Sequence analysis revealed the cloned GPR54 ORF to be 1 19 1 bp in length encoding a

protein of 396 arnino acids, identical to the previous phage clones and the original probe

and sharing significant identities with the galanin receptors (Fig. 10). Conserved residues

and consensus sequences of the GPCR family present in GPR54 included an asparagine

Fig. 10. Schematic representation of the GPR 54 receptor. GPR54 (string of yellow and blue spheres) in a ce11 membrane. Blue spheres represent amino acids conserved with the galanin receptors GdR1, GalR2, and GalR3. Green branches = glycosylation sites; red spheres = P K A P K C sites and orange spirals = palmitoyaltion sites. A disdphide bride is indicated between extracellular loops 1 and 2.

in TM 1, an aspartate in TM2, prolines in TM'S 4 through 7, three consensus sequences

for N-linked glycosylation in the amino terminus, cysteines in the first and second

extracellular loops, a PKNPKC consensus sequence in the second intracellular loop, a

PKC consensus sequence in the third intracellular loop and three possible pdmitoylation

cysteine sites in the carboxy tail.

A BLAST search with the rat GPR54 sequence revealed high identity with a

hurnan 3.5 Mb contig located in chromosome 19p13.3 containing a serine protease gene

cluster (Genbankm accession # AC005379). Sequence analysis revealed a previously

unrecognised 3.3 kb intron-containing human orthologue of GPR54 encoding a protein

398 amino acids in length and sharing a translated arnino acid identity of 8 1% ( 1 0 %

identity in the TM regions). The genomic sequence revealed four introns located in TM2

(-800 bp, interrupting the translated FYI..ANL sequence), TM3 (-800 bp, interrupting

IQQ..VSV), TM4 (-250 bp, interrupting WVG..SAA) and in the third intracellular loop

(- 180 bp, interrupting ALQ..GQV).

In a collaboration with Dr. Gary O'Neill of the Department of Biochemistry and

Molecular Biology, Merck Frosst Center for Therapeutic Research, GPR54 was tested as

a possible galanin receptor. GPR54 expressed in COS cells revealed no specific binding

with 125~-hurnan galanin (Lee et al., 1999). As a control experiment, the galanin

receptor, GaIR1, was expressed in COS cells and revealed high affinity and specific

binding for l251-human galanin. Despite the high level of sequence identity between

GPR54 and GalRI, ~ ~ k 5 4 does not appear to be another galanin receptor.

3.6 mRNA Tissue Distribution of the GPR54 Receptor

Both northem blot and in situ hybridization analyses of GPR54 were performed

with a DNA probe encoding GPR54 from TM3 to TM7. The tissue distribution of

GPR54 was obtained by northem blot analysis using poly(A)' RNA isolated from various

rat tissues (Fig. 1 1). In the brain, multiple RNA transcripts with a complex pattern were

detected in the medulla pons, midbrain, hippocampus, cortex, frontal cortex. and

striatum. The most intense band was approximateiy 3.7 Kb in length, with a single,

larger transcript of approximately 12 Kb length detected in the liver and intestine only.

No transcripts were revealed in the cerebellum or kidney tissues.

Using in situ hybndization of rat brain sections. the distribution of GPR54 mRNA

was found to be discretely localized to many areas (Fig. 12). The highest levels of

expression were seen in hypothalamic and amygdaloid nuclei. GPR54 mRNA was

highly expressed in the zona incerta, ventral tegrnental area, dentate gyrus, hypothalarnic

arcuate nucleus, dorsomedial hypothalamic nucleus, primary olfactory cortex, lateral

habenular nucleus, lateral hypothalamic area, locus coenileus, and the corticai and mediai

nuclei of the amygdala. GPR54 mRNA was also concentrated in the superior colliculus.

medial preoptic area, anterior hypothalamic area, posterior hypothalamic nucleus,

periaqueductal gray, parafascicular thalamic nucleus, parabrachial nucleus, and ventral

premammillary nucleus. The signals detected in the septohypothalamic nucleus, inferior

colliculus, media1 nucleus of the amygdala, mesencephalic reticular nucleus and

retrosplenial cortex were diffuse and less abundant.

Figure 11. Northern blot analyses of rat GPR54 mRNA. Northern blot analysis of the tissue distribution of GPR54 rnRNA in rat brain. Each lane contained 5 pg of poly(A)' RNA isolated from various rat tissues.

Figure 12. In situ hybridization of rat GPR54 mRNA. Darkfield autoradiograms of sagittal and coronal sections of rat brain showing the localization of GPR54 receptor mRNA showing in distance according to the stereotactic coordinates (Paxinos and Watson, 1982). (A) shows a lateral representative section at 0.9 mm. Also shown are representitive sections at levels relative to the bregma at -3.3 mm (B), -3.8 mm (C) and - 6.3 mm (D). Aco, cortical nucleus of the amygdala; AHy, anterior hypothalamic area; Arc, hypothalamic arcuate nucleus; IC, inferior colliculus; CA, field of Ammon's horn; DG, dentate gyrus; DM, dorsornedial hypothalamic nucleus; LC, locus coeruleus; LH, lateral hypothalamic area, LHb, lateral habenular nucleus; MeA, media1 nucleus of the amygdala; MPO, medial preoptic area; M N , mesencephalic reticular nucleus; PAG, periaqueductal gray; PB, parabrachial nucleus; PF, parafascicular thalarnic nucleus; PH. posterior hypothalamic nucleus; PMV, ventral premammillary nucleus; PO, primary olfactory cortex; RSpl, retrosplenial cortex; SC, superior colliculus; SHy, septohypothalamic nucleus; VTA, ventral tegmental area; 21, zona incerta.

Fig. 12. (Continued)

Fig. 12. (Continued)

3.7 Discovery and Cioning of the GPR57 and GPR58 Receptor Genes and a

Pseudogene yrGPR57

Recently, various novel genes have been found in the patent literature, including

those encoding novel GPCRs. However, certain patents are limited to only sequence

data, ofter, from a non-human source, contain little if any data on expression distributions

or function, and are generally unknown to the scientific community. In my efforts to

characterize novel GPCR-encoding genes, 1 have begun investigation of several patented

GPCR genes.

Human genornic DNA was PCR amplified using primers P5 and P6, based upon a

patent (#EP 0859055-Ml) gene sequence HNHCI32. 1 obtained a gene very similar to

HNHC132 with the exception of two single base-pair substitutions and two single base-

pair deletions. The first deletion resulted in a stop codon in TM3 and an ORF of only

309 bp encoding a 103 amino acid protein. In order to search for a gene identical to the

patent sequence containing an ORF encoding a complete GPCR, this DNA was used to

screen a human genomic library. Two phage were identified and PCR amplified with the

P5 and P6 primers and a sequence analysis revealed both the amplified genomic DNA

and genomic library gene products to be identical and both encoded a pseudogene,

vGPR57 (Fig. 13), sharing a 99.6% bp identity with HNHCI32 (which was renamed

GPR57).

1 obtained a rabbit sequence as revealed in a patent (# JP 1997051 795-Ail,

GenbankTM Accession # E12664) which only partially encoded a novel GPCR. This

sequence was used in a BLAST (Altschul et al., 1997) search of the GenbankTM GSS

database to reveal human genomic clone 200SD7 (GenbankTM Accession # B52458)

5'- CTCATCC~CC~GGAAAGM~CAAGGGATAAAGCACC 1 98 ATG GAT C ï A ACT TAT AIT' CCC GAA GAC CïA TCC AGT TGT CCA M TIT GTA M T AA* ATC Met Asp Leu Thr Tyr Lle Pro Glu Asp Leu Ser Ser Cys Pro Lys Phe Vai Asn Ile 30 158 CTG TCC TCC CAC CAA CCG CTC TTT TCA TGT CCA GCrT GAT AAT GTA TTC GGT TAT GAC TGG Leu Ser Ser His Gln Pro Leu Phe Ser Cys Pro Gly Asp Asn Val Phe Giy Tyr Asp Trp 30 TM 1 217 AGC CAT GAT TAT 'CA CTA TTt GGA AAC TI% GlT ATA ATG GTT TCC ATA TCG CAT lTC AAA Ser His Asp Tyr Leu Phe Gly Am Leu Val Ile Met Val Ser ïie Ser His Phe Lys 59 TM 2 277 CAG CIT CAC TCT CCC ACA AAC TIT CTG ATC CTC TCC ATG GCA ACC ACG CAC TTT CTG CïG GIn Leu His Ser Pro Thr Asn Phe Leu Ile Leu Ser Met Ala Thr Thr Asp Phe Leu Leu 79 337 GGT I7-ï GTC ATT ATG CCA TAC AGC ATA ATG CGA TCA GTG GAG AGT TGC TGG TAC TIT GGG Gly Pht Vd [Ir: &Met Pro Tyr Ser Ile Met Arg S a Val Glu Ser Cys Trp Tyr Phe Giy 99 TM 3 397 GAT GGC TTT TGT AAA TTC CAC ACA AGC TTT GAC ATG ATG CTC AGA CTG ACC TCC A I T ITC Asp Gly Phe Cys Lys Phc His Thr Ser Phe Asp Met Met Leu Arg Leu Thr Ser Ile Phe 119 457 CAC CTC TGT TCC ATT C i C i AïT GAC CGA TTT TAT GCC GTG TGT TAC CCT l'TA CAT TAC ACA His Leu Cys Ser Ile N a Ile Asp Arg Phe Tyr A h Val Cys Tyr Pm Leu His Tyr Thr 139 TM4 5 17 ACC .a ATG ACG M C TCC ACC ATA AAG CAA CTG CI% GCA TiT TGC TGG TCA GTT CCT GCT Thr Lys Mct Thr Asn Ser Thr Ile Lys Gh Leu Leu Ala Phe Cys Trp Ser Val Pm AIa 159 577 CIT ITT TCT TTT GGT W A GIT CTA TCT GAG GCC GAT G T ï TCC GGT ATG CAG AGC TAT AAG Leu Phe Ser Phe Gly Leu Val k u Ser Glu Ala Asp VaI Ser Gly met Gln Ser Tyr L-vs 1 79 63 7 ATA CTT GTT GCT TGC TI% AAT TTC TGT GCC C l T ACT 'ITC AAC h4A TTC TGG GGG ACA ATA Ile Leu Val Ala Cys Phe Asn Phe Cys N a Leu Thr Phe Asn Lys Phe Trp Gly ?hr Ile 199 TM 5 697 I T G T C ACT ACA TGT TTC T T ï ACC CCT GGC TCC ATC ATG GïT GGT A R TAT GGC AAA ATC Leu Phe Thr Thr Cys Phe Phe ïhr Pro Gly S a Ile Met Val Gly IIe Tyr Gly Lys Ile 219 757 TIT ATC G'IT TCC M CAG CAT GCT CGA GTC ATC AGC CAT GTG CCT GAA M C ACA AAG GGG Phe Ile Val Ser Lys GIn His Ala k g Val Ile Ser His Vd Pro Glu Am Thr Lys Gly 23 9 817 GCX GTG -4iU Ai\A CAC CTA TCC M G AAA AAG GAC -4GG AI\A GCA GCG AAG ACA CTG GGT ATA .Ma Vd Lys Lys His Leu Ser Lys Lys Lys Asp Arg Lys Ala Ah Lys Thr Leu Gly [Ie 259 TM 6 877 GTA ATG GGG GTG TTT C ï G GCT TGC TGG T'TG C m TGT TIT ClT GCT GTT CTG A l T GAC CCA Vai Met GIy Val Phe Leu Ala Cys Trp Leu Pro Cys Phe Leu Ala Vai Leu Ile Asp Pm 279 937 TAC CT.4 GAC TAC TCC ACT CCC ATA CTA ATA TTG GAT CTT ï T A GTG TGG C ï C CGG TAC T C Tyr Leu Asp Tyr S a ïhr Pro [le Leu Ile Leu Asp Leu Leu Val Trp Leu Arg Tyr Phe 299 TLM 7 997 AAC TCT ACT TGC AAC CCT CTT ATT CAT GGC T[T T?T AAT CCA TGG TTT CAG AAA GCA TTC Asn Ser Thr Cys Asn Pro Leu [le His Gly Ph<: Phe Asn Pro Trp Phe Gln Lys N a Phe 319 1 057 AAG TAC ATA GTG TCA GGA M t ATA TLT AGC TCC CAT TCA GAA ACT GCA .4AT TTG TIT C m Lys T-yr Ile Val Ser Gly Asn IIe Phe Ser Ser His Ser Glu Thr Ala Asn Leu Phe Pro 339 I I I 3 GAA GCA CAT T.UTMGCTITGCAAAAGTGAATAGAATA?TGCAAATGAGAT~G -3' Glu Ala His

Fig. 13. Sequence of the vGPR.57 pseudogene. Differences and deletions from GPEZ57 nucleotide sequence are indicated in lower case bold and by a "*" respectively. Without the frame shifts, the predicted TMç are shaded and labeled. Amino acids are numbered on the Ieft and nucleotides on the right.

which partiaily encoded a receptor from the third intracellular loop to the stop codon with

92% identity to the rabbit sequence. Human genomic DNA was PCR amplified using a

primer based on the TM6 encoded by clone 2005D7 (P8) and a degenerate primer

designed from the sequence encoding TM3 of PNR, 5-HT, pseudogene, and the rabbit

€12664 sequence (P7). A PCR product (-400 bp) revealed an 89% identity to the

patented rabbit sequence, confirming a hurnan ortholog. This DNA was used to screen a

human genomic library, revealing a phage encoding a novel GPCR, which 1 named

GPR58. The sequence obtained was from the first extracellular loop to the stop codon,

indicating the presence of an intron at the TMZ/first extracellular loop junction. Dunng

the course of my work, a patent was released (# IP 1997238686-NI, Genbankm

Accession # E13892) which included a gene named phBL5 encoding the human GPR58

ORF. To isolate the GPR58 ORF, two sets of human GPR58 primers (P9 through P12)

were used to arnplify the two exons flanking the TM2Ifirst intracellular loop junction

intron. The amplified fragments were joined in one round of PCR by the extension of

primers whose sequences overlapped the two fragments (Pl3 and P14) and the O W was

amplified in a second PCR round with GPR58 5' and 3' UTR-specific primers P9 and

P 12.

GPR57 and GPR58 encoded proteins of 343 and 306 amino acids respectively.

The sequences for GPR57 and GPR58 were aligned and found to have signifiant

sequence similarity to each other (Fig. 14). Conserved residues and consensus sequences

of the GPCR farnily present in the GPR57 and GPR58 receptors included an asparagine

in TMI, an aspartate in TM2, prolines in TMs 4 through 7, cysteines in the first and

second extracellular loops, and PKC consensus sequences in the second and third

S . . . a . . . .a . . . . - n i . . m

-

O X . O 0

intracellular loops. The sequence of the GPR58 ORF varied only by two thymine to

cytosine substitutions compared with the phBL5 patent sequence, resulting in a conserved

substitution of Mal3' in place of Val and a silent nucleotide variation encoding ~ y r ' ?

Given that these arnino acid differences are either silent or very well conserved and cause

no shifts in the translational reading frames, they would probably have little if any effect

on the mature GPR58 protein function.

3.8 GPR57 and GPR58 Expression

Northern analysis was also performed for GPR57 and GPR58 on human tissue.

Since the GPR57 and yGPR57 sequences shared 99.6% identity, the vGPR57 probe

wouId also detect GPR57 mRNA transcnpts. For GPR57, revealed in the patent (# EP

0859055-Ail) to have been cloned from human hippocampus cDNA, no visible

transcripts were detected in the pons, thalamus, globus pallidus, caudate, putamen or

cerebellum. For GPR58 (revealed in patent # JP 199705 l795-AA to have been cloned

from rabbit smooth muscle cDNA and in patent # JP 19972386864/1 from human

cerebellum cDNA) no visible transcripts were detected in the pons, thalamus,

hypothalamus, hippocampus, caudate, putamen, frontal cortex, basal forebrain, midbrain

or Iiver.

3.9 Attempted Pharmacological Characterization of the GPRSS Receptor

COS-7 cells were transfected with a construct encoding GPR58 and membranes

harvested for ligand binding assays. For the receptor encoded by GPR58, [)H]-SHT was

tested for binding in cornpetition expenments with three compounds: methiothepin (a 5-

HT, antagonist), mianserin (a non-selective 5-HT antagonist) and SDZ-205,557 HCl (a 5-

HT, antagonist). In each experirnent, no specific binding of serotonin to the GPR58

receptor was detected.

3.10 Chromosomal Localization of the GPR58 Receptor Genes and Pseudogene

yGPR57

A Southern blot of a hurnan monochromosornal somatic ce11 hybrid panel with a

probe encoding yGPR57 was performed. Given the 99.6% sequence identity between

yGPR57 and GPR57.1 expected to see bands revealing the chromosornal localization of

both vGPR57 and GPR57. A single band was detected (data not shown) suggesting that

both vGPR57 and GPR57 localized to chromosome 6.

FISH anaiysis of human metaphase spread chromosomes was used to identify the

chromosomal localization of yGPR57 and GPR58. The phage clones were biotinylated

and used for FISH mapping. The detailed positions were deterrnined based on the

summary from 20 photographs of human chromosome 6 region q23-q24 for vGPR57 and

chromosome 6 region q24 for GPR58 (Fig. 15).

3.11 Discovery and Cloning of the GPR61 and GPR62 Receptor Genes

Another patented gene encoding a novel GPCR was patent # J P 8245697, which

only partially encoded a rabbit GPCR. This sequence was used to design prirners

encoding TM3 (PH) and TM6 (P16), which were used to amplify human and rat

genomic DNA. PCR products -500 bp in length were subcloned and sequenced to reveal

hurnan and rat DNA with >80% identity to the rabbit gene, comfirming them to be the

Fig. 15. FISH analysis for yGPRS7 and GPR58. (A) represents yGPR57 and (B) GPR58, showing results of metaphase spread chromosomes probed with phage clones encoding vGPR57 or GPR58 and an ideogram summarizing the results of both FISH analyses. Each dot represents the location of a fluorescent signal on the chromosome using phage containing the yGPRS7 or GPR58 clone as a probe.

human and rat orthologues. The human sequence was used to design primers for RACE

(Frohman et al., 1988) to ampli@ Marathon-readyThf human whole brin cDNA. Both 3'

and 5' cDNA ends were amplified successfully of 1.6 and 1.1 kb in respective size to

elucidate the ORF encoding a 417 amino acid protein. The novel GPCR encoded shared

identical sequence with the TM3-TM6 previously amplified, and was narned GPR6 1.

The sequence for GPR61 was used to search the GenBankm databases. A search of the

HTGS database retrieved what appeared to be a novel, intronless GPCR-encoding gene in

a PAC clone (Accession #AC006255) localized to chromosome 3p2 1.1-2 1.9. The

proposed start methionine for this ORF conformed to an adequate Kozak consensus

sequence (Kozak, 1996) and was preceded by an in-frame stop codon. Primers were

designed to arnplify human genomic DNA which amplified a product - 1.2 kb in length.

This product was subcloned, sequenced, confirmed to encode a full-length ORF of 367

amino acid length with an identicai sequence to the GenBankTM retrieved PAC clone, and

the GPCR encoded was named GPR62.

The amino acid sequences for GPR61 and GPR62 were aligned and found to

share low but significant sequence similarity, particularly within TM2, 5, 6 and 7 (Fig.

16). Conserved residues and consensus sequences of the GPCR family present in GPR6 1

and GPR62 included an asparagine in TMI, aspartate in TM2, prolines in TM'S 5 through

7 (and in TM 4 for GPR61), cysteines in the first and second extracellular loops, a

PKA/PKC consensus sequence in the second intracellular loop, and a possible

palmitoylation cysteine site in the carboxy tail. GPR61 had one consensus sequence

while GPR62 had two consensus sequences for N-linked glycosylation in the amino

terminus.

Fig. 17. Northern blot analyses of human and rat GPR61. Each lane contains 10 pg

of p o l y ( ~ ) + RNA isolated from various human (A) and rat (B) tissues. The molecular s i x is indicated on the right.

3.12 mRNA Tissue Distribution of the GPR61 Receptor

To determine the expression patterns of GPR61, northem blots of human and rat

tissue were performed using the human 3' RACE amplified DNA and rat DNA fragments

encoding GPR61 as probes. As shown in Fig. 17, GPR61 reveais clear mRNA

expression in certain regions of the brain. In the human, GPR61 mRNA was found as a

single transcript in the caudare, putamen and thalamus of approximately 4.3 kb length. In

rat tissues, GPR6l mRNA saw three transcripts of approximately 5.1, 3.8 and 3-2 kb

length (3.8 kb being most prominent) in a variety of brain regions including the

cerebellurn, frontal cortex, cortex, striatum, midbrain, hypothalamus, medulla pons,

hippocampus, olfactory tubercle, pituitary and septa.

4.0 DISCUSSION

4.1 Apelin: Characterization of the Endogenous Peptide Ligand for the APJ

Receptor

Originally cloned in Our laboratory, the APJ gene encodes a receptor that most

closely resembled the angiotensin receptor ATI, sharing an amino acid identity of 54% in

the TM regions. However, the receptor displayed no specific binding for angiotensin II

(O'Dowd et al., 1993). In a successful application of the "reverse pharmacology"

technique, the apelin peptide was isolated from bovine stomach extracts using the bovine

APJ receptor expressed in CHO cells, and its sequence used to obtain human and bovine

cDNAs encoding preproapelin (Tatemoto et al., 1998). Subsequently, the entire protein

sequences of both bovine and human preproapelin were deduced from the cDNAs.

However, no cDNA sequences, detailed tissue distributions or physiological functional

data for preproapeIin were reported. To further investigate the physiological functions of

apelin I first endeavored to characterize the apelin gene to determine its genomic

organization, sequence, and mRNA distribution. Previously, cDNA sequences and

preliminary studies using synthetic peptides revealed the hurnan and bovine C-terminal

regions of preproapelin to be identical and essential for specific binding to the APJ

receptor (Tatemoto et al., 1998). Sequence analysis revealed high identities between

human, bovine and rat preproapelin with 100% identity in the C-terminal 13 amino acids,

which encodes the mature apelin peptide (Fig. 3a). In addition, a cornparison of the

mature apelin peptide with angiotensin II revealed several conserved amino acids (Fig.

3b). perhaps not unexpected given the degree of similarity between their receptors,

AT 1 /AT2 and APJ. Interes tingl y, the human apeiin genomic structure revealed an intron

in the 3' UTR, which is rare. Both human and rat cDNAs displayed a lack of Kozak

confonning start codons. While non-Kozak conforming start codons are rare, several

cases have been reported (Kozak, 1996).

In situ analysis of the brain revealed APJ mRNA to be more discretely expressed

than preproapelin rnRNA, especially dong the cerebroventrïcular system, suggesting

important CNS vascular functions. Future immunohistochemical studies will be aimed at

cornparing the distributions of the APJ receptor and apelin peptide. A cornparison of

CNS distributions of the APJ and AT1 receptors (Ferguson and Washburn, 1998)

revealed high levels of both APJ and AT1 in the choroid plexus. Given the sequence

similarities between angiotensin II and the apelin peptide, 1 also compared

angiotensinogen, the precursor to angiotensin II, and preproapelin expression patterns.

Apelin mRNA brain expression correlated well with angiotensinogen, which has been

shown to possess a distinct expression pattern within the limbic and sensorimotor areas of

the brain (Semia, 1995). Both preproapelin and angiotensinogen showed expression in

the hippocarnpus, as well as such hypothalamic regions as the dorsal hypothalamic area,

media1 preoptic area, and the ventrornedial hypothalamic nucleus. Two adjacent regions,

the periventricular and paraventricular nuclei expressed preproapelin mRNA and

angiotensinogen respectively. In the thalamus and midbrain, shared expression was seen

in the venuoposterior thalamic nucleus, medial habenular nuclei, anterior pretectal area,

periaqueductal gray, inferior and superior colliculi and the interpeduncular nucleus. Both

preproapelin and angiotensinogen were seen in the anterior and posterior cingulate

cortex, caudate putamen, media1 and lateral septal areas, and the primary olfactory cortex.

In view of the sequence and distribution similarities between apelin and

angiotensin II (as well as APJ and ATl/AT2), 1 predicted apelin would have similar

physiological effects as angiotensin II, known to have a role in regulating blood pressure,

fluid and electrolyte balance (Phillips et al., 1993; Saavedra, 1992) in addition to acting

in various roles as a neurotransmitter (Ferguson and Washburn, 1998). In the periphery,

apelin was widely expressed in many tissues with a particularly dense mRNA signal in

the hem. indicating possible roles for apelin in blood pressue and h e m rate. In a

collaboration with Dr. Daniel H. Osmond of the Department of Physiology, University of

Toronto, preliminary results of i.v. injections in rats reveaied apelin to have hypotensive

properties, eliciting a brief drop in blood pressure (both systolic and diastolic), in contrast

to the weI1-known vasopressor effects of angiotensin II (Lee et al., in press). Preliminary

behavioural data was also collected, which demonstrated i-p. injected apelin ellicited

short-term increases in drinking behaviour, in parallel with the thirst-promoting actions of

angiotensin iI (Lee et ai., in press). In the future, further behavioural and physiological

investigations will be conducted to fully characterize apelin-induced thirst and

cardiovascular effects.

4.2 TRH-R2: Discovery and Characterization of a Second GPCR for

Thyrotropin-Releasing Hormone

TRH is a tripeptide (pGlu-His-ProNH,) that functions as a hormone, a paracrine

regulatory factor and a neurotransmitter/neuromodulator. Until recently, only a single

GPCR for TRH was known (TRH-RI) (Gershengom and Osman, 1996). While our

laboratory independently discovered TRH-R2, during the course of this work, TM-R2

was reported by two other groups (Cao et al., 1998; Itadani et al., 1998). Within the ORF

of TRH-EU, the published sequences varied by two single nucleotide differences located

in the regions encoding the N-terminal portion of TM4 and the carboxy terminus.

Specificdly, one group reported an isoleucine and valine at arnino acid positions 143 and

347 respectively (Itadani et al., 1998), while the ocher reported a methionine and glutamic

acid at these respective positions (Cao et al., 1998). By cornparison, our TRH-R2

sequence agreed with 11e143 in accordance with the fint report by Itadani's group

(Itadani et al., 1998) but with ~11,1347 as reported by Cao's group (Cao et ai.. 1998).

In terms of ligand binding, it is noteworthy that of four amino acid residues within

the transmernbrane helices and two within the extrace1Iula.r loops of mouse TRH-R1

(Straub et al., 1990) previously identified as sites of direct interaction with TRH

(Gershengorn and Osman, 1996; Perlman et al., 1997), ail six residues are conserved in

rat (de la Pena et al., 1992; Zhao et al., 1993), human (Duthie et al., 1993) and chicken

TRH-RI (Sun et al., 1998) and rat TM-R2 (Cao et al., 1998; Itadani et al., 1998). This

would suggest a likely similarity between the binding of TRH analogs by these receptors,

with implications that differences could possibly exist in receptor tissue distribution.

expression or down-regulation to necessitate the expression of a second TRH receptor.

In fact, a collaboration with Dr. Marvin C. Gershengorn of the Department of

Medicine, Weill Medicd ColIege of Corne11 University, reveded no differences in TRH

and TRH-analog acute binding with TRH-RI or TRH-R2 (O'Dowd et al., in press).

However, tissue distribution analyses and down-regulation studies did find differences

between TRH-R 1 and TRH-R2. In situ hybridization analysis revealed TM-R2 mRNA

exhibited a distinct brain distribution with especially abundant levels of expression in

areas of the cortex, thaiarnus and the pontine nucleus (Fig. 9). In addition, other areas of

the midbrain including the medial and lateral geniculate nuclei, superior colliculus,

periaqueductal gray, mesencephalic reticular nucleus, and central raphe nucleus displayed

discrete levels of TRH-R2 mRNA expression. Together with strong distinct signals from

various sensory and motor control areas in the cortex and thaiamus (e-g. the striate areas

of the primary visual cortex, the paraventricular, centromedial, anteroventral and

ventroposterior thdamic nuclei), TM-R2 may play roles in nociception, motor control

and regdation of somatosensory transmission. Unlike the previous report (Cao et al.,

I998), expression of srnaIl arnounts of TM-R2 mRNA were found in the anterior lobe of

the pituitary (as seen clearly against the absence of signal in the neurointermediate lobe

of the pituitary ), suggesting possible roles for TRH-R2 in hormone regulation.

Furthermore, dense IabeIing was seen in the hypothalamus in the antenor and

lateral hypothalamic nuclei, as well as moderate levels of expression in the media1

preoptic area, paraventricular nucleus, posterior hypothalamic nucleus, and the ventral

and dorsai premarnmillary nuclei, suggesting that TRH-R2 may play a role in appetite

regulation, motivation or other hypothalamic functions. In addition, TRH-R2 was shown

to ex h i bit more rapid agonist-stimulated intemalization kinetics and a greater degree of

downregulation with prolonged agonist exposure than TRH-R1 (O'Dowd et al., in press).

Finally, TRH-R2 expression levels appeared to be less variable than for TRH-R 1. While

TRH-R2 exhibited a higher basal signaling activity than TRH-RI, chronic exposure (24

hr) of 1 p M TRH caused lesser induction of reporter gene transcription in cells

expressing TRH-R2 than in cells expressing TRH-RI (O'Dowd et al., in press).

4.3 GPR54: Discovery and Characterization of a Novel GPCR related to the

Galanin Receptors

Using Gf RS4 in a BLAST search (Altschul et al., 1997), the highest identity was

observed with the galanin receptor family of GPCRs. Specifically, GPR54 shared

significant arnino acid sequence identities in the TM regions with rat galanin receptors

GalRl(45%), GalR.3 (45%), and GalR2 (44%) (Fig. 10). Significantly, various residues

in the human GalRl receptor shown to be important for high-affinity galanin binding

(corresponding to His262, His265, Glu269, and Phe280 in rat GalR1; (Berthold et al.,

1997; Kask et al., 1996)) were not conserved in GPR54. Among these however, only

His262 is conserved among the three galanin receptors. In addition, the substitution of a

tyrosine residue found in GPR54, GalR2 and GalR3 in place of Phe280 in GalRl was

shown to have no significant effect on galanin binding (Berthoid et al., 1997) as opposed

to previous studies where Phe280 was replaced by alanine in GalRl (Kask et al., 1996).

A collaboration with Dr. Gary O'Neill of the Merck-Frosst Center for Therapeutic

Research revealed GPR54 expressed in COS cells displayed no specific binding for

galanin. However, the high levels of sequence identity between GPR54, GalR1. GalR2

and GalR3 suggest that GPR54's endogenous ligand is peptidergic in nature, and most

likely has sequence and structural similarities to gaianin.

Despite GPR54's inability to bind galanin, GPR54's CNS expression pattern was

found to resemble those of galanin receptors. Specifically, rat GalRl mRNA expression

is abundant in severai brain regions including the hypothalamus, arnygdala, hippocampus

and locus coeruleus (Parker et al., 1995). Rat GalR2 mRNA expression is found in the

mammilary nuclei, the dentate gyrus and posterior hypothalamic and arcuate nuclei

(Kolakowski et al-, 1998). Rat GalR3 is found to be abundantly expressed in the C A

regions of Ammon's horn and the dentate gyms with transcripts also detected in thalamic,

hypothalamic, marnmilary and amygdaloid nuclei (Kolakowski et al., 1998). Overall, the

significant levels of sequence sirnilarities between GPR54 with the galanin receptors, as

well as the significant degree of overlap in mRNA tissue distribution, suggests the

cognate ligand for GPR54 is a peptide neurotransmitter, perhaps involved with similar

physiulogical systems as gdanin, making GPR54 a prime candidate for current reverse

pharmacology ligand-discovering techniques.

4.4 GPR57 and GPRSS: Discovery of a Novel Subfamiiy of GPCRs

Using GPR57 and GPR58 protein sequences in BLAST searches, the highest

identities were observed with the PNR and serotonin 5-HT, receptors and a reported 5-

HT, pseudogene (Fig. 14). The GPR57 encoded receptor shared identities with the

GPR58 (59%). PNR (37%), the 5-HT, (30%) receptors and a 5-HT, pseudogene (35%).

The GPM8 encoded receptor shared identities with the PNR (42%), and 5-HT, (34%)

receptors and the 5-HT, pseudogene (49%).

The vGPR57 sequence varied from GPR57 by two single nucleotide deletions

and two nucleotide substitutions, maintaining an overall nucleotide identity of 99.6%

(Fig. 13). The two deletions resulted in two frame shifts relative to GPR57 in the regions

of the gene corresponding to the extracellular N-terminal segment and TM 1 respectively,

the first leading to a stop codon in TM3 and an ORF of only 309 bp encoding a 103

amino acid protein. We have previously reported other GPCR pseudogenes vDRD5- 1,

yDRD5-2 (Nguyen et al., 1991), @-HTR,, (Nguyen et al., I993), wGPR32 and

yGPR33 (Marchese et al., 1998b), and @PR53 (Sawzdargo et al., 1999).

The GPR58 receptor does not appear to encode an extraceIlular N-terminal

segment. While other GPCRs with very short extracellular N-terminal segments have

been identified (e-g. the adenosine A l receptor (Townsend-Nicholson and Shine. 1993,

A2b receptor (Pierce et al., 1992) and histamine H2 receptor (Gantz e t al., 1991)). the

ORF of GPR58 appeared to start within the first TM with the identified initiation

methionine conformed to an adequate Kozak sequence (Kozak, 1996).

Both GPR57 and GPR58 displayed residues important for ligand binding

comparable to those in biogenic amine-binding receptors. Specifically, an aspartate in

TM3, threonine in TM5 and phenylalanine in TM6 shown to be important for ligand

binding and stability in biogenic amine-binding receptors (Strader et al., !994) were

conserved in both the GPR57 and GPR58 receptors. Previous reports have localized the

receptor PNR at 6q23 (Zeng et al.. 1998) and the 5-HT, pseudogene at 6q22.1 (Liu et al.,

1998). Together, vGPR57, GPR57, GPR58, PNR and the 5-HT, pseudogene appear to

compose a family of oGPCR genes by sequence identity and that localize between q22-

q24 on chromosome 6. While GPR58 did not bind serotonin; based on sequence

identities and the conservation of several important residues with amine-binding GPCRs,

1 predict these receptors (and the functional receptor potentially encoded by a closely

related paralog to the 5-HT, pseudogene) will bind the same endogenous amine-like

ligand, perhaps of a type yet to be discovered.

4.5 GPR61 and GPR62: Discovery of a Novel Subfamily of GPCRs

Overall, GPR6 1 and GPR62 were seen to share the greatest amino acid sequence

with each other at 34%, with notable conservation of sequence in TM2, 5, 6, 7 and,

surprisingly, within the intracellular loops (Fig. 16). Given these regions of conservation,

GPR6 1 and GPR62 may bind the same cognate ligand, despite their low overall sequence

identity. Using GPR6 1 and GPR62 amino acid sequences in BLAST searches (Altschul

et al., 1997), sequence identities were seen, albeit low (c 30 %), with other GPCRs. Of

these, the highest levels of sequence identity were seen with various biogenic amine

receptors (eg. the serotonin 5-HT6, adrenergic al A, fl3, and histamine H2 receptors),

GPR54 (a galanin-like GPCR) and GPRlO (the receptor for the prolactin-releasing

peptide (Hinuma et al., 1998)). NormaIIy, a routine BLAST search of newly cloned

oGPCRs would suggest the nature of their cognate ligand by the sequence identities

shared with known GPCRs. However, GPR61 and GPR62 do not seem to be close to any

particular group of GPCRs, with sequence identities matching biogenic amine receptor as

well as peptide receptors. Sequence analyses also revealed no short motif or even single

amino acids known to be specifically conserved betweer? the various subfamilies of

GPCRs. For example, the biogenic amine receptors, which were by far the largest group

of GPCRs retrieved by GPR61 and GPR62 comparing BLAST searches, have a

conserved aspartic acid found in TM3 (Ji et al., 1998) which is found in neither GPR6 1 or

GPR62. Strategies to elucidate the cognate ligand for this novel subfamily of oGPCRs

will have to stem from their tissue distribution patterns. GPR61 northern analysis of rat

and human probes reveal strong expression in the brain, and indicates its cognate ligand

is most likely a neurotransmitter. Future studies will include in situ hybridization

analysis of rat GPR6 1 in rat brain, cloning the rat orthologue of GPR62 and determining

the tissue distribution of rat and human GPR62, to further elucidate the potential

physiological functions and identities of the cognate ligands for the two receptors.

4.6 Conclusions

This thesis describes the characterization of the novel cognate ligand apelin for

the APJ receptor as well as the discovery, isolation and characterization of six novel

GPCR-encoding genes and cDNAs for TRH-R2, GPR54, GPR57, GPR58, GPR6 1,

GPR62 and a pseudogene, yGPR57.

The initial characterization of each gene has provided a basis for future

investigations into the physiological roles for the proteins they encode. For apelin,

sequence and tissue distribution analyses revealed strong cornparisons with the

angiotensin II systern, which led to preliminary studies showing apelin to have roles in

drinking behaviour and blood pressure. For TRH-R2, the cloning and characterization of

a second thyrotropin-releasing hormone receptor has provided a new target to study the

multiple physiologicd roles of TRH, which already include the stimulation of thyrotropin

and prolactin from the pituitary gland, neurotransmitter and neurostimulatory effects, and

links to certain diseases and disorders of the brain including Huntington's chorea,

schizophrenia, Alzheimer's disease and depression. The oGPCRs presented in this thesis

provide new targets for the reverse pharmacology techniques currently employed to

assign cognate ligands to orphan receptors. For GPR54, sequence analysis and tissue

distribution suggest its cognate ligand to be a galanin-like peptide. The two novel GPCR

subfarnilies represented by GPR57lGPR58 and GPR61/GPR62 most likely, by their low

sequence identities with other GPCRs, bind novel endogenous ligands yet to be

discovered. With recent advances in reverse pharmacology, it is expected that these

orphans will be assigned their endogenous ligands in the near hture, with subsequent

studies elucidating their Function, increasing our knowledge of GPCR-mediated signaling

and offering potential targets for the development of novel therapeutic agents for diseases

and disorders.

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