10
Discovery of Three Novel Orphan G-Protein-Coupled Receptors Adriano Marchese,* Marek Sawzdargo,* Tuan Nguyen,² Regina Cheng,² Henry H. Q. Heng,Thomas Nowak,§ Dong-Soon Im,§ Kevin R. Lynch,§ Susan R. George,* , ² , and Brian F. O’Dowd* , ² ,1 *Department of Pharmacology, and Department of Medicine, University of Toronto, Medical Sciences Building, Toronto, Ontario M5S 1A8, Canada; ²Centre for Addiction and Mental Health, 33 Russell Street, Toronto, Ontario M5S 2S1, Canada; SeeDNA Biotech Inc., Farquharson Building, 4700 Keele Street, Downsview, Ontario M3J 1P3, Canada; and §Department of Pharmacology, University of Virginia Health Sciences Center, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908 Received April 9, 1998; accepted November 4, 1998 We have discovered three novel human genes, GPR34, GPR44, and GPR45, encoding family A G-pro- tein-coupled receptors (GPCRs). The receptor en- coded by GPR34 is most similar to the P2Y receptor subfamily, while the receptor encoded by GPR44 is most similar to chemoattractant receptors. The recep- tor encoded by GPR45 is the mammalian orthologue of a putative lysophosphatidic acid receptor from Xeno- pus laevis. Partial sequence of GPR34 was discovered during a search of the GenBank database of expressed sequence tags (ESTs). This sequence information was used both to isolate the full-length translational open reading frame from a human genomic library and to assemble a contig from additional GPR34 EST cDNAs. Northern blot and in situ hybridization analyses re- vealed GPR34 mRNA transcripts in several human and rat brain regions. Also, we used polymerase chain reaction (PCR) to amplify human genomic DNA using degenerate oligonucleotides designed from sequences encoding transmembrane domains 3 and 7 of opioid and somatostatin receptors. Two PCR products par- tially encoding novel GPCRs, named GPR44 and GPR45, were discovered and used to isolate the full- length translational open reading frames from a hu- man genomic library. Both GPR44 and GPR45 are ex- pressed in the central nervous system and periphery. For chromosomal localization, fluorescence in situ hy- bridization analysis was performed to assign GPR34 to chromosomes 4p12 and Xp11.3, GPR44 to chromosome 11q12– q13.3, and GPR45 to chromosome 2q11.1– q12. © 1999 Academic Press INTRODUCTION G-protein-coupled receptors (GPCRs) are integral membrane proteins containing seven putative trans- membrane domains. These proteins mediate signals to the interior of the cell via activation of heterotrimeric G proteins that in turn activate various effector pro- teins, ultimately resulting in a physiological response. GPCRs respond to a diverse array of endogenous ago- nists including biogenic amines, lipids, neuropeptides, glycoprotein hormones, tethered ligands and also re- spond to photons of light. GPCRs are involved in a number of behaviors and have been implicated in such processes as drug addiction and some neuropsychiatric diseases (see chapters in Blum and Noble, 1997). Therefore, cloning novel GPCRs potentially implicated in these processes could further our insight into the understanding of the molecular mechanisms involved. In this light, we have been engaged in a search for novel GPCRs, in particular those expressed in the cen- tral nervous system (CNS). The cognate ligands for many GPCRs have been assigned, but remain unknown for many orphan GPCRs (oGPCRs), and the difficulty in identifying li- gands is exacerbated by the paucity of small molecules known to signal cells through the heterotrimeric G protein pathways. Although typically the amino acid identity of oGPCRs with known receptors is low (usu- ally ,30%), which obviates a priori ligand assignment, sequence similarity clearly places them within the GPCR family. The number of oGPCRs identified has grown considerably to at least 74 currently cited in publications and in GenBank submissions. Here we report the sequence of three additional oGPCRs, GPR34, GPR44, and GPR45, each most closely related with three different subfamilies of GPCRs. We have detected GPR34, GPR44, and GPR45 expression in the CNS and peripheral tissues. We report also the chro- mosomal localization for each gene. MATERIALS AND METHODS EST database searching. GenBank’s database of expressed se- quence tags (dbEST) was queried with the complete amino acid sequence of several GPCRs using the TBLASTN algorithm (Altschul Sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF118265, AF118266, and AF118670. 1 To whom correspondence should be addressed. Telephone: (416) 978-7579. Fax: (416) 978-2733. E-mail: [email protected]. Genomics 56, 12–21 (1999) Article ID geno.1998.5655, available online at http://www.idealibrary.com on 12 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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Page 1: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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Genomics 56, 12–21 (1999)Article ID geno.1998.5655, available online at http://www.idealibrary.com on

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Discovery of Three Novel Orphan G-Protein-Coupled Receptors

Adriano Marchese,* Marek Sawzdargo,* Tuan Nguyen,† Regina Cheng,†Henry H. Q. Heng,‡ Thomas Nowak,§ Dong-Soon Im,§ Kevin R. Lynch,§

Susan R. George,* ,† , ¶ and Brian F. O’Dowd* ,† ,1

Department of Pharmacology, and ¶Department of Medicine, University of Toronto, Medical Sciences Building, Toronto, Ontario M5S1A8, Canada; †Centre for Addiction and Mental Health, 33 Russell Street, Toronto, Ontario M5S 2S1, Canada; ‡SeeDNA Biotech Inc.,

Farquharson Building, 4700 Keele Street, Downsview, Ontario M3J 1P3, Canada; and §Department of Pharmacology,University of Virginia Health Sciences Center, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908

Received April 9, 1998; accepted November 4, 1998

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We have discovered three novel human genes,PR34, GPR44, and GPR45, encoding family A G-pro-

ein-coupled receptors (GPCRs). The receptor en-oded by GPR34 is most similar to the P2Y receptorubfamily, while the receptor encoded by GPR44 isost similar to chemoattractant receptors. The recep-

or encoded by GPR45 is the mammalian orthologue ofputative lysophosphatidic acid receptor from Xeno-

us laevis. Partial sequence of GPR34 was discovereduring a search of the GenBank database of expressedequence tags (ESTs). This sequence information wassed both to isolate the full-length translational openeading frame from a human genomic library and tossemble a contig from additional GPR34 EST cDNAs.orthern blot and in situ hybridization analyses re-ealed GPR34 mRNA transcripts in several humannd rat brain regions. Also, we used polymerase chaineaction (PCR) to amplify human genomic DNA usingegenerate oligonucleotides designed from sequencesncoding transmembrane domains 3 and 7 of opioidnd somatostatin receptors. Two PCR products par-ially encoding novel GPCRs, named GPR44 andPR45, were discovered and used to isolate the full-

ength translational open reading frames from a hu-an genomic library. Both GPR44 and GPR45 are ex-

ressed in the central nervous system and periphery.or chromosomal localization, fluorescence in situ hy-ridization analysis was performed to assign GPR34 tohromosomes 4p12 and Xp11.3, GPR44 to chromosome1q12–q13.3, and GPR45 to chromosome 2q11.1–q12.1999 Academic Press

INTRODUCTION

G-protein-coupled receptors (GPCRs) are integralembrane proteins containing seven putative trans-

Sequence data from this article have been deposited with theenBank Data Library under Accession Nos. AF118265, AF118266,nd AF118670.

1 To whom correspondence should be addressed. Telephone: (416)78-7579. Fax: (416) 978-2733. E-mail: [email protected].

12888-7543/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

embrane domains. These proteins mediate signals tohe interior of the cell via activation of heterotrimeric

proteins that in turn activate various effector pro-eins, ultimately resulting in a physiological response.PCRs respond to a diverse array of endogenous ago-ists including biogenic amines, lipids, neuropeptides,lycoprotein hormones, tethered ligands and also re-pond to photons of light. GPCRs are involved in aumber of behaviors and have been implicated in suchrocesses as drug addiction and some neuropsychiatriciseases (see chapters in Blum and Noble, 1997).herefore, cloning novel GPCRs potentially implicated

n these processes could further our insight into thenderstanding of the molecular mechanisms involved.n this light, we have been engaged in a search forovel GPCRs, in particular those expressed in the cen-ral nervous system (CNS).

The cognate ligands for many GPCRs have beenssigned, but remain unknown for many orphanPCRs (oGPCRs), and the difficulty in identifying li-ands is exacerbated by the paucity of small moleculesnown to signal cells through the heterotrimeric Grotein pathways. Although typically the amino aciddentity of oGPCRs with known receptors is low (usu-lly ,30%), which obviates a priori ligand assignment,equence similarity clearly places them within thePCR family. The number of oGPCRs identified hasrown considerably to at least 74 currently cited inublications and in GenBank submissions. Here weeport the sequence of three additional oGPCRs,PR34, GPR44, and GPR45, each most closely relatedith three different subfamilies of GPCRs. We haveetected GPR34, GPR44, and GPR45 expression in theNS and peripheral tissues. We report also the chro-osomal localization for each gene.

MATERIALS AND METHODS

EST database searching. GenBank’s database of expressed se-uence tags (dbEST) was queried with the complete amino acidequence of several GPCRs using the TBLASTN algorithm (Altschul

Page 2: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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Page 3: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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14 MARCHESE ET AL.

Page 4: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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Page 5: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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16 MARCHESE ET AL.

t al., 1997). EST sequences that were returned having statisticallyignificant scores (E , 0.01) were examined further. The conceptu-lized amino acid sequences of the EST sequences were used to queryhe GenBank sequence databases (Benson et al., 1998) to determinehether the EST cDNAs encoded known GPCRs. Oligonucleotidesere designed (sense, 59-ACTACTGTCCCATGGATGAA; antisense,9-TAGCGATCCAAACTGATG), based on the sequence of one ESTDNA that partially encoded a novel putative GPCR, and used tomplify human genomic DNA to obtain a fragment to use as aybridization probe to screen a human genomic l phage DNA libraryo isolate a fragment encoding the full-length translational openeading frame, as previously described (Marchese et al., 1994).

PCR. Human genomic DNA was subjected to amplification byolymerase chain reaction (PCR) using Taq polymerase and degen-rate oligonucleotide primers designed based on sequences encodingransmembrane domains (TM) 3 and 7 of the mouse d-opioid receptornd human somatostatin receptors, sst1 and sst2. The cycling con-itions were as follows: denaturation at 94°C for 1 min, annealing at5 or 38°C for 2 min, and extension at 72°C for 1 min, for 30 cycles,ollowed by a 7-min extension at 72°C. PCR products in the expectedize range (approximately 500 bp) were subcloned into the plasmidBluescript (Stratagene), Escherichia coli cells were transformed,nd DNA was prepared and subjected to sequence analysis usinglasmid based primers, essentially as previously described

Marchese et al., 1994). The conceptualized amino acid sequencesere used to query the GenBank sequence database (Benson et al.,998) by performing BLAST searches (Altschul et al., 1997). PCRroducts that were found to encode novel GPCRs were radiolabelednd used to screen a l phage human genomic library to isolate aull-length clone, as previously described (Marchese et al., 1994).

Northern blot analysis. The mRNAs from several human and ratissues were extracted as described previously (Marchese et al.,994). Briefly, total RNA was extracted by the method of Chomczyn-ki and Sacchi (1987), and poly(A)1 RNA was isolated using oligo(dT)ellulose spin columns (Pharmacia). RNA was denatured and size-ractionated on a 1% formaldehyde agarose gel, transferred ontoylon membrane, and immobilized by UV irradiation. In addition, aommercial blot (MTN blot II, Clontech) of human immune systemissues [2 mg poly(A)1 RNA/lane], specifically from spleen, lymphode, thymus, peripheral blood leukocytes, bone marrow, and fetal

iver, was also probed. The blots were hybridized with a 32P-labeledNA fragment, washed with 23 SSPE and 0.1% SDS at 50°C for 20in and with 0.13 SSPE and 0.1% SDS at 50°C for 2 h, and exposed

o X-ray film at 270°C in the presence of an intensifying screen.

In situ hybridization. Male rats (Jackson Laboratories) were sac-ificed by decapitation, and brains were removed within 30 s androzen in crushed dry ice. Frozen brains were sectioned at 14 mmhickness on a Reichert–Jung cryostat at 220°C and thaw-mountednto microscope slides. Sections were fixed in freshly prepared 4%araformaldehyde in 0.02% diethylpyrocarbonate-treated water for0 min at 4°C in an ice bath and then washed for 5 min in coldhosphate-buffered saline, pH 7.4, before dehydration in an alcoholeries, essentially as described previously (Zastawny et al., 1994).ixed sections were stored at 270°C until used.A fragment encoding the rat orthologue of GPR34 was labeled by

andom priming using 35S-dCTP (NEN-Dupont). Rat brain sectionsere prehybridized for 2 h in buffer containing 50% deionized form-mide, 0.6 M NaCl, 10 mM Tris–Cl, pH 7.5, 10% dextran sulfate, 1%olyvinyl pyrollidone, 2% SDS, 100 mM dithiothreitol, 200 mg/mlerring sperm DNA, hybridized with the labeled probe (106 dpm/lice) for 16 h, and washed under conditions of increasing tempera-ure and decreasing salt concentration. The hybridized sections wereehydrated in a graded alcohol series, exposed to X-ray film (DupontRF-34) for 4–6 weeks at 270°C, and developed. For use as con-

rols, adjacent sections were hybridized following treatment withNase, to confirm the specificity of hybridization.

Chromosomal mapping. Mitotic figures were prepared from hu-an male lymphocytes and subjected to fluorescence in situ hybrid-

zation (FISH) analysis by probing with a biotinylated phage clone

ncoding each respective gene according to Heng et al. (1992) andeng and Tsui (1993).

RESULTS AND DISCUSSION

loning of Human GPR34

A human EST (clone ID 42j10, GenBank Accessiono. H97311) partially encoding a novel oGPCR was

dentified in the dbEST using thrombin (PAR-1) and2Y receptors as query sequences. Human DNA con-aining the full translational ORF was generated inwo ways. We amplified a fragment of human genomicNA using oligonucleotide primers designed from the

equence of the 42j10 EST and used this to screen auman genomic DNA library. Thus we isolated fourositive phage clones, and after restriction endonucle-se digestion and Southern analysis, a 5-kb SacI–XbaIragment was identified, subcloned, sequenced, andound to contain an uninterrupted translational openeading frame of 1132 bp encoding a 376-amino-acidrotein. In keeping with orphan GPCR nomenclature,e named this gene GPR34. This ORF began with aethionine in the context of the initiation consensus

equence described by Kozak (1989) and was precededy an in-frame termination codon.Alternately, the 42j10 EST sequence was used to

dentify (BLASTN) additional, overlapping ESTs.hese were retrieved from the IMAGE Consortium

Lennon et al., 1996), and their DNA sequences wereetermined and used for further searches of thebEST. Ultimately, we identified five additional ESTsclone IDs 244661, 838079, 701227, 503462, and42865), and from the sequence of these we were ableo construct a contig encoding the identical protein.he most complete EST clone (ID 244661) is a “head-o-head” fusion of two cDNAs consisting of about 350ucleotides of unknown sequence (including an Aluamily repeat) and about 1000 bp of GPR34 sequencextending from the near 59 untranslated sequence tohe region encoding TM 7.

In addition to the seven TMs characteristic ofPCRs, GPR34 contained consensus sequences for N-lycosylation sites in the amino terminus (Asn28,sn36, and Asn42), the second (Asn200) and third

Asn295) extracellular loops, consensus sequences forotential protein kinase C phosphorylation sites in thehird intracellular loop (Ser249 and Ser253), and autative palmitoylation site near the carboxyl termi-us (Cys339). The conceptualized GPR34 sequence wassed to query the GenBank database (Benson et al.,998) by performing BLASTP searches. GPR34 is mostlosely related to several orphan receptors, includingSC338 (31%), RBintron (29%), GPR17 (27%), andPR23 (26%). Within putative TM regions, the identity

s 42% with RSC338, 41% with RB intron, 41% withPR17, and 38% with GPR23. Of the GPCRs for whichcognate ligand has been assigned, GPR34 was most

losely related to human P2Y4 (UTP) receptor (26%verall and 35% within the TM domains). An align-

Page 6: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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17NOVEL ORPHAN GPCRs

ent of GPR34 and the five related sequences is shownn Fig. 1A.

Interestingly, the oGPCRs most similar to GPR34 lieithin the large subfamily of GPCRs that contains the

even P2Y receptors. Although GPR34 contains twoharged basic residues (His279 and Arg282) withinM6 located at the analogous position to the HXXRotif proposed to be important in P2Y receptors for

gonist potency, GPR34 does not contain the chargedasic residue within TM7 that is essential for agonistctivity (Communi and Boeynaems, 1997; Jiang et al.,997). Thus GPR34 is unlikely to be a P2Y receptorubtype. However, an unequivocal answer will have toait until this is demonstrated experimentally.

loning of GPR44

Another search strategy involved the use of PCR as aeans of isolating genes encoding novel GPCRs. Hu-an genomic DNA was subjected to amplification byCR using degenerate oligonucleotides designed basedn sequences encoding TM3 and TM7 of the mouse-opioid receptor and somatostatin receptor subtypes,st1 and sst2. We have used these primers previouslyo isolate 10 novel GPCRs, under different annealingonditions (Marchese et al., 1994, 1995; Heiber et al.,995; O’Dowd et al., 1995), and here we report twodditional novel GPCRs amplified using two different

FIG. 2. Tissue distribution analysis of GPR34, GPR44, and GPR4oted in text, isolated from human or rat tissues as indicated. (A) Rancoding rat GPR34. (B) Human central tissue distribution of GPR44C) GPR45 mRNA distribution in human central tissues and liver.

nnealing temperatures (45 and 38°C). A PCR productbtained from the amplification of human genomicNA at an annealing temperature of 45°C was found toncode a novel GPCR most closely related to the che-oattractant receptors. This PCR product was radio-

abeled and used to screen a human l phage genomicibrary, as previously described (Marchese et al., 1994).

single clone was isolated, and after restriction andouthern analysis, a 4-kb SacI fragment was sub-loned into pBluescript and a consensus sequence forn initiation methionine was revealed (Kozak, 1989),receded by an in-frame stop codon, followed by anninterrupted translational open reading frame of419 bp encoding a 472-amino-acid protein. The pro-ein encoded by GPR44 clearly shows the characteristiceven transmembrane domains and also contains twoonsensus sequences for potential glycosylation in themino terminus (Asn81 and Asn102), and the carboxyerminus contains a large number of serine and thre-nine residues that are part of consensus sequences forutative phosphorylation by protein kinases A and Cnd G-protein-coupled receptor kinases.The amino acid sequence encoded by GPR44 was

sed to query the GenBank sequence database (Bensont al., 1998) by performing BLAST searches. ClonePR44 is not represented in the GenBank databases

including the dbEST) but it was found to be related

xpression. Northern blot of poly(A)1 RNA (10 mg/lane), except wheresue distribution of GPR34, hybridized with a radiolabeled fragmentRNA. 5 mg of poly(A)1 RNA extracted from hypothalamus was used.

5 et tis

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Page 7: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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18 MARCHESE ET AL.

ost closely to members of the chemoattractant GPCRubfamily, including 29% with the C5a anaphylatoxineceptor, 29% with formylpeptide receptors 1 and 2FPR1 and FPR2), 29% with the oGPCR FPRL2, and7% with oGPCR, Dez. When only the transmembraneomains were compared, the identity rose to 35% with5aR, 35% with FPR1, 35% with FPR2, 36% withPRL2, and 37% with Dez. An amino acid comparisonetween GPR44 and the five related sequences ishown in Fig. 1B. It is likely that the cognate ligand forPR44 is either a known chemoattractant peptide mol-cule or a yet undiscovered peptide that shares se-uence identity with known chemoattractant mole-ules or a leukotriene.

loning of GPR45

The same oligonucleotides used in the isolation ofPR44 were used in PCR at a reduced annealing tem-erature (38°C) so that other gene fragments encodingore distantly related GPCRs would be amplified. ACR product that was found to encode a novel GPCRas identified and was used to identify a 11-kb KpnI

ragment from a human genomic library, which weave named GPR45. Sequence analysis revealed an

nitiation methionine (Kozak, 1989), preceded by anpstream in-frame stop codon, followed by an openeading frame of 1119 bp encoding a 372-amino-acidrotein. The protein encoded by GPR45 shows the typ-cal seven-transmembrane domain feature and alsoontains three consensus sequences for potential gly-osylation sites in the amino-terminal end (Asn4,sn17, Asn20) and a putative palmityolation site

Cys336) within the carboxyl-terminal end.When the amino acid sequence of GPR45 was used to

uery the GenBank database (Benson et al., 1998), weound that GPR45 was 70% identical to a putativeysophosphatidic acid (LPA) receptor from Xenopus lae-

FIG. 3. Autoradiograms of coronal sections of rat brain showingevels relative to the bregma at 1.3 mm (left) and 2.8 mm (righnteroventral thalamic nucleus; PO, primary olfactory cortex; SO,eriventricular hypothalamic nucleus; LOT, nucleus lateral olfactorentate gyrus; MHb, medial habenular nucleus; PO, primary olfactorypothalamic nucleus; Me, medial amygdaloid nucleus; Arc, arcuate

is oocytes named PSP24 (Guo et al., 1996). This highegree of similarity suggested that we had cloned theuman orthologue of the amphibian GPCR, PSP24.oth of these receptors are only distantly related tother GPCRs, for example, the receptors most closelyelated to GPR45, other than PSP24, are the neuroki-in 2 (21% identical amino acids overall) and the b1-drenergic (21%) receptors. An amino acid comparisonf GRP45 with its Xenopus orthologue, PSP24, neuro-inin 2, and b1-adrenergic receptors is presented inig. 1C.In view of Guo et al.’s (1996) suggestion that

SP24 is a functional LPA receptor, which is basedn shifts in dose–response curves to LPA in X. laevisocytes either overexpressing (injection of PSP24RNA) or underexpressing (i.e., antisense oligonu-

leotide or PSP24 cRNA injection) the PSP24 pro-ein, we tested the GPR45 DNA for its facility inonferring LPA responsiveness on cells lacking dis-ernible LPA responsiveness. On repeated trials, weere unable to observe calcium fluxes or inhibition ofdenylyl cyclase activation in GPR45 DNA-trans-ected K562 cells or HepG2 cells, respectively, chal-enged with doses of LPA up to 10 mM. [These exper-ments are described fully in another paper focusedn LPA receptor cloning (Im et al., in preparation).]lthough it is less than optimal to base the argumentn negative data, our complete lack of results inupport of clone GPR45 as a LPA receptor coupledith the failure of yeast expressing amphibianSP24 to respond to LPA (Erickson et al., 1998) andlaims that a very distantly related GPCR [i.e.,dg-2 (Hecht et al., 1996; An et al., 1997)] is a LPA

eceptor [although that claim is not without contro-ersy; see Zondag et al. (1998)] make us, at present,eluctant to accept that GPR45 (or PSP24) is a LPAeceptor.

localization of GPR34 mRNA. Shown are representative sections ataccording to the coordinates by Paxinos and Watson (1982). AV,raoptic hypothalamic nucleus; SCh, suprachiasmatic nucleus; Pe,act; CA, field CA of Ammon’s horn; PCG, postcingulate cortex; DG,rtex; DMH, dorsomedial hypothalamic nucleus; VMH, ventromedialpothalamic nucleus.

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Page 8: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

19NOVEL ORPHAN GPCRs

Page 9: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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20 MARCHESE ET AL.

issue Distribution Analyses

The mRNA tissue expression pattern for GPR34 wasetermined by Northern blot and in situ hybridizationnalyses. Northern analysis revealed high levels of twoRNA transcripts in many rat brain regions including

rontal cortex, cortex, striatum, midbrain, hippocam-us, medulla pons, and lower levels of both transcriptsn the cerebellum and spleen, with even lower levels inhe kidney (Fig. 2A). No mRNA transcripts for GPR34ere detected in rat liver and intestine. Northern anal-sis of human tissues revealed a single mRNA tran-cript in caudate, frontal cortex, putamen, thalamus,ypothalamus, and pons, a double transcript in liver,nd no transcripts in cerebellum or hippocampus (dataot shown). GPR34 was also expressed in peripheralissues as evidenced by the tissue sources of the sixST cDNAs: white adipose tissue, placenta, uterus,

onsillar germinal center B cells, fetal liver/spleen, andetina.

A 3.5-kb mRNA transcript for GPR44 was detectedrimarily in human thalamus, frontal cortex, pons, andippocampus and also at lower levels in hypothalamusnd caudate/putamen (Fig. 2B). In the periphery, a.4-kb transcript for GPR44 was detected in fetal liver,eripheral blood leukocytes, and thymus (data nothown). No mRNA transcripts were detected in spleen,ymph node, or bone marrow (data not shown). Highevels of mRNA transcripts for GPR45 were detected iniscrete areas of periphery and CNS. A 4.25-kb mRNAranscript was detected in basal forebrain, frontal cor-ex, and caudate, and no transcripts were detected inhalamus, hippocampus, or putamen (Fig. 2C). A.0-kb mRNA transcript for GPR45 was detected in theiver (Fig. 2C).

To obtain a more comprehensive expression patternor GPR34 mRNA within the central nervous system,e performed in situ hybridization experiments in ratrain. In general, GPR34 is diffusely expressed in therain. Levels of expression were detected in the pri-ary olfactory cortex, the supraoptic nucleus, the nu-

leus lateral olfactory tract, and the suprachiasmaticucleus (Fig. 3, left). Moderate, but diffuse, expressionas observed in the anteroventral thalamic nucleusnd the periventricular hypothalamic nucleus (Fig. 3,eft). High levels were also detected in Ammon’s hornnd the dentate gyrus of the hippocampus, as well ashe medial habenular nucleus (Fig. 3, right). Moderateevels were observed in several hypothalamic nuclei,ncluding the ventromedial, dorsomedial, and arcuateuclei (Fig. 3, right).

hromosomal Mapping

FISH analysis was performed to determine the chro-osome locus for the genes encoding each of these

FIG. 4. FISH analysis of (A) GPR34, (B) GPR45, and (C) GPR44robed with each respective biotinylated phage clone, as well as idio

eceptors, using methods described previouslyMarchese et al., 1994). Briefly, the phage containinghe genomic DNA were used to probe mitotic figuresrepared from human male metaphase spread chromo-omes isolated from lymphocytes. For GPR34, of the00 mitotic figures examined, 92 showed signals on oneair of chromosomes and on a single chromosome. Thehromosomes were identified by DAPI banding andurther localized by superimposing the photographsrom 10 figures, resulting in the signals being assignedo chromosome 4, region p12, and X chromosome, re-ion p11.3 (Fig. 4A).This is interesting because thisuggests that a sequence closely related to GPR34 ex-sts in the human genome, possibly encoding a highlyelated pseudogene or a closely related gene, possiblyncoding a receptor subtype. Since the genomic librarycreening resulted in the isolation of four positivehage clones, we examined them to determine whetherhey were different in sequence from GPR34. We sub-ected the phage DNA to PCR amplification using oli-onucleotides designed based on the sequence ofPR34 predicted to amplify the coding region and sub-

ected the PCR products to sequence analysis. All wereound to be identical, and all corresponded exactly inegions of overlap to the six EST cDNAs, indicatinghat we describe here only one of the two genomictructures predicted to exist based on the FISH anal-sis. In any case, GPR34 appears to be the first mem-er of a novel paralogous group identified. In the casef GPR44, 96 of the 100 mitotic figures probed showedignals on one pair of homologous chromosomes andere further localized as above to chromosome 11, re-ion q12–q13.3 (Fig. 4C). For GPR45, the signals ap-eared on one pair of homologous chromosomes on 94f the 100 mitotic figures examined and then furtherocalized as above to chromosome 2, region q11.2–q12Fig. 4B).

CONCLUSION

We have identified and cloned three additional mem-ers of the GPCR family, most closely related to threeistinct receptor families. The receptor encoded byPR34 is most closely related to P2Y receptors, the

eceptor encoded by GPR44 is most closely related tohemoattractant receptors, and the receptor encodedy GPR45 is most closely related to a X. laevis oocyteeceptor suggested to respond to LPA. Transcripts forPR34, GPR44, and GPR45 were detected in the CNSnd periphery. GPR34 was localized to two nonhomolo-ous chromosomes, 4p12 and Xp11.3, indicating thathere is another sequence closely related to GPR34 inhe human genome, while GPR44 was localized to chro-osome 11q12–q13.3, and GPR45 was localized to

hromosome 2q11.2–q12.

hown for each is a representative photograph of the mitotic figuresms summarizing the results of the FISH analysis.

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Page 10: Discovery of Three Novel Orphan G-Protein-Coupled Receptors

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21NOVEL ORPHAN GPCRs

The paucity of endogenous ligands has spurred aearch for novel endogenous molecules that may rep-esent cognate ligands for oGPCRs. Recent resultsave shown that oGPCRs stably transfected in null cell

ines are excellent targets for use in agonist responsessays for testing biological tissue extracts in efforts toiscover the cognate ligand (Reinscheid et al., 1995;eunier et al., 1995; Sakurai et al., 1998) and likewise

an prove to be excellent targets for drug discoveryStadel et al., 1997). Future experiments will be aimedt discovering the endogenous ligands for the receptorsncoded by GPR34, GPR44, and GPR45 and also atreating mice wherein the respective genes have beenisrupted, which together should help determine therecise physiological role for each of these receptors. Ashe functions of these orphan receptors are uncovered,hey are potential targets for the development of newherapeutics.

ACKNOWLEDGMENTS

This research was supported by grants from the Addiction Re-earch Foundation (Ontario), the National Institute on Drug AbuseNIDA), the National Institute for General Medical Sciences, the

edical Research Council of Canada, a PMAC grant from Merckrosst, and the Smokeless Tobacco Research Council Inc. A.M. isupported by the Health Research Personnel Development Programf the Ontario Ministry of Health. M.S. is supported by a Student-hip from the Medical Research Council of Canada.

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