cloning and chromosomal mapping of three novel genes, gpr9, gpr10, and gpr14, encoding receptors...

GENOMICS 29, 335–344 (1995)

Cloning and Chromosomal Mapping of Three Novel Genes, GPR9,GPR10, and GPR14, Encoding Receptors Related to Interleukin 8,

Neuropeptide Y, and Somatostatin Receptors

ADRIANO MARCHESE,*,1 MICHAEL HEIBER,*,1 TUAN NGUYEN,† HENRY H. Q. HENG,‡VICTOR R. SALDIVIA,* REGINA CHENG,† PHILIP M. MURPHY,§ LAP-CHEE TSUI,‡ XIAOMEI SHI,‡

PAUL GREGOR,\ SUSAN R. GEORGE,*,†,Ø BRIAN F. O’DOWD,*,†,2 AND JOHN M. DOCHERTY*

†Addiction Research Foundation, 33 Russell Street, Toronto, Ontario, M5S 2S1, Canada; Departments of *Pharmacology andØMedicine, University of Toronto, Toronto, Ontario, M5S 1A8, Canada; §Department of Health and Human Services, Laboratory of

Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892;‡Department of Genetics, Research Institute, The Hospital for Sick Children, Toronto, Ontario, M5G IX8, Canada;

and \Bayer, 400 West Morgan Lane, West Haven, Connecticut 06516

Received April 21, 1995; accepted June 27, 1995

peptide-binding receptors. We have used the polymeraseWe employed the polymerase chain reaction and ge- chain reaction (PCR) to amplify human genomic DNA

nomic DNA library screening to clone novel human with sets of primers designed based on certain highlygenes, GPR9 and GPR10, and a rat gene, GPR14. GPR9, conserved regions in the GPCRs. Genomic DNA was se-GPR10, and GPR14 each encode G protein-coupled re- lected as a template for the PCR since many genes encod-ceptors. GPR10 and GPR14 are intronless within their ing GPCRs lack introns in their coding region (reviewedcoding regions, while GPR9 contains at least one in- in O’Dowd et al., 1991), and the PCR fragments obtainedtron. The receptor encoded by GPR9 shares the high- were used to obtain the corresponding full-length genesest identity with human IL-8 receptor type B (38% from a genomic library. Using this approach, we haveoverall and 53% in the transmembrane regions), fol- reported the cloning of several novel human genes encod-lowed by IL-8 receptor type A (36% overall and 51% in ing novel GPCRs, including GPR1, GPR2, and GPR3the transmembrane domains). GPR10 encodes a recep- (Marchese et al., 1994), GPR4, GPR5, and GPR6 (Heibertor that shares highest identity with the neuropeptide

et al., 1995), GPR7 and GPR8 (O’Dowd et al., 1995), andY receptor (31% overall and 46% in the transmembraneAGTRL1 (APJ) (O’Dowd et al., 1993). In the presentdomains). The receptor encoded by GPR14 sharesstudy, we report the cloning and chromosomal localizationhighest identity with the somatostatin receptor SSTRof another two novel human genes, named GPR9 and4 (27% overall and 41% in the transmembrane do-GPR10, and a novel rat gene, GPR14, which encode recep-mains). Fluorescence in situ hybridization analysis lo-tors that share significant identity with interleukin-8,calized GPR9 to chromosome 8p11.2–p12 and GPR10neuropeptide Y, and somatostatin receptors, respectively.to chromosome 10q25.3–q26. q 1995 Academic Press, Inc.

GPR9 encodes a receptor that shares highest identitywith the IL-8 receptors and the receptor encoded by

INTRODUCTION the gene GPR2, which also encodes a receptor resem-bling the IL-8 receptors (Marchese et al., 1994). GPR9

A wide spectrum of intercellular signals is mediated may therefore represent a novel receptor belonging toby peptide ligands interacting with specific cell surface the family of chemokine receptors with a role in im-receptors. Many peptide-binding receptors belong to the mune responses in the body. GPR10 encodes a receptorG protein-coupled receptor (GPCR) family and exhibit that shares high identity with the neuropeptide Y typecommon structural features, including the presence of 1 receptor and an orphan receptor induced by glucocor-seven transmembrane domains (TM) and a number of ticoids (Harrigan et al., 1991). GPR10 may represent aconserved amino acid residues (Probst et al., 1992). Fol- member of the neuropeptide Y receptor family, there-lowing the cloning of the d opioid receptor (Evans et al., fore mediating the effects of the peptide neuropeptide1992; Kieffer et al., 1992), we conducted a search for novel Y. The effects of NPY are reported to be mediated by

at least three neuropeptide Y receptor subtypes defined1 The first and second authors have made an equal contribution. pharmacologically that belong to the G protein-coupled2 To whom correspondence should be addressed at the Department receptor family of receptors. To date only one neuropep-of Pharmacology, University of Toronto, Toronto, Ontario, M5S 1A8,

tide Y receptor has been cloned, designated the Y1 re-Canada. Telephone: (416) 978-7579. Fax: (416) 978-2733. E-mail:[email protected]. ceptor subtype (Larhammar et al., 1992). GPR14 en-

3350888-7543/95 $12.00

Copyright q 1995 by Academic Press, Inc.All rights of reproduction in any form reserved.

/ m3709$3612 09-08-95 09:47:42 gnma AP-Genomics

MARCHESE ET AL.336

agarose gel to size fractionate the RNA, and transferred to a posi-codes a receptor that shares highest identity with thetively charged nylon membrane (Hybond-N; Amersham) on whichsomatostatin and opioid receptors.the RNA was immobilized by UV crosslinking. Probes were labeledIn our ongoing search for novel genes encoding pep- with [a-32P]dCTP, washed twice for 60 min in a solution containing

tide-binding receptors, GPR9, GPR10, and GPR14 rep- 0.11 SSC and 0.1% SDS at 507C and then exposed for 2 weeks at0707C to Kodak XAR film with an intensifying screen.resent examples of the growing number of identified

Northern blot analysis was performed on various human cell lines.subtypes of known GPCR gene families.Poly(A)/ RNA was isolated from the following cell lines: normal hu-man peripheral blood-derived neutrophils, eosinophils, monocyte/

MATERIALS AND METHODS macrophages, PHA-activated tumor infiltrating T lymphocytes, anda number of transformed lines, including Jurkat (T lymphocytes),U937 (myelomonocytic), Daudi (B lymphocyte), K562 (erythroleuke-PCR amplification of genomic DNA. An aliquot of human genomic

DNA isolated from peripheral blood lymphocytes was subjected to mia), and RAJI (B lymphocytes). The 2.6-kb SacI DNA fragment thatcontained the coding region of GPR9 was isolated and labeled with [a-PCR amplification using primers designed based on a comparison of

the sequences in the third and seventh TM regions of the d opioid 32P]dCTP using the random primer labeling kit (Boehringer). Threeseparate Northern blots were incubated overnight at 377C in hybrid-receptor and somatostatin receptors (as described in Marchese et al.,

1994). Briefly, the conditions used for the PCR were 30 cycles of ization buffer containing 1 million cpm ml01 of denatured labeledprobe. The blots were washed at 657C in 0.51 SSC for 30 min and1.5 min denaturation at 937C, 2 min annealing at 387C, and 4 min

extension at 727C, followed by a 7-min extension at 727C. The DNA then exposed for 2 days at 0707C to Kodak XAR film with an intensi-fying screen.amplified from this reaction was precipitated with ethanol, phosphor-

ylated with T4 polynucleotide kinase, and blunt-ended with the Fluorescence in situ hybridization detection. Human lymphocytesKlenow enzyme. The DNA was electrophoresed on 0.5% agarose gel, were cultured in an a-minimal essential medium (a-MEM) supple-and a gel slice containing DNA fragments of 550 bp was subcloned mented with 10% fetal calf serum and phytohemagglutinin (PHA)into pBluescript (Stratagene) and transformed in D10B bacteria. at 377C for 68–72 h. The lymphocyte cultures were treated withPlasmid DNA was isolated from the transformants, and the inserts BrdU (0.18 mg ml01, Sigma) for an additional 16 h to synchronizewere sequenced using the T7 polymerase sequencing kit (Pharmacia) the cell population. The synchronized cells were washed three timeswith primers specific for the vector. Two PCR products, clone 9 and with serum-free medium and incubated at 377C for 6 h in a-MEMclone 10, were identified containing nucleotide sequences character- with thymidine (2.5 mg ml01, Sigma). Cells were harvested and slidesistic of GPCRs. were made using standard procedures, including hypotonic treat-

In another PCR experiment human genomic DNA was subjected to ment and fixation followed by air drying. The procedure for fluores-amplification using primers designed based on the sequence encoding cence in situ hybridization (FISH) analysis was performed as pre-TM2 (P1: 5*-CTC AAT/C C/T TA/G GC G/C ATA/T GC G/C GA) and viously described by Heng et al. (1992) and Heng and Tsui (1993).TM7 (P2: 5*-TAAA G C/G ATG GGG TTC AA/TG CAG/A CA/T A/G Slides that had been aged a few days were baked at 557C for 1 h.TT) of the recently cloned human orphan receptor GPR1 (Marchese After RNase A treatment, the slides were denatured in a solution ofet al., 1994) and the related C5a anaphylatoxin receptor. The condi- 70% formamide in 21 SSC for 1 min at 707C followed by dehydrationtions for the PCR were as follows: 2 min denaturation at 947C, 2 min with ethanol. Purified bacteriophage DNA containing either GPR9annealing at 557C, 3 min extension at 727C for 30 cycles followed or GPR10 was biotinylated with dATP using a Bionick labeling kitby 7 min extension at 727C. The amplified DNA was cloned into (BRL) for use as probes. The labeled probes were denatured at 757CpBluescript and analyzed as described above. This search process for 5 min in a hybridization mix consisting of 50% formamide andresulted in the isolation of PCR product, clone 11, which also ap- 10% dextran sulfate and loaded onto the slides. After overnight hy-peared to encode a GPCR. bridization, detection, and amplification, the FISH signals and the

Rat genomic DNA was subjected to amplification in the PCR using diamidinophenylindole (DAPI) banding pattern were visualized byspecific primers designed based on the sequence encoding TM3 and fluorescence microscopy. Pictures were taken using Kodak slide filmTM6 of human 11. The conditions for the PCR were as follows: 2 ASA 800/1600 in one single operation by simply switching the filtersmin denaturation at 947C, 2 min annealing at 457C, 3 min extension on the microscope (Heng and Tsui, 1993).at 727C for 30 cycles followed by 7 min extension at 727C. The ampli-fied DNA product, rat clone 11, was cloned into pBluescript and

RESULTSsequenced.Library screening. Each of the three human insert fragments,

In experiments designed to search for genes encodingclones 9, 10, and 11, was extracted from the pBluescript vector, radio-labeled with [a-32P]dCTP (NEN) by nick-translation (Amersham), novel receptors, we amplified human genomic DNA inand used as probe to screen a human lEMBL3 SP6/T7 genomic li- the PCR using primers designed based on the TM re-brary (Clontech) under conditions that we have previously described gions of the genes encoding the d opioid and somato-(Heiber et al., 1995; Marchese et al., 1994). In total, 1 million indepen-

statin receptors. Sequence analysis of several hundreddent phage clones were screened, and DNA from the resultant posi-tive probe-binding phage was purified, digested with several restric- PCR products revealed some that encoded partial seg-tion endonucleases, subjected to agarose gel electrophoresis, and ana- ments (TM3–TM7) of novel GPCRs (Marchese et al.,lyzed by Southern blotting with the same probe used to screen the 1994; Heiber et al., 1995; O’Dowd et al., 1995) and twolibrary. Probe-binding restriction fragments were isolated, subcloned

new clones, clones 9 and 10. In addition, primers de-into pBluescript, and sequenced.signed based primarily on the TM regions (TM2 andOne million independent phage clones from a rat lEMBL3 SP6/

T7 genomic library (Clontech) were screened using rat clone 11 as a TM7) of GPR1 were used to generate clone 11, whichprobe. The conditions for screening were identical to those used in also encodes a novel GPCR.the screening of the human library listed above.

Northern blot analysis of the human genes GPR9, GPR10, and Cloning of GPR9GPR14. Poly(A)/ RNA was isolated from the human brain regionsputamen, pons, hypothalamus, hippocampus, frontal cortex, thala- To obtain the full-length gene corresponding to clonemus, and cerebellum, as previously described (Zastawny et al., 1994). 9, a human lEMBL3 SP6/T7 genomic DNA library wasPoly(A)/ RNA was also isolated from the rat tissues brain, heart,

screened. Seven positive probe-binding signals of vary-spleen, liver, lung, kidney, and ovary in the same manner as above.Samples were denatured, electrophoresed on a 1% formaldehyde ing intensity were obtained, and the phage DNA in the


CLONING NOVEL G PROTEIN-COUPLED RECEPTORS 337

FIG. 1. The nucleotide and deduced amino acid sequence of (A) GPR9 and (B) GPR10. Nucleotides are numbered on the right andamino acids on the left. Nucleotides are numbered beginning immediately following the putative intron acceptor splice site (underlined).Lowercase letters indicate intronic sequence. Putative N-linked glycosylation sites (,), phosphorylation sites for protein kinase C (j) andprotein kinase A (h), and the carboxy-terminal palmitoylation site (l) are indicated. The transmembrane domains are shaded and numbered.

corresponding plaques was purified. The DNA isolated ing a long open reading frame encoding 364 amino acidswith seven hydrophobic regions. The clone appeared tofrom one of these phage, which corresponded to the

strongest autoradiographic signal, was purified and re- lack an initiation methionine, suggesting that theGPR9 gene contains a second exon in the 5* region, asstriction digested with AccI and ApaI, Southern blot-

ted, and probed with radiolabeled clone 9 to detect a indicated by the presence of a putative intron acceptorsplice site in the amino terminal region (Fig. 1A).diagnostic 400-bp fragment present in clone 9. Further

restriction and Southern analysis with the clone 9probe was performed, resulting in the detection of a 2.6- Cloning of GPR10kb SacI fragment, which was excised from the purifiedphage and subcloned into pBluescript. A contiguous The PCR of human genomic DNA resulted in the

isolation of another product, named clone 10, with1092-bp region of this fragment was sequenced, reveal-


MARCHESE ET AL.338

FIG. 1—Continued

novel sequence characteristic of a GPCR. Clone 10 was Cloning of GPR14used to screen the lEMBL3 SP6/T7 genomic library,and this resulted in the isolation of two probe-binding The amplification of human genomic DNA with prim-phage clones. After restriction and Southern blot anal- ers designed based primarily on the sequence of GPR1ysis a 6-kb KpnI fragment, named GPR10, was isolated resulted in the isolation of a PCR product, clone 11,and found to contain sequence identical to that of clone encoding sequence of a potentially novel GPCR and10. Sequence analysis of GPR10 revealed a Kozak con- showing highest identity to the somatostatin and opioidsensus sequence for an initiation methionine followed receptors. Initially clone 11 was used to screen a hu-by an open reading frame of 1107 bp encoding a protein man genomic library; however, no positive probe-bind-of 369 amino acids also with seven hydrophobic regions ing phage clones were obtained. Subsequently, rat ge-

nomic DNA was amplified in the PCR with primers(see Fig. 1B).



designed based on the sequence encoding TM3 and graphed, and these data are summarized in Fig. 2B.Since a higher frequency of FISH signals was obtainedTM6 of human clone 11. We obtained a clone encoding

the partial coding sequence of the rat orthologue of from chromosome 10, the human GPR10 gene is locatedon chromosome 10 q25.3–q26.1, and a related sequenceclone 11, and this was used to screen a rat genomic

library. This screening resulted in the isolation of three is located on chromosome 13 q14.3–q21.1.positive probe-binding phage clones, and DNA pre-pared from these clones was subjected to restriction DISCUSSIONand Southern analysis, leading to the isolation of a 1.3-kb PstI fragment and a 7-kb SacI fragment, both of In an effort to isolate genes encoding novel peptide-

binding GPCRs, we have used the same set of primerswhich were subcloned into the plasmid Bluescript andsequenced. Sequence from each of these clones con- previously employed successfully in the cloning of hu-

man genes GPR1 to 8 (Heiber et al., 1995; Marchese ettained sequence identical to that of the rat PCR productclone 11. The PstI fragment did not encode the full- al., 1994; O’Dowd et al., 1995). We used lower strin-

gency annealing temperature conditions in the PCR,length gene; the 5* end was truncated in the receptorcoding region at TM3. The SacI fragment did encode and we obtained novel PCR products with distinct ho-

mology to other GPCR genes; with these fragments wethe full-length gene named GPR14. The SacI fragmentcontained an intronless open reading frame of 1158 bp screened a human genomic library and obtained the

genes GPR9 and GPR10. We have also used a secondencoding a receptor of 386 amino acids.set of primers designed based on the sequence of GPR1

Northern Analysis of the Human Genes GPR9, to isolate the novel gene GPR14 from rat.GPR10, and GPR14 Hydrophobic analysis of the deduced amino acid se-

quences encoded by the genes GPR9, GPR10, andNorthern blot analysis of poly(A)/ RNA isolated from GPR14 demonstrated the seven TMs characteristic ofseveral human brain regions was performed to investi- GPCRs and the protein sequences also contained agate the tissue distribution of GPR9, GPR10, and number of specific amino acid residues generally con-GPR14. Using radiolabeled clones 9, 10, and 11 as served among GPCRs (as reviewed in Probst et al.,probes, no transcripts were detected in putamen, pons, 1992). The proteins encoded by GPR9, GPR10, andhypothalamus, hippocampus, frontal cortex, thalamus, GPR14 possessed small third intracellular loops con-or cerebellum following a 2-week exposure to X-ray sidered to be somewhat characteristic of peptide-bind-film. Northern blot analysis was also performed on ing receptors (Richter et al., 1991). Three consensuspoly(A)/ RNA isolated from various human cell lines, sequences for N-linked glycosylation were present inusing the radiolabeled 2.6-kb SacI DNA fragment that the receptor encoded by GPR9, two in the amino termi-contained GPR9 as a probe. GPR9-derived RNA was nus (i.e., at Asn18 and Asn28) and one in the secondnot detected in neutrophils, eosinophils, or monocyte/ extracellular loop (i.e., at Asn195) (Fig. 1A). Consensusmacrophages derived from human peripheral blood, sequences for phosphorylation by both protein kinasePHA-activated tumor infiltrating T lymphocytes, or a C (PKC) and protein kinase A (PKA) at specific serinenumber of transformed human cell lines including Jur- and threonine residues (i.e., PKC, Thr79, Ser347; PKA,kat, U937, Daudi, K562, and RAJI. Also, GPR14 tran- Ser351 and Ser352) were present in the intracellular re-scripts were not detected in rat brain, heart, spleen, gions, and a cysteine (i.e., Cys334) in the carboxy tail iskidney, ovary, lung, and liver. a potential site of palmitoylation (Fig. 1A). The receptorencoded by GPR10 also contained consensus sequencesChromosomal Localization: FISH Mapping of thefor posttranslational modifications, including two as-Human Genes GPR9 and GPR10paragine residues in the N-terminus (Asn27 and Asn36)as potential sites for glycosylation and a potential siteFor GPR9, a total of 100 mitotic chromosome struc-

tures were examined, 71 of which showed double hy- for phosphorylation by PKA in the third intracellularloop (Thr275) (Fig. 1B). The receptor encoded by GPR14bridization signals banded on chromosome 8, one on

each of the two sister chromatids; therefore, GPR9 was also contained sites for phosphorylation by PKA andPKC in the intracellular portions of the receptor.mapped to chromosome 8 p11.2–p12. A total of 10 mi-

totic figures were photographed, and these data are The receptor encoded by GPR9 had the greatest over-all amino acid identity with the IL-8B receptor ‘‘low-summarized in Fig. 2A.

For GPR10, a total of 104 mitotic figures were exam- affinity’’ subtype (IL-8rB; 38%), followed by the IL-8Areceptor ‘‘high-affinity’’ subtype (IL-8rA; 36%) and theined, 54 of which showed double hybridization signals

on banded chromosome 10, one on each of the two sister GPR2 orphan receptor (36%) (Fig. 3A). When the con-served amino acid substitutions were taken into consid-chromatids (52%). However, under less stringent wash-

ing conditions for GPR10, FISH signals were also de- eration, the percentage identity between the proteinencoded by GPR9 and each of the receptors encoded bytected on chromosome 13 q14.3–q21.1, but at lower

frequency (33% of the signals; data not shown). This IL-8rB, IL-8rA, and GPR2 increased significantly. Themajority of the identical amino acid residues sharedcrosshybridization site may represent a sequence re-

lated to GPR10. A total of 10 mitotic figures were photo- between the GPR9 protein and each of these receptors


MARCHESE ET AL.340

FIG. 2. Summary of the FISH data for genes (A) using the phage probe containing GPR9 and (B) using the phage probe containingGPR10 on human chromosomes. Each dot represents the location of a fluorescent signal on the chromosome.

was concentrated within the seven TMs. In each case, in the transmembrane domains was higher than theoverall identity and was 46% for the neuropeptide Ythe TM identity shared with the protein encoded by

GPR9 was: IL-8rB, 53%; IL-8rA, 51%; GPR2, 44%. A receptor and 46% for the glucocorticoid induced recep-tor, GIR.distinctive feature of the IL-8 receptors, not common

among other GPCRs, is a high content (approximately The receptor encoded by GPR14 shared high aminoacid identity with the somatostatin and opioid recep-20%) of acidic amino acids in the amino terminus

(Ahuja and Murphy, 1993). An abundance of the acidic tors. Compared with somatostatin receptor SSTR4 (De-mchyshyn et al., 1993), the overall amino acid identityresidues glutamate and aspartate is also seen in the

amino terminus of the receptor encoded by GPR9 as was approximately 27% and within the transmem-brane domains 41%. When compared with the k opioidwell as GPR2 (Fig. 3A).

Although the receptor encoded by GPR9 shared the receptor the overall identity was approximately 25%,and within the TM domain the identity increased tohighest identity with the IL-8 receptors, it also showed

significant identity to other GPCRs, including the Bur- approximately 34%, shown aligned in Fig. 3C. Theidentities shared between the receptor encoded bykitt lymphoma receptor 1 (BLR 1; Dobner et al., 1992),

the Epstein–Barr virus-induced G protein-coupled re- GPR14 and other receptors overall and within the TMdomains, respectively, are GPR7, 26 and 32%; GPR8,ceptor 1 (EBI 1; Birkenbach et al., 1993), and the MIP-

1a/Rantes receptor, also known as C-C chemokine re- 26 and 34%; d, 26 and 34%; and m, 26 and 34%. Signifi-cantly, an aspartic acid in TM 3, important in ligandceptor 1 (Gao et al., 1993; Neote et al., 1993). The GPR9-

encoded receptor may represent yet another chemokine binding, that is present in all somatostatin and opioidreceptors is also conserved in GPR14.receptor with a potential role in inflammatory and im-

mune responses in the body. Future studies will involve In summary, we report the cloning and chromosomallocalization of two novel human GPCR genes and abinding assays to test which chemokines are functional

ligands for the receptor encoded by GPR9. novel rat GPCR. Comparison of the primary structureof the receptors encoded by these genes with that ofThe receptor encoded by GPR10 showed highest

overall amino acid identity with the neuropeptide Y1 other known receptors indicated that the endogenousligands are likely peptidergic. Endogenous mammalianreceptor subtype (31%; Larhammar et al., 1992) and

an orphan receptor induced by glucocorticoids (GIR) peptides have been shown to function in a wide varietyof important physiological processes, including thermo-(30%; Harrigan et al., 1991). The amino acid identity



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MARCHESE ET AL.344

expression of the human macrophage inflammatory protein 1a/regulation, motor activity, the perception of reward andRANTES receptor. J. Exp. Med. 177: 1421–1427.pain, and immunoregulatory responses. Furthermore,

Harrigan, M. T., Campbell, N. F., and Bourgeois, S. (1991). Identifi-their roles in specific peptide neurotransmitter systemscation of a gene induced by glucocorticoids in murine T-cells: A

are among the most complicated integrated neuronal potential G protein-coupled receptor. Mol. Endocrinol. 5: 1331–systems known. Therefore, the molecular cloning of 1338.multiple forms of the many types of peptide-binding Heiber, M., Docherty, J. M., Shah, G., Nguyen, T., Cheng, R., Heng,

H. H. Q., Marchese, A., Tsui, L.-C., Shi, X., George, S. R., andreceptors will greatly advance our knowledge of theO’Dowd, B. F. (1995). Isolation of three novel human genes encod-specific structural features of receptors, promote theing G protein-coupled receptors. DNA Cell Biol. 14: 25–35.identification of the endogenous ligands with which

Heng, H. H. Q., Squire, J., and Tsui, L.-C. (1992). High-resolutionthey interact, and ultimately enable greater under- mapping of mammalian genes by in situ hybridization to free chro-standing of the molecular basis of cellular communica- matin. Proc. Natl. Acad. Sci. USA 89: 9509–9513.tion. Heng, H. H. Q., and Tsui, L.-C. (1993). Modes of DAPI banding and

simultaneous in situ hybridization. Chromosoma 102: 325–332.Kieffer, B. L., Befort, K., Gaveriaux-Ruff, C., and Hirth, C. (1992).ACKNOWLEDGMENTS The d-opioid receptor: Isolation of a cDNA by expressing cloning

and pharmacological characterization. Proc. Natl. Acad. Sci. USAFunding for this work was provided by the Addiction Research 89: 12048–12052.

Foundation of Ontario, the Medical Research Council of Canada, and Larhammar, D., Blomqvist, A. G., Yee, F., Jazin, E., Yoo, H., andNIDA (National Institute on Drug Abuse), in the form of Grant 1 Wahlestedt, C. (1992). Cloning and functional expression of a hu-RO1-DA07223-01. We thank Girsh Shah and Yang Shen for their man neuropeptide Y/peptide YY receptor of the Y1 type. J. Biol.excellent technical assistance. Chem. 267: 10935–10938.

Marchese, A., Docherty, J. M., Nguyen, T., Heiber, M., Cheng, R.,Heng, H. H. Q., Tsui, L.-C., Shi, X., George, S. R., and O’Dowd, B. F.REFERENCES(1994). Cloning of human genes encoding novel G protein-coupledreceptors. Genomics 23: 609–618.Ahuja, S. K., and Murphy, P. M. (1993). Molecular piracy of mamma-

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cloning and chromosomal mapping of three novel genes, gpr9, gpr10, and gpr14, encoding receptors...

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