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Cloning Genes Encoding Receptors Relatedto Chemoattractant Receptors

Adriano Marchese,* Tuan Nguyen,† Preeti Malik,‡ Shijie Xu,‡ Regina Cheng,†Zhidong Xie,* Henry H. Q. Heng,§ Susan R. George,*,†,

Ø

Lee F. Kolakowski, Jr.,‡,\ and Brian F. O’Dowd*,†,1

*Department of Pharmacology and ØDepartment of Medicine, University of Toronto, Medical Sciences Building,Toronto, Ontario M5S 1A8, Canada; ‡Department of Pharmacology and \Department of Biochemistry,

University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284;†Addiction Research Foundation, 33 Russell Street, Toronto, Ontario M5S 2S1,Canada; and §SeeDNA Biotech, Inc., Farquharson Building, 4700 Keele Street,

Downsview, Ontario M3J 1P3, Canada

Received February 11, 1998; accepted March 3, 1998

been utilized to isolate novel genes or cDNAs encodingWe report the cloning of a novel human gene GPCRs (11), resulting in the isolation of many genes

(GPR32) encoding a putative G-protein-coupled re- and cDNAs encoding receptors for which the endoge-ceptor (GPCR) of 356 amino acids and a related pseu- nous ligands have not been identified. To date we (4–dogene cGPR32. The deduced amino acid sequence of 7, 9, 10, 12–17) and others have identified approxi-GPR32 shares 35–39% identity with members of the mately 70 novel genes encoding orphan receptors. Wechemoattractant receptor family. cGPR32 shares 93% report here the discovery of two additional members ofnucleotide identity with GPR32. We identified a the GPCR family, GPR32 and GPR33, encoding recep-mouse EST encoding a putative GPCR (GPR33) of 309 tors showing identity to chemoattractant receptorsamino acids. The deduced amino acid sequence of also. We report related pseudogenes, as well as theGPR33 shares 30–35% identity with members of the chromosomal localization.chemoattractant receptor family and 36% identity Human genomic DNA was subjected to amplificationwith the receptor encoded by GPR32. The human or-

by PCR with Pfu DNA polymerase (Strategene, Lathologue of GPR33 contains a single basepair substi-Jolla, CA) using degenerate oligonucleotide primers de-tution with respect to the mouse, resulting in the pres-signed based on sequences encoding TM2 [5*-ATC-ence of an in-frame stop codon within the predicted(TC)TCAACCT(GT)GC(TC)(CA)T(GC)GC(AC)GA] andsecond intracellular loop, demonstrating that it is aTM7 [5*-CAGGAAGGCGTA(GA)AG(GA)A(AC)(TG)G-pseudogene. Through fluorescence in situ hybridiza-G(AG)TT] of the opioid-like receptors GPR7 and GPR8,tion and physical mapping of YACs, both GPR32 andthe opioid receptors, and the somatostatin receptorscGPR32 were mapped to chromosomal 19, regionunder the following conditions for 30 cycles: 1 min atq13.3, while cGPR33 was mapped to chromosome

14q12. q 1998 Academic Press 947C, 2 min at 507C, 3 min at 727C, followed by a 5-min extension at 727C. PCR products were subjected toagarose gel electrophoresis and stained with ethidiumbromide, fragments in the 700 bp range were subclonedA wide array of endogenous molecules, includinginto the EcoRV site of the plasmid pBluescript, andneurotransmitters, neuropeptides, chemokines, andbacterial cells were transformed and selected on ampi-hormones, interact with integral membrane proteinscillin plates. Fifty colonies were selected, and DNA wascoupled to G proteins conveying an intracellular signalprepared and subjected to sequence analysis. A singlethat leads to a biological response. The genes or cDNAs

encoding several hundred G-protein-coupled receptors PCR product encoding a novel GPCR displaying se-(GPCRs) have now been cloned and can be subdivided quence identity to formylpeptide receptors was identi-based on pharmacology and sequence conservation. fied and used to screen a human genomic library toThis shared sequence conservation of the receptors has isolate a full-length gene, as previously described (9).

Three phage clones were isolated, and after restriction,Southern blot, and sequence analyses it was deter-Sequence data from this article have been deposited with the

EMBL/GenBank Data Libraries under Accession Nos. AF045764– mined that two of the clones were identical, but differ-AF045767. ent from the third clone. A 10-kb XhoI fragment, named

1 To whom correspondence should be addressed at the Department GPR32, isolated from the third clone, contained se-of Pharmacology, University of Toronto, Medical Sciences Building,quence identical to the PCR product, as well as a con-Toronto, Ontario M5S 1A8, Canada. Telephone: (416) 978-7579. Fax:

(416) 978-2733. E-mail: brian.odowd@utoronto.ca. sensus sequence for a putative initiation methionine

281 GENOMICS 50, 281–286 (1998)ARTICLE NO. GE985297

0888-7543/98 $25.00Copyright q 1998 by Academic Press

All rights of reproduction in any form reserved.

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followed by an open reading frame of 1071 nucleotides by GPR32 and GPR33 with sequences within the Gen-Bank database using BLAST (1) revealed greatestencoding a 356-amino-acid protein (Fig. 1).

A comparison of the sequence from the other two identity to the chemoattractant receptors. The receptorencoded by GPR32 shares 39% identity with formylpep-clones, named cGPR32, with GPR32 revealed a 90%

identity at the nucleotide level, indicating that we had tide receptor FPR1, 35% with formylpeptide receptor-like 2 (FPRL2), 35% with orphan receptor Dez, andisolated a gene closely related to GPR32. cGPR32 con-

tained a frame shift in the region of the gene corre- 34% with formylpeptide receptor-like 1 (FPRL1). Thereceptor encoded by GPR33 shared 35.5% identity withsponding to transmembrane domain 6 caused by a 4-

nucleotide-basepair deletion relative to GPR32, leading orphan receptor Dez, 33% with FPRL2, 32% withFPR1, 31% with FPRL1, and 35.6% with the receptorto the introduction of a premature in-frame stop codon,

likely precluding it from encoding a functional receptor encoded by GPR32. A comparison between the deducedamino acid sequence of GPR32 and that of GPR33 and(Fig. 2A). Amino acid sequence colinear with the recep-

tor encoded by GPR32 appears on a different reading selected receptors is shown in Fig. 1.We were unable to detect mRNA transcripts forframe. GPR32 and cGPR32 share 80% amino acid iden-

tity, increasing to 93% at the nucleotide level over the GPR32 in human liver, medulla oblongata, hypothala-mus, amygdala, basal forebrain, frontal cortex, cau-entire open reading frame, suggesting a recent gene

duplication event. To determine whether cGPR32 en- date–putamen, midbrain, thalamus, and hippocampusby Northern analysis when a fragment encodingcoded a polymorphic variant or a pseudogene, we exam-

ined the genome of four unrelated humans by PCR us- GPR32 was used as a probe, as described by Marcheseet al. (9). Similarly, mRNA transcripts for cGPR32ing primers designed to amplify specifically cGPR32.

Each individual examined contained the same 4-bp de- were not detected in human liver, medulla oblongata,amygdala, basal forebrain, frontal cortex, caudate–pu-letion, demonstrating that cGPR32 is a pseudogene

(data not shown). tamen, midbrain, thalamus, and hippocampus when afragment encoding cGPR32 was used as a probe.A search of the dbEST, as previously described (15),

identified a mouse EST (clone ID 575313; GenBank mRNA transcripts for cGPR33 were not detected inhuman caudate putamen, thalamus, frontal cortex, andAccession No. AA125130) encoding a novel GPCR

showing identity to the receptor encoded by GPR32. midbrain by Northern blot analysis. A mRNA tran-script of 2.4 kb was detected in whole mouse brain byThe EST fragment, named GPR33, was requested from

the IMAGE Consortium (8) and sequenced in both di- Northern analysis (data not shown).GPR32, cGPR32, and cGPR33 were assigned to arections, revealing a consensus sequence for an initia-

tion methionine followed by an open reading frame of human chromosome by fluorescence in situ hybridiza-tion (FISH) on metaphase-spread chromosomes pre-1020 bp encoding a 339-amino-acid protein (Fig. 1).

Using the mouse EST as a probe, a human phage clone pared from lymphocytes and probed with a biotinylatedphage encoding each respective genomic structure, es-was isolated from a human genomic library as pre-

viously described (9) and washed under the following sentially as previously described (9). For each phage atotal of 100 mitotic figures were analyzed, with theconditions: 457C for 15 min in a solution containing

21 SSC and 1% SDS. After restriction and Southern hybridization efficiency being 97% for the phage con-taining GPR32, 97% for the phage containing cGPR32,analyses, a 7-kb SacI fragment was isolated from a

single positive phage and sequenced, revealing a con- and 94% for the phage containing cGPR33. DAPI band-ing was used to identify the specific chromosome hy-sensus sequence for an initiation methionine followed

by an open reading frame of 417 nucleotides inter- bridizing to each probe, and a detailed position wasachieved by summarizing 10 photos. No additional locirupted by the presence of a stop codon in what corre-

sponds to the second intracellular loop (Fig. 2B). A com- were detected by FISH, allowing for the assignment ofGPR32 to chromosome 19q13.3, cGPR32 to 19q13.3,parison of the mouse and human nucleotide sequences

at this region indicated that the mouse cDNA contained and cGPR33 to chromosome 14q12.Previous studies of formylpeptide and formylpeptide-a codon for arginine (CGA), whereas in the human gene

the first nucleotide of the codon was substituted to a like receptor genes have shown that these genes mapto human chromosome 19q13.1–q13.3, as does the hu-T, changing it to a stop codon. Overall the nucleotide

identity between mouse and human GPR33 was 79.5%, man C5a receptor gene (3). To explore further the rela-tionship among FPR1 and the related receptors FPRL1indicating that they may be orthologous. To investigate

whether the base substitution in the human gene was and FPRL2, we first queried the physical map of hu-man chromosome 19 available at URL http://www-due to cloning a variant of GPR33, we amplified this

region from several individuals by PCR and subjected bio.llnl.gov/ genome/ html/chrom_map.html (2). Thisphysical map contains the position of the FPR1 genethe products to sequence analysis. The sequence in four

unrelated individuals all contained the same basepair on cosmid 17663 and overlapping YACs 929g8, 806c7,859d11, and 774e3. YAC 746c11 extends toward thesubstitution leading to the presence of a stop codon,

indicating that GPR33 is a pseudogene in human, and telomeric side of FPR1. Each of the YACs was utilizedas template in touch PCR using primer pairs specificit is thus referred to as cGPR33 (data not shown).

A comparison of the amino acid sequences encoded for GPR32, cGPR32, FPR1, FPRL1, and FPRL2 and

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FIG. 2. Sequences of (A) cGPR32 and (B) cGPR33. The position of the in-frame stop codon in each pseudogene is boxed and indicatedwith an asterisk in B. Translation of nucleotide sequence after the stop codons is relative to the functional counterparts. The predictedtransmembrane domains are shaded and numbered. Amino acids are numbered on the left and nucleotides are numbered on the right.The sequences reported here have been deposited into the GenBank database under Accession Nos. AF045765 (cGPR32) and AF045767(cGPR33).

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FIG. 2—Continued

designed to amplify products from each of these genes the PCR products is derived from the appropriate tar-get. Attempts to utilize primers from one gene to an-at sizes 290, 350, 400, 450, and 495 bp, respectively.

The expected sizes were observed in human genomic other using LA PCR were unsuccessful, suggesting thatthese genes are distributed within the region moreDNA for each primer pair, while each of the respective

genes was amplified from YACs 859d11 and 929g8, in- than 10 kb apart from each other.In conclusion, we have identified two novel chemoat-dicating the presence of all five genes (data not shown).

To confirm the specificity of each of the primer pairs, tractant-like GPCRs, encoded by GPR32 and GPR33.The human genome also contains a pseudogene closelyPCR products from the YAC and human genomic DNA

experiments were subcloned and analyzed by DNA se- related to GPR32, while the human orthologue ofmouse GPR33 encodes a pseudogene. Both GPR32 andquencing. Sequencing data demonstrate that each of

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to genes encoding somatostatin receptors. FEBS Lett. 398: 253–cGPR32 have been colocalized on human chromosome258.19 with the genes encoding other chemoattractant re-

8. Lennon, G., Auffray, C., Polymeropoulos, M., and Soares, M. B.ceptors, while GPR33 has been mapped on chromo-(1996). The I.M.A.G.E. Consortium: An integrated molecular

some 14. Future experiments will be aimed at identi- analysis of genomes and their expression. Genomics 33: 151–fying a ligand that binds to these receptors, which 152.should help us determine the precise physiological role 9. Marchese, A., Docherty, J. M., Nguyen, T., Heiber, M., Cheng,

R., Heng, H. H. Q., Tsui, L.-C., Shi, X., George, S. R., andfor these GPCRs.O’Dowd, B. F. (1994). Cloning of human genes encoding novelG protein-coupled receptors. Genomics 23: 609–618.

ACKNOWLEDGMENTS10. Marchese, A., Heiber, M., Nguyen, T., Heng, H. H. Q., Saldivia,

V. R., Cheng, R., Murphy, P. M., Tsui, L.-C., Shi, X., Gregor, P.,This research was supported by grants from the Addiction Re-George, S. R., O’Dowd, B. F., and Docherty, J. M. (1995). Clon-search Foundation, the National Institute on Drug Abuse, and theing and chromosomal mapping of three novel genes, GPR9,Medical Research Council of Canada and by a PMAC/MRC grantGPR10, and GPR14, encoding receptors related to interleukinfrom Merck Frosst Research Laboratories, Inc. We thank Nancy Ni-8, neuropeptide Y, and somatostatin receptors. Genomics 29:cholls, Mai Nguyen, Victor Saldivia, and Yang Shen for excellent335–344.technical assistance and Dr. Susan L. Naylor and Dr. Kevin R. Lynch

11. Marchese, A., George, S. R., and O’Dowd, B. F. (1998). Cloningfor helpful suggestions. A.M. is supported by the Health Researchof novel G protein-coupled receptor genes: The use of homologyPersonnel Development Program of the Ontario Ministry of Health.screening and the polymerase chain reaction. In ‘‘Identificationand Expression of G Protein-Coupled Receptors’’ (K. R. Lynch,

REFERENCES Ed.), Wiley-Liss, New York, pp. 1–26.12. O’Dowd, B. F., Heiber, M., Chan, A., Heng, H. H. Q., Tsui,

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