Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes

Gene 187 (1997) 75–81

Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes Brian F. O’Dowd a,b,*, Tuan Nguyen a, Benjamin P. Jung b, Adriano Marchese b, Regina Cheng a, Henry H.Q. Heng d, Lee F. Kolakowski, Jr.e, Kevin R. Lynch f, Susan R. George a,b,c a Addiction Research Foundation, 33 Russell St, Toronto, Ontario M5S 2S1, Canada b Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada c Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada d SeeDNA Biotech Inc., Farquharson Building, 4700 Keele Street, Downsview, Ontario M3J 1P3, Canada e Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78284, USA f Department of Pharmacology, University of Virginia Health Sciences Center, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA Received 18 June 1996; revised 16 September 1996; accepted 17 September 1996; Received by J.L. Slightom

Abstract We report the discovery of four novel human putative G-protein-coupled receptor (GPCR) genes. Gene GPR20 was isolated by amplifying genomic DNA with oligos based on the opioid and somatostatin related receptor genes and subsequent screening of a genomic library. Also, using our customized search procedure of a database of expressed sequence tags (dbEST ), cDNA sequences that partially encoded novel GPCRs were identified. These cDNA fragments were obtained and used to screen a genomic library to isolate the full-length coding region of the genes. This resulted in the isolation of genes GPR21, GPR22 and GPR23. The four encoded receptors share significant identity to each other and to other members of the receptor family. Northern blot analysis revealed expression of GPR20 and GPR22 in several human brain regions while GPR20 expression was detected also in liver. Fluorescence in situ hybridization (FISH ) was used to map GPR20 to chromosome 8q, region 24.3–24.2, GPR21 to chromosome 9, region q33, GPR22 to chromosome 7, region q22–q31.1, and GPR23 to chromosome X, region q13–q21.1. [ 1997 Elsevier Science B.V. All rights reserved. Keywords: Polymerase chain reaction; Chromosome; Northern blot; Intronless

1. Introduction The characterization of novel receptor systems in the brain and peripheral regions continues in parallel with the discovery of novel endogenous peptides (Reinscheid * Corresponding author at the Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Tel. +1 416 9787579; Fax +1 416 9782733; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; DAPI, 4∞,6-diamidino-2-phenylindole; dbEST, database of expressed sequence tags; ENase, restriction endonuclease; EST, expressed sequence tag; FISH, fluorescence in situ hybridization; GPCR, G-protein-coupled receptor(s); GPR, gene (DNA, RNA) encoding GPCR; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PCR, polymerase chain reaction; SSC, 0.15 M NaCl/0.015 M Na · citrate pH 7.6; 3 SSPE, 3 M NaCl/0.2 M NaHPO · H 0/0.02 M EDTA pH 7.4; TM, 4 2 transmembrane; UV, ultraviolet.

et al., 1995; Meunier et al., 1995). The endogenous ligands for many of these newly discovered receptors, particular in the GPCR family, remain to be identified. Our research plan has been to identify novel genes encoding GPCR genes and in particular those that are related to the opioids and other peptide binding receptors. Knowledge of the distribution of the novel receptor genes will assist in the discovery of novel endogenous ligands, which will aid in elucidating the function of newly discovered receptor systems. Our methods have previously been successful in finding several genes encoding opioid-related receptors namely GPR7 and GPR8 (O’Dowd et al., 1995). In total our search for novel GPCRs has now revealed 19 genes encoding receptors, for which the ligands have not been identified: APJ (O’Dowd et al., 1993), GPR1, GPR2, GPR3 (Marchese et al., 1994), GPR4, GPR5, GPR6 (Heiber et al., 1995), GPR7, GPR8 (O’Dowd

0378-1119/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 7 22 - 6

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et al., 1995), GPR9, GPR10, GPR14 (Marchese et al., 1995), GPR15 (Heiber et al., 1996), GPR19 (O’Dowd et al., 1996), GPR24 ( Kolakowski et al., 1996). The GPR symbol, used in the naming of many of these genes, is used in accordance with the Genome Data Base nomenclature. Other groups have reported the isolation of genes GPR11 (Nystedt et al., 1995), GPR12 (Song et al., 1995) and GPR13 (Raport et al., 1995). We have continued the search for additional members of this receptor gene family and we now report the cloning of four additional members named GPR20, GPR21, GPR22 and GPR23.

2. Materials and methods 2.1. Amplification of genomic DNA

sequences that were returned having statistically significant scores were examined further. The conceptualized aa sequences of the EST sequences were used to query (Pearson and Lipman, 1988; Pearson, 1995) our GPCR database using the FastA algorithm to determine whether the EST cDNAs represented known GPCRs. The aa sequences thus filtered were used to query the SwissProt (release 31) data base using the FastA algorithm (BLOSUM 50 matrix, ktup=1) (Pearson and Lipman, 1988; Pearson, 1995). Two EST cDNAs that met these criteria were requested from the I.M.A.G.E. Consortium (Lennon et al., 1996) (cloneID: 31997; GenBank accession No. R17870) and Prof. C.C. Liew (cloneID: G3141; GenBank accession No. R58357). When the clones were received, the reported nt sequences were verified by single pass sequencing of both ends of the cDNA.

Human genomic DNA was amplified by the polymerase chain reaction (PCR) using degenerate oligos designed based on the sequences encoding transmembrane ( TM ) domains 3 (P1: 5∞-GTSATGAGYGYVGACCGMTA; S=C or G, Y=C or T, V=G or C or A, M=C or A, H=A or C or T, W=T or A) and 7 (P2: 5∞-GGGGTTSAGGCASSWGTT ) that are conserved among the opioid and somatostatin receptors and the related receptors encoded by genes GPR7 and GPR8. PCR conditions were as follows: denaturation at 94°C for 3 min, annealing at 50°C for 2 min and extension at 72°C for 3 min, for 30 cycles, followed by a 7 min extension at 72°C (Nguyen et al., 1995). In another experiment, human genomic DNA was amplified by PCR using degenerate oligos designed based on sequences encoding a highly conserved region following TM domain 2 (P3: 5∞-TGGGAHHSTGGCCVTTYGG), and the sequences encoding TM domain 3 (P4: 5∞-TGGGAHHSTGGCCVTTYGG) of the opioid and somatostatin receptors, and GPR7 and GPR8. The PCR conditions were as follows: denaturation at 94°C for 1 min, annealing at 50°C for 2 min and extension at 72°C for 3 min, for 30 cycles, followed by a 7 min extension at 72°C. In each experiment the resultant amplified PCR products were extracted with phenol/chloroform, precipitated with ethanol and electrophoresed on a low melting point agarose gel. PCR-amplified DNA in the expected size range was excised from the gel, ligated into the EcoRV site of pBluescript SK(−) (Stratagene, La Jolla, CA, USA) and sequenced.

Human mRNAs from several human tissues were extracted as described previously (Marchese et al., 1994). Briefly, total RNA was extracted by the method of Chomczynski and Sacchi (1987), and poly(A)+ RNA was isolated using oligo(dT ) cellulose spin columns (Pharmacia, Uppsala, Sweden). RNA was denatured and size fractionated on a 1% formaldehyde agarose gel, transferred onto nylon membrane and immobilized by UV irradiation. The blots were hybridized with a 32P-labeled DNA fragment, washed with 2×SSPE and 0.1% SDS at 50°C for 20 min and with 0.1×SSPE and 0.1% SDS at 50°C for 2 h and exposed to X-ray film at −70°C in the presence of an intensifying screen.

2.2. Searching the dbEST

2.5. FISH detection

We queried the dbEST maintained by the National Center for Biotechnology Information (NCBI ), with the complete aa sequence of, for example, the a -adrenoceptor, using the TBLASTN algorithm 2 (Altschul et al., 1990). Expressed sequence tag ( EST )

Metaphase spread chromosomes derived from human lymphocytes were used for chromosomal assignment of the genes GPR20, GPR21, GPR22 and GPR23. Slides were prepared and FISH was performed as previously described (Heng et al., 1992; Heng and Tsui, 1993).

2.3. Library screening DNA fragments used as probes were labeled with [32P]dCTP (ICN ) by nick translation (Amersham, Arlington Heights, IL, USA) and used to screen a lEMBL3 SP6/T7 human genomic library (Clontech, Palo Alto, CA, USA). Positive phage clones were plaque purified, DNA was prepared, ENase digested, electrophoresed on an agarose gel, transferred to nylon membrane, and hybridized with the same probe used to screen the library, as described in Marchese et al. (1994). Probe binding fragments were subcloned into pBluescript (Stratagene), and the coding regions of each gene were sequenced in both directions. 2.4. Northern blot analysis

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3. Results 3.1. Cloning of gene GPR20 This novel GPCR gene was detected by PCR amplification of genomic DNA using oligos based on sequences of the opioid/somatostatin receptor genes and the related genes GPR7 and GPR8. We have used these oligos previously to discover the uridine nucleotide receptor (UNR) gene (Nguyen et al., 1995) and GPR15 (Heiber et al., 1996). One of the sequenced PCR products partially encoded a GPCR and was used to screen a genomic library to obtain the full-length gene. Seven positive phage clones were plaque purified, and after ENase digestion and Southern analysis a 5.5-kb NcoI fragment was isolated. This genomic clone, named GPR20, with overlapping sequence identical to the PCR product, contained an intronless ORF of 1062 bp, encoding a putative GPCR protein of 354 aa (Fig. 1). 3.2. Cloning of genes GPR21 and GPR22 In our continuing search for novel genes that belong to the GPCR gene superfamily we searched the dbEST for the presence of GPCR-encoding sequences. A search of the dbEST revealed a number of sequences. Some of these represented GPCRs described previously, but two EST cDNAs represented novel sequences only partially encoding GPCRs. One of these EST cDNA clones (clone ID: 31997) was obtained from the I.M.A.G.E. Consortium and the other (clone ID: G3141) from Prof. C.C. Liew at the University of Toronto. EST cDNA (ID: 31997; 1.5 kb) was isolated originally from a human infant brain cDNA library. A human genomic library was screened with this fragment to obtain the full-length ORF. Three positive phage clones were isolated from the screening, plaque purified, and after ENase digestion and Southern blot analysis, a 2.3-kb HindIII-EcoRI fragment was isolated and sequenced. This genomic fragment contained overlapping sequence identical to the EST cDNA and continued an additional 500 bp upstream from the 5∞ end of the EST cDNA clone. The genomic clone, called GPR21, contained the complete intronless ORF of 1047 bp encoding a putative GPCR protein of 349 aa (Fig. 1). The filters were screened again with EST cDNA (ID: G3141; 1 kb), to obtain the full-length ORF. Five positive clones were isolated, plaque purified, and after ENase digestion and Southern blot analysis, were determined to be the same. A 3-kb BglI fragment that contained overlapping sequence identical to the EST cDNA was isolated. This genomic clone, called GPR22, encoding a putative GPCR contained an ORF that was truncated in the putative carboxy terminus of the receptor because it represented the end of the genomic insert in the vector. The full-length translational ORF (433

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aa) was obtained by splicing together the genomic sequence with the cDNA sequence so that the sequence 3∞ of the third nt of codon 394 was obtained from the cDNA (Fig. 1).

3.3. Cloning of gene GPR23 Also in our search for opioid or peptide binding receptors, we designed degenerate oligos based on sequences following TM2 and TM3 of the opioid receptors. PCR of human genomic DNA with these oligos resulted in the isolation of a clone (approximately 100 bp), that shared significant identity with GPCR genes. The sequence of this clone was used to query the GenBank database and was found to be identical to R12 (GenBank accession No. U33447), a previously cloned gene encoding an orphan GPCR (Raport et al., 1995). The isolated clone also shared high identity to an EST cDNA (ID: 51646, 1.8 kb) sequence that partially encoded a novel GPCR, truncated in the putative TM2 domain. This EST cDNA, was requested from the I.M.A.G.E. Consortium (Lennon et al., 1996) and used to screen a human genomic library in order to obtain the full-length ORF. Five positive phage clones were isolated, plaque purified and DNA was prepared. Following ENase digestion and Southern blot analysis, a 4.5-kb SacI fragment was isolated, sequenced, and found to contain identical overlapping sequence to the EST cDNA. This genomic clone, named GPR23, contained an intronless ORF of 1110 bp encoding for a putative GPCR protein of 370 aa (Fig. 1).

3.4. Northern blot analysis of the genes GPR20, GPR21, GPR22 and GPR23 Tissue distribution for the expression of genes GPR20, GPR21, GPR22 and GPR23 was examined by northern blot analysis using poly(A)+RNA isolated from several adult human brain regions and human liver. A 4.4 kb mRNA was detected for GPR20 in thalamus, putamen, caudate (Fig. 2A), and liver (not shown), while no hybridization signals were detected in the frontal cortex, pons, or hypothalamus. A 3.0 kb mRNA was detected for GPR22 in four of the brain regions examined including frontal cortex, caudate, putamen and thalamus ( Fig. 2B), but expression was not detected in pons, hypothalamus and hippocampus. The EST cDNA encoding GPR22 was isolated originally from human heart, and the EST cDNAs encoding GPR21 and GPR23 were isolated originally from human brain. However, transcripts for these genes, GPR21 and GPR23, were not detected in the brain regions examined: thalamus, putamen, caudate, frontal cortex, pons, hypothalamus, hippocampus.

Fig. 1. Amino-acid comparison of the receptor proteins encoded by genes GPR20, GPR21, GPR22 and GPR23. Amino acids identical with any two of the four receptors are boxed and shaded. The putative TM domains are noted. Gaps (-) have been introduced to maximize the alignment between the sequences. Sequence data reported in this article have been deposited into GenBank under accession Nos. U66578 (GPR23), U66579 (GPR20), U66580 (GPR21) and U66581 (GPR22).

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Fig. 2. Northern blot analysis of the tissue distribution of (A) GPR20 and (B) GPR22. Each lane contains 5 mg of poly(A)+ RNA.

3.5. Chromosomal assignments of the genes GPR20, GPR21, GPR22 and GPR23 FISH of human metaphase spread chromosomes was used to identify the specific chromosomal localization of the four novel genes. Biotinylated phages containing either GPR20, GPR21, GPR22 or GPR23 were used as the probes for FISH mapping. For each phage, of the 100 mitotic figures checked, a signal appeared on only one pair of chromosomes 94 to 100% of the time, indicating a very high hybridization efficiency. DAPIbinding (4∞,6-diamidino-2-phenylindole) patterns on the mitotic chromosomes were used to identify the specific chromosomes to which each phage hybridized. For higher resolution, a summary from ten photographs was taken in order to identify the specific region on the chromosome to which each phage hybridized. No additional loci were detected by FISH analysis for any of the phage under the conditions used. Therefore, GPR20 was assigned to chromosome 8, region q24.3-24.2 (Fig. 3A), GPR21 was assigned to chromosome 9, region q33 (Fig. 3B), GPR22 was assigned to chromosomes 7, region q22–q31.1 (Fig. 3C ) and GPR23 was assigned to chromosome X, region q13–q21.1 ( Fig. 3D).

4. Discussion Hydropathy analysis of the aa sequence encoded by GPR20, GPR21, GPR22 and GPR23 each demonstrated the seven putative TM regions characteristic of GPCRs. A comparison of the aa sequence of the receptor encoded by GPR20 with other functionally defined GPCRs revealed that this receptor was related to both the UNR (43% in TM domains) and the kappa opioid receptor

(30% in TM domains). The protein encoded by GPR20 contains two N-linked glycosylation consensus sites in the amino terminus, and a consensus sequence for phosphorylation by protein kinase A in the carboxyterminal region ( Fig. 1). The tissue distribution of GPR20 expression overlapped with the sigma receptor binding sites (Debonnel, 1993); however, COS7 membranes transiently expressing the receptor protein (as previously described by Zastawny et al., 1994) encoded by GPR20 did not show specific binding to [3H ]DTG, [3H ]haloperidol, [3H ]GBR 12909, [3H ]fluphenazine and [3H ]YM-09151. Identifying ligands that bind to the orphan receptors may be elusive, but these orphan receptors should prove useful in discovering novel ligand systems. The aa sequence of the receptor encoded by GPR21 was compared with that of other GPCRs and was found to be most similar to the b -adrenergic receptor (33% 1 in TM domains), the histamine H2 receptor (32% in TM domains), and the adenosine A3 receptor (32% in TM domains). The receptor encoded by GPR21 had a lysine residue in an almost equivalent position as the aspartic acid (characteristic of the biogenic amine receptors) in the third TM region of the b -adrenergic recep1 tor. A lysine residue found in an analogous position in the endothelin receptor is important for binding of peptide agonists to the receptor (Zhu et al., 1992). The receptor encoded by GPR21 has two N-linked glycosylation consensus sites in the amino terminus, potential sites for modification by protein kinase C and protein kinase A in the carboxy terminus and a cysteine residue for potential modification by palmitoylation in the carboxy terminal region ( Fig. 1). Comparing the aa sequence of the receptor encoded by GPR22 with other GPCRs revealed that it shared

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Fig. 3. Summary of the FISH data on human chromosomes using phage probes containing each of the genes (A) GPR20, (B) GPR21, (C ) GPR22 and (D) GPR23. Each dot represents the location of a fluorescent signal on the chromosome.

highest identity with the human cholecystokinin-B receptor (34% in TM domains). The receptor encoded by GPR22 has one N-linked glycosylation consensus site in the amino terminus and one in the second extracellular loop, and two potential sites for modification by protein kinase A in the third intracellular loop ( Fig. 1). An aa comparison of the receptor encoded by GPR23 with other GPCRs revealed that it shared highest identity with the chicken purinoreceptor (P2Y5) receptor (66% in TM domains), and the UNR (40% in TM domains). The receptor encoded by GPR23 has four N-linked glycosylation consensus sites, three in the N terminus and one in the second extracellular loop, and one consensus sequence for potential phosphorylation by protein kinase A in the third intracellular loop. The high homology to P2Y prompted us to check for 5 binding with purinergic ligands. A 4.5 kb fragment encoding GPR23 was inserted into the expression vector pLXSN and used to infect 1321N1 cell lines, as described previously (Miller and Rosman, 1989). To assay for receptor activity, intracellular calcium flux was measured after the addition of various nucleotides (ATP, UTP, ADP, or UDP), as previously described ( Erb et al., 1995). However, no calcium flux was detected in response to any of the nucleotides. In summary, we have identified and cloned an additional four novel members of the human GPCR gene family, GPR20, GPR21, GPR22 and GPR23. These receptor genes have unique chromosomal localizations

and GPR20 and GPR22 were each shown to be expressed in discrete human brain regions. We have yet to identify any ligands that may bind to these receptors, therefore, these receptors are currently known as orphan receptors.

Acknowledgement This research was supported by grants from the Addiction Research Foundation (Ontario), the National Institutes of Drug Abuse (NIDA), the Medical Research Council of Canada, and the Smokeless Tobacco Research Council to B.F.O. and S.R.G. and the National Institute of General Medical Sciences (GM52722) to K.R.L. A.M. is a Fellow of the Health Research Personnel Development Program of the Ontario Ministry of Health. We would like to acknowledge Victor Saldivia and Yang Shen for their excellent technical assistance. For the functional expression studies on the receptor encoded by GPR23, we would like to thank Drs. Gary A. Weisman, John T. Turner and Laurie Erb from the University of Missouri.

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