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Identification of a Novel Human Eicosanoid Receptor Coupled to Gi/o

2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês

10.1074/jbc.m203194200

1083-351X

Takeshi Hosoi, Yutaka Koguchi, Emiko Sugikawa, Aiko Chikada, Koji Ogawa, Naoki Tsuda, Naoki Suto, Shiho Tsunoda, Tomoyasu Taniguchi, Tetsuo Ohnuki,

Neuropeptides and Animal Physiology

We have conducted an in silico data base search for and cloned a novel G-protein-coupled receptor (GPCR) named TG1019. Dot and Northern blotting analyses showed that transcripts of the novel GPCR were expressed in various tissues except brain, and the expression was more intense in liver, kidney, peripheral leukocyte, lung, and spleen than in other tissues. By GTPγS binding assay using the TG1019-Gαi1-protein fusion expressed in insect cells, eicosanoids, and polyunsaturated fatty acids such as 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE), 5(S)-hydroperoxy-6E,8Z, 11Z,14Z-eicosatetraenoic acid, and arachidonic acid were identified to exhibit agonistic activities against TG1019. 5-oxo-ETE was the most potent to enhance the specific binding by 6-fold at a maximum effect dose of submicromolar to micromolar order with an ED50 value of 5.7 nm. Conversely, polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid showed antagonistic activities against TG1019. In Chinese hamster ovary cells transiently expressing TG1019, the forskolin-stimulated production of cAMP was inhibited up to ∼70% by 5-oxo-ETE, with an IC50 value of 33 nm. This inhibition was sensitive to pretreatment of the cells with pertussis toxin. We have conducted an in silico data base search for and cloned a novel G-protein-coupled receptor (GPCR) named TG1019. Dot and Northern blotting analyses showed that transcripts of the novel GPCR were expressed in various tissues except brain, and the expression was more intense in liver, kidney, peripheral leukocyte, lung, and spleen than in other tissues. By GTPγS binding assay using the TG1019-Gαi1-protein fusion expressed in insect cells, eicosanoids, and polyunsaturated fatty acids such as 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE), 5(S)-hydroperoxy-6E,8Z, 11Z,14Z-eicosatetraenoic acid, and arachidonic acid were identified to exhibit agonistic activities against TG1019. 5-oxo-ETE was the most potent to enhance the specific binding by 6-fold at a maximum effect dose of submicromolar to micromolar order with an ED50 value of 5.7 nm. Conversely, polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid showed antagonistic activities against TG1019. In Chinese hamster ovary cells transiently expressing TG1019, the forskolin-stimulated production of cAMP was inhibited up to ∼70% by 5-oxo-ETE, with an IC50 value of 33 nm. This inhibition was sensitive to pretreatment of the cells with pertussis toxin. G-protein-coupled receptors 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid 5(S)-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid 5Z,6Z,11Z,14Z-eicosatetraenoic acid 5Z,8Z,11Z-eicosatrienoic acid 5(±)-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid 5(S)-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid 5(R)-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid 5(S)-hydroxy-6E,8Z,11Z-eicosatrienoic acid 11Z,14Z,17Z-eicosatrienoic acid 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid amino acid(s) nucleotide(s) Krebs-Ringer Hepes buffer prostaglandin leukotriene guanosine 5′-O-(thiotriphosphate) Chinese hamster ovary transmembrane domain Dulbecco's modified Eagle's medium The G-protein-coupled receptor (GPCR)1 superfamily is the largest known receptor family, characterized by seven transmembrane domains with an extracellular N terminus and a cytoplasmic C terminus (1Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (685) Google Scholar, 2Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 3Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). GPCRs transduce a variety of extracellular signals such as photons, odorants, biogenic amines, peptides, hormone proteins, proteases, nucleotides, and lipids into activation of G-proteins for effectors to generate intracellular second messengers. They are divided into distinct subfamilies according to their various types of ligands and according to sequence homologies. Recently, vigorous in silico search for and cloning of novel genes with sequence motifs characteristic of GPCRs have outpaced identification of novel endogenous ligands, accumulating a group of putative GPCR genes (by the criterion of sequence similarity), for which the ligands are not known (4Stadel J.M. Wilson S. Bergsma D.J. Trends Pharm. Sci. 1997; 18: 430-437Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 5Marchese A. George S.R. Kolokowski Jr., L.F. Lynch K.R. O'Dowd B.F. Trends Pharm. Sci. 1999; 20: 370-375Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). These GPCRs, commonly known as orphans, are likely to mediate heretofore unidentified signaling and might represent a fruitful source for new drugs, judging from the precedents established by the numerous GPCRs used as targets of important drugs in use today (6Wilson S. Bergsma D.J. Chambers J.K. Muir A.I. Fantom K.G.M. Ellis C. Murdock P.R. Herrity N.C. Stadel J.M. Br. J. Pharmacol. 1998; 125: 1387-1392Crossref PubMed Scopus (115) Google Scholar). There have been several reports on identification of cognate ligands for orphan GPCRs by using the recombinant orphan receptors as the specific sensors in bioassays (7Sakurai T. Amemiya A. Ishii M. Matsuzaki I. Chemelli R.M. Tanaka H. Williams S.C. Richardson J.A. Kozlowski G.P. Wilson S. Arch J.R.S. Buckingham R.E. Haynes A.C. Carr S.A. Annan R.S. MacNulty D.E. Liu W.-S. Terrett J.A. Elshourbagy N.A. Bergsma D.J. Yanagisawa M. Cell. 1998; 92: 573-585Abstract Full Text Full Text PDF PubMed Scopus (3200) Google Scholar, 8Hinuma S. Habata Y. Fujii R. Kawamata Y. Hosoya M. Fukusumi S. Kitada C. Masuo Y. Asano T. Matsumoto H. Sekiguchi M. Kurokawa T. Nishimura O. Onda H. Fujino M. Nature. 1998; 393: 272-276Crossref PubMed Scopus (541) Google Scholar, 9Ames R.S. Sarau H.M. Chambers J.K. Willette R.N. Aiyar N.V. Romanic A.M. Louden C.S. Foley J.J. Sauermelch C.F. Coatney R.W., Ao, Z. Disa J. Holmes S.D. Stadel J.M. Martin J.D. Liu W.-S. Glover G.I. Wilson S. MucNulty D.E. Ellis C.E. Elshourbagy N.A. Shabon U. Trill J.J. Hay D.W.P. Ohlstein E.H. Bergsma D.J. Douglas S.A. Nature. 1999; 401: 282-286Crossref PubMed Scopus (790) Google Scholar). Here, we describe discovery of a novel Gi/o-coupled receptor, tentatively denoted as TG1019, whose ligands were identified to be eicosatetraenoic acids and polyunsaturated fatty acids, including 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE), 5(S)-hydroxyperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5(S)-HPETE), 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE), and arachidonic acid. Arachidonic acid is metabolized into biologically and pharmacologically important mediators, which include prostaglandins, leukotrienes, prostacyclins, thromboxanes, and eicosatetraenoic acids such as 5(S)-HPETE, 5(S)-HETE, and 5-oxo-ETE (10Needleman P. Turk J. Jakschik B.A. Morrison A.R. Lefkowith J.B. Annu. Rev. Biochem. 1986; 55: 69-102Crossref PubMed Google Scholar). Although GPCRs for the preceding four kinds of mediators have been already known (11Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar), there have been no reports on GPCR for the eicosatetraenoic acids. 5-oxo-ETE, 5(S)-HPETE, 5Z,8Z,11Z-eicosatrienoic acid (Mead acid), 5(R)-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5(R)-HETE), 5(S)-hydroxy-6E,8Z,11Z-eicosatrienoic acid (5(S)-HETrE), 11Z,14Z,17Z-eicosatrienoic acid, 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid (DHA), 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid (EPA), and 8Z,11Z,14Z-eicosatrienoic acid (dihomo-γ-linolenic acid) were purchased from Cayman Chemical Company (Ann Arbor, MI). All of the other lipids used in this study were obtained from BIOMOL Research Laboratories, Inc. (Plymouth, PA). Rabbit anti-human Gαi1, anti-human Gαq, and anti-human Gαs antisera were from Calbiochem. Pertussis toxin and fetal bovine serum were from Sigma Chemical Co. Rolipram and forskolin were from Tocris (Avonmouth, United Kingdom) and Nacalai Tesque (Japan), respectively. [α-32P]dCTP and [35S]GTPγS were from Amersham Biosciences. DMEM/F-12 media for cell culture was from Invitrogen. We created a consensus amino acid sequence (IYSIVFVVGLLGNALVIWVLLRHKKMRTVTNIYILNLAIX26LCKIVSFLYXVNMYASIFTLTAISIDRYLAIVHPLX65FX33RVVRMILVVVVVFAICWLPYHIX14AX10LAYLNSCINPIIYAFLSKNFR; the amino acid sequence is shown in one-letter designation, in whichX represents any amino acid) of a GPCR family recognizing peptide ligands by aligning the GPCRs registered in the GPCR data base (12Horn F. Weare J. Beukers M.W. Horsch S. Bairoch A. Chen W. Edvardsen O. Campagne F. Vriend G. Nucleic Acids Res. 1998; 26: 275-279Crossref PubMed Scopus (341) Google Scholar), using BlockMaker (13Henikoff S. Henikoff J.G. Alford W.J. Pietrokovski S. Gene. 1995; 163: GC17-GC26Crossref PubMed Scopus (300) Google Scholar). Putative orphan GPCR sequences were searched for among public data bases by tblastn (14Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) using the consensus sequence as a query. Specific oligonucleotide primers were designed on the basis of the sequence of GenBankTM accession number AC013396.3: a sense primer, 5′-ttctcagtggctgcgagaatgctgat-3′ corresponding to nucleotides (nt) 97,735–97,760 of AC013396.3 and an antisense primer, 5′-acccacctgagtcctgccagtgcttt-3′ corresponding to nt 96,299–96,324 ofAC103396.3. A PCR with these primers was performed on a human cDNA library (Human Universal Quick-Clone cDNA, CLONTECH) by using Advantage 2 Polymerase Mix (CLONTECH). The amplification conditions were as follows: 30 s at 94 °C, 30 cycles of 30 s at 94 °C, 30 s at 64 °C, and 2.5 min at 72 °C, and then 2.5 min at 72 °C. The amplification gave a DNA fragment of ∼1.5 kb, which was purified by agarose gel electrophoresis and cloned into pGEM-T Easy Vector (Promega Corp.), resulting in pGEM-TG1019. The cloned DNA was sequenced on both strands with ThermoSequenase Cycle sequencing kit (USB Corp., Cleveland, OH), and DNA sequencer Long Read IR 4200 (Aloka, Japan). We read all parts of the cloned sequence at least three times each. Human Multiple Tissue Expression (MTE) Array2 (CLONTECH), a nylon membrane on which poly(A) RNAs extracted from various human tissues and cell lines were dotted, and Human 12-Lane MTN blot (CLONTECH) were used for analysis of distribution of TG1019 expression in human tissues and for Northern hybridization analysis, respectively. [α-32P]dCTP-labeled probe was prepared by random priming of the 0.9-kb fragment corresponding nt 371–1270 encoding a large part of TG1019 (Fig. 1) with Prime-a-Gene labeling system (Promega Corp.). The DNA fragment was generated by PCR using a sense primer (5′-tttggccctcttcatcttctgcat-3′), an antisense primer (5′-ccctgcactttcagcttccctatg-3′), and pGEM-TG1019 as a template. Hybridization was conducted as described in the manufacturer's protocols. The membranes were prehybridized in ExpressHyb hybridization solution (CLONTECH) supplemented with 0.1 mg of salmon testes DNA (Sigma) per milliliter at 65 °C for 30 min and hybridized with the 32P-labeled probe in ExpressHyb solution supplemented with 0.1 mg of salmon testes DNA (Sigma) per milliliter at 65 °C for 14 h. In the case of dot blotting hybridization, 6 μg of human COT-1 DNA (Roche Molecular Biochemicals) per milliliter and 0.2× SSC were also added to the hybridization solution. The bolt of MTE Array2 was washed four times in 2× SSC containing 1% SDS at 65 °C and twice in 0.1× SSC containing 0.5% SDS at 55 °C for 20 min. The MTN blot was washed four times in 2× SSC containing 0.05% SDS at room temperature and twice in 0.1× SSC containing 0.1% SDS at 50 °C for 20 min. The membranes were exposed to an imaging plate (BAS-III, Fujifilm, Japan) and analyzed by using a BAS2000 imaging analyzer (Fujifilm). Identification of a ligand for the orphan GPCR was conducted by measuring ligand-dependent GTPγS binding to Gα-protein. We constructed an assay system using fused GPCR-Gα-protein expressed in insect cells by referring to the reports of Wenzel-Seifert and Seifert (15Wenzel-Seifert K. Seifert R. Mol. Pharmacol. 2000; 58: 954-966Crossref PubMed Scopus (148) Google Scholar) and Bahia et al.(16Bahia D.S. Wise A. Fanelli F. Lee M. Rees S. Milligan G. Biochemistry. 1998; 37: 11555-11562Crossref PubMed Scopus (81) Google Scholar). cDNAs of Gαi1 with a mutation of Cys351 → Ile, Gαq, and GαsLwere amplified by PCR with the primers designed on the basis of the registered sequences for Gαi1 (GenBankTMaccession number AF055013), Gαq (GenBankTMU43083), and GαsL (GenBankTMX04408) genes and templates of Marathon-Ready cDNA Brain (CLONTECH) for Gαi1(Cys351 → Ile), Marathon-Ready cDNA Prostate (CLONTECH) for Gαq, and Marathon-Ready cDNA Bone marrow (CLONTECH) for GαsL, respectively. The mutation in Gαi1, known to enhance the GTPγS binding activated by ligands as compared with the native (16Bahia D.S. Wise A. Fanelli F. Lee M. Rees S. Milligan G. Biochemistry. 1998; 37: 11555-11562Crossref PubMed Scopus (81) Google Scholar), was introduced by replacing the nucleotide in the antisense primer used. CpoI and BamHI restriction recognition sequences were attached to the sense and antisense primers, respectively. The amplified DNAs were cloned into pGEM-T Easy vector and sequenced to be confirmed the same as the registered sequences. The plasmids obtained above were cut with NotI and BamHI, and the DNA fragments of the coding regions for Gαi1(Cys351 → Ile), Gαq, and GαsL were purified by agarose gel electrophoresis and inserted into a baculovirus vector plasmid pVL1392 (BD PharMingen). The DNA fragment encoding TG1019 was amplified by using 5′-agatctatgttgtgtcaccgtggtggccagc-3′ as a sense primer, 5′-aagcttcggtccgccctgggaggagccttccttttcca-3′ as an antisense primer, and pGEM-TG1019 as a template, and cloned into pGEM-T Easy. By the PCR reaction, a stop codon of the TG1019 gene was replaced by a Gly codon, and a CpoI site was added to the 3′-end of the gene. The resultant plasmid was cut with NotI and CpoI, and the TG1019 gene was inserted into pVL1392 carrying each of the Gα genes, generating pVL1392/TG1019-Gαi1(Cys351→ Ile), pVL1392/TG1019-Gαq, and pVL1392/TG1019-GαsL, respectively. Two amino acids, -Gly-Pro-, were inserted between TG1019 and each of the Gα-proteins. Transfection of Sf9 cells with the baculovirus expression vectors and preparation of the recombinant virus were carried out with a BaculoGold kit (BD PharMingen) or a Bac-N-Blue transfection kit (Invitrogen) as recommended by the manufacturer. The Sf9 cells grown in a dish of 10-cm diameter were transfected by the recombinant virus and cultured at 27 °C for 4 days. The cells were harvested in 3.6 ml of 20 mmTris-HCl (pH 7.5), 1 mm EDTA, 0.2 mmphenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, and 2 μg/ml aprotinin and homogenized with a Teflon homogenizer. The homogenate was centrifuged at 600 × g for 10 min, and the supernatant was subjected to ultracentrifugation at 50,000 × g for 20 min. The precipitate was suspended in 450 μl of a reaction buffer containing 20 mm Tris-HCl (pH 7.5), 50 mm NaCl, and 10 mm MgCl2. The membrane suspension was diluted to 16 ml with the reaction buffer and added to 6 μl of 10 mm GDP just before use in the reaction. Expression of the fused TG1019-Gα-proteins was confirmed by Western blotting analysis using the antisera against the respective species of Gα subunits. Reactions of GTPγS binding were done in 200 μl of reaction mixture consisting of 160 μl of the diluted membrane suspension, 20 μl of 5 nm [35S]GTPγS (5 nCi/μl), and 20 μl of a sample to be tested: The radioisotope and sample were prepared in the reaction buffer. After incubation at 30 °C for 60 min, the reaction mixture was filtrated through UniFilter-96GF/B (Packard), and the filter was washed three times with the ice-cold reaction buffer, and dried 60 °C for 30 min. The radioactivities that remained on the filter were measured by liquid scintillation counting. The specific binding was defined as the remainder of the radioactivity bound on membrane after incubation with 500 pm[35S]GTPγS minus that after incubation with 500 pm [35S]GTPγS and cold 10 μmGTPγS. Results are reported as the percentage of the specific [35S]GTPγS binding in the presence of the sample above the binding in the absence of the sample. The full-length cDNA encoding TG1019 on pGEM-TG1019 was subcloned into a NotI site of pcDNA3.1 expression vector (Invitrogen), resulting in pcDNA3.1-TG1019. CHO cells (1 × 106 cells) were seeded in a dish of 6-cm diameter (Sumilon, Japan) and cultured in DMEM/F-12 supplemented with 10% fetal bovine serum at 37 °C for 20 h in a humidified atmosphere of 5% CO2. The cells were transfected by 5 μg of pcDNA3.1-TG1019 DNA or pcDNA3.1 for a mock experiment with LipofectAMINE (Invitrogen), as recommended by the manufacturer. After incubation for 24 h, the cells were harvested with cell-dissociation buffer (enzyme-free/phosphate-buffered saline-based, Invitrogen) and washed twice with Krebs-Ringer Hepes (KRH) buffer (124 mm NaCl, 5 mm KCl, 1.25 mmMgSO4, 1.45 mm CaCl2, 1.25 mm KH2PO4, 25 mm HEPES (pH 7.4), and 8 mm glucose). After incubation at 37 °C for 30 min with KRH buffer supplemented with 25 μmrolipram, 90 μl of the cell suspension (5–7.5 × 104 cells/well) was seeded in a 96-well plate. Then, 90 μl of KRH buffer containing 1 μm forskolin and 2× final concentration of 5-oxo-ETE was added, and the plates were incubated at room temperature for 10 min. The cells were lysed by adding 20 μl of lysis buffer 1A (a component of the cAMP enzyme immunoassay system, Amersham Biosciences), and 80 μl of the cell lysate was used for measurement of cAMP produced during the incubation with the kit, as recommended by the manufacturer. To examine effect of pertussis toxin treatment on the cAMP production, the transfected CHO cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum for 20 h and then in the culture medium supplemented with 100 ng of pertussis toxin per ml for 4 h. A tblastn search with the consensus sequence for the peptide-ligand GPCRs identified an intronless coding sequence on a genomic clone RP11-489G24 (GenBankTM accession number AC013396.3) mapped in the 2p21 region. This 1272-bp sequence putatively coded for a protein with 423 amino acids, which was predicted to have seven transmembrane domains by HMMTOP analysis (17Tusnady G.E. Simon I. J. Mol. Biol. 1998; 283: 489-506Crossref PubMed Scopus (949) Google Scholar). We designed the primers based on the registered DNA sequence and cloned a 1462-bp sequence containing the open reading frame coding for the putative GPCR from the cDNA library by PCR. Fig.1 shows the nucleotide and deduced amino acid sequences of the cloned cDNA. The nucleotide sequence had differences of four nucleotides at positions of nucleotide (nt) 487 (A → G), nt 771 (A → G), nt 1022 (A → C), and nt 1038 (A → G) as compared with the sequence registered in GenBankTMaccession number AC013396.3, which resulted in the changes of amino acids encoded at positions of amino acids (aa) 150 (Asp → Gly), aa 245 (Lys → Glu), and aa 334 (Thr → Ala) (Fig. 1). To investigate whether these differences were due to errors of PCR or nucleotide polymorphisms, we carried out cloning an ApaI fragment (nt 283–1173) (Fig. 1) by PCR using 5′-tccctctgcctttaccactgtggg-3′ as a sense primer, 5′-gtaggagctctcgtcgctcactg-3′ as an antisense primer, and Marathon-Ready cDNA library (human fetal spleen, CLONTECH), constructed by using a mixture of mRNA preparations from 29 persons as a template, and sequenced the amplified DNA fragments. We conducted two separate PCR reactions and analyzed a total of 10 clones, that is six and four clones obtained by each of the reactions. Regarding the nucleotide positions of nt 487, 771, and 1038, all clones sequenced gave the nucleotides identical to the registered sequence, i.e. A at nt 487, 771, and 1038, whereas at nt 1022, five clones (two and three clones from each PCR) had C as the clone shown in Fig. 1, and five clones (four and one clone from each PCR) had A as the registered sequence. Therefore, it would be conceivable that the sequence registered in GenBankTMaccession number AC013396.3 was accurate and that the position of nt 1022 exhibited a nucleotide polymorphism. For further studies, we used the clone with the sequence depicted in Fig. 1, otherwise stated below. HMMTOP predicted that the putative gene might contain seven transmembrane regions of from Ala93 to Phe117, from Pro123 to Leu147, from Ala164 to Leu188, from Ser205 to Leu229, from Ala255 to Ile279, from Leu297 to Ala321, and from Leu341to Phe362 (Fig. 1). The sequence contained one potential site for N-linked glycosylation (Asn44) in the extracellular N-terminal domain, one potential site for phosphorylation by protein kinase C (Ser398), and oneN-myristoylation site (Gly371) in the C-terminal region. The GPCR family 1 (rhodopsin) signature was found in the boundary region between transmembrane III and the second intracellular loop (from Ala178 to Val194). A prominent feature of the putative GPCR was a Ser-rich stretch from Ser46 to Ser92 in the N-terminal region. There were two candidates for a start codon of translation, Met1and Met40. Further studies are needed to identify the start codon. We tentatively denoted the novel GPCR as TG1019. TG1019 displayed a low homology with orphan GPCRs, HM74 (18Nomura H. Nielsen B.W. Matsushima K. Int. Immunol. 1993; 5: 1239-1249Crossref PubMed Scopus (141) Google Scholar) (41%, identities = 121 aa/294 aa) and GPR31 (19Zingoni A. Rocchi M. Storlazzi C.T. Bernardini G. Santoni A. Napolitano M. Genomics. 1997; 42: 519-523Crossref PubMed Scopus (20) Google Scholar) (33%, identities = 104 aa/306 aa), but not a significant homology with GPCRs for which ligands have been identified, as far as searched by BLAST. Dot-blotting hybridization analysis showed that the gene encoding TG1019 was expressed in various tissues except brain, although somewhat more intense signals were observed in mRNA from kidney and liver (Fig.2A). Northern hybridization identified three hybridizing bands with molecular lengths of 6.5, 3.3, and 1.8 kb (Fig. 2B). The transcripts of 6.5 and 3.3 kb were expressed in liver and kidney and in skeletal muscle, respectively, whereas the 1.8-kb transcript was major in peripheral leukocyte, lung, placenta, small intestine, spleen, thymus, colon, and heart, and also found in skeletal muscle, liver, and kidney. In Northern blot analysis, hybridization bands of peripheral leukocyte, lung, liver, kidney, and spleen seemed to be more intense than those of the other tissues (Fig.2B). At first, we estimated how good our GPCR-Gα-protein fusion system worked by using human β2-adrenoreceptor (20Ostrowski J. Kjelsberg M.A. Caron M.G. Lefkowitz R.J. Annu. Rev. Pharmacol. Toxicol. 1992; 32: 167-183Crossref PubMed Google Scholar)-GαsL, human UDP-glucose receptor (21Chambers J.K. Macdonald L.E. Sarau H.M. Ames R.S. Freeman K. Foley J.J. Zhu Y. McLaughlin M.M. Murdock P. MacMillan L. Trill J. Swift A. Aiyar N. Taylor P. Vawter L. Naheed S. Szekeres P. Hervieu G. Scott C. Watson J.M. Murphy A.J. Duzix E. Klein C. Bergsma D.J. Wilson S. Livi G.P. J. Biol. Chem. 2000; 275: 10767-10771Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar)-Gαi1(Cys351 → Ile), and human endothelin A receptor (22Adachi M. Yang Y.Y. Furuichi Y. Miyamoto C. Biochem. Biophys. Res. Commun. 1991; 180: 1265-1272Crossref PubMed Scopus (94) Google Scholar)-Gαq as model experiments. Binding of GTPγS to the GPCR-Gα fusions was enhanced by the respective ligands with the following maximal activations and ED50 values: 800% of basal and 8 nm for β2-adrenoreceptor-GαsL by isoproterenol; 170% of basal and 210 nm for UDP-glucose receptor-Gαi1 by UDP-glucose; and 180% of basal and 1 nm for endothelin A receptor-Gαq by endothelin-1 (data not shown). We screened a library of natural bioactive compounds and their relatives for ligands of TG1019 using the TG1019-Gα fusions. It was found that some kinds of eicosanoids and unsaturated fatty acids activated binding of GTPγS to the membrane fraction expressing TG1019-Gαi(Cys351 → Ile). The most potent ligand with agonistic activity was 5-oxo-ETE, which enhanced the specific binding of GTPγS by 5- to 6-fold at 0.1–1 μmconcentration (Fig. 3A). In contrast, 5-oxo-ETE did not significantly activate the binding of GTPγS to TG1019-GαsL and TG1019-Gqa, although a small degree (∼40%) of activation of TG1019-Gαq could be detected by 5-oxo-ETE (Fig.3A) as well as by the other active eicosanoids and unsaturated fatty acids (data not shown). Another GPCR-Giafusion, UDP-glucose receptor-Gαi1(Cys351 → Ile), was not activated by 5-oxo-ETE at all (Fig. 3A). The identified agonists included 5-oxo-ETE, 5(S)-HPETE, arachidonic acid, Mead acid, 5(±)-HETE, and 5(S)-HETrE (Fig. 3B). Their rank order of the potencies for the activation was 5-oxo-ETE (maximal activation = 600 ± 26% of basal, ED50 = 5.7 ± 2.2 nm) ≫ 5(S)-HPETE (maximal activation = 480 ± 25% of basal, ED50 = 69 ± 10 nm) > arachidonic acid (maximal activation = 270 ± 13% of basal, ED50 = 240 ± 100 nm) = Mead acid (maximal activation = 230 ± 8.0% of basal at 3 μm) = 5(±)-HETE (maximal activation = 270 ± 13% of basal at 3 μm) = 5(S)-HETrE (maximal activation = 290 ± 21% of basal at 3 μm). Both 5(S)-HETE and 5(R)-HETE had essentially the same potency of activation as the racemic sample (data not shown). 5,8,11-Eicosatriynoic acid, and 5Z,8Z-eicosadienoic acid were also found to be weakly active as 5(±)-HETE and 5(S)-HETrE (data not shown). Among metabolites of arachidonic acid and the related compounds, the following compounds were tested and found to have no activity at 0.2 μm in the reaction mixture: prostaglandin (PG) A2, PG B2, PG D2, PG E2, PG F2α, 15-keto-PG 2α, 6-keto-PG 1α, PG J2, Δ12-PG J2, leukotriene (LT) B4, 20-hydroxy-LT B4, 20-carboxy-LT B4, LT C4, LT D4, LT E4, thromboxane B2, 11-dehydrothroboxane B2, lipoxin A4, lipoxin B4, 8(S)-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid, 9(S)-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid, 11(S)-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid, 12(S)-hydroxy-5Z,8Z, 10E,14Z-eicosatetraenoic acid, 15(S)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid, 12(S)-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid, 15(S)-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid, 15-oxo-6Z,8Z,11Z,13E-eicosatetraenoic acid, and 11Z,14Z-eicosadienoic acid. Antagonists against TG1019, if found, would be useful for elucidation of functions of the GPCR. We screened lipid compounds for the antagonists using the GTPγS binding assay. The GPCR was activated with 0.1 μm5-oxo-ETE, achieving the submaximal activation. 4Z,7Z,10Z,13Z,16Z,19Z-Docosahexaenoic acid (DHA), 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid (EPA), dihomo-γ-linolenic acid, and 11Z,14Z,17Z-eicosatrienoic acid were found to antagonize the ligand against TG1019 with the following IC50 values: DHA, 1.6 ± 0.2 μm; EPA, 6.0 ± 1.2 μm; dihomo-γ-linolenic acid, 3.7 ± 0.7 μm; and 11Z,14Z,17Z-eicosatrienoic acid, 5.1 ± 0.6 μm (Fig.4). In the GTPγS binding assay, TG1019 was markedly activated by the eicosanoids and polyunsaturated fatty acids only when fused to Gαi-protein but not to Gαs or Gαq. This result suggested that TG1019 would probably be coupled to a type of Gαi/o-protein. To confirm this idea, we transiently overexpressed TG1019 in CHO cells and tested the effect of 5-oxo-ETE on forskolin-stimulated cAMP production. In the cells transfected by the expression plasmid encoding TG1019 (pcDNA3.1-TG1019), the cAMP production was clearly inhibited by 5-oxo-ETE, as compared with that in the cells transfected by pcDNA3.1 (mock) (Fig. 5). The distinct inhibition (∼30% inhibition) was observed at 10 nm 5-oxo-ETE, and the inhibition dose-dependently increased to ∼70% at 1 ∼ 3 μm 5-oxo-ETE with IC50 value = 33 ± 19 nm. Pretreatment of the cells transfected by pcDNA3.1-TG1019 with pertussis toxin completely abolished the inhibition of the forskolin-stimulated cAMP production by 5-oxo-ETE. This result supported the idea that TG1019 would be coupled to a Gαi/o-protein. The amino acid sequence of TG1019 shown in Fig. 1 had the differences of three amino acids (aa 150, Asp → Gly; aa 245, Lys → Glu; and aa 334, Thr → Ala) in comparison with the sequence deduced from the nucleotide sequence registered in GenBankTM accession number AC013396.3, as described under "Data Base Search and Cloning of TG1019." To investigate whether or not the differences affected its biological function, we cloned the ApaI fragment of 0.9 kb encoding the same amino acid sequence as the registered one by PCR and constructed the expression plasmid by substituting theApaI fragment of pcDNA3.1-TG1019 with the newly cloned fragment. Forskolin-stimulated cAMP accumulation was inhibited in CHO cells transiently expressing the receptor with the registered sequence like in those expressing TG1019 with the sequence of Fig. 1 (data not shown). Therefore, the changes of amino acids would not affect the biological function of TG1019. We described in this paper the cloning of a novel G-protein-coupled receptor, tentatively named as TG1019, and identification of the eicosanoids and polyunsaturated fatty acids such as 5-oxo-ETE, 5(S)-HPETE, and arachidonic acid as its ligands by using the GPCR-Gα fusion system. The novel GPCR would be coupled to a Gαi/o-protein, because the marked enhancement of GTPγS binding to TG1019-Gα-protein by the ligands was observed only when TG1019 was fused to Gαi1(Cys351 → Ile). This coupling was confirmed by the observation of the inhibition of forskolin-stimulated cAMP production by 5-oxo-ETE in the cells transiently overexpressing