Identification of a G Protein-coupled Receptor Specifically Responsive to β-Alanine
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m314240200
ISSN1083-351X
AutoresTokuyuki Shinohara, Masataka Harada, Kazuhiro Ogi, Minoru Maruyama, Ryo Fujii, Hideyuki Tanaka, Shoji Fukusumi, Hidetoshi Komatsu, Masaki Hosoya, Yuko Noguchi, Takuya Watanabe, Takeo Moriya, Yasuaki Itoh, Shuji Hinuma,
Tópico(s)Neuropeptides and Animal Physiology
ResumoWe isolated a cDNA encoding an orphan G protein-coupled receptor, TGR7, which has been recently reported to correspond to MrgD. To search for ligands for TGR7, we screened a series of small molecule compounds by detecting the Ca2+ influx in Chinese hamster ovary cells expressing TGR7. Through this screening, we found that β-alanine at micromolar doses specifically evoked Ca2+ influx in cells expressing human, rat, or mouse TGR7. A structural analogue, γ-aminobutyric acid, weakly stimulated cells expressing human or rat TGR7, but another analogue, glycine, did not. In addition, β-alanine decreased forskolin-stimulated cAMP production in cells expressing TGR7, suggesting that TGR7 couples with G proteins Gq and Gi. In guanosine 5′-O-3-thiotriphosphate binding assays conducted using a membrane fraction of cells expressing TGR7, β-alanine specifically increased the binding of guanosine 5′-O-3-thiotriphosphate. When a fusion protein composed of TGR7 and green fluorescent protein was expressed in cells, it localized at the plasma membrane but internalized into the cytoplasm after treatment with β-alanine. In addition, we found that β-[3H]alanine more efficiently bound to TGR7-expressing cells than to control cells. From these results, we concluded that TGR7 functioned as a specific membrane receptor for β-alanine. Quantitative PCR analysis revealed that TGR7 mRNA was predominantly expressed in the dorsal root ganglia in rats. By in situ hybridization and immunostaining, we confirmed that TGR7 mRNA was co-expressed in the small diameter neurons with P2X3 and VR1, both in rat and monkey dorsal root ganglia. Our results suggest that TGR7 participates in the modulation of neuropathic pain. We isolated a cDNA encoding an orphan G protein-coupled receptor, TGR7, which has been recently reported to correspond to MrgD. To search for ligands for TGR7, we screened a series of small molecule compounds by detecting the Ca2+ influx in Chinese hamster ovary cells expressing TGR7. Through this screening, we found that β-alanine at micromolar doses specifically evoked Ca2+ influx in cells expressing human, rat, or mouse TGR7. A structural analogue, γ-aminobutyric acid, weakly stimulated cells expressing human or rat TGR7, but another analogue, glycine, did not. In addition, β-alanine decreased forskolin-stimulated cAMP production in cells expressing TGR7, suggesting that TGR7 couples with G proteins Gq and Gi. In guanosine 5′-O-3-thiotriphosphate binding assays conducted using a membrane fraction of cells expressing TGR7, β-alanine specifically increased the binding of guanosine 5′-O-3-thiotriphosphate. When a fusion protein composed of TGR7 and green fluorescent protein was expressed in cells, it localized at the plasma membrane but internalized into the cytoplasm after treatment with β-alanine. In addition, we found that β-[3H]alanine more efficiently bound to TGR7-expressing cells than to control cells. From these results, we concluded that TGR7 functioned as a specific membrane receptor for β-alanine. Quantitative PCR analysis revealed that TGR7 mRNA was predominantly expressed in the dorsal root ganglia in rats. By in situ hybridization and immunostaining, we confirmed that TGR7 mRNA was co-expressed in the small diameter neurons with P2X3 and VR1, both in rat and monkey dorsal root ganglia. Our results suggest that TGR7 participates in the modulation of neuropathic pain. G protein-coupled receptors (GPCR) 1The abbreviations used are: GPCR, G protein-coupled receptor; Mrg(s), Mas-related gene(s); VR1, vanilloid receptor subtype-1; PTX, pertussis toxin; CHO, Chinese hamster ovary; DRG, dorsal root ganglia; GABA, γ-aminobutyric acid; GABAA, GABA type A; GTPγS, guanosine 5′-O-3-thiotriphosphate; IB4, Griffonia simplicifolia isolectin B4; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GFP, green fluorescent protein; hTGR7, human TGR7; rTGR7, rat TGR7; mTGR7, mouse TGR7.1The abbreviations used are: GPCR, G protein-coupled receptor; Mrg(s), Mas-related gene(s); VR1, vanilloid receptor subtype-1; PTX, pertussis toxin; CHO, Chinese hamster ovary; DRG, dorsal root ganglia; GABA, γ-aminobutyric acid; GABAA, GABA type A; GTPγS, guanosine 5′-O-3-thiotriphosphate; IB4, Griffonia simplicifolia isolectin B4; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GFP, green fluorescent protein; hTGR7, human TGR7; rTGR7, rat TGR7; mTGR7, mouse TGR7. constitute a family with hundreds of members and have a variety of physiological functions. We have established previously (1Hinuma 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 (529) Google Scholar, 2Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1308) Google Scholar) a strategy widely applicable to identify orphan GPCR ligands. Our strategy involves searching for ligands by monitoring signal transductions, such as changes in cAMP or Ca2+ mobilization, in cells expressing orphan GPCRs (3Hinuma S. Onda H. Fujino M. J. Mol. Med. 1999; 7: 495-504Crossref Scopus (54) Google Scholar). By applying this approach, we have succeeded in identifying various orphan GPCR ligands (4Itoh Y. Kawamata Y. Harada M. Kobayashi M. Fujii R. Fukusumi S. Ogi K. Hosoya M. Tanaka Y. Uejima H. Tanaka H. Maruyama M. Satoh R. Okubo S. Kizawa H. Komatsu H. Matsumura F. Noguchi Y. Shinohara T. Hinuma S. Fujisawa Y. Fujino M. Nature. 2003; 422: 173-176Crossref PubMed Scopus (1226) Google Scholar, 5Kawamata Y. Fujii R. Hosoya M. Harada M. Yoshida H. Miwa M. Fukusumi S. Habata Y. Itoh T. Shintani Y. Hinuma S. Fujisawa Y. Fujino M. J. Biol. Chem. 2003; 278: 9435-9440Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar, 6Fujii R. Yoshida H. Fukusumi S. Habata Y. Hosoya M. Kawamata Y. Yano T. Hinuma S. Kitada C. Asami T. Mori M. Fujisawa Y. Fujino M. J. Biol. Chem. 2002; 277: 34010-34016Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 7Hinuma S. Shintani Y. Fukusumi S. Iijima N. Matsumoto Y. Hosoya M. Fujii R. Watanabe T. Kikuchi K. Terao Y. Yano T. Yamamoto T. Kawamata Y. Habata Y. Asada M. Kitada C. Kurokawa T. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Nat. Cell Biol. 2000; 2: 703-708Crossref PubMed Scopus (499) Google Scholar, 8Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Habata Y. Hinuma S. Onda H. Nishimura O. Fujino M. J. Biol. Chem. 2000; 275: 21068-21074Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 9Hosoya M. Moriya T. Kawamata Y. Ohkubo S. Fujii R. Matsui H. Shintani Y. Fukusumi S. Habata Y. Hinuma S. Onda H. Nishimura O. Fujino M. J. Biol. Chem. 2000; 275: 29528-29532Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 10Fukusumi S. Yoshida H. Fujii R. Maruyama M. Komatsu H. Habata Y. Shintani Y. Hinuma S. Fujino M. J. Biol. Chem. 2003; 278: 46387-46395Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar).In the course of our search for orphan GPCRs, we isolated TGR7, which was recently found to correspond to MrgD (11Dong X. Han S. Zylka M.J. Simon M.I. Anderson D.J. Cell. 2001; 106: 619-632Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). TGR7 has been classified as a member of the GPCR subfamily referred to as Mas-related genes (Mrgs), also known as sensory neuron-specific GPCRs. These GPCRs are known to be expressed in the sensory neurons of the dorsal root ganglia (DRG). To date, the following ligands have been identified for Mrgs: several neuropeptides, with an RFamide structure at their C termini, act as ligands for mouse MrgA1, MrgA4, MrgC11, and MAS1 (11Dong X. Han S. Zylka M.J. Simon M.I. Anderson D.J. Cell. 2001; 106: 619-632Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 12Han S.K. Dong X. Hwang J.I. Zylka M.J. Anderson D.J. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14740-14745Crossref PubMed Scopus (139) Google Scholar); proenkephalin A gene products, especially BAM22, that stimulate human MrgX1 (hSNSR4) and SNSR3 (13Lembo P.M. Grazzini E. Groblewski T. O'Donnell D. Roy M.O. Zhang J. Hoffert C. Cao J. Schmidt R. Pelletier M. Labarre M. Gosselin M. Fortin Y. Banville D. Shen S.H. Strom P. Payza K. Dray A. Walker P. Ahmad S. Nat. Neurosci. 2002; 5: 201-209Crossref PubMed Scopus (299) Google Scholar); adenine, which activates a member of the Mrg family found in rats (14Bender E. Buist A. Jurzak M. Langlois X. Baggerman G. Verhasselt P. Ercken M. Guo H.Q. Wintmolders C. Van den Wyngaert I. Van Oers I. Schoofs L. Luyten W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8573-8578Crossref PubMed Scopus (110) Google Scholar); and a recent report indicates that cortistatin-14 is a potent agonist for human MrgX2 (15Robas N. Mead E. Fidock M. J. Biol. Chem. 2003; 278: 44400-44404Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). However, no ligand has yet been identified for TGR7 (MrgD). Because TGR7 is co-expressed with major nociceptors, a purinergic receptor (P2X3) and a vanilloid receptor (VR1), in the sensory neurons of the DRG in rats, it has been thought to play some role in pain sensation or modulation (11Dong X. Han S. Zylka M.J. Simon M.I. Anderson D.J. Cell. 2001; 106: 619-632Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 16Zylka M.J. Dong X. Southwell A.L. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10043-10048Crossref PubMed Scopus (192) Google Scholar).β-Alanine is a small amino acid structurally related to the major inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Various studies (17DeFeudis F.V. Martin del Rio R. Gen. Pharmacol. 1977; 8: 177-180Crossref PubMed Scopus (59) Google Scholar, 18Usherwood P.N.R. Adv. Comp. Physiol. Biochem. 1978; 7: 227-309Crossref PubMed Google Scholar) indicate that β-alanine acts as a depressant in the central nervous system. It has been reported that β-alanine pharmacologically activates the glycine and GABA receptors with less efficacy than their native ligands (19Wu F.S. Gibbs T.T. Farb D.H. Eur. J. Pharmacol. 1993; 246: 239-246Crossref PubMed Scopus (64) Google Scholar, 20Rajendra S. Lynch J.W. Schofield P.R. Pharmacol. Ther. 1997; 73: 121-146Crossref PubMed Scopus (259) Google Scholar). It has also been reported that β-alanine decreases glutamatergic excitation by binding to the glycine co-agonist site on the N-methyl-d-aspartate receptor (21Ogita K. Suzuki T. Yoneda Y. Neuropharmacology. 1989; 11: 1263-1270Crossref Scopus (54) Google Scholar 22Pullan L. M. Powel R. J. Neurosci. Lett. 1992; 148: 199-201Crossref PubMed Scopus (31) Google Scholar). These dual effects of β-alanine to both decrease excitation and increase inhibition are very unique. The high affinity uptake of β-alanine has been detected in various types of the neurons (23Schon F. Kelly J.S. Brain Res. 1975; 86: 243-257Crossref PubMed Scopus (248) Google Scholar, 24Kontro P. Neuroscience. 1983; 8: 153-159Crossref PubMed Scopus (24) Google Scholar, 25Larsson O.M. Griffiths R. Allen I.C. Schousboe A. J. Neurochem. 1986; 47: 426-432Crossref PubMed Scopus (93) Google Scholar, 26Saransaari P. Oja S.S. Neuroscience. 1993; 53: 475-481Crossref PubMed Scopus (18) Google Scholar). In addition, a sodium-dependent transporter for β-alanine has been identified in the mouse brain (27Hosli E. Hosli L. Neuroscience. 1980; 5: 145-152Crossref PubMed Scopus (36) Google Scholar, 28Liu Q.R. Lopez-Corcuera B. Nelson H. Mandiyan S. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12145-12149Crossref PubMed Scopus (243) Google Scholar). There is therefore the possibility that β-alanine acts as a neurotransmitter and/or neuromodulator. However, no receptor specific for β-alanine has yet been identified.Here we report that an orphan GPCR, TGR7, which is highly expressed in the DRG, acts as a specific receptor for β-alanine. Our findings raise the possibility that β-alanine modulates pain sensation through TGR7.EXPERIMENTAL PROCEDURESCloning of TGR7 cDNAs—We designed primers (5′-GTCGACATGAACTACACTCCTTATAGCAGCCCAGCC-3′ and 5′-ACTAGTTCAGACCCCATCATTAGTACATGTGGATG-3′) to isolate an entire rat TGR7 cDNA by reverse transcriptase PCR. PCR was performed in a reaction mixture (25 μl in total) containing a 0.2 μm concentration of each primer, a template cDNA synthesized from rat cerebellum cDNA, 0.2 mm dNTPs, 1.25 units of Advantage 2 polymerase mix (Clontech), and 2.5 μl of a buffer provided by the manufacturer. The mixture was heated once at 95 °C for 30 s, 5 cycles at 94 °C for 5 s and at 70 °C for 5 min, 5 cycles at 94 °C for 5 s and at 68 °C for 5 min, 35 cycles at 94 °C for 5 s and at 65 °C for 5 min, and finally at 65 °C for 5 min for an extension reaction. By this means, we obtained an ∼1-kb product containing a full coding region and then determined its nucleotide sequence. In a similar manner, we isolated human, mouse, and cynomolgus monkey TGR7 DNA fragments by PCR from human liver cDNA, mouse genomic DNA, and cynomolgus monkey genomic DNA, respectively. To obtain these DNA fragments, we designed the following primer sets: 5′-GTCGACATGAACCAGACTTTGAATAGCAGT-3′and 5′-ACTAGTTCAAGCCCCCATCTCATTGGTGCC-3′ for human TGR7; 5′-GGAACACTCGAGCCACCATGAACTCCACTCTTGACAGCAGC-3′ and 5′-GGATCAGCTAGCTCAGACCCCATCATTAGTACACGTG-3′for mouse TGR7; and 5′-GCCTCCCGGTCATGGTCTGTTACAT-3′ and 5′-CGATGGCGACACCGTTTACTTGC-3′ for cynomolgus monkey TGR7.Preparation of Chinese Hamster Ovary (CHO) Cells Expressing TGR7—The entire coding region of human, rat, or mouse TGR7 cDNA was inserted downstream of the SRα promoter in the expression vector pAKKO-111H (29Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Crossref PubMed Scopus (36) Google Scholar). The resultant expression plasmids were transfected into dhfr- CHO cells, and transformed dhfr+ CHO cells expressing human, rat, or mouse TGR7 (CHO-hTGR7, CHO-rTGR7, and CHO-mTGR7 cells, respectively) were selected as described elsewhere (29Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Crossref PubMed Scopus (36) Google Scholar).Ca2+ Mobilization Assays—CHO cells expressing TGR7 and mock-transfected (i.e. the empty expression vector was transfected) CHO cells were seeded in black-walled clear-bottomed 96-well tissue culture plates (Costar) at 3 × 104 cells/well and cultured overnight. The cells were then incubated at 37 °C for 1 h in HEPES-buffered Hanks' balanced salt solution (pH 7.4) containing 2.5 mm probenecid and 4 μm fluo-3AM (Dojindo). Next, the cells were washed four times with the solution without fluo-3AM. Intracellular Ca2+ concentrations ([Ca2+]i) were measured with a fluorometric imaging plate reader system (Molecular Devices) both before and after samples were added.Guanosine 5′-O-3-Thiotriphosphate (GTPγS) Binding Assays—Membrane fractions prepared from CHO-hTGR7 and mock-transfected CHO cells as described elsewhere (29Hinuma S. Hosoya M. Ogi K. Tanaka H. Nagai Y. Onda H. Biochim. Biophys. Acta. 1994; 1219: 251-259Crossref PubMed Scopus (36) Google Scholar) were suspended at 500 μg/ml in a binding buffer (pH 7.4) containing 50 mm Tris, 150 mm NaCl, 5 mm MgCl2, 1 mm EGTA, 30 μm GDP, and 0.05% CHAPS. The membrane fractions (196 μl) were mixed with β-alanine (2 μl) and 200 nm [35S]GTPγS (Amersham Biosciences) (2 μl). After incubation at 25 °C for 60 min, the reaction mixtures were diluted with 2 ml of a chilled washing buffer, which was a modified binding buffer without GDP, and then filtered through GF/F filters (Whatman). The filters were washed with 2 ml of the washing buffer, dried, and subjected to a liquid scintillation counter to measure [35S]GTPγS bound to the membrane fractions.cAMP Production-inhibitory Assays—The inhibitory activities of β-alanine on cAMP production in CHO-hTGR7 cells in the presence of forskolin were examined according to our method described previously (30Fukusumi S. Habata Y. Yoshida H. Iijima N. Kawamata Y. Hosoya M. Fujii R. Hinuma S. Kitada C. Shintani Y. Suenaga M. Onda H. Nishimura O. Tanaka M. Ibata Y. Fujino M. Biochim. Biophys. Acta. 2001; 1540: 221-232Crossref PubMed Scopus (153) Google Scholar). For pretreatment with pertussis toxin (PTX), the cells were incubated with 100 ng/ml of PTX (Sigma) overnight before performing the cAMP production-inhibitory assays.Internalization of TGR7—An expression vector (pAKKO-hTGR7-GFP) with a fusion protein composed of human TGR7 and green fluorescent protein (hTGR7-GFP) was constructed by the insertion of a fused DNA (human TGR7- and GFP-coding regions were connected in tandem) into pAKKO-111H. CHO cells stably expressing hTGR7-GFP were seeded onto chambered coverglasses (Nalgene) and cultured overnight. After treatment with 0.3 mm β-alanine for 30 min, the fusion protein in the cells was observed under a confocal fluorescence microscope.Tritiated β-Alanine Binding Assay—β-[3H]Alanine (60 Ci/mmol) was purchased from Muromachi (ART-205, Tokyo, Japan). CHO cells expressing TGR7 and mock-transfected CHO cells were harvested with phosphate-buffered saline(-)-EDTA and suspended in a HEPES buffer. β-[3H]Alanine binding was performed in a final volume of 200 μl/tube consisting of 100 μl of cell suspension (0.6-10 × 105 cells/tube), 50 μl of β-[3H]alanine (1.5-150 nm), and 50 μl of a Hanks' balanced salt solution buffer with or without β-alanine (a final concentration of 1 mm). After incubation at 37 °C for 30 min, the cells were collected by centrifugation and washed three times with 3 ml of the HEPES buffer chilled in ice. The cells were then suspended in a lysis buffer (Hanks' balanced salt solution containing 1% SDS), and radioactivity retained in the cells was quantified with a liquid scintillation counter.Quantitative Reverse Transcriptase PCR Analyses for TGR7 mRNA—Poly(A)+ RNA fractions were prepared from the tissues of 8-12-week-old Wistar rats, and cDNAs were synthesized from them as described previously (31Fujii R. Fukusumi S. Hosoya M. Kawamata Y. Habata Y. Hinuma S. Sekiguchi M. Kitada C. Kurokawa T. Nishimura O. Onda H. Sumino Y. Fujino M. Regul. Pept. 1999; 31: 1-10Crossref Scopus (99) Google Scholar). Total RNA fractions were prepared from the DRG and spinal cord of a 3-year-old female cynomolgus monkey, and cDNAs were synthesized using SuperScript II reverse transcriptase (Invitrogen). Rat and monkey TGR7 mRNA expressions were determined with a Prism 7700 sequence detector (PE Biosystems) with primers and probes (5′-CTGTCGAGTTTCCACAGGTTCC-3′, 5′-TTGCGCAGAGGTACGGTTCC-3′, and 5′-5-carboxyfluorescein-ATCCACGCGACGTTCCGAGTCTCCA-5-carboxytetramethylrhodamine-3′ for rat TGR7; 5′-TGGCTGCCATGCTCAGC-3′, 5′-CTCGTGGACCTTGTCAGTGGT-3′, and 5′-5-carboxyfluorescein-TGGAAACCCAGCCCCTGGTCAG-5-carboxytetramethylrhodamine-3′ for monkey TGR7).In Situ Hybridization and Immunohistochemistry—We isolated DRG from 10-week-old male Wistar rats and an adult female cynomolgus monkey. The DRG were embedded in optimal cutting temperature (OCT) compound frozen in liquid nitrogen. Fresh frozen sections (6 μm) were attached to silanized slides and fixed with 4% paraformaldehyde. In situ hybridization was carried out using digoxygenin-labeled riboprobes prepared from full-length rat and monkey TGR7 cDNAs. To be visualized as purple, TGR7 mRNA was treated with alkaline phosphatase-conjugated anti-digoxygenin antibody, using 4-nitrotetrazolium blue chloride and 5-bromo-4-chloro-3-indolyl phosphate (which changes to red under a confocal laser microscope). For double staining in immunohistochemistry, we used anti-neurofilament 200 kDa (Chemicon), anti-calcitonin gene-related peptide (Affiniti Research Products), anti-substance P (American Research Products), anti-somatostatin (Biogenesis), anti-P2X3 (Chemicon), and anti-VRl (Chemicon) primary antibodies, and Alexa 488-labeled secondary antibodies (Molecular Probes). For the labeling of Griffonia simplicifolia isolectin B4 (IB4), the sections were incubated with fluorescein isothiocyanate-conjugated IB4 (Sigma). After processing, the sections were mounted and then examined by light and confocal laser microscopy.RESULTSDemonstration of β-Alanine as a Ligand for TGR7—We isolated TGR7 cDNAs from human, rat, mouse, and monkey. The proteins from these cDNAs encoded had amino acid lengths of 321, 319, 321, and 320, respectively, and showed 80% (human versus monkey), 58% (human versus rat), and 84% (rat versus mouse) amino acid identity. Although the TGR7 amino acid sequences were not highly conserved among the species, a phylogenetic analysis of all known GPCRs showed that they were the closest counterparts (data not shown). To search for ligands of TGR7, we transiently expressed human TGR7 cDNA in CHO cells and then examined changes in [Ca2+]i by adding various test samples. Through our screening of over 1500 compounds, we discovered that β-alanine induced the rapid mobilization of [Ca2+]i in CHO cells expressing human TGR7 in a dose-dependent manner, whereas β-alanine did not induce an increase of [Ca2+]i in mock-transfected CHO cells (Fig. 1A). To confirm this receptor-ligand relationship in other animal species, we prepared CHO cells stably expressing human, rat, or mouse TGR7 (i.e. CHO-hTGR7, CHO-rTGR7, and CHO-mTGR7 cells, respectively) and examined changes in [Ca2+]i in response to β-alanine. The EC50 values of β-alanine in CHO-hTGR7, CHO-rTGR7, and CHO-mTGR7 cells were 15, 14, and 44 μm, respectively (Table I). Slight stimulatory activity was detected for GABA in CHO-hTGR7 and CHO-rTGR7 cells. l-Carnosine was found to be active only in CHO-rTGR7 cells. Other compounds structurally related to β-alanine, that is l-glycine, l-alanine, taurine, and anserine, did not induce an evident increase of [Ca2+]i when examined in CHO-hTGR7 cells. We subsequently examined the effect of β-alanine on cAMP production in CHO cells expressing TGR7. As shown in Fig. 1B, β-alanine suppressed forskolin-induced cAMP production in CHO-hTGR7 cells; however, this decrease did not occur after pretreatment with PTX. Similar suppressive effects of β-alanine on cAMP production in CHO-rTGR7 and CHO-mTGR7 cells were also observed (data not shown). These results suggest that human TGR7 couples to Gq and Gi in CHO cells. To confirm further that the interaction of β-alanine and TGR7 occurred at the plasma membrane, we prepared membrane fractions from CHO-hTGR7 cells and examined [35S]GTPγS binding to these fractions (Fig. 1C). Significant levels of binding were detected with 3 μm of β-alanine, the EC50 value was at 25 μm, and maximum levels were reached with 100-300 μm. Such increases in [35S]GTPγS binding were not detected in the membrane fractions of mock-transfected CHO cells.Table IAgonistic potency to induce intracellular Ca2+ rise in CHO cells expressing TGR7LigandEC50 (μm)HumanRatMousel-GlycineInactiveInactiveInactiveβ-Alanine15 ± 114 ± 244 ± 6l-AlanineInactiveInactiveInactiveGABA191 ± 12165 ± 2>300l-CarnosineInactive34 ± 1>300AnserineInactive>300InactiveTaurineInactiveNDND Open table in a new tab To confirm that TGR7 functions as a cell surface receptor, we stably expressed a fusion protein (hTGR7-GFP) in CHO cells and then examined its subcellular localization. In the absence of a ligand, hTGR7-GFP was localized typically at the plasma membrane (Fig. 1D, left panel). However, in the presence of β-alanine, hTGR7-GFP was internalized into the cytoplasm (Fig. 1D, right panel). Treatment with l-alanine or l-glycine had no effect on hTGR7-GFP localization (data not shown). We examined the binding of β-[3H]alanine to intact CHO-hTGR7 cells. As shown in Fig. 1 (E and F), β-[3H]alanine more effectively bound to CHO-hTGR7 cells than to mock-transfected CHO cells in a manner dependent on the cell number and β-[3H]alanine concentration, indicating that β-alanine specifically binds to CHO-hTGR7 cells.Tissue Distribution of TGR7 mRNA in Rat and Monkey—We first analyzed the tissue distribution of TGR7 mRNA in rats. TGR7 mRNA was primarily expressed in the DRG (Fig. 2A). Moderate levels of expression were detected in the testes, urinary bladder, arteries, and uterus (Fig. 2A). To examine whether TGR7 mRNA was highly expressed not only in rodents but also in primates, we analyzed the expression of TGR7 and GABAB1a mRNAs in monkey DRG and the spinal cord. A high level of TGR7 mRNA was detected in the DRG, whereas only a low amount was found in the spinal cord (Fig. 2B, left panel). In contrast, a high level of GABAB1a mRNA was detected in both the DRG and spinal cord. These results suggest that the high level of TGR7 mRNA expression in the DRG is conserved between rodents and primates.Fig. 2Tissue distribution of TGR7 mRNA in the rat and monkey. Poly (A)+ RNA from rat tissues and total RNA from monkey tissues were subjected to quantitative reverse transcriptase PCR analyses using an ABI Prism 7700 sequence detector. A, rat; B, monkey. Each column represents a mean value in duplicate determinations.View Large Image Figure ViewerDownload (PPT)Expression of TGR7 mRNA in DRG—To compare the detailed expression of TGR7 mRNA in the neurons of the DRG between rodents and primates, we performed in situ hybridization using digoxygenin-labeled riboprobes. TGR7 mRNA signals were detected in small diameter DRG neurons in both the rat and monkey (Fig. 3), suggesting that TGR7 is expressed in C-fibers mediating pain or nociceptive responses in both rodents and primates.Fig. 3Expression of TGR7 in DRG neurons in the rat and monkey. In situ hybridization for TGR7 using digoxygenin-labeled cRNA probe showed that TGR7 was expressed in DRG small diameter neurons in a rodent and primate. A, rat TGR7 antisense probe; B, rat TGR7 sense probe; C, cynomolgus monkey TGR7 antisense probe; D, cynomolgus monkey TGR7 sense probe. Scale bar = 50 μm.View Large Image Figure ViewerDownload (PPT)To determine whether TGR7 co-localized with neuronal markers, we conducted double-staining in situ hybridization and immunostaining for various markers in rat and monkey DRG neurons. We employed seven kinds of neuronal markers (i.e. neurofilament 200 kDa, calcitonin gene-related peptide, substance P, IB4, somatostatin, P2X3, and VR1) to distinguish the subgroups of rat DRG neurons. Anti-neurofilament 200 kDa antibody recognizes high molecular weight neurofilaments and therefore is one of the markers for myelinated large diameter neurons. TGR7 mRNA was not detected in neurofilament 200 kDa immunoreactive neurons in the rat DRG (Fig. 4, A-C). TGR7 mRNA was also not expressed in calcitonin gene-related peptide-, somatostatin-, and substance P-immunoreactive neurons (images not shown). Most of the TGR7 mRNA-positive neurons were labeled with IB4, a marker of non-myelinated small diameter neurons in rat DRG (Fig. 4, D-F). These results suggest that TGR7 mRNA expresses in non-peptidergic nociceptive neurons. Likewise, most of the TGR7 mRNA-positive neurons were co-localized P2X3-immunoreactive and VR1-immunoreactive neurons in the rat DRG (Fig. 4, G-L). These results are highly consistent with those reported by other groups (11Dong X. Han S. Zylka M.J. Simon M.I. Anderson D.J. Cell. 2001; 106: 619-632Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 16Zylka M.J. Dong X. Southwell A.L. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10043-10048Crossref PubMed Scopus (192) Google Scholar).Fig. 4Expression pattern of TGR7 in the rat and monkey DRG neurons. Expression patterns of TGR7 mRNA were examined by double labeling of in situ hybridization with several neuronal makers. Co-localization between TGR7 and nociceptive neuronal makers was examined in the rat and monkey. The left column shows TGR7 mRNA in situ hybridization, the middle column shows immunostaining for neural markers or lectin staining with fluorescent IB4, and the right column shows merged images. A-C, rat TGR7 mRNA is not present in neurofilament 200 kDa-immunoreactive neurons. D-F, rat TGR7 and IB4 are co-labeled in small diameter neurons. G-I, rat TGR7 and P2X3 are co-expressed. J-L, rat TGR7 and VR1 are co-expressed. M-O, monkey TGR7 and IB4 are co-labeled. P-R, monkey TGR7 and P2X3 are co-expressed. S-U, monkey TGR7 and VR1 are co-expressed. Scale bar = 50 μm.View Large Image Figure ViewerDownload (PPT)To compare with the localization of TGR7 mRNA in rat DRG neurons, we also performed the double-staining hybridization of TGR7 and neuronal markers with monkey DRG neurons. We found that most TGR7 mRNA-positive neurons were also positive for IB4 (Fig. 4, M-O), P2X3 (Fig. 4, P-R), and VR1 (Fig. 4, S-U) indicating that the expression pattern of TGR7 is similar between rats and monkeys. Because P2X3 and VR1 are important molecules for mediating pain (32Deleted in proofGoogle Scholar, 33Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6943) Google Scholar), these data suggest that TGR7 plays a role in pain modulation both in rodents and primates.DISCUSSIONIn this paper, we have demonstrated that an orphan GPCR, TGR7, is specifically responsive to β-alanine. We found that β-alanine induced intracellular Ca2+ influx and suppressed cAMP production in CHO cells expressing TGR7. The responsiveness of TGR7 to β-alanine was detected in all species examined (i.e. human, rat, and mouse) even though the amino acid sequence of TGR7 among these species was not always highly conserved. We showed through [35
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