Kynurenic Acid as a Ligand for Orphan G Protein-coupled Receptor GPR35
Local catabolism of the essential amino acid tryptophan is considered an important mechanism in regulating immunological and neurological responses. The kynurenine pathway is the main route for the non-protein metabolism of tryptophan. The intermediates of the kynurenine pathway are present at micromolar concentrations in blood and are regulated by inflammatory stimuli. Here we show that GPR35, a previously orphan G protein-coupled receptor, functions as a receptor for the kynurenine pathway intermediate kynurenic acid. Kynurenic acid elicits calcium mobilization and inositol phosphate production in a GPR35-dependent manner in the presence of Gqi/o chimeric G proteins. Kynurenic acid stimulates [35S]guanosine 5′-O-(3-thiotriphosphate) binding in GPR35-expressing cells, an effect abolished by pertussis toxin treatment. Kynurenic acid also induces the internalization of GPR35. Expression analysis indicates that GPR35 is predominantly detected in immune cells and the gastrointestinal tract. Furthermore, we show that kynurenic acid inhibits lipopolysaccharide-induced tumor necrosis factor-α secretion in peripheral blood mononuclear cells. Our results suggest unexpected signaling functions for kynurenic acid through GPR35 activation. Local catabolism of the essential amino acid tryptophan is considered an important mechanism in regulating immunological and neurological responses. The kynurenine pathway is the main route for the non-protein metabolism of tryptophan. The intermediates of the kynurenine pathway are present at micromolar concentrations in blood and are regulated by inflammatory stimuli. Here we show that GPR35, a previously orphan G protein-coupled receptor, functions as a receptor for the kynurenine pathway intermediate kynurenic acid. Kynurenic acid elicits calcium mobilization and inositol phosphate production in a GPR35-dependent manner in the presence of Gqi/o chimeric G proteins. Kynurenic acid stimulates [35S]guanosine 5′-O-(3-thiotriphosphate) binding in GPR35-expressing cells, an effect abolished by pertussis toxin treatment. Kynurenic acid also induces the internalization of GPR35. Expression analysis indicates that GPR35 is predominantly detected in immune cells and the gastrointestinal tract. Furthermore, we show that kynurenic acid inhibits lipopolysaccharide-induced tumor necrosis factor-α secretion in peripheral blood mononuclear cells. Our results suggest unexpected signaling functions for kynurenic acid through GPR35 activation. G protein-coupled receptors (GPCRs) 3The abbreviations used are: GPCR, G protein-coupled receptor; GTPγS, guanosine 5′-O-(3-thiotriphosphate); [Ca2+]i, intracellular Ca2+ concentration; EC50, medium effective concentration; NMDA, N-methyl-d-aspartate; CHO, Chinese hamster ovary; LPS, lipopolysaccharides; TNFα, tumor necrosis factor α.3The abbreviations used are: GPCR, G protein-coupled receptor; GTPγS, guanosine 5′-O-(3-thiotriphosphate); [Ca2+]i, intracellular Ca2+ concentration; EC50, medium effective concentration; NMDA, N-methyl-d-aspartate; CHO, Chinese hamster ovary; LPS, lipopolysaccharides; TNFα, tumor necrosis factor α. constitute one of the largest gene families yet identified (1Fredriksson R. Schioth H.B. Mol. Pharmacol. 2005; 67: 1414-1425Crossref PubMed Scopus (454) Google Scholar, 2Fredriksson R. Lagerstrom M.C. Lundin L.G. Schioth H.B. Mol. Pharmacol. 2003; 63: 1256-1272Crossref PubMed Scopus (2086) Google Scholar). It has been estimated that half of all modern drugs target these receptors (3Flower D.R. Biochim. Biophys. Acta. 1999; 1422: 207-234Crossref PubMed Scopus (218) Google Scholar, 4Wise A. Gearing K. Rees S. Drug Discov. Today. 2002; 7: 235-246Crossref PubMed Scopus (329) Google Scholar). GPCRs contain seven transmembrane domains and are activated by a wide variety of ligands, including light, ions, metabolic intermediates, amino acids, nucleotides, lipids, peptides, and proteins. In addition to ∼250 characterized receptors, ∼120 human genes encode non-olfactory GPCRs whose ligands and function remain to be determined (1Fredriksson R. Schioth H.B. Mol. Pharmacol. 2005; 67: 1414-1425Crossref PubMed Scopus (454) Google Scholar). These orphan receptors are expected to play important roles in the regulation of a diversity of physiological functions. In the past decade an increasing number of GPCRs have been de-orphanized. Many of the identified ligands are metabolic intermediates, including succinate (ligand for GPR91) (5He W. Miao F.J. Lin D.C. Schwandner R.T. Wang Z. Gao J. Chen J.L. Tian H. Ling L. Nature. 2004; 429: 188-193Crossref PubMed Scopus (614) Google Scholar), α-ketoglutarate (ligand for GPR99) (5He W. Miao F.J. Lin D.C. Schwandner R.T. Wang Z. Gao J. Chen J.L. Tian H. Ling L. Nature. 2004; 429: 188-193Crossref PubMed Scopus (614) Google Scholar), fatty acids (ligands for GPR40/41/43/120) (6Briscoe C.P. Tadayyon M. Andrews J.L. Benson W.G. Chambers J.K. Eilert M.M. Ellis C. Elshourbagy N.A. Goetz A.S. Minnick D.T. Murdock P.R. Sauls Jr., H.R. Shabon U. Spinage L.D. Strum J.C. Szekeres P.G. Tan K.B. Way J.M. Ignar D.M. Wilson S. Muir A.I. J. Biol. Chem. 2003; 278: 11303-11311Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar, 7Brown A.J. Goldsworthy S.M. Barnes A.A. Eilert M.M. Tcheang L. Daniels D. Muir A.I. Wigglesworth M.J. Kinghorn I. Fraser N.J. Pike N.B. Strum J.C. Steplewski K.M. Murdock P.R. Holder J.C. Marshall F.H. Szekeres P.G. Wilson S. Ignar D.M. Foord S.M. Wise A. Dowell S.J. J. Biol. Chem. 2003; 278: 11312-11319Abstract Full Text Full Text PDF PubMed Scopus (1473) Google Scholar, 8Le Poul E. Loison C. Struyf S. Springael J.Y. Lannoy V. Decobecq M.E. Brezillon S. Dupriez V. Vassart G. Van Damme J. Parmentier M. Detheux M. J. Biol. Chem. 2003; 278: 25481-25489Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar, 9Itoh 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 (1213) Google Scholar, 10Hirasawa A. Tsumaya K. Awaji T. Katsuma S. Adachi T. Yamada M. Sugimoto Y. Miyazaki S. Tsujimoto G. Nat. Med. 2005; 11: 90-94Crossref PubMed Scopus (1115) Google Scholar), ketone body (ligand for HM74a) (11Taggart A.K. Kero J. Gan X. Cai T.Q. Cheng K. Ippolito M. Ren N. Kaplan R. Wu K. Wu T.J. Jin L. Liaw C. Chen R. Richman J. Connolly D. Offermanns S. Wright S.D. Waters M.G. J. Biol. Chem. 2005; 280: 26649-26652Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar), and bile acids (ligand for BG37) (12Maruyama T. Miyamoto Y. Nakamura T. Tamai Y. Okada H. Sugiyama E. Nakamura T. Itadani H. Tanaka K. Biochem. Biophys. Res. Commun. 2002; 298: 714-719Crossref PubMed Scopus (688) Google Scholar). We have built a library of ∼300 biochemical intermediates to test their ability to activate orphan GPCRs. We identified kynurenic acid, one of the first metabolites of tryptophan isolated and characterized in mammals (13Ellinger A. Hoppe-Seyler's Z. Physiol. Chem. 1904; 43: 325-337Crossref Scopus (18) Google Scholar, 14Homer A. J. Biol. Chem. 1914; 17: 509-518Abstract Full Text PDF Google Scholar), as a ligand for GPR35. Cloned as an orphan GPCR in 1998 (15O'Dowd B.F. Nguyen T. Marchese A. Cheng R. Lynch K.R. Heng H.H. Kolakowski Jr., L.F. George S.R. Genomics. 1998; 47: 310-313Crossref PubMed Scopus (248) Google Scholar), GPR35 shares 30% amino acid identity with GPR55. Expression analysis revealed prominent expression of GPR35 in immune and gastrointestinal tissues, suggesting potential physiological roles for GPR35 in these organs. Tryptophan metabolites such as serotonin and melatonin are well known ligands for GPCRs. Kynurenic acid has been reported to play important physiological roles in the brain (16Stone T.W. Darlington L.G. Nat. Rev. Drug Discov. 2002; 1: 609-620Crossref PubMed Scopus (617) Google Scholar, 17Schwarcz R. Curr. Opin. Pharmacol. 2004; 4: 12-17Crossref PubMed Scopus (213) Google Scholar). Most biological effects associated with kynurenic acid, such as neuroprotective activities, have been attributed to its antagonism on N-methyl-d-aspartate (NMDA) receptor (16Stone T.W. Darlington L.G. Nat. Rev. Drug Discov. 2002; 1: 609-620Crossref PubMed Scopus (617) Google Scholar, 18Stone T.W. Mackay G.M. Forrest C.M. Clark C.J. Darlington L.G. Clin. Chem. Lab. Med. 2003; 41: 852-859Crossref PubMed Scopus (130) Google Scholar). Here we have identified a novel mechanism by which kynurenic acid may regulate peripheral cellular responses through activation of GPR35. Cloning and Cell Culture—Full-length human, mouse, and rat GPR35 were cloned by PCR from human universal cDNA, mouse spleen cDNA, and rat spleen cDNA (BD Bioscience Clontech), respectively. Sequence-confirmed cDNAs were inserted into the mammalian expression vector pcDNA3.1 (Invitrogen). Chinese hamster ovary (CHO) cells were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (Cellgro) containing 10% fetal bovine serum and antibiotics. HeLa and HEK293 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. All cell lines were cultured at 37 °C with 5% CO2. CHO-GPR35 stable cells were generated by transfecting CHO cells with N-terminal-FLAG-tagged human GPR35 and subsequently selected in 1 mg/ml G418 (Invitrogen). Flow cytometry analysis was carried out on FACSCalibur (BD Biosciences) after staining with anti-FLAG M2 monoclonal antibody (Sigma) and goat anti-mouse IgG-fluorescein isothiocyanate secondary antibody (Caltag). All compounds tested were from Sigma. Aequorin Assay—CHO cells were transfected with either empty vector or vector expressing GPR35 together with the aequorin reporter plasmid using Lipofectamine 2000 reagent (Invitrogen) (5He W. Miao F.J. Lin D.C. Schwandner R.T. Wang Z. Gao J. Chen J.L. Tian H. Ling L. Nature. 2004; 429: 188-193Crossref PubMed Scopus (614) Google Scholar, 19Stables J. Green A. Marshall F. Fraser N. Knight E. Sautel M. Milligan G. Lee M. Rees S. Anal. Biochem. 1997; 252: 115-126Crossref PubMed Scopus (179) Google Scholar). For each 10-cm dish, 5 μg of GPR35 and 5 μg of aequorin reporter plasmids were used. When indicated, 2 μg of plasmids expressing small G proteins (Gα16, Gqo5, Gqi9, and/or Gqs5) (20Amatruda T.T. II I Steele D.A. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5587-5591Crossref PubMed Scopus (238) Google Scholar, 21Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (599) Google Scholar, 22Coward P. Chan S.D. Wada H.G. Humphries G.M. Conklin B.R. Anal. Biochem. 1999; 270: 242-248Crossref PubMed Scopus (202) Google Scholar, 23Milligan G. Marshall F. Rees S. Trends Pharmacol. Sci. 1996; 17: 235-237Abstract Full Text PDF PubMed Scopus (107) Google Scholar) were also included. 24 h after transfection cells were harvested and resuspended in Hanks' buffered salt solution containing 0.01% bovine serum albumin and 20 mm HEPES (Cellgro), loaded with 1 μg/ml coelenterazine f (P. J. K. Industrievertetungen, Handel, Germany) at room temperature for 1 h, and stimulated with compounds. Ligand-induced calcium mobilization, as indicated by an increase in aequorin luminescence, was recorded over a period of 20 s with a Microlumat luminometer (Berthold). Inositol Phosphate Accumulation Assay—HEK293 cells seeded in 96-well plates were transfected with GPR35 (100 ng/well) and small G proteins (Gα16 or Gqo5, 20 ng/well). After labeling with [3H]myoinositol (Amersham Biosciences) for 16 h, cells were stimulated with compounds in Hanks' buffered salt solution, 25 mm Hepes (pH 7.4), 10 mm LiCl, 0.01% bovine serum albumin at 37 °C for 1 h. 20 mm acid was used to the cells at °C for h. (Amersham Biosciences) were to the cell and in the was recorded on a stable cells were with or pertussis toxin for 16 h were resuspended and in 10 mm (pH 7.4), 1 mm by at for 10 at °C to and cellular were by the at for and resuspended in 20 mm HEPES (pH and 5 mm 25 μg of was at room temperature for 1 h in mm 5 mm bovine serum albumin (pH containing and in the or presence of kynurenic acid. were by through and the were on cells were seeded on in plates and transfected with N-terminal-FLAG-tagged human, mouse, or rat GPR35. For cells were with with 5% goat serum in (Cellgro), and with anti-FLAG monoclonal antibody (Sigma) for 1 h on After in cells were with goat anti-mouse secondary antibody for an of GPR35 was by kynurenic acid for at 37 After ligand cells were with and with staining with were with a to a from human or mouse (BD were with I was on an using PCR of GPR35 to were using a and were using and for human GPR35 were and and for the and for mouse GPR35 were and and for the In GPR35 cDNA cloned in vector (Invitrogen) was used as a for using and or GPR35 were to mouse The mm 20 mm (pH 10 mm 10% solution, mg/ml as T. 2003; 17: PubMed Scopus Google Scholar). After were to in and after were with for peripheral blood mononuclear cells and were from blood mononuclear cells were seeded at a of 2 and were seeded at 2 in plates in Kynurenic acid was 1 h at 10 were at 37 °C for h, and was for cells were used as concentrations were determined with the for ligands for orphan we tested a of ∼300 biochemical intermediates for their ability to an increase in intracellular Ca2+ using the aequorin J. Green A. Marshall F. Fraser N. Knight E. Sautel M. Milligan G. Lee M. Rees S. Anal. Biochem. 1997; 252: 115-126Crossref PubMed Scopus (179) Google Scholar). CHO cells were transfected with plasmids human GPR35, aequorin and a mixture of or chimeric small G proteins (Gα16, Gqo5, and which have been reported to with GPCRs that are to Ca2+ signaling and their to calcium mobilization B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (599) Google Scholar, 22Coward P. Chan S.D. Wada H.G. Humphries G.M. Conklin B.R. Anal. Biochem. 1999; 270: 242-248Crossref PubMed Scopus (202) Google Scholar, 23Milligan G. Marshall F. Rees S. Trends Pharmacol. Sci. 1996; 17: 235-237Abstract Full Text PDF PubMed Scopus (107) Google Scholar). Kynurenic acid a in in cells expressing human GPR35 and G with a medium effective was in cells Kynurenic acid in in the tryptophan metabolic pathway Kynurenic acid also activated mouse and rat of GPR35 acid, of the tryptophan pathway and indicated to have functions to kynurenic acid (16Stone T.W. Darlington L.G. Nat. Rev. Drug Discov. 2002; 1: 609-620Crossref PubMed Scopus (617) Google Scholar), kynurenic acid is on GPR35 on human GPR35, with of and for mouse and rat GPR35, respectively. GPR35 was activated by kynurenic acid by tryptophan metabolic pathway intermediates such as acid and that are in cell regulation M. S. P. H. R. Lee L. 2005; PubMed Scopus Google Scholar). ∼300 biochemical intermediates, kynurenic acid was to be the in GPR35 Furthermore, kynurenic acid activate including the of GPR35 metabolites in aequorin indicates the of a that of the and is from were generated from CHO cells transfected with GPR35, and chimeric G plasmids in aequorin at in a the signaling of GPR35, CHO cells were transfected with plasmids human GPR35 and small G proteins and tested in aequorin The Gqi/o P. Chan S.D. Wada H.G. Humphries G.M. Conklin B.R. Anal. Biochem. 1999; 270: 242-248Crossref PubMed Scopus (202) Google Scholar), and Gqi9, the activation of GPR35 by kynurenic acid, and the G The of Gqi/o was reported to the GPCRs to the to calcium mobilization B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (599) Google Scholar). These results suggest that GPR35 may through in CHO cells. Kynurenic acid the of inositol phosphate in HEK293 cells transfected with GPR35 and inositol phosphate was detected in the of G suggesting that GPR35 may through the pathway These results with the that the Gqi/o GPR35 activation in the aequorin Flow cytometry analysis expression of GPR35 on CHO cells expressing N-terminal-FLAG-tagged human GPR35 vector cells Kynurenic acid stimulated in from CHO-GPR35 cells, an effect abolished by with pertussis toxin cells to kynurenic acid The for kynurenic activation of human GPR35 in binding was to the from the aequorin These together with the for Gqi/o in the aequorin and inositol phosphate suggest that GPR35 activation by kynurenic acid to a pertussis Ligand-induced receptor internalization is of GPCR activation and M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). staining of cells expressing N-terminal-FLAG-tagged human, mouse, or rat GPR35 revealed that GPR35 proteins from were to the In a Gao X. Z. M. 1997; 278: PubMed Scopus Google Scholar) an expected intracellular Kynurenic acid the of GPR35 from to intracellular a of receptor Expression analysis by PCR revealed that human GPR35 and mouse GPR35 were predominantly in immune and gastrointestinal tissues, with expression in In GPR35 was detected in the peripheral small and In of GPR35 expression were detected in the spleen and gastrointestinal results were using and to of GPR35 of immune cells, GPR35 was detected in cells, and cells, with expression in cells, and In using mouse with were detected in the spleen and gastrointestinal including and GPR35 in these In of the in in and in GPR35 was in the cells in the of with expression in the were detected in and the potential biological functions of kynurenic acid on immune cells expressing GPR35, we tested the effect of kynurenic acid on secretion in human peripheral blood mononuclear cells. Kynurenic acid by secretion in these cells kynurenic acid was to secretion in a manner results were using peripheral blood In the we have identified kynurenic acid, an intermediate in the tryptophan metabolic as a ligand for GPR35. Kynurenic acid activated GPR35 in aequorin and inositol phosphate in the presence of Gqi/o chimeric G proteins. Kynurenic acid also stimulated binding in a GPR35-dependent manner and the internalization of GPR35. The of kynurenic acid as an ligand for GPR35 the of tryptophan catabolism in regulating biological functions. The tryptophan metabolic pathway to the of kynurenine is the main route for non-protein metabolism of the essential amino acid tryptophan. the tryptophan that is into is by the kynurenine pathway (16Stone T.W. Darlington L.G. Nat. Rev. Drug Discov. 2002; 1: 609-620Crossref PubMed Scopus (617) Google Scholar). its roles in and the the kynurenine pathway has as an target for (16Stone T.W. Darlington L.G. Nat. Rev. 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Drug Discov. 2002; 1: 609-620Crossref PubMed Scopus (617) Google Scholar). It is that kynurenic acid by activated immune cells the of gastrointestinal cells expressing GPR35, to in these we have identified the tryptophan kynurenic acid as an ligand for GPR35. together with (5He W. Miao F.J. Lin D.C. Schwandner R.T. Wang Z. Gao J. Chen J.L. Tian H. Ling L. Nature. 2004; 429: 188-193Crossref PubMed Scopus (614) Google Scholar, C.P. Tadayyon M. Andrews J.L. Benson W.G. Chambers J.K. Eilert M.M. Ellis C. Elshourbagy N.A. Goetz A.S. Minnick D.T. Murdock P.R. Sauls Jr., H.R. Shabon U. Spinage L.D. Strum J.C. Szekeres P.G. Tan K.B. Way J.M. Ignar D.M. Wilson S. Muir A.I. J. Biol. Chem. 2003; 278: 11303-11311Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar, 7Brown A.J. Goldsworthy S.M. Barnes A.A. Eilert M.M. Tcheang L. Daniels D. Muir A.I. Wigglesworth M.J. Kinghorn I. Fraser N.J. Pike N.B. Strum J.C. Steplewski K.M. Murdock P.R. Holder J.C. Marshall F.H. Szekeres P.G. Wilson S. Ignar D.M. Foord S.M. Wise A. Dowell S.J. J. Biol. Chem. 2003; 278: 11312-11319Abstract Full Text Full Text PDF PubMed Scopus (1473) Google Scholar, 8Le Poul E. Loison C. Struyf S. Springael J.Y. Lannoy V. Decobecq M.E. Brezillon S. Dupriez V. Vassart G. Van Damme J. Parmentier M. Detheux M. J. Biol. Chem. 2003; 278: 25481-25489Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar, 9Itoh 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 (1213) Google Scholar, 10Hirasawa A. Tsumaya K. Awaji T. Katsuma S. Adachi T. Yamada M. Sugimoto Y. Miyazaki S. Tsujimoto G. Nat. Med. 2005; 11: 90-94Crossref PubMed Scopus (1115) Google Scholar) that metabolic intermediates previously to be The signaling functions of these as a for The identified receptors for these metabolic intermediates present novel for and We and for and We also for of the