Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins

GPR20 was isolated as an orphan G protein-coupled receptor from genomic DNA by PCR amplification. Although GPR20 was closely related to nucleotide or lipid receptors, the functional role of this receptor, as well as its endogenous ligand, remains unclear. Here we demonstrate that GPR20 is constitutively active in the absence of ligand, leading to continuous activation of its coupled G proteins. When GPR20 was exogenously expressed in HEK293 cells, both the basal level and the prostaglandin E2-induced production of cAMP were significantly decreased. A remarkable increase in [35S]guanosine 5′-(γ-thio)triphosphate (GTPγS) binding to membrane preparations was also observed in GPR20-expressing cells. These effects of GPR20 overexpression were diminished in cells treated with pertussis toxin, suggesting that the expression of GPR20 results in the activation of Gi/o proteins. Involvement of GPR20 in the activation of Gi/o proteins was also supported by evidence that the disruption of a conserved DRY motif in GPR20 attenuated both [35S]GTPγS incorporation and inhibition of the prostaglandin E2-induced cAMP production. Knockdown of GPR20 in PC12h cells resulted in an elevation of the basal cAMP level, suggesting that the endogenous GPR20 achieves a constitutively or spontaneously active conformation. Furthermore, enhancement of [3H]thymidine incorporation was also observed in the GPR20-silencing cells, implying that the GPR20 expression seems to attenuate PC12h cell growth. Taken together, these data indicate that GPR20 constitutively activates Gi proteins without ligand stimulation. The receptor may be involved in cellular processes, including control of intracellular cAMP levels and mitogenic signaling. GPR20 was isolated as an orphan G protein-coupled receptor from genomic DNA by PCR amplification. Although GPR20 was closely related to nucleotide or lipid receptors, the functional role of this receptor, as well as its endogenous ligand, remains unclear. Here we demonstrate that GPR20 is constitutively active in the absence of ligand, leading to continuous activation of its coupled G proteins. When GPR20 was exogenously expressed in HEK293 cells, both the basal level and the prostaglandin E2-induced production of cAMP were significantly decreased. A remarkable increase in [35S]guanosine 5′-(γ-thio)triphosphate (GTPγS) binding to membrane preparations was also observed in GPR20-expressing cells. These effects of GPR20 overexpression were diminished in cells treated with pertussis toxin, suggesting that the expression of GPR20 results in the activation of Gi/o proteins. Involvement of GPR20 in the activation of Gi/o proteins was also supported by evidence that the disruption of a conserved DRY motif in GPR20 attenuated both [35S]GTPγS incorporation and inhibition of the prostaglandin E2-induced cAMP production. Knockdown of GPR20 in PC12h cells resulted in an elevation of the basal cAMP level, suggesting that the endogenous GPR20 achieves a constitutively or spontaneously active conformation. Furthermore, enhancement of [3H]thymidine incorporation was also observed in the GPR20-silencing cells, implying that the GPR20 expression seems to attenuate PC12h cell growth. Taken together, these data indicate that GPR20 constitutively activates Gi proteins without ligand stimulation. The receptor may be involved in cellular processes, including control of intracellular cAMP levels and mitogenic signaling. Mammalian G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCR, G protein-coupled receptor; GTPγS, guanosine 5′-(γ-thio) triphosphate; BSA, bovine serum albumin; PGE2, prostaglandin E2; PTX, pertussis toxin; BLT1, leukotriene B4 type 1 receptor; qRT, quantitative reverse-transcription; shRNA, short hairpin RNA; LPA, lysophosphatidic acid; ERKs, extracellular signal-regulated kinases; EIA, enzyme immunoassay; ANOVA, analysis of variance; h, human; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein. are a diverse superfamily of proteins with hundreds of members, and these receptors regulate many processes in vivo via interactions with a variety of ligands, including small organic molecules, lipids, protons, hormones, short and large polypeptides, glycoproteins, and even photons. Despite the vast and longstanding efforts of academic and industrial researchers to pair GPCRs with potential ligands, more than 150 nonsensory GPCRs still remain orphan receptors, for which the cognate ligands have not yet been identified (1Civelli O. Trends Pharmacol. Sci. 2005; 26: 15-19Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Because GPCRs have proven particularly amenable to modulation by small molecules and are the targets of approximately half of currently marketed prescription drugs, the nonsensory GPCRs are clearly important therapeutic targets (2Drews J. Science. 2000; 287: 1960-1964Crossref PubMed Scopus (2307) Google Scholar). Consequently, the orphan GPCRs constitute a vast reservoir of potential drug targets for therapeutic development. Indeed, numerous research groups, including ours, work on characterizing these receptors. GPR20 is one of the orphan GPCRs that has been identified from human genomic DNA by PCR amplification using primers based on the sequences of the opioid/somatostatin-related receptors, GPR7 and GPR8 (3Odowd B.F. Nguyen T. Jung B.P. Marchese A. Cheng R. Heng H.H.Q. Kolakowski L.F. Lynch K.R. George S.R. Gene (Amst.). 1997; 187: 75-81Crossref PubMed Scopus (38) Google Scholar). The expression of human GPR20 has been detected in several brain regions, including the caudate nuclei, putamen, and the thalamus (3Odowd B.F. Nguyen T. Jung B.P. Marchese A. Cheng R. Heng H.H.Q. Kolakowski L.F. Lynch K.R. George S.R. Gene (Amst.). 1997; 187: 75-81Crossref PubMed Scopus (38) Google Scholar). A recently disclosed patent demonstrated that GPR20-deficient mice exhibited a hyperactivity disorder characterized by an increase in total distance traveled in an open field test (4Brennan, T. J., Matthews, W., and Moore, M. (January 23, 2003) U. S. Patent 0018989Google Scholar), implying a substantial role of GPR20 in neurophysiological function. However, the physiological mechanisms of GPR20 action, including the identification of natural ligands for GPR20, have not yet been elucidated. In this study, we show that GPR20 has the potential to constitutively activate Gi-type G proteins without extracellular ligands. Furthermore, the physiological relevance of this constitutive activation of GPR20 was confirmed by RNA interference experiments and mitogenic response assays. Materials—Prostaglandin E2 (PGE2) was purchased from Cayman Chemical (Ann Arbor, MI). 3-Isobutyl-1-methylxanthine, GDP, GTPγS, bovine serum albumin (BSA, fatty acidfree grade), and the M5 mouse monoclonal anti-FLAG antibody were purchased from Sigma. Pertussis toxin (PTX) was obtained from List Biological Laboratories (Campbell, CA). Ex-TaqDNA polymerase and restriction enzymes were purchased from Takara Bio (Tokyo, Japan). [35S]GTPγS and the AlphaScreen cAMP assay kit were obtained from PerkinElmer Life Sciences. Phycoerythrin-labeled anti-mouse IgG was obtained from Beckman Coulter (Miami, FL). Alexa Fluor 488-labeled anti-mouse IgG, Lipofectamine 2000, Opti-MEM I, and SuperScriptII reverse transcriptase were purchased from Invitrogen. G418 was purchased from Wako (Osaka, Japan). KOD Dash DNA polymerase and KOD Plus DNA polymerase were purchased from TOYOBO (Tokyo, Japan). Complete protease inhibitor mixture was purchased from Roche Diagnostics. The cAMP Biotrak EIA system was purchased from GE Healthcare. Construction of a Phylogenetic Tree—Amino acid sequences of selected human GPCRs were obtained from GenBank™ and SwissProt. The phylogenetic tree was generated from the amino acid sequences of these GPCRs using the all-against-all matching method (available on line). The tree was constructed on the basis of point-accepted mutation distances between each pair of sequences estimated by the dynamic programming algorithm. Construction of the Human GPR20 and BLT1-expressing Plasmids—A DNA fragment containing the human GPR20 (hGPR20) gene (GenBank™ accession number NM_005293) was obtained from human genomic DNA by PCR amplification using the sense primer, 5′-ATGGAGAAGGGGGATGCTGGGC-3′, and the antisense primer, 5′-TTCAGGCCACCACATCCCATCG-3′. For construction of the hGPR20 expression plasmid, the entire open reading frame of hGPR20 was amplified from the above DNA fragment by PCR using the sense primer, 5′-CAGGATATCCCCTCTGTGTCTCCAGCGGG-3′, and the antisense primer, 5′-CAGGAATTCCTAAGCCTCGGGCCCATTAG-3′, and subcloned into the EcoRV and EcoRI sites of the pCXN2.1-FLAG vector, a modified version of pCXN2 (5Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4666) Google Scholar). The resultant plasmid, designated pCXN2.1-FLAG-GPR20, was used to express an N-terminally FLAG-tagged hGPR20. For construction of the expression plasmid for human leukotriene B4 type 1 receptor (hBLT1, GenBank™ accession number NM_181657), an entire open reading frame of hBLT1 was subcloned into the pCXN2.1-FLAG vector. Construction of Short Hairpin RNA-expressing Plasmids–Oligonucleotides for the construction of three short hairpin RNA (shRNA) plasmids directed against rat GPR20 (rGPR20) mRNA were designed using the siDirect small interfering RNA design system provided by the RNAi Co. Ltd. (Tokyo, Japan). The designed sequences are as follows: sh1 sense, 5′-GATCCCCCATCGTCTACTGTTTTATTCAAGAGATAAAACAGTAGACGATGGGGTA-3′, and sh1-antisense, 5′-AGCTTACCCCATCGTCTACTGTTTTATCTCTTGAATAAAACAGTAGACGATGGGG-3′; sh2 sense, 5′-GATCCGACCCTGTCTGTATTGGGTTTCAAGAGAACCCAATACAGACAGGGTCACA-3′, and sh2 antisense, 5′-AGCTTGTGACCCTGTCTGTATTGGGTTCTCTTGAAACCCAATACAGACAGGGTCG-3′; and sh3 sense, 5′-GATCCCTCAGCACCGGTTCTCACATTATTCAAGAGATGTGAGAACCGGTGCTGAGGAA-3′, and sh3 antisense, 5′-AGCTTTCCTCAGCACCGGTTCTCACATCTCTTGAATAATGTGAGAACCGGTGCTGAGG-3′. After annealing the sense and antisense oligonucleotides, the resultant DNA fragments were subcloned into the BamHI and HindIII sites of the pSilencer 4.1-CMV hygro vector (purchased from Ambion (Austin, TX), abbreviated pSilencer), giving rise to three rGPR20-shRNA plasmids, pSilencer-sh1, pSilencer-sh2, and pSilencer-sh3. We used a negative control-shRNA plasmid supplied by Ambion as a control scrambled shRNA. Construction of a Mutant hGPR20 (R148A) Plasmid—Mutagenesis of GPR20 was performed via site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions, with the exception that the KOD Plus DNA polymerase (TOYOBO) was used for PCR amplification. pCXN2.1-FLAG-GPR20 was used as a template plasmid, and the primers for mutagenesis were as follows: R148A sense, 5′-CTGCATCTGCGTGGACgccTACCTGGCCATCGTG-3′, and R148A antisense, 5′-CACGATGGCCAGGTAggcGTCCACGCAGATGCAG-3′. The resultant construct was verified through complete DNA sequencing of both DNA strands. Generation of PTX-resistant Gi-GFP Fusion Proteins—Expression plasmids for GFP-fused PTX-resistant Gi1, Gi2, and Gi3 proteins (6Hunt T. Carroll R. Peralta E. J. Biol. Chem. 1994; 269: 29565-29570Abstract Full Text PDF PubMed Google S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google A. M. S. M. J. 1997; PubMed Scopus Google M. M. H. J. Pharmacol. PubMed Scopus Google T. S. E. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google were constructed as The of the and proteins and the in the Gi3 were with a by PCR amplification. an EcoRI was between and the of each Gi by site-directed A the and the M. M. H. J. Pharmacol. PubMed Scopus Google Scholar), was into the EcoRI in each Gi protein. and cells were in in modified with bovine serum PC12h cells were in modified with serum and bovine were with expression plasmids using Lipofectamine in Opti-MEM to the manufacturer's of cells were with pCXN2.1-FLAG-GPR20 and for in the of These cells were with anti-FLAG in containing serum for by with Alexa Fluor 488-labeled anti-mouse IgG in serum for designated cells, were as a by cell using an and in the of of the cells were performed using an cAMP and PC12h cells were on or for In cells were treated with for were with A containing and and in of A containing a in for of in A were and cells were for The were by the of by After for the cAMP in the were using an AlphaScreen cAMP assay kit Life to the manufacturer's For of the basal cAMP in cells, a cAMP Biotrak EIA system was were as with the exception of the of of supplied in the EIA kit to the cells were with containing In cells were with for The cells were by in containing a complete protease inhibitor mixture and for The were and for 1 The membrane were in and to [35S]GTPγS binding The in each were by the method M. PubMed Scopus Google using the assay kit [35S]GTPγS preparations of were in of [35S]GTPγS binding 1 1 GDP, and containing [35S]GTPγS for was to the binding mixture to a of [35S]GTPγS was from [35S]GTPγS by through and with of and was using a from human were purchased from and the expression of hGPR20 in human the of the GPR20 in each was using a gene expression assay assay and the modified PCR containing The of was estimated using a and the method in the analysis For each a was using a of from the total were from using an RNA kit and were using PCR were in in of of DNA and each of the and the reverse A fragment of the mouse GPR20 was generated using following primer, and reverse primer, of GPR20 in RNA was isolated from cell using by of using a kit was generated from of the mRNA using and A fragment of a fragment of and a fragment of were generated by The primers used in these were as follows: hGPR20 sense primer, and antisense primer, and sense primer, and antisense primer, The primers for were the as The sequences for were as follows: sense primer, and antisense primer, sense primer, and antisense primer, and sense primer, and antisense primer, of RNA the of the by pSilencer-sh1, pSilencer-sh2, or quantitative was of these plasmids into PC12h cells, mRNA and were performed as the resultant the of expressed and were estimated using DNA to the manufacturer's In these the primers for were the as For each a was using a of of the cells were on for well was with 1 of in the of a the of the cells were using and [3H]thymidine was from [3H]thymidine by through that were with of The was with a of [3H]thymidine into PC12h DNA was expressed as of the hGPR20 human GPR20 from human genomic DNA by PCR amplification. However, several sequences in hGPR20 were from the GPR20 (3Odowd B.F. Nguyen T. Jung B.P. Marchese A. Cheng R. Heng H.H.Q. Kolakowski L.F. Lynch K.R. George S.R. Gene (Amst.). 1997; 187: 75-81Crossref PubMed Scopus (38) Google Scholar). In and in the GPR20 were and in We used hGPR20 in this study, we verified the sequences of several isolated hGPR20 and the was recently in the human total data number The of hGPR20 exhibited and amino acid to mouse and rat GPR20, Phylogenetic analysis that hGPR20 was closely related to functional receptors for or and receptors, the that GPR20 may or as ligands. and of GPR20 from human was performed to the expression of hGPR20. In these hGPR20 was with the expression in small mouse GPR20 mRNA was detected in a of with expression in as small and GPR20 mRNA was detected in cell including and PC12h and expressed levels of the GPR20 mRNA of these cell was used for the experiments of cAMP by the of GPR20 to a of receptors, we a ligand using the (purchased from and this we constructed an expression plasmid an N-terminally FLAG-tagged hGPR20 and a HEK293 cell to expression of on the cell of cells was by analysis Although we the of and using cells by intracellular and cAMP of was an for hGPR20 not Furthermore, not to in the extracellular not even GPR20 is closely related to the and M. M. J. U. H. R. K. PubMed Scopus Google T. T. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. J. M. A. K. M. E. T. A. K. H. T. H. T. A. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google S. T. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), in the phylogenetic we not activation of GPR20 by several hormones, including and not the of these we detected a in basal cAMP level in cells, with that of cells we observed that the expression resulted in attenuated cAMP in with cells effects were observed in HEK293 cells implying that these were not an of the cell Because the above effects by expression were by with and expression of hGPR20 may the activation of G proteins in the absence of ligand stimulation. [35S]GTPγS to from we Gi-type G proteins are in the of hGPR20. membrane preparations from HEK293 cells with of the expression plasmid, we of by [35S]GTPγS binding Because exogenously expressed GPCRs activate G in the absence of ligands, we the of hGPR20 with which is well as a receptor T. T. K. T. 1997; PubMed Scopus Google K. T. K. M. T. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in of hGPR20 [35S]GTPγS incorporation into the in a even hBLT1 not the [35S]GTPγS incorporation these The of [35S]GTPγS binding was diminished by which is with the of cAMP in cells evidence for the activation of Gi proteins by we [35S]GTPγS incorporation into exogenously expressed PTX-resistant of the Gi Gi1, Gi2, and (6Hunt T. Carroll R. Peralta E. J. Biol. Chem. 1994; 269: 29565-29570Abstract Full Text PDF PubMed Google S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google A. M. S. M. J. 1997; PubMed Scopus Google M. M. H. J. Pharmacol. PubMed Scopus Google T. S. E. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in cells. in even in cells with PTX, [35S]GTPγS binding to the PTX-resistant of Gi proteins was observed in the cells. These results that hGPR20 activates Gi-type G proteins. Involvement of the DRY of GPR20 in of Gi have the of a conserved of amino acid in the intracellular of in G activation J. S. DNA Biol. PubMed Scopus Google T. J. PubMed Scopus Google Scholar). is designated the DRY more evidence for the constitutive activation of Gi proteins by GPR20, we the disruption of the DRY motif in hGPR20 Gi We constructed a the R148A receptor, in which is by The R148A receptor was expressed on the cell levels to the as by analysis in [35S]GTPγS incorporation into membrane preparations from R148A cells was with from cells the the R148A receptor was not to cAMP these results indicate that GPR20 is constitutively on the cell leading to activation of Gi and that the DRY motif in hGPR20 a role in activation of in cAMP by of we that overexpression of GPR20 resulted in constitutive activation of Gi data were obtained by using overexpression we or not a physiological expression level of GPR20 is to the constitutive activation of Gi proteins. For this we performed an RNAi using PC12h cells that express GPR20 We constructed three plasmids, pSilencer-sh1, pSilencer-sh2, and the mRNA of of each plasmid significantly the endogenous mRNA in PC12h cells the plasmid, into PC12h cells the of mRNA by with control In PC12h cells these rGPR20-shRNA plasmids, we observed in the basal cAMP level with levels in the of the control plasmid The results that endogenous GPR20 also constitutively or spontaneously active conformation. Furthermore, we that of GPR20 in PC12h cells resulted in an increase in [3H]thymidine incorporation These that GPR20 is involved in the of cell growth. the endogenous GPR20 the mitogenic of PC12h cells by control cellular cAMP level through Gi We the expression and of an orphan GPCR, We demonstrated that exogenously expressed hGPR20 continuous activation of Gi proteins in HEK293 cells in the absence of as intracellular and cAMP were not by with of lipids, or in extracellular Although is still the that a for hGPR20 in the we observed of of fatty BSA, which is to and in the on the continuous activation of Gi proteins by hGPR20 not Furthermore, mutation of the in the hGPR20 DRY motif Gi activation Because the DRY motif is to the receptor J. S. K. R. J. H. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. A. S. U. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the of this motif has been to be for R. Pharmacol. PubMed Scopus Google Scholar), these results the that GPR20 has the for continuous activation of Gi proteins. are several of evidence that GPCRs are in the absence of and to a of into cells M. H. R. A. H. R. Pharmacol. PubMed Scopus Google Scholar). Here we evidence that the exogenously expressed GPR20 activates Gi proteins without ligand in HEK293 cells. The activation of GPR20 was observed not in HEK293 cells also in the rat cell not Although the constitutive activation of the exogenously expressed was observed of an of the receptor in cells J. S. K. T. Pharmacol. 2000; PubMed Scopus Google Scholar), as in we demonstrated that GPR20 a basal than Furthermore, GPR20 a spontaneously active in physiological from the results of RNA using PC12h cells that express GPR20 In these cells, GPR20 may have a in the intracellular cAMP levels via of we observed the mitogenic of the GPR20-silencing in PC12h cells. demonstrated that cAMP cell by extracellular signal-regulated in diverse cell T. R. PubMed Scopus Google S. J. PubMed Scopus Google J. PubMed Scopus Google Scholar). to in cells, activate and the and of in these cells activates J. Trends Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The expression of was confirmed in PC12h cells not and that GPR20-silencing resulted in of cAMP production and cell are with the above cell is by activation of GPCRs that are coupled to protein. is still are in the GPR20-silencing cells, and results a that GPR20 expression in PC12h cells the mitogenic by the The expression of GPR20 was in human and mouse with the levels of GPR20 mRNA detected in S. M. J. A. Google that GPR20 is expressed in which are well as the of the The expression of GPR20 was with expression of the activation is a for in gene expression GPR20 may be involved in the of cells. analysis also demonstrated that GPR20 is expressed in the brain and in of cells In a recently disclosed patent (4Brennan, T. J., Matthews, W., and Moore, M. (January 23, 2003) U. S. Patent 0018989Google Scholar), a role for GPR20 in the system has been GPR20-deficient mice a hyperactivity disorder characterized by an increase in total distance traveled in an open field Because cAMP production in brain in GPR20-deficient the cAMP levels in these may be related to the hyperactivity disorder (4Brennan, T. J., Matthews, W., and Moore, M. (January 23, 2003) U. S. Patent 0018989Google Scholar). has been demonstrated that several orphan including J. O. S. M. J. PubMed Scopus Google S. T. A. J. Science. PubMed Scopus Google Scholar), S. K. K. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), S. K. A. E. J. J. M. PubMed Scopus Google Scholar), and S. S. E. A. J. H. M. J. M. PubMed Scopus Google Scholar), have the to cAMP by constitutive activation of in a and are expressed in of the including the caudate nuclei, putamen, and the thalamus S. K. A. E. J. J. M. PubMed Scopus Google S. S. E. A. J. H. M. J. M. PubMed Scopus Google Scholar), suggesting that may GPR20 in these Because the intracellular cAMP level is for cellular including cell and the of intracellular cAMP may be by the in expression of these and receptors. a large number of GPCRs are as orphan receptors for which the cognate ligands have not yet been identified (1Civelli O. Trends Pharmacol. Sci. 2005; 26: 15-19Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The in of orphan GPCRs is identification of the natural for these receptors, of GPCRs has many of research through of natural ligands. We have identified a lysophosphatidic acid receptor, K. S. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the of T. T. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), S. T. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), and acid as a of leukotriene B4 type receptor, and T. J. in Scholar). The for ligands for orphan receptors is to ligand or by cellular using cells the orphan be is to the assay for a orphan receptor, of intracellular or cAMP However, is still which of G proteins are coupled to these orphan are still for this ligand In this study, we demonstrate not that GPR20 is coupled to Gi-type G also that the coupled Gi proteins are constitutively by GPR20 without ligand in a of basal cAMP However, we the that ligands for GPR20 in GPR20 a potential for of Gi activation via the binding of the obtained in this be to assay for ligands or In work that GPR20 has the to activate Gi proteins without extracellular the role of this receptor in the basal of cAMP in cells. Furthermore, that the expression of GPR20 is involved in the of cell by cellular cAMP In GPR20 has the potential to G activation by ligand efforts to or ligands for GPR20 be to the of this receptor in We and for and and for mouse and and for We also Miyazaki for