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• W1989085193 abstract "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. 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 (105) 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 (2200) 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 (33) 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 (33) 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 (4524) 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 Scholar, 7Senogles S. J. Biol. Chem. 1994; 269: 23120-23127Abstract Full Text PDF PubMed Google Scholar, 8Wise A. Watson-Koken M. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar, 9Ayoub M. Maurel D. Binet V. Fink M. Prézeau L. Ansanay H. Pin J. Mol. Pharmacol. 2007; 71: 1329-1340Crossref PubMed Scopus (75) Google Scholar, 10Hynes T. Mervine S. Yost E. Sabo J. Berlot C. J. Biol. Chem. 2004; 279: 44101-44112Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) were constructed as follows. The Cys351 residues of the Gi1 and Gi2 proteins and the Cys352 residue in the Gi3 protein were substituted with a Gly residue by PCR amplification. Next, an EcoRI site was created between Ile93 and Asp94 within the helical domain of each Gi protein by site-directed mutagenesis. A GFP sequence, bearing two Gly-rich linker sequences, Gly-Asn-Ser-Gly-Gly at the N-terminal end and Gly-Gly-Gly-Asn-Ser at the C-terminal end (9Ayoub M. Maurel D. Binet V. Fink M. Prézeau L. Ansanay H. Pin J. Mol. Pharmacol. 2007; 71: 1329-1340Crossref PubMed Scopus (75) Google Scholar), was inserted into the EcoRI site created in each Gi protein. Cell Culture and Transfections—Human embryonic kidney (HEK) 293 cells were maintained under 5% CO2 in air at 37 °C in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% (v/v) fetal bovine serum (Invitrogen). PC12h cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 5% fetal bovine serum. Cells were transfected with expression plasmids using Lipofectamine 2000 in serum-free Opti-MEM I according to the manufacturer's description. Establishment of Cells Stably Expressing hGPR20—HEK293 cells were transfected with pCXN2.1-FLAG-GPR20 and cultured for 30 days in the presence of 1.0 mg/ml G418. These cells were incubated with 10 μg/ml anti-FLAG (M5) in phosphate-buffered saline (PBS) containing 2% goat serum for 30 min, followed by staining with Alexa Fluor 488-labeled anti-mouse IgG in PBS/2% goat serum for 15 min. Transformants highly expressing FLAG-hGPR20, designated HEK293-GPR20 cells, were collected as a polyclonal population by cell sorting using an EPICS ALTRA instrument (Beckman Coulter) and maintained in the presence of 0.3 mg/ml G418. Flow cytometric analyses of the FLAG-hGPR20-expressing cells were performed using an EPICS XL instrument (Beckman Coulter). cAMP Measurement—HEK293 and PC12h cells (5–8 × 104 cells) were cultured on poly-d-lysine-coated 96-well plates (for HEK293, BD Biosciences) or collagen-coated 96-well plates (for PC12h, Iwaki) for 24 h. In some experiments, cells were treated with 100 ng/ml PTX for 6 h. Cells were washed twice with buffer A (Hanks' balanced salt solution containing 25 mm HEPES-NaOH, pH 7.4, and 0.1% BSA) and preincubated in 100 μl of buffer A containing 0.5 mm 3-isobutyl-1-methylxanthine (from a 500 mm stock in dimethyl sulfoxide stored at –30 °C) for 15 min at 37 °C. Subsequently, various concentrations of PGE2 in buffer A (100 μl) were added, and cells were incubated for 30 min at 37 °C. The reactions were terminated by the addition of 10% Tween 20 (20 μl), followed by overnight storage at 4 °C. After centrifugation at 800 × g for 5 min, the cAMP concentrations in the supernatants (10 μl) were measured using an AlphaScreen cAMP assay kit (PerkinElmer Life Sciences) according to the manufacturer's instructions. For determination of the basal cAMP concentration in nonstimulated cells, a cAMP Biotrak EIA system was used. Cells were processed as described above, with the exception of the addition of 25 μl of lysis buffer supplied in the EIA kit to terminate the reactions. Membrane Preparation—Two days after transfection, cells were harvested with PBS containing 2 mm EDTA. In some experiments, cells were pretreated with 100 ng/ml PTX for 6 h. The cells were disrupted by sonication in ice-cold sonication buffer (20 mm Tris-HCl, pH 7.5, 0.25 m sucrose, 10 mm MgCl2, 2mm EDTA) containing a complete protease inhibitor mixture and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatants were collected and centrifuged at 100,000 × g for 1 h at 4 °C. The resulting membrane pellets were resuspended in sonication buffer and subjected to [35S]GTPγS binding experiments. The protein concentrations in each sample were determined by the Bradford method (11Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) using the protein assay kit (Bio-Rad). [35S]GTPγS Binding Assay—Membrane preparations (10 μg of protein) were incubated in 100 μl of [35S]GTPγS binding buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 5 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 5 mm GDP, and 0.1% BSA) containing 0.5 nm [35S]GTPγS for 30 min at 30 °C. To determine nonspecific binding, unlabeled GTPγS was added to the binding mixture to a final concentration of 10 μm. Bound [35S]GTPγS was separated from free [35S]GTPγS by rapid filtration through GF/C filters and washed with 2 ml of ice-cold TMN buffer (10 mm Tris-HCl, pH 7.5, 25 mm MgCl2, and 100 mm NaCl). Radioactivity was measured using a Top Count scintillation counter (Packard Instrument Co.). Quantitative Real Time RT-PCR Analysis—First-strand cDNAs from 16 human tissues were purchased from Clontech (human multiple tissue cDNA panel I and II). To examine the expression of hGPR20 in human tissues, the amount of the GPR20 cDNA in each sample was evaluated using a TaqMan gene expression assay (Applied Biosystems, Foster City, CA; assay code Hs00271049_s1) and the modified TaqMan Universal PCR Master Mix containing 0.01% BSA. The amount of cDNA was estimated using a LightCycler apparatus (Roche Diagnostics) and the Fit Points method in the LightCycler analysis software. For each evaluation, a specific standard curve was made using a series of dilutions from the corresponding cDNA. Mouse total RNAs were prepared from various tissues using an Absolutely RNA miniprep kit (Stratagene, La Jolla CA), and first strand cDNAs were synthesized using Superscript II. PCR amplifications were carried out in microcapillary tubes, in 20-μl reaction volumes consisting of 2 μl of cDNA solution, 1× FastStart DNA Master SYBR Green I (Roche Diagnostics), and 0.5 μm each of the forward and the reverse primers. A 194-bp fragment of the mouse GPR20 (mGPR20) cDNA was generated using following primers: forward primer, 5′-AAACCCAACCAGAAGAGTTCACC-3′, and reverse primer, 5′-AAATGCCAACCAGATACCCAAGT-3′. Evaluation of GPR20 Expression in Various Cell Lines—Total RNA was isolated from cell lines using Isogen (Nippon Gene, Toyama, Japan), followed by purification of mRNAs using a poly (A)+ isolation kit (Nippon Gene). cDNA was generated from 100 ng of the purified mRNA using Superscript II and random hexamer primers. A 187-bp fragment of hGPR20, a 194-bp fragment of mGPR20, and a 197-bp fragment of rGPR20 were generated by PCR. The primers used in these analyses were as follows: hGPR20 sense primer, 5′-GCTGGCGCTGTACGTCTTCT-3′, and antisense primer, 5′-CATGTTGAGGAAGTAACCGAGGA-3′; and rGPR20, sense primer, 5′-GTCCTGGGCTACTTCCTCAACAT-3′, and antisense primer, 5′-CCACCAGTCTTCACACCCAATAC-3′. The primers for mGPR20 were the same as described above. The primer sequences for β-actin were as follows: human, sense primer, 5′-CAGGATGCAGAAGGAGATCACTG-3′, and antisense primer, 5′-TACTCCTGCTTGCTGATCCACAT-3′; rat, sense primer, 5′-AGACCTCTATGCCAACACAGTGC-3′, and antisense primer, 5′-ATAGAGCCACCAATCCACACAGA-3′; and mouse, sense primer, 5′-CACAGGCATTGTGATGGAC-3′, and antisense primer, 5′-CTTCTGCATCCTGTCAGC-3′. Validation of RNA Interference—To validate the efficiency of the rGPR20-knockdown by pSilencer-sh1, pSilencer-sh2, or pSilencer-sh3, quantitative real time RT-PCR (qRT-PCR) was performed. Seven days after transfection of these plasmids into PC12h cells, mRNA isolation and cDNA synthesis were performed as described above. Using the resultant cDNAs, the amounts of expressed rGPR20 and β-actin mRNAs were estimated using LightCycler Fast Start DNA MasterPLUS SYBR Green I (Roche Diagnostics) according to the manufacturer's protocol. In these experiments, the primers for β-actin were the same as described above. For each evaluation, a specific standard curve was made using a series of dilutions of the corresponding cDNA. [3H]Thymidine Incorporation—PC12h cells (1 × 104 cells) were cultured on collagen-coated 96-well plates for 48 h. Each well was pulsed with 1 μCi of [3H]methyl thymidine in the last 6 h of a 48-h culture. At the end of incubation, the cells were lysed using water, and incorporated [3H]thymidine was separated from free [3H]thymidine by rapid filtration through GF/C filters that were washed with 2 ml of water. The radioactivity was measured with a Top Count scintillation counter (Packard Instrument Co.). Incorporation of [3H]thymidine into PC12h DNA was expressed as dpm per well. Cloning of the hGPR20 Gene—We cloned human GPR20 from human genomic DNA by PCR amplification. However, several sequences in our cloned hGPR20 were different from the reported GPR20 sequence (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 (33) Google Scholar). In particular, Cys53–Val54 and Ala157–Pro158–Ala159–Ala160 in the reported GPR20 were Trp53–Leu54 and Gly157–Ser158–Arg159–Arg160, respectively, in our clone. We used our hGPR20 clone in this study, because we verified the sequences of several independently isolated hGPR20 clones, and the same sequence was recently deposited in the human total genome sequence data base at NCBI (accession number NT_008046). The primary structure of hGPR20 exhibited 82.8 and 81.4% amino acid identity to mouse and rat GPR20, respectively (Fig. 1A). Phylogenetic analysis revealed that hGPR20 was closely related to functional receptors for nucleotides or lipids (e.g. TG1019, CysLT1, CysLT2, LPA4, LPA5, GPR34, and PAF receptors, Fig. 1B), raising the possibility that GPR20 may recognize nucleotides or lipids as ligands. Tissue and Cellular Expression of GPR20 mRNA—Using cDNAs prepared from 16 human tissues, qRT-PCR was performed to evaluate the expression of hGPR20. In these tissues, hGPR20 was ubiquitously expressed, with the highest expression in small intestine (Fig. 2A). Similarly, mouse GPR20 mRNA was detected in a broad range of tissues with abundant expression in intestinal tissues, such as small intestine and colon (Fig. 2B). GPR20 mRNA was detected in various cell lines, including N1E115 (mouse neuroblastoma cells), B103 (rat neuroblastoma cells), PC12 and PC12h (rat pheochromocytoma cells), U937 (human histiocytic lymphoma cells), HL60 (human acute promyelocytic leukemia cells), and COLO320 (human colon adenocarcinoma cells), expressed detectable levels of the GPR20 mRNA (Fig. 2C). One of these cell lines, PC12h, was used for the GPR20-knockdown experiments shown below. Reduction of cAMP Formation by the Expression of hGPR20—Because GPR20 belongs to a family of lipid/nucleotide-recognizing receptors, we carried out a ligand screening using the BioMol Screen-Well™ Bioactive Lipid Library (purchased from BioMol) and 17 nucleotides (Sigma). To facilitate this screening, we constructed an expression plasmid producing an N-terminally FLAG-tagged hGPR20 (FLAG-hGPR20) and established a HEK293 cell line stably expressing FLAG-hGPR20 (HEK293-GPR20 cells). Prior to screening, expression of FLAG-hGPR20 on the cell surface of HEK293-GPR20 cells was validated by flow cytometric analysis (Fig. 3A). Although we evaluated the agonistic activity of 198 lipids and 17 nucleotides using HEK293-GPR20 cells by monitoring intracellular calcium mobilization and cAMP formation, none of them was an effective agonist for hGPR20 (data not shown). Furthermore, FLAG-hGPR20 did not respond to changes in the extracellular pH (data not shown), even though GPR20 is closely related to the four proton-sensing GPCRs, i.e. G2A, GPR4, OGR1, and TDAG8 (12Ludwig M. Vanek M. Guerini D. Gasser J. Jones C. Junker U. Hofstetter H. Wolf R. Seuwen K. Nature. 2003; 425: 93-98Crossref PubMed Scopus (501) Google Scholar, 13Murakami N. Yokomizo T. Okuno T. Shimizu T. J. Biol. Chem. 2004; 279: 42484-42491Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 14Wang J. Kon J. Mogi C. Tobo M. Damirin A. Sato K. Komachi M. Malchinkhuu E. Murata N. Kimura T. Kuwabara A. Wakamatsu K. Koizumi H. Uede T. Tsujimoto G. Kurose H. Sato T. Harada A. Misawa N. Tomura H. Okajima F. J. Biol. Chem. 2004; 279: 45626-45633Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 15Ishii S. Kihara Y. Shimizu T. J. Biol. Chem. 2005; 280: 9083-9087Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), in the phylogenetic tree. Additionally, we did not observe any activation of GPR20 by several steroid hormones, including hydrocortisone, corticosterone, β-estradiol, and progesterone (data not shown). During the course of these experiments, we unexpectedly detected a modest but significant decrease in basal cAMP level in HEK293-GPR20 cells, compared with that of vector-transfected cells (Fig. 3B). Moreover, we observed that the FLAG-hGPR20 expression resulted in attenuated PGE2-stimulated cAMP formation in comparison with vector-transfected cells (Fig. 3C). Similar effects were observed in HEK293 cells transiently expressing FLAG-hGPR20 (Fig. 3E), implying that these observations were not an artifact of the cloned cell lines. Because the above effects by FLAG-hGPR20 expression were abolished by treatment with PTX (Fig. 3, B, D and E), exogenous expression of hGPR20 may cause the activation of PTX-sensitive G proteins in the absence of ligand stimulation. Enhanced [35S]GTPγS Binding to Membranes Prepared from hGPR20-expressing Cells—Next we examined whether Gi-type G proteins are indeed activated in the presence of hGPR20. Using membrane preparations from HEK293 cells transiently transfected with various amounts of the FLAG-hGPR20 expression plasmid, we evaluated GDP-GTP exchange of Gα-subunits by [35S]GTPγS binding assay. Because exogenously expressed GPCRs occasionally activate G protein in the absence of ligands, we compared the effect of hGPR20 with hBLT1, which is well known as a Gi-coupled receptor (16Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (845) Google Scholar, 17Kuniyeda K. Okuno T. Terawaki K. Miyano M. Yokomizo T. Shimizu T. J. Biol. Chem. 2007; 282: 3998-4006Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). As shown in Fig. 4A, transfection of hGPR20 enhanced [35S]GTPγS incorporation into the membranes in a dose-dependent manner, even though hBLT1 did not affect the [35S]GTPγS incorporation at these doses. The augmentation of [35S]GTPγS binding was diminished by PTX treatment (Fig. 4B), which is consistent with the PTX sensitivity of PGE2-induced cAMP formation in GPR20-transfected cells (Fig. 3, B–E). To provide further evidence for the activation of Gi proteins by hGPR20, we examined whether [35S]GTPγS incorporation into exogenously expressed PTX-resistant mutants of the Gi protein (the Gi1, Gi2, and Gi3) (6Hunt T. Carroll R. Peralta E. J. Biol. Chem. 1994; 269: 29565-29570Abstract Full Text PDF PubMed Google Scholar, 7Senogles S. J. Biol. Chem. 1994; 269: 23120-23127Abstract Full Text PDF PubMed Google Scholar, 8Wise A. Watson-Koken M. Rees S. Lee M. Milligan G. Biochem. J. 1997; 321: 721-728Crossref PubMed Scopus (67) Google Scholar, 9Ayoub M. Maurel D. Binet V. Fink M. Prézeau L. Ansanay H. Pin J. Mol. Pharmacol. 2007; 71: 1329-1340Crossref PubMed Scopus (75) Google Scholar, 10Hynes T. Mervine S. Yost E. Sabo J. Berlot C. J. Biol. Chem. 2004; 279: 44101-44112Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) increased in FLAG-hGPR20 expressing cells. As shown in Fig. 4C, even in cells pretreated with PTX, increasing [35S]GTPγS binding to the PTX-resistant mutants of Gi proteins was observed in the FLAG-hGPR20-expressing cells. These results suggest that hGPR20 continuously activates Gi-type G proteins. Involvement of the DRY Motif of GPR20 in Activation of Gi Proteins—Several reports have described the significance of a highly conserved stretch of amino acid residues located in the second intracellular loop of various GPCRs, (Glu/Asp)-Arg-Tyr, in G protein activation (18Probst W. Snyder L. Schuster D. Brosius J. Sealfon S. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (676) Google Scholar, 19Savarese T. Fraser C. Biochem. J. 1992; 283: 1-19Crossref PubMed Scopus (439) Google Scholar). This domain is generally designated the DRY motif. To obtain more evidence for the constitutive activation of Gi proteins by GPR20, we examined whether the disruption of the DRY motif in hGPR20 (amino acids 147–149) affects Gi activation. We constructed a mutant FLAG-hGPR20, the R148A receptor, in which Asp148 is substituted by Ala. The R148A receptor was expressed on the cell surface at levels similar to the wild-type FLAG-hGPR20, as judged by flow cytometric analysis (Fig. 5A). As shown in Fig. 5B, [35S]GTPγS incorporation into membrane preparations from R148A receptor-expressing cells was remarkably reduced compared with membranes from cells expressing the wild-type receptor. Additionally, the R148A receptor was not able to inhibit PGE2-induced cAMP formation (Fig. 5C). All these results indicate that GPR20 is constitutively activated on the cell surface, leading to sustained activation of Gi proteins, and that the DRY motif in hGPR20 plays a key role in activation of Gi. Increase in Basal cAMP Levels by Silencing of Endogenously Expressed GPR20—Although we showed that overexpression of GPR20 resulted in constitutive activation of Gi proteins, those data were obtained by using overexpression systems. Therefore, we next examined whether or not a physiological expression level of GPR20 is enough to evoke the constitutive activation of Gi proteins. For this purpose, we performed an RNAi experiment using PC12h cells that endogenously express GPR20 (Fig. 2C). We constructed three shRNA plasmids, pSilencer-sh" @default.
• W1989085193 created "2016-06-24" @default.
• W1989085193 creator A5008474125 @default.
• W1989085193 creator A5008934626 @default.
• W1989085193 creator A5068984150 @default.
• W1989085193 creator A5076042354 @default.
• W1989085193 date "2008-05-01" @default.
• W1989085193 modified "2023-10-16" @default.
• W1989085193 title "Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins" @default.
• W1989085193 cites W1483147211 @default.
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