G2A Is a Proton-sensing G-protein-coupled Receptor Antagonized by Lysophosphatidylcholine
2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês
10.1074/jbc.m406561200
ISSN1083-351X
AutoresNaoka Murakami, Takehiko Yokomizo, Toshiaki Okuno, Takao Shimizu,
Tópico(s)Ion Transport and Channel Regulation
ResumoG2A (from G2 accumulation) is a G-protein-coupled receptor (GPCR) that regulates the cell cycle, proliferation, oncogenesis, and immunity. G2A shares significant homology with three GPCRs including ovarian cancer GPCR (OGR1/GPR68), GPR4, and T cell death-associated gene 8 (TDAG8). Lysophosphatidylcholine (LPC) and sphingosylphosphorylcholine (SPC) were reported as ligands for G2A and GPR4 and for OGR1 (SPC only), and a glycosphingolipid psychosine was reported as ligand for TDAG8. As OGR1 and GPR4 were reported as proton-sensing GPCRs (Ludwig, M. G., Vanek, M., Guerini, D., Gasser, J. A., Jones, C. E., Junker, U., Hofstetter, H., Wolf, R. M., and Seuwen, K. (2003) Nature 425, 93–98), we evaluated the proton-sensing function of G2A. Transient expression of G2A caused significant activation of the zif 268 promoter and inositol phosphate (IP) accumulation at pH 7.6, and lowering extracellular pH augmented the activation only in G2A-expressing cells. LPC inhibited the pH-dependent activation of G2A in a dose-dependent manner in these assays. Thus, G2A is another proton-sensing GPCR, and LPC functions as an antagonist, not as an agonist, and regulates the proton-dependent activation of G2A. G2A (from G2 accumulation) is a G-protein-coupled receptor (GPCR) that regulates the cell cycle, proliferation, oncogenesis, and immunity. G2A shares significant homology with three GPCRs including ovarian cancer GPCR (OGR1/GPR68), GPR4, and T cell death-associated gene 8 (TDAG8). Lysophosphatidylcholine (LPC) and sphingosylphosphorylcholine (SPC) were reported as ligands for G2A and GPR4 and for OGR1 (SPC only), and a glycosphingolipid psychosine was reported as ligand for TDAG8. As OGR1 and GPR4 were reported as proton-sensing GPCRs (Ludwig, M. G., Vanek, M., Guerini, D., Gasser, J. A., Jones, C. E., Junker, U., Hofstetter, H., Wolf, R. M., and Seuwen, K. (2003) Nature 425, 93–98), we evaluated the proton-sensing function of G2A. Transient expression of G2A caused significant activation of the zif 268 promoter and inositol phosphate (IP) accumulation at pH 7.6, and lowering extracellular pH augmented the activation only in G2A-expressing cells. LPC inhibited the pH-dependent activation of G2A in a dose-dependent manner in these assays. Thus, G2A is another proton-sensing GPCR, and LPC functions as an antagonist, not as an agonist, and regulates the proton-dependent activation of G2A. G-protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G-protein-coupled receptor; ANOVA, analysis of variance; BSA, bovine serum albumin; CHO, Chinese hamster ovary; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; EPPS, N-2-hydroxyethylpiperazine-N′-3-propane-sulfonic acid; G2A, G2 accumulation; HEK, human embryonic kidney; IP, inositol phosphate; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; MES, 2-(N-morpholino)ethanesulfonic acid; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; PTX, pertussis toxin; S1P, sphingosine 1-phosphate; SLE, systemic lupus erythematosus; SPC, sphingosylphosphorylcholine; WT, wild type. mediate various cellular functions including proliferation, differentiation, adhesion, and migration and play pivotal roles in development, homeostasis, inflammation, immunity, oncogenesis, and cancer metastasis (1Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. 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Oncogene. 2000; 19: 3866-3877Crossref PubMed Scopus (60) Google Scholar). Recently, TDAG8 and GPR4, other members of the G2A family (see below), were reported to be also oncogenic and overexpressed in human cancers (12Sin W.C. Zhang Y. Zhong W. Adhikarakunnathu S. Powers S. Hoey T. An S. Yang J. Oncogene. 2004; 23: 6299-6303Crossref PubMed Scopus (82) Google Scholar). It has been reported that expression of G2A caused constitutive activation of small GTPase Rho through G13 (13Kabarowski J.H. Feramisco J.D. Le L.Q. Gu J.L. Luoh S.W. Simon M.I. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12109-12114Crossref PubMed Scopus (63) Google Scholar), making the identification of G2A ligands difficult. There are several GPCRs with structural similarities to G2A (14Xu Y. Biochim. Biophys. Acta. 2002; 1582: 81-88Crossref PubMed Scopus (197) Google Scholar). Ovarian cancer G-protein-coupled receptor 1 (OGR1/GPR68) (15Xu Y. Zhu K. Hong G. Wu W. Baudhuin L.M. Xiao Y. Damron D.S. Nat. 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Science. 2001; 293: 702-705Crossref PubMed Scopus (277) Google Scholar) and chemotaxis in T cells that intrinsically express G2A (27Radu C.G. Yang L.V. Riedinger M. Au M. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 245-250Crossref PubMed Scopus (164) Google Scholar). Very recently, however, Ludwig et al. (28Ludwig M.G. Vanek M. Guerini D. Gasser J.A. Jones C.E. Junker U. Hofstetter H. Wolf R.M. Seuwen K. Nature. 2003; 425: 93-98Crossref PubMed Scopus (530) Google Scholar) reported that OGR1 and GPR4 are activated by increasing concentrations of extracellular protons, leading to the accumulation of inositol phosphates (IPs) and cAMP, respectively. They also described that LPC and SPC failed to activate OGR1 and GPR4, contrary to previous reports (15Xu Y. Zhu K. Hong G. Wu W. Baudhuin L.M. Xiao Y. Damron D.S. Nat. Cell Biol. 2000; 2: 261-267Crossref PubMed Scopus (176) Google Scholar, 26Zhu K. Baudhuin L.M. Hong G. Williams F.S. Cristina K.L. Kabarowski J.H. Witte O.N. Xu Y. J. Biol. Chem. 2001; 276: 41325-41335Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). These observations led us to investigate the proton-sensing ability of G2A using G2A-overexpressing cells. In this report, we show that lowering extracellular pH caused G2A activation determined by reporter gene assay using zif 268 promoter and measuring IP accumulation, both of which were inhibited by LPC. Materials—LPC (18:1, 18:0, and 16:0), lysophosphatidic acid (LPA, 18:1), lysophosphatidylserine (lyso-PS, 18:1), and lysophosphatidylethanolamine (lyso-PE, 18:1) were purchased from Avanti Polar Lipids (Alabaster, AL), S1P and sphingomyelin (SM) were from Biomol Research Laboratories (Plymouth Meeting, PA), and platelet-activating factor (PAF), lyso-PAF, PGE2, and SPC were from Cayman Chemical (Ann Arbor, MI). Alexa Fluor 488-phalloidin was from Molecular Probes, Inc. (Eugene, OR). Myo-[3H]inositol was from Amersham Biosciences. Dulbecco's modified Eagle's medium (DMEM) was from Sigma. M5 anti-FLAG antibody and phosphatidylethanolamine-conjugated anti-mouse IgG were from Sigma and Coulter (Fullerton, CA), respectively. HEPES, EPPS, and MES were from Wako (Osaka, Japan). ECL was from Amersham Biosciences. Buffers and pH—To cover a wide range of pH, DMEM containing 0.1% BSA (bovine serum albumin, Fraction V, Sigma) was buffered with HEPES/EPPS/MES (7.5 mm each, designated below as HEM), and the concentration of sodium bicarbonate in DMEM was reduced to 0.5 mg/ml. The pH shown in the results was adjusted under stimulating conditions using a carefully calibrated pH meter (DKK-TOA Corp., Tokyo, Japan). Subcloning and Site-directed Mutagenesis of Human G2A—Wild type human G2A was subcloned from an expressed sequence tag clone (GenBank™/EBI identification number 5455247) into EcoRI and KpnI sites of the pCXN2-FLAG vector (29Miyazaki J. Takaki S. Araki K. Tashiro F. Tominaga A. Takatsu K. Yamamura K. Gene (Amst.). 1989; 79: 269-277Crossref PubMed Scopus (370) Google Scholar) by PCR using KOD-Plus (Toyobo, Osaka, Japan) and primers (sense primer, 5′-GGGGTACCCTACTGAAAAACGGTTACAATGG-3′, and antisense primer, 5′-GGAATTCAGCAGGACTCCTCAATCAG-3′) and designated as pCXN2.1-FLAG-G2A. Site-directed mutagenesis was performed by an overhang method (as described in Molecular Cloning, 3rd Ed. (49Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar)) using KOD-Plus. The primers for mutagenesis were 5′-CTGCTTCGCCCCGTACTTCCTGGTTCTCCTCGTC-3′ for H259F and 5′-CCTCGTCGGGATCGTTTTCTACCCGGTGTTCCAGACG-3′ for H174F and their complementary oligonucleotides. Entire open reading frames (ORFs) for WT and mutant G2A were sequenced using an ABI PRISM 3100 genetic analyzer (Applied Biosystems). Cell Culture, Transfection, and Flow Cytometry—PC12h cells (donated by the late Professor H. Hatanaka of Osaka University) were maintained in DMEM supplemented with 10% heat-inactivated horse serum and 5% fetal calf serum (FCS), 100 IU/ml penicillin, and 100 μg/ml streptomycin. NIH-3T3, human embryonic kidney (HEK) 293, and COS-7 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum, penicillin, and streptomycin. NIH-3T3 cells were transfected with pCXN2.1-FLAG-G2A using LipofectAMINE PLUS reagent (Invitrogen) according to the manufacturer's protocol and were cultured for 2 weeks under 1.0 mg/ml G418 (Wako). For staining, cells were incubated with 10 μg/ml anti-FLAG antibody (M5) in PBS (–) containing 2% goat serum (PBS/goat serum) for 30 min, followed by staining with phosphatidylethanolamine-conjugated anti-mouse IgG in PBS/goat serum for 15 min. Cells highly expressing G2A were collected as a polyclonal population by cell sorting using EPICS ALTRA (Coulter Electronics Ltd.) and maintained under 0.3 mg/ml G418. EPICS XL (Coulter Electronics Ltd.) was used for flow cytometry. Reporter Gene Assay—pH-induced responses were determined by reporter gene assay (Naito et al., Japanese patent publication number JP2000-354500) with modifications. 1.5 × 105 PC12h cells were transfected with 400 ng of pCXN2.1-FLAG-G2A or an empty vector, 480 ng of zif 268-firefly luciferase-pGL2 (a kind gift from Dr. T. Naito, Japan Tobacco, Tokyo, Japan), 20 ng of cytomegalovirus (CMV) promoterdriven Renilla luciferase-pRL (Promega, Madison, WI), and/or other constructs of interest using SuperFect (Qiagen, Hiden, Germany) according to the manufacturer's protocol. An expression vector for C3 exoenzyme (pEF-C3) was a kind gift from Dr. S. Narumiya (Kyoto University), and an expression vector for a dominant negative form of RhoA (pCMV5-RhoA-T19N) was from Dr. H. Ito (Nara Institute of Science and Technology). 50 ng of pEF-C3 and 100 ng of pCMV5-RhoA-T19N were used for a single transfection. We equalized the total amount of DNA for transfection (1.0 μg/1.5 × 105 cells) among samples by adding empty vectors (pCXN2.1(+), pEF-BOS, or pCMV5). Transfected cells were cultured on a collagen-coated 24-well plate for 45 h. Cells were then stimulated for 9 h with HEM-buffered DMEM, 0.1% BSA adjusted to various pH values under 5% CO2 at 37 °C. As for cell-conditioned medium, PC12h cells were seeded at 1.5 × 105 cells/well on a 24-well plate and incubated for 45 h, followed by stimulation with HEM-buffered DMEM, 0.1% BSA adjusted to pH 7.0 for 9 h. Thereafter, the supernatant was recovered, adjusted to pH 7.6 or 7.0, filtrated to remove cell debris, and used for assay. Firefly and Renilla luciferase activities were measured using PICAGENE Dual Seapansy (Toyo Ink, Tokyo, Japan) and MiniLumat LB 9506 luminometer (Berthold, Bundoora, Australia). The relative luciferase activity is represented as a firefly luciferase value/a Renilla luciferase value (30Noguchi K. Ishii S. Shimizu T. J. Biol. Chem. 2003; 278: 25600-25606Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). Confocal Microscopic Observation of Actin Polymerization—NIH-3T3 cells were seeded onto a poly-l-lysine-coated glass bottom dish (MATSUNAMI, Tokyo, Japan) at the density of 2 × 105 cells/dish and were serum-starved for 24 h with DMEM containing 0.1% BSA (Fraction V, Sigma). Cells were then stimulated for 20 min with HEM-buffered DMEM containing 0.1% BSA adjusted to pH 7.6 or 7.0 with or without LPA (18:1), PGE2, and LPC (18:1) and subsequently fixed by 3.7% formaldehyde, PBS for 30 min at 37 °C. Cells were washed in PBS, permeabilized by incubation with PBS containing 0.4% Triton X-100 for 5 min at room temperature, and incubated with Alexa Fluor 488-phalloidin (2 units/ml, Molecular Probes, Inc.) in 0.1% Triton X-100, PBS for 15 min at room temperature. Cells were washed three times with PBS, followed by observation using a confocal microscope LSM510 (Carl Zeiss, Germany) equipped with a ×63 objective lens (Carl Zeiss, Germany). For quantitative analysis of stress fiber formation, the percentage of cells that exhibited strong actin stress fiber was evaluated. Eight images were randomly obtained from one culture dish, and at least 150 cells were evaluated for stress fiber formation in a blinded manner. Immunoprecipitation of FLAG-tagged G2A—NIH-3T3 cells highly expressing G2A were grown to subconfluency on a 10-cm dish, scraped off with 1 ml of lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and Complete protease inhibitor mixture (Roche Diagnostics)), homogenized through a 25-gauge needle, and incubated at 4 °C for 30 min with rotation. The cell suspension was centrifuged for 10 min at 10,000 × g at 4 °C, and the supernatant was bound with prewashed protein A/G-agarose (Santa Cruz Biotechnology) for 4 h at 4 °C with rotation to preclear the nonspecific binding protein. After centrifugation for 10 min at 10,000 × g at 4 °C, the supernatant was incubated with prewashed M2-agarose (Sigma) overnight at 4 °C with rotation, followed by 3 min of centrifugation at 10,000 × g at 4 °C. Thereafter, the pellet was washed twice with wash buffer (50 mm Tris-HCl, pH 7.5, 500 mm NaCl, 0.1% Nonidet P-40, and 0.05% sodium deoxycholate) and twice with wash buffer without NaCl. For Western blotting analyses, half of the immunoprecipitate was applied to SDS-PAGE (10% polyacrylamide) and transferred to ECL membranes, and FLAG-tagged G2A was detected using biotin-conjugated anti-FLAG antibody (M5) and horseradish peroxidase-conjugated streptavidin. The signal was visualized using the ECL chemiluminescence detection system. Inositol Phosphate Accumulation Assay—COS-7 cells were seeded at 1 × 105 cells/well on a 12-well plate. Twenty-four h after transfection, the culture medium was replaced with fresh medium containing 1 μCi/ml myo-[3H]inositol (Amersham Biosciences) and 100 ng/ml pertussis toxin (PTX) if mentioned, followed by incubation for another 18–24 h. Cells were washed twice and incubated for 45 min in HEM-buffered DMEM containing 0.1% BSA and 20 mm LiCl adjusted to various pH values. Accumulated IPs were purified by ion exchange chromatography (AG 1-X8 resin, Bio-Rad), and radioactivities were measured by scintillation counting (31Fukunaga K. Ishii S. Asano K. Yokomizo T. Shiomi T. Shimizu T. Yamaguchi K. J. Biol. Chem. 2001; 276: 43025-43030Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Statistical Analysis—Data were analyzed for statistical significance using Student's unpaired t test or ANOVA using Prism 4 software (GraphPad Software, Inc., San Diego, CA). Differences were considered significant at p < 0.05 or 0.01, as indicated. pH-dependent Activation of the zif 268 Promoter through G2A—As G2A was reported to activate RhoA in a ligand-independent manner (13Kabarowski J.H. Feramisco J.D. Le L.Q. Gu J.L. Luoh S.W. Simon M.I. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12109-12114Crossref PubMed Scopus (63) Google Scholar), we performed a reporter gene assay using zif 268-luciferase as a reporter gene (32Hirabayashi T. Saffen D. Eur. J. Biochem. 2000; 267: 2525-2532Crossref PubMed Scopus (8) Google Scholar). zif 268 promoter contains four serum-responsive elements (SREs), one Sp1-binding site, and one cAMP-responsive element (CRE) and is activated through RhoA (1Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (196) Google Scholar). Expression of the receptor protein on the plasma membrane of G2A-transfected PC12h cells was confirmed by flow cytometry 2 days after transfection (data not shown). pH-dependent activation of zif 268 promoter was observed in transiently G2A-expressing cells but not in mock-transfected cells (Fig. 1A). At pH 7.6, G2A exhibited significant activity that was double the value of the mock transfectant. However, this “constitutive” activity was actually proton-dependent activation through G2A, as G2A did not show activity at pH 8.8 (Fig. 1D). Because the basal pH of the culture medium (DMEM, 10% fetal calf serum or 0.1% BSA without any buffer) under 5% CO2 is 7.6, we describe pH 7.6 as the basal pH and describe the activity of G2A at pH 7.6 as constitutive activity hereafter. Although lowering extracellular pH evoked zif promoter activation through G2A, it is possible that PC12h cells secrete under the acidic conditions some compounds or metabolites that activate G2A. Thus, we examined zif 268 promoter activation using cell-conditioned medium. Cell-conditioned medium was obtained as culture supernatant after a 9-h incubation of PC12h cells at pH 7.0 and was adjusted to pH 7.6 or 7.0 prior to assay as described under “Experimental Procedures.” As shown in Fig. 1B, the same level of zif promoter activation through G2A was observed with the cell-conditioned medium as with the control (cell-free) medium at pH 7.0. Cell-conditioned medium adjusted to pH 7.6 did not activate the zif promoter. This result excludes the possibility that pH-dependent activation of G2A is a result of the products secreted from the cells. Because overexpression of G2A was reported to activate Rho GTPase through G13 (13Kabarowski J.H. Feramisco J.D. Le L.Q. Gu J.L. Luoh S.W. Simon M.I. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12109-12114Crossref PubMed Scopus (63) Google Scholar), we examined whether this pH-dependent activation of zif 268 promoter through G2A is inhibited by coexpression of C3 exoenzyme (33Sekine A. Fujiwara M. Narumiya S. J. Biol. Chem. 1989; 264: 8602-8605Abstract Full Text PDF PubMed Google Scholar) or a dominant negative form of RhoA (RhoA-T19N) (34Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). zif 268 promoter activation through G2A both at pH 7.6 and pH 7.2 was inhibited completely by C3 exoenzyme and partially (∼50%) by RhoA-T19N (Fig. 1C). This result demonstrates that the pH-dependent activation of zif 268 promoter through G2A requires activation of Rho GTPase. pH-dependent Inositol Phosphate Production in G2A-expressing Cells—G2A was reported previously to activate IP accumulation in a ligand-independent manner (35Lin P. Ye R.D. J. Biol. Chem. 2003; 278: 14379-14386Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We first examined IP formation using NIH-3T3 cells stably expressing G2A (G2A-NIH-3T3). As reported previously (36de Martin R. Schmid J.A. Hofer-Warbinek R. Mutat. Res. 1999; 437: 231-243Crossref PubMed Scopus (73) Google Scholar), IP formation in G2A-NIH-3T3 cells was 5-fold higher than in mock-transfected cells at pH 7.6, and no additional IP accumulation was observed by lowering pH (data not shown). Next, we measured IP formation using COS-7 cells 48 h after transfection and found that IP accumulation in G2A-transfected cells increased in a pH-dependent manner (Fig. 2A). This pH-dependent IP accumulation was not observed in mock-transfected cells. Pretreatment of the cells with PTX partially inhibited pH-dependent IP accumulation (Fig. 2B), showing that both PTX-sensitive and -insensitive G-proteins are responsible for phospholipase C activation through G2A. Antagonistic Effects of LPC on G2A Activation at Low pH— LPC and SPC were reported previously as ligands for G2A (25Kabarowski J.H. Zhu K. Le L.Q. Witte O.N. Xu Y. Science. 2001; 293: 702-705Crossref PubMed Scopus (277) Google Scholar). Thus, we examined the effect of LPC and SPC at various pH values. At pH 7.6, 1 μm LPC (18:1, 18:0, and 16:0), SPC, and other lipid species did not enhance nor inhibit zif promoter activation through G2A (Fig. 3A). At pH 7.2, however, only LPC (18:1, 18:0, and 16:0) showed an inhibitory effect upon pH-dependent zif promoter activation through G2A (Fig. 3B), and there was no difference observed among three LPCs with different acyl moieties at the sn-1 position in their antagonistic effects. We treated the cells with various concentrations of LPC at pH 7.2 and found that LPC inhibited zif promoter activation in a dose-dependent manner (Fig. 3C). To investigate whether LPC or SPC inhibits pH-dependent IP formation, we performed an IP accumulation assay in the presence of LPC or SPC. As observed in the zif-luciferase reporter gene assay, 1 μm LPC (18:1, 18:0, and 16:0) or SPC did not promote nor inhibit IP formation through G2A at pH 7.6 (Fig. 3D), and LPC (18:1) inhibited IP production at acidic pH in a dose-dependent manner only in G2A-expressing cells (Fig. 3E). One μm LPC (18:1) partially (∼50%) inhibited and 10 μm LPC completely inhibited IP accumulation at various pH values (Fig. 3F). In both zif-luciferase reporter gene assay and IP accumulation assay, LPC inhibited the activation of G2A at acidic pH. Thus, LPC appears to act on G2A as an antagonist rather than an agonist. Actin Stress Fiber Formation—As shown in the reporter gene assay, the G2A signaling pathway is coupled to Rho GTPase. Activation of Rho GTPase induces actin stress fiber formation in fibroblasts (37Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, 38Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). We therefore observed the rearrangement of fibrous actin in NIH-3T3 cells stably expressing G2A using a confocal microscope. These cells were collected as a polyclonal population by cell sorting as described under “Experimental Procedures.” Proper expression of G2A on the plasma membrane was confirmed by flow cytometry and Western blotting (Fig. 4, A and B). At pH 7.6, G2A-NIH-3T3 cells exhibited prominent actin stress fiber formation (Fig. 4C, top) that resembles the effect of LPA (Fig. 4C, bottom), and no significant additional rearrangement was detected at lower pH (Fig. 4C, middle). This shows that the constitutive activity of G2A at pH 7.6 is sufficient and saturable to cause stress fiber formation. Next, we determined whether LPC (18:1) inhibits the actin polymerization in G2A-NIH-3T3 cells. The stress fibers observed in G2A-NIH-3T3 cells were degraded partially by 1 μm LPC and completely degraded by 10 μm LPC (Fig. 4C). To rule out the possibility that any intrinsically expressed receptor for LPC on NIH-3T3 cells inhibits actin polymerization independently of G2A, we examined LPA (18:1)-induced actin stress fiber formation in mock transfectant in the presence of LPC at pH 7.6 (Fig. 4C). In mock-transfected NIH-3T3 cells, LPA-induced actin rearrangement was not inhibited by 1 or 10 μm LPC (18:1), suggesting that LPC alone could not inhibit actin rearrangement in NIH-3T3 cells. Surprisingly, LPA-induced actin polymerization in G2A-NIH-3T3 cells was inhibited partially by 1 μm LPC and almost completely inhibited by 10 μm LPC (18:1). For quantitative analysis of stress fiber formation, the percentage of cells that exhibited strong actin stress fiber formation was evaluated (Fig. 4D). In mock-transfected cells, stress fiber formation was enhanced by LPC. This might be because of intrinsically expressed LPC receptors (yet unidentified) that transmit positive signals to cause actin bundling. We observed a significant increase in stress fiber-positive cells in G2A-NIH-3T3 cells at pH 7.6. This was not enhanced by lowering the extracellular pH to 7.2. Ten μm LPC inhibited G2A-dependent actin polymerization both at pH 7.6 and 7.2. We further investigated inhibitory effects of LPC (18:1) on the stress fiber formation caused by other ligands. We used LPA (4Ishii I. Fukushima N. Ye X. Chun J. Annu. Rev. Biochem. 2004; 73: 321-354Crossref PubMed Scopus (653) Google Scholar), PGE2 (39Hasegawa H. Negishi M. Katoh H. Ichikawa A. Biochem. Biophys. Res. Commun. 1997; 234: 631-636Crossref PubMed Scopus (30) Google Scholar), thrombin (40Wang Q. Liu M. Kozas
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