Regulation of Rac and Cdc42 Pathways by Gi during Lysophosphatidic Acid-induced Cell Spreading

Abstract

The pertussis toxin-sensitive G protein, Gi, has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that Gi-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of Gαi and Gα11 and Gβγ subunits enhanced spreading of pertussis toxin-treated cells. Gβ1 with Gγ12, a major Gγ form in fibroblasts, was more effective for increasing cell spreading than Gβ1γ2 or Gβ1plus Gγ12S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished Gβ1γ12-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both Gαi and Gβγ stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin. The pertussis toxin-sensitive G protein, Gi, has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that Gi-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of Gαi and Gα11 and Gβγ subunits enhanced spreading of pertussis toxin-treated cells. Gβ1 with Gγ12, a major Gγ form in fibroblasts, was more effective for increasing cell spreading than Gβ1γ2 or Gβ1plus Gγ12S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished Gβ1γ12-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both Gαi and Gβγ stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin. lysophosphatidic acid heterotrimeric guanine nucleotide-binding regulatory protein pertussis toxin bisindolylmaleimide I glutathione S-transferase guanine nucleotide exchange factor p21-activated kinase Cdc42/Rac interactive binding domain protein kinase C N-tris(hydroxymethyl)methylglycine Most cell types respond to extracellular matrix proteins by adhering and then spreading out to acquire a flattened morphology. The cell spreading is also regulated by soluble factors from serum such as lysophosphatidic acid (LPA)1and platelet-derived growth factor, which then induce cell growth and migration (1Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, 2Anand-Apte B. Zetter B.R. Stem Cells. 1997; 15: 259-267Crossref PubMed Scopus (77) Google Scholar). The process is accomplished by complex dynamic rearrangements of the actin cytoskeleton, and the dynamics appear to be coordinated in space and time by intracellular signaling pathways involving Rho family small GTP-binding proteins (3Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, 4Howe A. Aplin A.E. Alahari S.K. Juliano R.L. Curr. Opin. Cell Biol. 1998; 10: 220-231Crossref PubMed Scopus (587) Google Scholar), although the precise mechanisms are poorly understood. Rho family members such as Rho, Rac, and Cdc42 are part of the Ras superfamily of small GTP-binding proteins that act as guanine nucleotide-regulated switches (3Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). Their activity is regulated primarily by two groups of proteins: guanine nucleotide exchange factors (GEF), which catalyze exchange of GDP for GTP; and GTPase-activating proteins, which stimulate hydrolysis of GTP to GDP. Upon binding to GTP, Rho family GTP-binding proteins interact with and activate various downstream effector proteins such as Rho kinases and p21-activated kinases (PAK). Rho stimulates the formation of actin stress fibers and focal adhesions, whereas Cdc42 activation triggers the extension of filopodia and Rac controls growth factor-stimulated membrane ruffling and formation of lamellipodia (3Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar). It has been defined how soluble extracellular factors induce the assembly of focal adhesions and actin filaments in serum-starved adherent fibroblasts through activation of Rho. However, there are several reports that Rho family small GTP-binding proteins are also involved in the generation of intracellular morphological structures during cell spreading on an extracellular matrix (5Price L.S. Leng J. Schwartz M.A. Bokoch G.M. Mol. Biol. Cell. 1998; 9: 1863-1871Crossref PubMed Scopus (529) Google Scholar, 6Clark E.A. King W.G. Brugge J.S. Symons M. Hynes R.O. J. Cell Biol. 1998; 142: 573-586Crossref PubMed Scopus (535) Google Scholar, 7Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar). Activation of Rac and Cdc42 and consequent stimulation of PAK have been observed in NIH 3T3 cells in response to plating cells on fibronectin (5Price L.S. Leng J. Schwartz M.A. Bokoch G.M. Mol. Biol. Cell. 1998; 9: 1863-1871Crossref PubMed Scopus (529) Google Scholar). With Rat1 cells, cell spreading was significantly reduced by expression of dominant negative mutants of Rac, Cdc42, and Rho (6Clark E.A. King W.G. Brugge J.S. Symons M. Hynes R.O. J. Cell Biol. 1998; 142: 573-586Crossref PubMed Scopus (535) Google Scholar). More recently, it was shown that plating Swiss 3T3 cells on fibronectin-coated dishes elicited a transient inhibition of Rho activation, suggesting the existence of an adhesion-dependent negative feedback loop (7Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar). LPA induces multiple cellular responses through a G protein-coupled receptor and activates Ras and Rho family GTP-binding proteins via different pathways (1Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). In fibroblasts, LPA stimulates cell growth and lowers cAMP levels in a pertussis toxin (PTX)-sensitive manner, suggesting coupling of the LPA receptor to Gi family G proteins (8van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (684) Google Scholar). LPA-induced activation of the Ras/mitogen-activated protein kinase cascade also occurs in a PTX-sensitive fashion, and Gβγ subunits of G proteins transduce this effect (9van Corven E.J. Hordijk P.L. Medema R.H. Bos J.L. Moolenaar W.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1257-1261Crossref PubMed Scopus (336) Google Scholar, 10Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (335) Google Scholar). In contrast, in a variety of cells, stimulation of phospholipase C by LPA was shown to be PTX-insensitive, so that LPA receptor coupling to Gq family G proteins may also be important (11Seufferlein T. Rozengurt E. J. Biol. Chem. 1994; 269: 9345-9351Abstract Full Text PDF PubMed Google Scholar). LPA induces the Rho-dependent formation of actin stress fibers and focal adhesions in quiescent Swiss 3T3 fibroblasts (12Buhl 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, 13Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Gα12 and Gα13 probably mediate this action, because microinjection of constitutively active mutants of Gα12 and Gα13 into fibroblasts triggers actin polymerization in a Rho-dependent manner (12Buhl 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,13Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). It is well known that LPA induces cell migration, which is blocked by PTX, suggesting the involvement of Gi. However, regulation of Rho family GTP-binding proteins by Gi has not been clearly shown to date. In the present study, we found that PTX blocks LPA-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. We show here Gi stimulation via the LPA receptor leads to Rac and Cdc42 activation during cell spreading. pCMV5-Gαi2Q205L, pCMV5-Gα11Q209L, pCMV5-GαsQ227L, pCMV5-Gα12Q229L, pCMV5-Gγ2, pCMV5-Gγ12, pCMV5-Gγ12S2A, pCMV5-Gβ1, the pCMV5 carboxyl terminus of β-adrenergic receptor kinase 1 (βARKct), pCMV5-FLAG-RhoAT19N, pCMV5-FLAG-Rac1T17N, pCMV5-FLAG-Cdc42HsT17N, pCMV5-FLAG-RhoAG14V, pCMV5-FLAG-Rac1G12V, and pCMV5-FLAG-Cdc42HsG12V were constructed as detailed previously (14Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 15Yamauchi J. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 7602-7607Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 16Ueda H. Yamauchi J. Itoh H. Morishita R. Kaziro Y. Kato K. Asano T. J. Biol. Chem. 1999; 274: 12124-12128Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 17Nagao M. Kaziro Y. Itoh H. Oncogene. 1999; 18: 4425-4434Crossref PubMed Scopus (66) Google Scholar, 18Kato J. Kaziro Y. Satoh T. Biochem. Biophys. Res. Commun. 2000; 268: 141-147Crossref PubMed Scopus (44) Google Scholar, 19Ueda H. Itoh H. Yamauchi J. Morishita R. Kaziro Y. Kato K. Asano T. J. Biol. Chem. 2000; 275: 2098-2102Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 20Yamauchi J. Kawano T. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 2000; 275: 7633-7640Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Antibodies against Gαi2, Gγ7, Gγ12, phospho-Gγ12, and Gβ subunits, generated by ourselves, have been described previously (21Morishita R. Kato K. Asano T. Eur. J. Biochem. 1988; 174: 87-94Crossref PubMed Scopus (46) Google Scholar, 22Asano T. Morishita R. Ohashi K. Nagahama M. Miyake T. Kato K. J. Neurochem. 1995; 64: 1267-1273Crossref PubMed Scopus (37) Google Scholar, 23Morishita R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 24Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar). Rabbit polyclonal antibodies against phosphotyrosine, Gαq/11, Gαs, and Gα12 were purchased from Santa Cruz Biotechnology and mouse monoclonal antibodies against human Rac1 and Cdc42 from Transduction Laboratories. Mouse monoclonal antibodies against the FLAG and Myc epitopes were purchased from Sigma and Roche Molecular Biochemicals, respectively. Tetramethylrhodamine isothiocyanate-phalloidin was obtained from Molecular Probe, PTX from Seikagaku Kogyo, bisindolylmaleimide I (BIM) from Calbiochem-Novabiochem Co., LPA from Avanti Polar Lipids Inc., platelet-derived growth factor and epidermal growth factor from Immunobiological Laboratories, glutathione-coupled Sepharose 4B beads from Amersham Pharmacia Biotech, and isopropyl-1-thio-β-d-galactopyranoside and fibronectin from Wako Pure Chemical Industries. PAK1 cDNA was a generous gift from L. Lim (National University of Singapore). The Cdc42/Rac interactive binding domain (CRIB) of PAK1 (amino acids 67–150) (25Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar) was amplified by polymerase chain reaction using PAK1 cDNA as a template, ligated into pCMV-Myc, and confirmed by DNA sequencing (20Yamauchi J. Kawano T. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 2000; 275: 7633-7640Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The cDNA of Myc-CRIB was cloned into a pGEX-2T vector (Amersham Pharmacia Biotech). Expression and purification of GST fusion protein were performed in accordance with the manufacturer's instructions and published protocols (26Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der F. J. Cell Biol. 1998; PubMed Scopus (587) Google Scholar). cells with the were and expression of protein was by of isopropyl-1-thio-β-d-galactopyranoside for were in 1 then on and for of to a of Cell were for and the was with glutathione-coupled Sepharose 4B beads for by for The beads were in and with fusion beads were in and was to a of and NIH 3T3 cells were in with transient cells were a of with to the manufacturer's instructions The of the DNA for a was to of of or vector pCMV5 and of with was performed and the cells were for in the or of PTX for the were then with with and with inhibitor and 1 serum were then in on fibronectin-coated or and for 1 in the or of platelet-derived growth factor, epidermal growth factor, and cells were in in for or with in 1 for cells were for with cell spreading, of cells were obtained using a and the of or cells were using the of cells were shown the of were by cell were to PubMed Scopus Google Scholar) or H. G. Biochem. PubMed Scopus Google Scholar) for with various This was performed as described previously (26Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der F. J. Cell Biol. 1998; PubMed Scopus (587) Google Scholar). on fibronectin-coated cells were with for in and and for were from the to protein this with fusion proteins to glutathione-coupled Sepharose 4B beads for was by in an of to the beads were in PubMed Scopus Google Scholar) and then for Rac and Cdc42 by using monoclonal mouse antibodies against human Rac1 and Cdc42, respectively. was with a and was performed using the the role of Gi signaling during cell spreading, serum-starved NIH 3T3 fibroblasts were for in the or of PTX, on fibronectin-coated then to LPA for various and with to in the of PTX a of This was diminished with PTX that PTX the of Gi, we the of PTX with by known to cell spreading or cell platelet-derived growth factor, and epidermal growth factor cell spreading, although the of in cell was than that observed in cells, and this was not by PTX These that PTX blocks Gi activation via the LPA The that spreading occurs on fibronectin in the or of but is by PTX in the of LPA be of PTX on cell spreading by LPA platelet-derived growth factor, and epidermal growth 3T3 cells were in the or of PTX for on fibronectin-coated in the or of platelet-derived growth factor or epidermal growth factor and for which of Rho family GTP-binding proteins to cell spreading by we with various Rho family mutants into NIH 3T3 cells. dominant negative mutants of Rac and Cdc42 were whereas dominant negative Rho was effect and the constitutively active mutants of Rac and Cdc42 enhanced spreading of cells and In contrast, the expression of the constitutively active form of Rho of both and cells The LPA receptor activates not Gi, but also G proteins such as and which are PTX-insensitive, and is involved in Rho-dependent stress formation (12Buhl 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). it is that Rho is also during spreading. In of a dominant negative mutant of Rho cell spreading the cells were by PTX These that the LPA receptor induces activation of and Rho through Gi and probably respectively. cell with inhibition of by PTX the activation of endogenous Rac and Cdc42 during cell spreading, we performed of Rac and Cdc42 using Sepharose to that to active forms of Rac and Cdc42 Bokoch G.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), Sepharose beads were with from NIH 3T3 cells FLAG mutants of Rho family GTP-binding beads constitutively active forms of Rac and Cdc42 but not dominant negative forms of Rac and Cdc42, indicating a for the constitutively active dominant negative mutants of Rho were by with published Bokoch G.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) Rac and Cdc42 were during cell spreading on cell were with beads and the proteins were by with antibodies against Rac and Cdc42 and In the cells, Rac activity was and Cdc42 activity In contrast, Rac and Cdc42 not significantly in cells and These also indicate that Rac and Cdc42 activation is mediated by the Gi during LPA-induced cell spreading. the involvement of Rac and Cdc42 activation in LPA-induced cell spreading, we Myc-CRIB into NIH 3T3 cells and on fibronectin-coated in the of Expression of Myc-CRIB cell spreading we which G protein or is for cell spreading. we with several constitutively active mutants of subunits of G proteins and the of spreading of cells in the of LPA active mutants of and cell spreading to the levels observed in cells in the of active Gα12 and and were not effective in cells These active cell spreading in the of PTX suggesting that Gα12 and cell in we Gβ1 with various Gγ forms into NIH 3T3 cells. Gβγ expression also spreading of cells we which and and this to the LPA-induced cell spreading of expression of subunits and on cell spreading. NIH 3T3 cells were with and pCMV5 or in the or of PTX for and on fibronectin-coated dishes or in the of cells were and for spreading. were with antibodies against and the against with and T. Morishita R. Ohashi K. Nagahama M. Miyake T. Kato K. J. Neurochem. 1995; 64: 1267-1273Crossref PubMed Scopus (37) Google Scholar), it was to and are from with cells. with cells. with We previously that the of the a major Gγ in fibroblasts, is by and that this is blocked by PTX R. Nakayama H. Isobe T. Matsuda T. Hashimoto Y. Okano T. Fukada Y. Mizuno K. Ohno S. Kozawa O. Kato K. Asano T. J. Biol. Chem. 1995; 270: 29469-29475Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 24Asano T. Morishita R. Ueda H. Asano M. Kato K. Eur. J. Biochem. 1998; 251: 314-319Crossref PubMed Scopus (13) Google Scholar), as shown in We have also that phosphorylation of migration of NIH 3T3 cells H. Yamauchi J. Itoh H. Morishita R. Kaziro Y. Kato K. Asano T. J. Biol. Chem. 1999; 274: 12124-12128Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Expression of was more effective cell spreading than that of Gβ1γ2 or Gβ1 with the mutant Gγ12S2A, in which a phosphorylation site is replaced with phosphorylation of by is involved in cell spreading, we the effect of the inhibitor E.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) It significantly cell spreading by as well as phosphorylation of Gγ12, effect on cell spreading. It be that Gβ1γ12-induced cell spreading in the of is to cell spreading. In cells, suppressed LPA-induced cell spreading and phosphorylation of is known to stimulate phospholipase C, which then activates Expression of phosphorylation of in cells and the with diminished cell spreading by but not by may to cell spreading. cell spreading with and Gβγ is mediated through Rac and Cdc42 we dominant negative mutants of Rac and Cdc42 with and Gβγ into cells The dominant negative mutant of Rac cell spreading by and However, the dominant negative form of Cdc42 to be with cell spreading. It has been that PTX blocks cell growth and migration by LPA (1Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar, T. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). In the present study, we that Gi LPA-induced cell spreading, of important of growth and migration LPA receptor is not with Gi but also with Gq and family G Gα12 and which activate Rho, induce the formation of actin stress fibers and focal adhesions (12Buhl 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, 13Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). The present that Gi activates Rac and Cdc42 during cell spreading by The that the dominant negative mutant of Rho enhanced spreading of cells that this protein is also with LPA reports that Rac Rho activity in NIH 3T3 cells E.E. ten Klooster J.P. van Delft S. van der J. Cell Biol. 1999; PubMed Scopus Google Scholar), or Rac may Rho F. Bokoch G.M. Science. 1999; PubMed Scopus Google Scholar). In the which is by Rac and Cdc42, was found to the phosphorylation of chain by Rho. (7Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar) have described that plating Swiss 3T3 cells on fibronectin-coated dishes a transient inhibition of Rho, by an activation suggesting the existence of an adhesion-dependent negative feedback In addition, cell observed by expression of that Rho activation cell in the that activation of Rac and Cdc42 by Gi cell spreading and probably with cell in cells the it has been shown that Rac, Cdc42, and Rho were during cell spreading on fibronectin (5Price L.S. Leng J. Schwartz M.A. Bokoch G.M. Mol. Biol. Cell. 1998; 9: 1863-1871Crossref PubMed Scopus (529) Google Scholar, 6Clark E.A. King W.G. Brugge J.S. Symons M. Hynes R.O. J. Cell Biol. 1998; 142: 573-586Crossref PubMed Scopus (535) Google Scholar, 7Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar), and cells also to probably to the of Rho by Rac activation The of spreading may be by a activation and Rho The that of Gi by PTX leads to cell on fibronectin in the of LPA that activation of Rho by and fibronectin is than activation of by fibronectin This the that spreading occurs on fibronectin in the or of but is by PTX in the of It has been shown that Gαi and Gβγ from Gi by receptor stimulation and E.J. 1997; PubMed Scopus Google Scholar). The present that both to LPA-induced cell spreading. The that expression of cell spreading an involvement of Gαi in to We have previously that of Gβ1 with various Gγ forms into NIH 3T3 cells cell migration H. Yamauchi J. Itoh H. Morishita R. Kaziro Y. Kato K. Asano T. J. Biol. Chem. 1999; 274: 12124-12128Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Cell of cells was than of and cells. the effect of was by of by we that protein phosphorylation cell migration H. Yamauchi J. Itoh H. Morishita R. Kaziro Y. Kato K. Asano T. J. Biol. Chem. 1999; 274: 12124-12128Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The present to be more cell spreading than Gβ1γ2 or with obtained for In to the expression of cell spreading in cells and this was blocked by an the existence of and pathways in cell spreading be of in cells to an involvement in a pathway. Gq family G proteins be to be by LPA in cells, cell spreading not The of cell spreading that of intracellular cAMP by or a cAMP inhibitor blocked LPA-induced cell spreading and migration of cells A.M. J. Cell Biol. 1998; PubMed Scopus Google Scholar). levels of intracellular cAMP by also of human cells the receptor T. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), and Gαi and Gβγ several types of E.J. 1997; PubMed Scopus Google Scholar). It been that LPA cAMP levels in Swiss 3T3 cells in a PTX-sensitive manner (8van Corven E.J. Groenink A. Jalink K. Eichholtz T. Moolenaar W.H. Cell. 1989; 59: 45-54Abstract Full Text PDF PubMed Scopus (684) Google Scholar). of cAMP levels by Gi may be important in LPA-induced cell spreading. In the expression of the constitutively active mutant cell spreading GTP-binding proteins are regulated by and GTPase-activating proteins (3Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, S. Biophys. 1997; PubMed Scopus Google Scholar), and reports have that Gα12 and Gα13 are to to T. G. Science. 1998; PubMed Scopus Google Scholar, T. G. Science. 1998; PubMed Scopus Google Scholar) or S. M. T. J.S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Gα13 but not Gα12 stimulates the exchange reaction of Gβγ may also be with the of a of K. Kaziro Y. Satoh T. 1999; PubMed Scopus Google Scholar), but activity Rho is not by Gβγ With to GTPase-activating proteins, Gαi to that for a of the Ras of the receptor from to the membrane and the of N. Y. E. T. T. T. A. K. Matsuda M. 1999; PubMed Scopus Google Scholar). that Gαi and Gβγ induce cell spreading in a manner the existence of or GTPase-activating proteins, which interact with Gαi or Gβγ We M. I. T. R. A. K. and L. Lim for the We are also to Y. Kaziro for