Selective Uncoupling of Gα12 from Rho-mediated Signaling

Abstract

The heterotrimeric G protein G12 has been implicated in such cellular regulatory processes as cytoskeletal rearrangement, cell-cell adhesion, and oncogenic transformation. Although the activated α-subunit of G12 has been shown to interact directly with a number of protein effectors, the roles of many of these protein-protein interactions in G12-mediated cell physiology are poorly understood. To begin dissecting the specific cellular pathways engaged upon G12 activation, we produced a series of substitution mutants in the regions of Gα12 predicted to play a role in effector binding. Here we report the identification and characterization of an altered form of Gα12 that is functionally uncoupled from signaling through the monomeric G protein Rho, a protein known to propagate several Gα12-mediated signals. This mutant of Gα12 fails to bind the Rho-specific guanine nucleotide exchange factors p115RhoGEF and LARG (leukemia-associated RhoGEF), fails to stimulate Rho-dependent transcriptional activation, and fails to trigger activation of RhoA and the Rho-mediated cellular responses of cell rounding and c-jun N-terminal kinase activation. Importantly, this mutant of Gα12 retains coupling to the effector protein E-cadherin, as evidenced by its ability both to bind E-cadherin in vitro and to disrupt E-cadherin-mediated cell-cell adhesion. Furthermore, this mutant retains the ability to trigger β-catenin release from the cytoplasmic domain of cadherin. This identification of a variant of Gα12 that is selectively uncoupled from one signaling pathway while retaining signaling capacity through a separate pathway will facilitate investigations into the mechanisms through which G12 proteins mediate diverse biological responses. The heterotrimeric G protein G12 has been implicated in such cellular regulatory processes as cytoskeletal rearrangement, cell-cell adhesion, and oncogenic transformation. Although the activated α-subunit of G12 has been shown to interact directly with a number of protein effectors, the roles of many of these protein-protein interactions in G12-mediated cell physiology are poorly understood. To begin dissecting the specific cellular pathways engaged upon G12 activation, we produced a series of substitution mutants in the regions of Gα12 predicted to play a role in effector binding. Here we report the identification and characterization of an altered form of Gα12 that is functionally uncoupled from signaling through the monomeric G protein Rho, a protein known to propagate several Gα12-mediated signals. This mutant of Gα12 fails to bind the Rho-specific guanine nucleotide exchange factors p115RhoGEF and LARG (leukemia-associated RhoGEF), fails to stimulate Rho-dependent transcriptional activation, and fails to trigger activation of RhoA and the Rho-mediated cellular responses of cell rounding and c-jun N-terminal kinase activation. Importantly, this mutant of Gα12 retains coupling to the effector protein E-cadherin, as evidenced by its ability both to bind E-cadherin in vitro and to disrupt E-cadherin-mediated cell-cell adhesion. Furthermore, this mutant retains the ability to trigger β-catenin release from the cytoplasmic domain of cadherin. This identification of a variant of Gα12 that is selectively uncoupled from one signaling pathway while retaining signaling capacity through a separate pathway will facilitate investigations into the mechanisms through which G12 proteins mediate diverse biological responses. Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) 1The abbreviations used are: G protein, guanine nucleotide-binding protein; Gα, α-subunit of the heterotrimeric G protein; QL, mutationally activated Gln-to-Leu variant of Gα protein; E-cadherin or E-cad, epithelial cadherin; p115RGS, region comprising amino acids 1–252 of p115RhoGEF and harboring its RGS domain; LARG, leukemia-associated RhoGEF; Δp115-Gα12QL, variant of Gα12QL that lacks the ability to interact with p115RGS; GFP, green fluorescent protein; PBS, phosphate-buffered saline; GST, glutathione S-transferase; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; TLCK, Nα-p-tosyl-l-lysine chloromethyl ketone; JNK, c-jun N-terminal kinase; PIPES, 1,4-piperazinediethanesulfonic acid; SRF, serum response factor. mediate cellular signaling from heptahelical cell surface receptors to a wide variety of downstream effector proteins that in turn propagate signals to elicit certain cellular responses and changes. The α-subunits of the G12 subfamily of G proteins, Gα12 and Gα13, have been linked to cellular events such as cytoskeletal rearrangements (1Buhl 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, 2Gohla A. Offermanns S. Wilkie T.M. Schultz G. J. Biol. Chem. 1999; 274: 17901-17907Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), cell proliferation (3Collins L.R. Ricketts W.A. Olefsky J.M. Brown J.H. Oncogene. 1997; 15: 595-600Crossref PubMed Scopus (29) Google Scholar), Na+/H+ exchange (4Dermott J.M. Wadsworth S.J. van Rossum G.D. Dhanasekaran N. J. Cell. Biochem. 2001; 81: 1-8Crossref PubMed Scopus (5) Google Scholar, 5Voyno-Yasenetskaya T. Conklin B.R. Gilbert R.L. Hooley R. Bourne H.R. Barber D.L. J. Biol. Chem. 1994; 269: 4721-4724Abstract Full Text PDF PubMed Google Scholar), activation of phospholipase C-ϵ (6Lopez I. Mak E.C. Ding J. Hamm H.E. Lomasney J.W. J. Biol. Chem. 2001; 276: 2758-2765Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), activation of phospholipase D (7Plonk S.G. Park S.K. Exton J.H. J. Biol. Chem. 1998; 273: 4823-4826Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), down-regulation of cell-cell adhesion (8Meigs T.E. Fedor-Chaiken M. Kaplan D.D. Brackenbury R. Casey P.J. J. Biol. Chem. 2002; 277: 24594-24600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), activation of radixin (9Vaiskunaite R. Adarichev V. Furthmayr H. Kozasa T. Gudkov A. Voyno-Yasenetskaya T.A. J. Biol. Chem. 2000; 275: 26206-26212Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and modulation of tight junction-mediated paracellular permeability (10Meyer T.N. Schwesinger C. Denker B.M. J. Biol. Chem. 2002; 277: 24855-24858Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Furthermore, mutationally activated Gα12 and Gα13 have been demonstrated to elicit oncogenic transformation of several cell lines (11Jiang H. Wu D. Simon M.I. FEBS Lett. 1993; 330: 319-322Crossref PubMed Scopus (101) Google Scholar, 12Xu N. Bradley L. Ambdukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar). Although a number of proteins involved in these processes have been shown to bind directly to activated (GTP-liganded) Gα12 and/or Gα13 (13Kurose H. Life Sci. 2003; 74: 155-161Crossref PubMed Scopus (91) Google Scholar), the roles of these effector proteins in mediating particular G12-dependent cellular events are still largely undefined. Two subsets of G12 effectors that are the best characterized are a class of Rho-specific guanine nucleotide exchange factors that includes p115RhoGEF, leukemia-associated RhoGEF (LARG), and PDZ-RhoGEF, as well as members of the cadherin superfamily of cell surface adhesion proteins (14Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Crossref PubMed Scopus (211) Google Scholar, 15Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 16Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar, 17Kaplan D.D. Meigs T.E. Casey P.J. J. Biol. Chem. 2001; 276: 44037-44043Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 18Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (740) Google Scholar, 19Meigs T.E. Fields T.A. McKee D.D. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 519-524PubMed Google Scholar). Cell-free studies using purified proteins have demonstrated that Gα13 directly stimulates the ability of p115RhoGEF to enhance guanine nucleotide exchange on the monomeric G protein RhoA and, reciprocally, that p115RhoGEF binding accelerates the rate of GTP hydrolysis by Gα13 and Gα12 (16Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar, 18Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (740) Google Scholar). Also, activated Gα12 and Gα13 have been demonstrated to bind the cytoplasmic domain of several cadherins in vitro, and expression of mutationally activated Gα12 and Gα13 in cells disrupts the extracellular adhesive function of epithelial cadherin (E-cadherin) in a manner that requires direct Gα12-cadherin interaction (8Meigs T.E. Fedor-Chaiken M. Kaplan D.D. Brackenbury R. Casey P.J. J. Biol. Chem. 2002; 277: 24594-24600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 19Meigs T.E. Fields T.A. McKee D.D. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 519-524PubMed Google Scholar). Furthermore, binding of activated Gα12 to cadherin results in release of the cytoplasmic protein β-catenin from cadherin, allowing β-catenin to act as a transcriptional activator of genes involved in cell proliferation, differentiation, and oncogenesis (17Kaplan D.D. Meigs T.E. Casey P.J. J. Biol. Chem. 2001; 276: 44037-44043Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 19Meigs T.E. Fields T.A. McKee D.D. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 519-524PubMed Google Scholar). As the number of known G12 binding partners has increased, so has the apparent complexity of the signaling networks that originate with the receptor-driven activation of G12 proteins. To elucidate the biological significance of the interactions between G12 proteins and their various effectors, reagents that selectively manipulate the interaction between Gα12/13 and individual target proteins would be of great value, as these tools could reveal the role of particular G12-effector interactions in specific signaling events. To this end, we have introduced a series of mutations into the primary sequence of Gα12 and from these have identified a mutant that is impaired both in binding Rho-specific guanine nucleotide exchange factors and in activating Rho-mediated signaling pathways. This mutant retains normal binding to E-cadherin as well as the ability to disrupt cadherin function when expressed in cells. This variant of Gα12 provides a novel reagent for dissecting the roles of distinct downstream effector pathways that are triggered following Gα12 activation and also provides important new structure-function information regarding the nature of the interaction between G12 proteins and Rho-specific guanine nucleotide exchange factors. Materials—The Myc-p115RGS plasmid and the plasmid containing a GST fusion to the RGS domain of LARG from Kozasa of The plasmid containing a GST fusion to the domain by The plasmid a from of and the plasmid and from and from and from Science. from and from specific for protein from from of introduced into mutationally activated Gα12 by a the for Gα12QL using the to the This to of the between and of the and as in and to by for with from to a of The containing the on by the sequence T.E. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google and harboring into of the by substitution mutants in Gα12QL as sequence for by the an that the of the sequence by the nucleotide sequence that the amino sequence by the Gα12QL downstream of the also and these used in the to the by The and of the harboring Gα12QL has been (8Meigs T.E. Fedor-Chaiken M. Kaplan D.D. Brackenbury R. Casey P.J. J. Biol. Chem. 2002; 277: 24594-24600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The the variant of Gα12QL into the which also the green fluorescent protein with downstream of a separate of the by The plasmid and the plasmid by into purified by The in cells and purified using the produced using the purified by the and used as a of glutathione fusion of the N-terminal domain of p115RhoGEF produced by of this domain from the Myc-p115RGS plasmid with into the to facilitate its into the plasmid This by of GST to p115RGS, to the cytoplasmic domain of E-cadherin T.E. Fields T.A. McKee D.D. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 519-524PubMed Google Scholar), and to the RGS domain of LARG produced in cells and purified using as T.E. Fields T.A. McKee D.D. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 519-524PubMed Google Scholar). domain purified as G. J. Biol. Chem. 280: Full Text Full Text PDF PubMed Scopus Google Scholar). in to with or various mutants of Gα12QL using reagent to the cells with phosphate-buffered by in PBS, by and in with the and and for This for and the using of this and the into that purified GST fusion proteins on for and by and with of and to and as (17Kaplan D.D. Meigs T.E. Casey P.J. J. Biol. Chem. 2001; 276: 44037-44043Abstract Full Text Full Text PDF PubMed Scopus (46) Google in to Gα12QL or its binding of the proteins produced using a in vitro to the into and with GST fusion proteins as and proteins by and in and by cells with the plasmid the for downstream of serum response and the plasmid the for downstream of a kinase a plasmid Gα12QL or a mutant cells with and in the for an with and in for by and by for and using a for the using a cells a of on and to for with harboring a a variant of or to for and cells for rounding using an cells a of in and to for with harboring a a variant or cells for and for an activation as G. J. Biol. Chem. 280: Full Text Full Text PDF PubMed Scopus Google Scholar). cells with while on and to the for and for for protein from to for RhoA and of protein with the GST fusion of the domain of on for and the by and with in by and to to c-jun N-terminal cells in and to for with harboring a a variant or to for and cells for with while on and c-jun N-terminal kinase TPCK, TLCK, to the for and for for protein and of protein from by and to to of cells and E-cadherin with harboring a variant of or a cells to as (8Meigs T.E. Fedor-Chaiken M. Kaplan D.D. Brackenbury R. Casey P.J. J. Biol. Chem. 2002; 277: 24594-24600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). and cells on in PIPES, and for in the containing by a in containing by in containing of containing serum cells for and primary to β-catenin a in an cells in and a in serum for as and for in a in with and using using an To of Gα12 for its coupling to downstream effector proteins, we a series of substitution mutants the mutationally activated form of To for of these of the proteins expressed in cells. To the expressed Gα12QL from a into Gα12QL the the domain of the of Gα proteins have that this region is from the and known of the and this region used to into interaction between and its effector protein, phospholipase T.E. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Gα12QL expressed in cells binding to the Gα12 effectors E-cadherin and p115RhoGEF which to that of Gα12QL to Gα12 from the RGS protein p115RhoGEF by a the region of Gα12 to a Gα in cells and have been uncoupled from RGS proteins by this G. M.I. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). this This as produced in cells and from of the and binding to GST of the cytoplasmic domain of E-cadherin or the N-terminal RGS domain of p115RhoGEF As shown in Gα12QL to both of these effector proteins, and the apparent of for both effector proteins the mutant of Gα12 the of altered function we we on a to mutant of Gα12 impaired in effector binding. To this end, we produced a series of substitution mutants the region of Gα12 that of the that are known to play a role in interactions H. Hamm H.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Gilman A.G. Science. 1997; PubMed Scopus Google Scholar). To a of amino acids in the primary sequence of Gα12QL by the sequence which is to be a well substitution in proteins of its in both and S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This has been to in the protein S. A. 1998; PubMed Scopus Google and in C. Cell. Biol. 2001; PubMed Scopus Google Scholar). The of Gα12 mutants expressed in cells and from The of plasmid used for cells to of Gα12 as by Gα12 for binding to p115RhoGEF and E-cadherin as that the primary sequence of Gα12 from of the region to downstream of the region of these proteins to GST a protein and The of these binding to the and proteins that from that of Gα12QL an results for the variant in are a in binding to both effector an of this is the variant shown in This a of the on the of and these one in a of binding to p115RhoGEF while retaining normal binding to This variant also to GST of the cytoplasmic of cadherin and The region of Gα12 altered in the variant downstream of the region of Gα12 To that the binding of Gα12QL or its to these effector proteins, several of the Gα proteins, and the produced the in a these proteins for binding to and the results in on these we this variant of Gα12QL as to of its interaction with this is used for the of this The impaired binding of the variant to p115RhoGEF that this protein would be functionally uncoupled from To this we the ability of to serum response in cells. Gα12 signaling to has been well characterized as a Rho-dependent pathway of its to specific of C. S. N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus (196) Google Scholar). cells with a plasmid harboring a downstream of an with a plasmid Gα12QL an in This by the of p115RGS, which the G domain of p115RhoGEF and as a by activated Gα12 T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (740) Google Scholar, S. H. Kozasa T. J.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). to the results with expression of to stimulate transcriptional activation of cell that expressed with Gα12QL in these The G12 subfamily α-subunit Gα13 stimulates the ability of p115RhoGEF to trigger guanine nucleotide exchange on the monomeric G protein Although Gα12 with the RGS domain of p115RhoGEF in vitro (16Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google are on the ability of Gα12 to stimulate activation through has that a Rho-specific guanine nucleotide exchange LARG, is functionally activated by Gα12 in cells N. S. H. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus (174) Google Scholar). we Gα12QL and the variant for interaction with As shown in Gα12QL by an GST fusion of the RGS domain of LARG, binding to this LARG fusion protein, of cell expression of these Gα proteins the variant is in binding to both p115RhoGEF and the Two well of cellular signaling through RhoA are cell rounding (8Meigs T.E. Fedor-Chaiken M. Kaplan D.D. Brackenbury R. Casey P.J. J. Biol. Chem. 2002; 277: 24594-24600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, U. Simon M.I. Schultz G. Offermanns S. J. Biol. 1999; PubMed Scopus Google Scholar, M. T.M. C. D. Brown J.H. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus (91) Google and the activation of L.R. A. M. Brown J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. Johnson G.L. Dhanasekaran N. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus (174) Google Scholar). as of the variant is uncoupled from Rho-mediated signaling we the of Gα12QL and to stimulate these responses when expressed in cells. As shown in and cells with a Gα12QL a from a to a a such cells with an the variant the that the cells cells expressed of the variant as of Gα12QL To for activation, cells with the and cells and to using an that the form of Although Gα12QL the of activated in these expression of the variant to an in the we also directly the ability of to RhoA in cells through using a GST fusion of the domain of Although expression of Gα12QL to an in the of activated RhoA as the of activated RhoA in cells to that in cells The that the variant normal binding to the cytoplasmic domain of E-cadherin that signaling to by this Gα12 mutant its ability to cadherin function would be To this in a cellular we cell-cell adhesion using cells cells have been shown to form in a that be upon of activated Gα12QL a that the ability of the activated Gα protein to trigger release of β-catenin from the cytoplasmic domain of E-cadherin (8Meigs T.E. Fedor-Chaiken M. Kaplan D.D. Brackenbury R. Casey P.J. J. Biol. Chem. 2002; 277: 24594-24600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). with cells E-cadherin in a in the of of these in cells E-cadherin Although of cells with a to the with an Gα12QL a in as (8Meigs T.E. Fedor-Chaiken M. Kaplan D.D. Brackenbury R. Casey P.J. J. Biol. Chem. 2002; 277: 24594-24600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Importantly, results when the variant expressed in these this protein of to the as Gα12QL that the mutant the ability to cadherin function in an of the in the and that the cell-cell interactions by As in the activated Gα12 disrupts the interaction between cadherin and its cytoplasmic protein β-catenin in a manner that requires direct Gα12-cadherin interaction (17Kaplan D.D. Meigs T.E. Casey P.J. J. Biol. Chem. 2001; 276: 44037-44043Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 19Meigs T.E. Fields T.A. McKee D.D. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 519-524PubMed Google Scholar). This of Gα12QL binding to cadherins be directly in cells by following the in of β-catenin from a to a the cell when activated Gα12 is introduced T.E. Fields T.A. McKee D.D. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 519-524PubMed Google Scholar). cells and cells with a β-catenin to the cell in cells Gα12QL this to the in the and cells Δp115-Gα12QL, the of β-catenin that of cells Gα12QL that the variant retains the ability to interact with the cadherin cytoplasmic domain in cells and trigger a of The of this to the heterotrimeric G protein Gα12 that are for its interaction with certain downstream effector proteins. To this end, a series of of Gα12 by of amino acids with the well amino This has in of the protein involved in interaction with the transcriptional activator and in important of S. A. 1998; PubMed Scopus Google Scholar, C. Cell. Biol. 2001; PubMed Scopus Google Scholar). this of proteins, we identified a mutant that is impaired in binding to the well characterized G12 effector proteins p115RhoGEF and LARG and This mutant is and functionally uncoupled from Rho-mediated as lacks the ability to bind to these exchange factors also to stimulate activation in cells as well as several known Rho-mediated cellular responses. The that uncoupled this Gα12 variant from p115RhoGEF is a of amino acids downstream of the region of the regions of Gα proteins are well as important for binding to many effectors H. Hamm H.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Gilman A.G. Science. 1997; PubMed Scopus Google Scholar), is that such an in to one of these regions would effector binding. a mutant of Gα13 in which a the region to an to be functionally uncoupled from Rho-mediated signaling pathways S. S. N. Kozasa T. PubMed Scopus Google Scholar). Importantly, the variant retains interaction with one of its effector proteins, This that the binding for p115RhoGEF is distinct or a the E-cadherin binding on the variant a for dissecting the roles of activation in particular Gα12-mediated cellular events such as cell proliferation, oncogenic and activation of phospholipase of in this is that the mutant produced the region of Gα12QL the ability of Gα12QL to bind the domain This amino substitution to a that has been shown to Gα and from R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and also a that the Gα protein, from its RGS protein, G. M.I. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). This RGS domain of p115RhoGEF has to RGS proteins, and also to Gα12 and Gα13 and accelerates GTP hydrolysis of these proteins (16Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (675) Google Scholar, 18Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (740) Google p115RhoGEF is a RGS protein Sci. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). that of this in the region of Gα12 to a interaction that the nature of this is from the between the subfamily proteins and their RGS proteins. is that proteins of the G12 subfamily are involved in a of cell biological signaling and and cytoskeletal Furthermore, these proteins have been shown to interact with an of signaling proteins. of the signaling networks from the activation of Gα12 will reagents that of this G protein from particular effector proteins. mutants of Gα12 that have the ability to interact with one or effectors while retaining normal for effectors will tools for such The a this in the form of a mutant of Gα12 that is uncoupled from a well characterized signaling Rho-mediated while retaining the ability to through the and for for with RhoA activation and Fields for and for