Title: 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. 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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 were gifts from Tohru Kozasa (University of Illinois, Chicago). The pGEX-2T plasmid containing a GST fusion to the rhotekin RhoA-binding domain (GST-RBD) was kindly provided by Robert Lefkowitz (Duke University, Durham, NC). The reporter plasmid SRE-L was a gift from Channing Der (University of North Carolina, Chapel Hill), and the internal control reporter plasmid pRL-TK and dual-luciferase system were purchased from Promega. Anti-Gα12 and anti-RhoA antibodies were purchased from Santa Cruz Biotechnology and anti-Myc antibody from Roche Applied Science. Anti-β-catenin antibody was purchased from Zymed Laboratories Inc., and Cy3-conjugated secondary antibody from Jackson Immunoresearch (West Grove, PA). Mouse monoclonal antibody specific for phospho-SAPK (stress-activated protein kinase)/JNK (Thr183/Thr185) was purchased from Cell Signaling Technology (Beverly, MA). Protease inhibitors were purchased from Sigma. Construction of Plasmids—The Myc epitope tag (EQKLISEEDL) was introduced into mutationally activated Gα12 (Gα12QL) by first creating a silent AgeI restriction site within the cDNA for Gα12QL using the QuikChange site-directed mutagenesis kit (Promega) according to the manufacturer's instructions. This site was positioned to allow insertion of the Myc tag between proline 139 and valine 140 of Gα12QL. Next, the oligonucleotides 5′-ccggtatcaggaggtggtggatctgagcagaagctgatcagcgaggaggacctgtcaggtggaggaggttca-3′ and 5′-ccggtgaacctcctccacctgacaggtcctcctcgctgatcagcttctgctcagatccaccacctcctgata-3′ were synthesized as 5′-end phosphorylated forms, combined at 1 μm each in 10 mm Tris, pH 7.5, 1 mm EDTA, and allowed to anneal by incubation at 95 °C for 5 min with subsequent cooling from 85 to 30 °C over a period of ∼4 h. The resulting double-stranded oligonucleotide, containing the Myc tag flanked on each side by the flexible linker sequence SGGGGS (20Hughes T.E. Zhang H. Logothetis D.E. Berlot C.H. J. Biol. Chem. 2001; 276: 4227-4235Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and harboring appropriate 4-base overhangs, was then ligated into AgeI-digested Gα12QL. Correct orientation of the Myc tag was verified by sequencing. NAAIRS substitution mutants were generated in Myc-tagged Gα12QL as follows. For each 6-amino acid sequence designated for replacement by the sextet Asn-Ala-Ala-Ile-Arg-Ser, an oligonucleotide was designed that contained the 15 nucleotides immediately upstream of the designated 6-codon sequence within Gα12QL, followed by the nucleotide sequence 5′-aatgctgctatacgatcg-3′ that encodes the amino acid sequence NAAIRS, followed by the 15 nucleotides within Gα12QL immediately downstream of the 6-codon sequence. An antiparallel, precisely complementary oligonucleotide was also synthesized, and these two oligonucleotides were used in the QuikChange procedure to introduce the desired NAAIRS substitution. All constructs were verified by sequencing. The construction and purification of the adenovirus harboring Gα12QL has been described previously (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 cDNA encoding the Δp115 variant of Gα12QL (see "Results") was subcloned into the adenoviral shuttle vector pAdTrak-CMV, which also contains the green fluorescent protein (GFP) cDNA, with each cDNA positioned downstream of a separate cytomegalovirus promoter. Correct subcloning of the cDNA was confirmed by sequencing. The recombinant pAdTrak plasmid and the adenoviral backbone plasmid pAdEasy-1 were co-transformed by electroporation into competent BJ5183 Escherichia coli. Recombinant viral DNA was purified by cesium chloride density centrifugation. The recombinant adenovirus was amplified in HEK293 cells and purified using the Adeno-X virus purification kit (BD Biosciences). A recombinant adenovirus produced using the empty pAdTrack-CMV vector was purified by the same procedure and used as a control. Production of Recombinant Proteins—The glutathione S-transferase (GST) fusion of the N-terminal domain of p115RhoGEF (p115RGS) was produced by PCR amplification of this domain (residues 1–252) from the Myc-p115RGS plasmid with restriction sites incorporated into the amplified product to facilitate its subcloning into the plasmid pGEX-2T (Amersham Biosciences). This construct was verified by sequencing. Fusions of GST to p115RGS, to the cytoplasmic domain of E-cadherin (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), and to the RGS domain of LARG were produced in BL21-DE3 Gold cells (Stratagene) and purified using glutathione-coated Sepharose (Amersham Biosciences) as described previously (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). GST-rhotekin RhoA-binding domain was purified as described previously (21Barnes W.G. Reiter E. Violin J.D. Ren X.R. Milligan G. Lefkowitz R.J. J. Biol. Chem. 2005; 280: 8041-8050Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In Vitro Binding Assays—HEK293 cells, grown in 6-well plates to ∼70% confluency, were transfected with either Myc-tagged Gα12QL, empty pcDNA3.1 plasmid, or various NAAIRS mutants of Myc-tagged Gα12QL using Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. Approximately 48 h post-transfection, cells were washed twice with phosphate-buffered saline (PBS), harvested by scraping in PBS, pelleted by centrifugation at 800 × g, and then resuspended in lysis buffer (50 mm HEPES, pH 8.0, 1 mm EDTA, 3 mm dithiothreitol, 10 mm MgSO4, 1% polyoxyethylene-10-lauryl ether) supplemented with the protease inhibitors TPCK (61 μm), TLCK (58 μm), and phenylmethylsulfonyl fluoride (267 μm), and continually inverted at 4 °C for 30 min. This lysate was centrifuged at 100,000 × g for 45 min at 4 °C, and the resulting supernatant was diluted 10-fold using lysis buffer lacking polyoxyethylene-10-lauryl ether. An aliquot of this solution was set aside, and the remainder was divided equally into tubes that received purified GST fusion proteins immobilized on glutathione-Sepharose (see above). Samples were continually inverted for 2 h at 4 °C, and then glutathione-Sepharose was pelleted by centrifugation at 1300 × g and washed four times with 1 ml of reduced-detergent lysis buffer (containing 0.1% polyoxyethylene-10-lauryl ether). Pelleted material was resuspended and subjected to SDS-PAGE and immunoblot analysis as described (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) in order to detect Gα12QL or its NAAIRS variants. For binding assays utilizing untagged 35S-labeled variants of Gα12QL, the proteins were produced using a TnT in vitro coupled transcription/translation system (Promega) according to the manufacturer's instructions. Reactions were diluted into reduced detergent lysis buffer (see above) and incubated with GST fusion proteins as described above, and then proteins were separated by SDS-PAGE and gels fixed in 10% acetic acid, 1% glycerol, dried under vacuum, and analyzed by autoradiography. Luciferase Reporter Assays—HEK293 cells were transfected with the SRE-L plasmid (containing the cDNA for firefly luciferase positioned downstream of serum response element) and the pRL-TK plasmid (containing the cDNA for Renilla luciferase positioned downstream of a thymidine kinase promoter) plus a plasmid encoding Gα12QL or a mutant variant. Approximately 36 h post-transfection, cells were washed with serum-free Dulbecco's modified Eagle's medium and then incubated in the same medium for an additional 16 h. Cells were washed with PBS and then incubated in Passive® lysis buffer (Promega) for 20 min. Lysates were cleared by centrifugation and then assayed by luminometry for firefly and Renilla luciferase activities using a dual-luciferase assay system (Promega). Firefly luciferase activity measurements were normalized for the corresponding Renilla luciferase values. Measurements were performed using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). Cell Rounding Assays—MDA-MB-231 cells were seeded at a density of 200,000 cells/dish on 35-mm glass-bottom Petri dishes (MatTek, Ashland, MA) and allowed to grow for 24 h. Cells were infected with adenovirus harboring a cDNA encoding GFP plus either Gα12QL, a variant of Gα12QL, or no cDNA. Infections were allowed to proceed for 4 h, and then cells were serum-starved for 15–16 h. Cell rounding phenotype was visualized using an Eclipse TE300 inverted microscope (Nikon). Rho Activation Assays—MDA-MB-231 cells were seeded at a density of ∼250,000 cells/well in 6-well plates and allowed to grow for ∼24 h. Cells were infected with adenovirus harboring a cDNA encoding GFP plus either Gα12QL, a variant thereof, or no cDNA. Infected cells were incubated for 4 h and then serum-starved for an additional 17–20 h. Rho activation assays were performed as described previously (21Barnes W.G. Reiter E. Violin J.D. Ren X.R. Milligan G. Lefkowitz R.J. J. Biol. Chem. 2005; 280: 8041-8050Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Briefly, cells were washed quickly with PBS while on ice, and cold lysis buffer (25 mm HEPES, pH 8.0, 150 mm NaCl, 10 mm MgCl2, 1 mm EDTA, 1% Nonidet P-40, 2% glycerol, 10 μg/ml leupeptin, 10 μg/ml aprotinin) was added to the wells. Cells were lysed for 10 min and then centrifuged at 16,000 × g for 5 min at 4 °C. Supernatants were assayed for protein concentration. An aliquot was saved from each lysate to assay for total endogenous RhoA levels, and equal amounts of protein incubated with the GST fusion of the RhoA-binding domain of rhotekin immobilized on glutathione-Sepharose. Samples were mixed for 1 h at 4 °C, and then the glutathione-Sepharose was pelleted by centrifugation at 700 × g and washed three times with cold lysis buffer. Pelleted material was resuspended in SDS-PAGE sample buffer, separated by gel electrophoresis, and subjected to immunoblot analysis to detect RhoA. c-jun N-terminal Kinase Activation Assays—MDA-MB-231 cells were seeded at ∼250,000 cells/well in 6-well plates and allowed to grow for 24 h. Cells were infected with adenovirus harboring a cDNA encoding GFP plus either Gα12QL, a variant thereof, or no cDNA. Infections were allowed to proceed for 4 h, and then cells were serum-starved for 24 h. Cells were then washed quickly twice with cold PBS while on ice, and cold c-jun N-terminal kinase (JNK) assay buffer (50 mm Tris, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mm EDTA, 50 mm NaF, 150 mm NaCl, 2 mm DTT, 0.2 mm sodium vanadate, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 3 μg/ml leupeptin, 4 μg/ml aprotinin, 30 μm TPCK, 29 μm TLCK, 133 mm phenylmethylsulfonyl fluoride) was added to the wells. Cells were lysed for 10 min and then centrifuged at 16,000 × g for 5 min at 4 °C. Supernatants were assayed for protein concentration, and then equal amounts of total protein from lysates were separated by SDS-PAGE and subjected to immunoblot analysis to detect levels of phospho-JNK. Cell Aggregation Assays—Parental MDA-MB-435 cells and those stably expressing E-cadherin were infected with recombinant adenoviruses harboring Gα12QL, a variant of Gα12QL, or a control adenovirus. Three days post-infection, cells were subjected to fast-aggregation assays as described previously (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). Indirect Immunofluorescence—DLD-1 and HEK293 cells grown on glass coverslips were washed twice in PHEM buffer (60 mm PIPES, pH 6.9, 25 mm HEPES pH 7.0, 10 mm EGTA, 4 mm MgSO4) and then incubated at 37 °C for 20 min in the same buffer containing 4% paraformaldehyde. Cells were then permeabilized by a 5-min incubation in PHEM buffer containing 0.5% Triton X-100 followed by three 5-min washes in PHEM containing 0.1% Triton X-100. A blocking solution of PHEM buffer containing 10% goat serum (Invitrogen) was added, cells were incubated for 30 min at 37 °C, and then primary antibody to β-catenin was applied at a 1:100 dilution in PHEM buffer plus 5% goat serum. Following an overnight incubation at 4 °C, cells were washed three times in PHEM plus 0.1% Triton X-100, and then Cy3-conjugated secondary antibody was applied at a 1:500 dilution in PHEM buffer plus 5% goat serum for 1 h. Cells were then washed three times as described above and incubated for 5 min in Hoechst stain (Molecular Probes, Eugene, OR) at a 1:1000 dilution in PHEM buffer, washed with distilled water, and mounted onto slides using ProLong antifade solution (Molecular Probes). Cells were visualized using an LSM-410 laser scanning confocal microscope (Carl Zeiss). To identify structural determinants of Gα12 necessary for its coupling to downstream effector proteins, we designed a series of substitution mutants within the mutationally activated (QL) form of Gα12. To allow for proper post-translational modification (e.g. acylation) of these variants of Gα12QL, the proteins were expressed in HEK293 cells. To distinguish the ectopically expressed Gα12QL variants from endogenous, wild-type Gα12, a Myc epitope tag was inserted into Gα12QL at the αB/αC loop within the helical domain of the protein. Structural analyses of other Gα subunit proteins have revealed that this highly conserved region is spatially removed from the GTP-binding and known effector-binding domains of the α-subunits, and this region was used to insert GFP into Gαq without disrupting interaction between Gαq and its effector protein, phospholipase C-β (20Hughes T.E. Zhang H. Logothetis D.E. Berlot C.H. J. Biol. Chem. 2001; 276: 4227-4235Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Myc-tagged Gα12QL expressed in HEK293 cells exhibited binding to the Gα12 effectors E-cadherin and p115RhoGEF (Fig. 1A), which was essentially identical to that of untagged Gα12QL (data not shown). We first attempted to uncouple Gα12 from the G12-specific RGS protein p115RhoGEF by converting a highly conserved glycine within the "Switch I" region of Gα12 to a serine, because other Gα subunits in mammalian cells and yeast have been uncoupled from RGS proteins by this alteration (22DiBello P.R. Garrison T.R. Apanovitch D.M. Hoffman G. Shuey D.J. Mason K. Cockett M.I. Dohlman H.G. J. Biol. Chem. 1998; 273: 5780-5784Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 23Lan K.L. Sarvazyan N.A. Taussig R. Mackenzie R.G. DiBello P.R. Dohlman H.G. Neubig R.R. J. Biol. Chem. 1998; 273: 12794-12797Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). We generated this Gly-to-Ser mutation within Myc-tagged Gα12QL. This variant, denoted as G208S-Gα12QL, was produced in HEK293 cells and extracted from membrane preparations of the cells, and then binding to GST fusions of either the cytoplasmic C-terminal domain of E-cadherin or the N-terminal RGS domain of p115RhoGEF was assessed. As shown in Fig. 1A, Myc-tagged Gα12QL bound strongly to both of these effector proteins, and the apparent affinity of G208S-Gα12QL for both effector proteins was not significantly changed. Because the single point mutant of Gα12 did not yield the type of altered function we sought, we embarked on a more global strategy to identify mutant forms of Gα12 impaired in effector binding. To this end, we produced a series of substitution mutants within the region of Gα12 that encompasses all three of the "Switch regions" that are known to play a critical role in other Gα-effector interactions (24Skiba N.P. Bae H. Hamm H.E. J. Biol. Chem. 1996; 271: 413-424Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 25Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (676) Google Scholar). To produce each mutant, a sextet of consecutive amino acids in the primary sequence of Myc-tagged Gα12QL was replaced by the sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS), which is believed to be a well tolerated substitution in proteins because of its appearance in both β-sheet and α-helical secondary structures (26Wilson I.A. Haft D.H. Getzoff E.D. Tainer J.A. Lerner R.A. Brenner S. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5255-5259Crossref PubMed Scopus (133) Google Scholar). This NAAIRS mutagenesis strategy has been successfully employed to dissect functional domains in the retinoblastoma protein (27Sellers W.R. Novitch B.G. Miyake S. Heith A. Otterson G.A. Kaye F.J. Lassar A.B. Kaelin Jr., W.G. Genes Dev. 1998; 12: 95-106Crossref PubMed Scopus (288) Google Scholar) and in telomerase (28Armbruster B.N. Banik S.S. Guo C. Smith A.C. Counter C.M. Mol. Cell. Biol. 2001; 21: 7775-7786Crossref PubMed Scopus (144) Google Scholar). The panel of Gα12 NAAIRS mutants was expressed in HEK293 cells and then extracted from membranes. The amount of plasmid DNA used for transfecting cells was varied to achieve similar levels of ectopic Gα12 expression, as determined by immunoblot analysis (data not shown). These Gα12 variants were screened for binding to p115RhoGEF and E-cadherin as described above. In all, 13 variants were analyzed that covered the primary sequence of Gα12 from just upstream of the Switch I region to just downstream of the Switch III region (see Fig. 1B). None of these proteins bound to immobilized GST lacking a protein adduct (Fig. 1A, and data not shown). The majority of these variants exhibited binding to the GST-p115RGS and GST-E-cadherin proteins that was not markedly different from that of parental Gα12QL (for an example, see results for the Δ238–243 variant in Fig. 1A; others are not shown). A few additional variants showed a marked decrease in binding to both effector proteins; an example of this is the Δ196–201 variant shown in Fig. 1A. This may reflect a nonspecific, global effect of the mutation on the structure of Gα12, and therefore these variants were not pursued further. However, one NAAIRS variant, designated Δ244–249 in Fig. 1A, showed a nearly complete loss of binding to p115RhoGEF while retaining normal binding to E-cadherin. This variant also bound to GST fusions of the cytoplasmic domains of neural cadherin and cadherin-14 (data not shown). The region of Gα12 altered in the Δ244–249 variant lies immediately downstream of the Switch II region of Gα12 (Fig. 1B). To ensure that the Myc epitope ta