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• W2040086538 abstract "The α-subunit of G proteins of the G12/13 family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of Gα12/13 to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between Gα13 and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of Gαi proteins. Using Gαi2, which does not bind RGL domains, as the backbone in which Gα13 sequences were swapped or mutated, we observed that the switch region of Gα13 is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of Gα13 was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of Gα13 and chimeric Gαi2 proteins was also necessary for Rho activation. These findings revealed the structural features by which Gα13 interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs. The α-subunit of G proteins of the G12/13 family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of Gα12/13 to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between Gα13 and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of Gαi proteins. Using Gαi2, which does not bind RGL domains, as the backbone in which Gα13 sequences were swapped or mutated, we observed that the switch region of Gα13 is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of Gα13 was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of Gα13 and chimeric Gαi2 proteins was also necessary for Rho activation. These findings revealed the structural features by which Gα13 interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs. Rho GTPases, which include Rho, Rac, and Cdc42, play a central role in the regulation of a number of basic cellular events such as cell movement and changes in cell shape, as well as in the control of gene expression regulation and cell growth (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3768) Google Scholar). These GTP-binding proteins act as molecular switches that are inactive in their GDP-bound form, and upon exchange of GDP for GTP, they adopt an active conformation in which they can interact with their specific effector molecules, thereby affecting their localization and/or activity (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3768) Google Scholar, 2Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (967) Google Scholar, 3Burridge K. Wennerberg K. Cell. 2004; 116: 167-179Abstract Full Text Full Text PDF PubMed Scopus (1492) Google Scholar). This nucleotide exchange is promoted by a large family of guanine nucleotide exchange factors (GEFs), 1The abbreviations used are: GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; DH, dbl-homology; GPCR, G protein-coupled receptor; SRE, serum responsive element; GAP, GTPase-activating protein; RGL, RGS-like; HEK, human embryonic kidney. 1The abbreviations used are: GEF, guanine nucleotide exchange factor; PH, pleckstrin homology; DH, dbl-homology; GPCR, G protein-coupled receptor; SRE, serum responsive element; GAP, GTPase-activating protein; RGL, RGS-like; HEK, human embryonic kidney. the vast majority of which are characterized by the presence of a dbl-homology (DH) and pleckstrin homology (PH) domain (2Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (967) Google Scholar, 4Erickson J.W. Cerione R.A. Biochemistry. 2004; 43: 837-842Crossref PubMed Scopus (115) Google Scholar). These GEFs also exhibit a number of additional regulatory regions by which they are strictly controlled by a diverse array of upstream signaling pathways, including those initiated by cell adhesion molecules, tyrosine kinase growth factor receptors, as well as by G protein-coupled receptors (GPCRs) (2Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (967) Google Scholar, 5Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (295) Google Scholar). In particular for Rho, this GTPase participates in many physiological and pathological processes that involve the activation of GPCRs. For example, GPCRs such as those for thrombin and lysophosphatidic acid (LPA) promote cytoskeletal changes and expression from serum responsive element (SRE)-regulated genes by activating Rho (6Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 7Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (195) Google Scholar). Rho also participates in platelet aggregation (8Moers A. Nieswandt B. Massberg S. Wettschureck N. Gruner S. Konrad I. Schulte V. Aktas B. Gratacap M.P. Simon M.I. Gawaz M. Offermanns S. Nat. Med. 2003; 9: 1418-1422Crossref PubMed Scopus (208) Google Scholar) and in smooth muscle contraction when elicited by a large number of vasoactive hormones that act on GPCRs (9Gohla A. Schultz G. Offermanns S. Circ. Res. 2000; 87: 221-227Crossref PubMed Scopus (195) Google Scholar). The pathway by which these GPCRs stimulate Rho involves the activation of α-subunits of the G12/13 and Gq family of heterotrimeric G proteins. Gα12/13 in turn stimulate Rho through the direct interaction with a group of Rho GEFs characterized by the presence of a RGS-like (RGL) domain (10Hart 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 (671) Google Scholar, 11Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Crossref PubMed Scopus (209) Google Scholar, 12Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar), whereas Gαq activates Rho through a still not fully understood mechanism (13Booden M.A. Siderovski D.P. Der C.J. Mol. Cell. Biol. 2002; 22: 4053-4061Crossref PubMed Scopus (147) Google Scholar, 14Chikumi H. Vazquez-Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The family of RGL-containing Rho GEFs comprises three members: PDZ-RhoGEF (PRG) and LARG, which contain an N-terminal PDZ domain, and p115-RhoGEF (p115), which lacks this N-terminal protein-protein interaction domain (10Hart 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 (671) Google Scholar, 11Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Crossref PubMed Scopus (209) Google Scholar, 12Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). The PDZ domain of PRG and LARG mediates the interaction of these GEFs with membrane receptors including plexins of the B family and insulin-like growth factor receptor (15Swiercz J.M. Kuner R. Behrens J. Offermanns S. Neuron. 2002; 35: 51-63Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 16Perrot V. Vazquez-Prado J. Gutkind J.S. J. Biol. Chem. 2002; 277: 43115-43120Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 17Taya S. Inagaki N. Sengiku H. Makino H. Iwamatsu A. Urakawa I. Nagao K. Kataoka S. Kaibuchi K. J. Cell Biol. 2001; 155: 809-820Crossref PubMed Scopus (95) Google Scholar, 18Aurandt J. Vikis H.G. Gutkind J.S. Ahn N. Guan K.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12085-12090Crossref PubMed Scopus (150) Google Scholar). The RGL domain is followed by DH and PH homology domains, by which they promote the nucleotide exchange on Rho, and a long C-terminal domain that harbors regulatory properties (19Fukuhara S. Chikumi H. Gutkind J.S. Oncogene. 2001; 20: 1661-1668Crossref PubMed Scopus (189) Google Scholar, 20Chikumi H. Barac A. Behbahani B. Gao Y. Teramoto H. Zheng Y. Gutkind J.S. Oncogene. 2004; 23: 233-240Crossref PubMed Scopus (88) Google Scholar, 21Barac A. Basile J. Vazquez-Prado J. Gao Y. Zheng Y. Gutkind J.S. J. Biol. Chem. 2004; 279: 6182-6189Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The RGL domain is directly recognized by receptor-stimulated Gα12/13, thus providing a molecular bridge for the activation of Rho by Gα12/13 (19Fukuhara S. Chikumi H. Gutkind J.S. Oncogene. 2001; 20: 1661-1668Crossref PubMed Scopus (189) Google Scholar), and in the case of p115, this domain also acts as a GTPase-activating protein (GAP) for Gα13 (22Kozasa 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 (733) Google Scholar). However, this GAP activity is not required to couple G13 to Rho activation, as p115 mutants that possess a reduced GAP activity toward Gα13 and a decreased binding ability for this α-subunit, still exhibit a normal ability to stimulate Rho exchange when activated by Gα13 (23Chen Z. Singer W.D. Wells C.D. Sprang S.R. Sternweis P.C. J. Biol. Chem. 2003; 278: 9912-9919Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The Gα subunit is composed by a α-helical domain and a GTPase domain, which includes a switch region that changes conformation upon nucleotide exchange, and is directly involved in binding and hydrolysis of GTP (24Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (929) Google Scholar, 25Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (745) Google Scholar). In the inactive heterotrimer the Gα subunit, with GDP bound to it, keeps stable interactions with the heterodimer Gβγ. Therefore, the effector motifs are covered in the heterotrimer, rendering both Gα and Gβγ unable to activate their respective effectors. Agonists acting on GPCRs induce a structural change that results in the nucleotide exchange and the dissociation of the heterotrimer, allowing the activation of Gα- and Gβγ-dependent effectors. Signaling is terminated by the hydrolysis of GTP to GDP by virtue of the intrinsic GTPase activity of Gα subunits, which is further stimulated by the interaction with regulators of G protein signaling (RGS proteins). In this regard, the crystal structure of the complex formed by RGS4-Gαi (26Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar), and that of the RGL domains of PRG and p115 (27Longenecker K.L. Lewis M.E. Chikumi H. Gutkind J.S. Derewenda Z.S. Structure (Camb.). 2001; 9: 559-569Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 28Chen Z. Wells C.D. Sternweis P.C. Sprang S.R. Nat. Struct. Biol. 2001; 8: 805-809Crossref PubMed Scopus (50) Google Scholar), have provided a model for the likely mechanism by which the activity of Gα subunits is terminated, as well as the process by which Gα13 and RGL-RhoGEFs might establish a functional interaction. Regarding the latter, in the proposed model the region including switch 1 and switch 2 within the GTPase domain of Gα12/13, would be predicted to participate in the interactions with the RGL domain from RGL-RhoGEFs, whereas the N-terminal α-helical domain from Gα12/13 would not establish direct contact interactions with them. As no structural information is yet available for Gα12/13 members, we have addressed in this study the structural requirements for Gα13-dependent stimulation of Rho by engineering chimeric G proteins using Gαi2, which does not activate RGL-RhoGEFs, as the backbone in which Gα13 sequences were swapped or mutated. The emerging results revealed that the entire switch region from Gα13 is necessary but insufficient to exert a Rho-stimulating activity when expressed in the context of Gαi2. In fact, Gα13 depends on most of its GTPase domain, excluding its C-terminal 36 amino acids, for a functional interaction with PRG. Within this region, the integrity of both switch 1 and switch 2 is strictly required for a maximal effect. Furthermore, membrane localization of Gα13 or the Rho-activating chimeric Gαi2 subunits is also necessary for Rho activation, gene expression, and cell transformation. These findings indicate that specific structural features present in Gα13 together with additional molecular interactions occuring at the level of the plasma membrane are required for the effective coupling of Gα13 to the RGL-containing family of RhoGEFs, and ultimately to stimulate Rho. Bioinformatic Tools—The structure of Gαi1/RGS4 (26Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar) was analyzed with the CN3D program (www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml) to identify amino acids in the Gαi-RGS4-contact interface within 3 Å. Sequence alignment corresponding to the switch region from representative members of each Gα protein family was performed using ClustalW (www.ch.embnet.org/software/ClustalW.html) and the figure prepared with Boxshade (www.ch.embnet.org/software/BOX_form.html). DNA Constructs—The cDNAs for Gαi2SWα13QL and Gα13SWαi2QL chimeras were obtained by two consecutive PCR reactions, using human Gαi2- and human Gα13 GTPase-deficient mutants as templates, and cloned into the mammalian expression vector pCEFL2 by EcoRI and XbaI restriction sites that were introduced with the 5′- and 3′-primers, respectively. The cDNAs corresponding to the N-terminal, switch, and C-terminal regions were first obtained by PCR in which the internal primers were designed to overlap with the sequence of the cDNA of the fragments to be fused with in the second PCR reaction. The corresponding chimeric cDNAs were obtained by a second PCR reaction in which the three initial fragments were mixed as templates. The second set of chimeras in which the content of Gα13 was extended from the switch region toward the extremes was also obtained by PCR, in this case the template for the second PCR was the mixture of two cDNA fragments corresponding to either N- or C-terminal domains, as indicated in the respective figures. From this second series, the chimera in which the content of Gα13 extended from the switch region toward the C-terminal end was used as template for further modifications, which included reduction of the contribution of Gα13 at the C-terminal end by substitution with the corresponding sequences from Gαi2; and point mutations at the putative RGL-contact sites within the switch region and at the N-terminal myristylation signal. Point mutations were performed either by PCR to mutate the N-terminal myristylation signal by substituting Gly for Ala in the second codon, and by the QuikChange mutagenesis kit from Stratagene to substitute the putative RGL contact sites, following the manufacturer's instructions. All chimeric and mutant molecules were sequenced at the NIDCR DNA sequencing facility, and their expression was confirmed by Western blot using antibodies detecting the corresponding N-terminal domain. The sequence for the different primers will be made available upon request. Cell Lines and Transfections—Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum. For transient transfections, tissue culture plates were treated for 10 min with phosphate-buffered saline containing 5 μg/ml poly-d-lysine before seeding the cells to prevent them from detaching from the plates during the transfection procedure and thereafter. Transient transfections in HEK 293T cells were performed using the Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. NIH 3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) calf serum, and were used to monitor the transforming potential of the different chimeras as described (7Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (195) Google Scholar). Western Blots and Protein-Protein Interactions—Transfected cells were lysed at 4 °C in a buffer containing 50 mm Tris, pH 7.4, 0.15 m NaCl, 1% Triton X-100, 20 mm β-glycerophosphate, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin, and insoluble material was removed by centrifugation. Lysates containing ∼50 μg of total cellular protein or affinity isolated proteins (see below) were analyzed by Western blotting after SDS-polyacrylamide gel electrophoresis and visualized by enhanced chemiluminescence detection (Amersham Biosciences) using rabbit anti-Gα13 (SC-410, Santa Cruz Biotechnology) or rabbit anti-Gαi2 (SC-7276, Santa Cruz Biotechnology) depending on whether the N-terminal domain of the transfected G protein chimeras was from Gαi2 or Gα13 and goat anti-rabbit (Cappel) IgGs coupled to horseradish peroxidase as a secondary antibody. To test the ability of the different chimeras to interact with RGL domain from PRG, lysates from HEK 293T transfected cells were incubated with bacterially expressed six histidine-tagged recombinant RGL isolated with talon beads (Clontech) as previously described (27Longenecker K.L. Lewis M.E. Chikumi H. Gutkind J.S. Derewenda Z.S. Structure (Camb.). 2001; 9: 559-569Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Luciferase Assays—Cells in 24-well plates were transfected with different expression plasmids together with 0.1 μg of pSRE luciferase reporter plasmid, pNull Renilla, and pcDNAIII-β-gal (a plasmid expressing β-galactosidase) to normalize for transfection efficiency. Fire-fly and Renilla luciferase activities present in cell lysates were assayed using a dual-luciferase reporter system (Promega), and light emission was quantitated using a Monolight 2010 luminometer (Analytical Luminescence Laboratory) as specified by the manufacturer (39Marinissen M.J. Chiariello M. Tanos T. Bernard O. Narumiya S. Gutkind J.S. Mol. Cell. 2004; 14: 29-41Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In Vivo Rho Activation Assay—HEK 293T cells were transfected using the Lipofectamine Plus™ reagent. The day after transfection, cells were cultured for 24 h in serum-free Dulbecco's modified Eagle's medium and assayed for Rho activity using the Rho-binding domain (RBD) of rhotekin bound to glutathione-Sepharose beads to isolate the GTP-bound forms of Rho, as previously described (14Chikumi H. Vazquez-Prado J. Servitja J.M. Miyazaki H. Gutkind J.S. J. Biol. Chem. 2002; 277: 27130-27134Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Briefly, serumstarved HEK-293T cells transfected with the indicated plasmids were lysed at 4 °C in a buffer containing 20 mm HEPES, pH 7.4, 0.1 m NaCl, 1% Triton X-100, 10 mm EGTA, 40 mm β-glycerophosphate, 20 mm MgCl2, 1mm Na3VO4, 1mm dithiothreitol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. Lysates were incubated with GST-rhotekin-RBD previously bound to glutathione-Sepharose beads and washed four times with lysis buffer, and associated GTP-bound forms of Rho were released with protein loading buffer and revealed by Western blot using a monoclonal antibody against RhoA (26C4, Santa Cruz Biotechnology). The content of Rho in total cell lysates was determined as a reference. Cell Fractionation—HEK 293T cells in 10-cm dishes were transfected with the indicated chimeras and grown for 48 h. Cells were washed once with phosphate-buffered saline and then dislodged from the plate by washing and pelleted at low speed. The cell pellet was suspended in 0.5 ml of lysis buffer (50 mm Tris-HCl, pH 8, 2.5 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin), and cells were lysed by 10 passages through a 27-gauge needle. Lysed cells were centrifuged at 2500 rpm for 5 min at 4 °C to remove nuclei and intact cells. The supernatant was centrifuged at 14,000 rpm for 60 min at 4 °C, and the pellet (particulate fraction) was suspended in an equal volume of lysis buffer. The supernatant was further centrifuged at 100,000 × g for 5 min in a Beckman airfuge to obtain the soluble fraction and mixed with Laemmli sample buffer. Fractions were analyzed by Western blotting using anti-Gαi2 antibody as indicated before. Focus Forming Assays—NIH 3T3 cells were transfected by the calcium phosphate precipitation technique with different expression plasmids together with 1 μg of pcDNAIII-β-gal, a plasmid expressing the enzyme β-galactosidase, adjusting the total amount of plasmid DNA with empty vector. The day after transfection, the cells were washed in medium supplemented with 5% calf serum and then maintained in the same medium until foci were scored, 2–3 weeks later. Duplicate plates were fixed with phosphate-buffered saline containing 2% (v/v) formaldehyde and 0.2% (v/v) glutaraldehyde and stained at 37 °C for β-galactosidase activity with a phosphate-buffered saline solution containing 2 mm MgCl2, 5 mm K3Fe(CN)6, 5 mm K4Fe(CN)6, and 0.1% 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) to evaluate the transfection efficiency. The Switch Region of Gα13 Is Required but Not Sufficient for Rho Activation—The structure of the RGL domain from two of the three known members of Gα12/13-responsive RhoGEFs has recently been solved (27Longenecker K.L. Lewis M.E. Chikumi H. Gutkind J.S. Derewenda Z.S. Structure (Camb.). 2001; 9: 559-569Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 28Chen Z. Wells C.D. Sternweis P.C. Sprang S.R. Nat. Struct. Biol. 2001; 8: 805-809Crossref PubMed Scopus (50) Google Scholar). The similarity of the structures of these RGL domains with the known RGS structures raised the possibility that the functional interaction between Gα12/13 and RGL-RhoGEFs might be similar to that of the complex formed by Gαi and RGS4 shown in Fig. 1A (26Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar). Residues in Gαi that provide surface areas in direct contact with RGS4 are indicated. In particular, this region can be subdivided into 3 switch areas, which are highly conserved among each G protein α-subunit family. Of interest, all 6 residues involved in the interaction between Gαi and RGS4 are also conserved in Gαs and Gαq, but 3 of them are quite distinct in Gα12 and Gα13, as depicted in Fig. 1B. Nonetheless, as no information regarding the three-dimensional characteristics of Gα13 family members is available, the molecular basis of their interaction with RGL-Rho-GEFs and the consequent activation of Rho remain to be determined. To begin addressing this issue, we first evaluated the possibility that the switch region from Gα13 contains all the structural elements required to elicit Rho activation. For this purpose, we engineered G protein α-subunits in which the switch region from active Gα13, Gα13QL, replaced the corresponding region from active Gαi2, Gαi2QL (Fig. 1B). The resulting chimera, named Gαi2SWα13QL, contains the first 173 amino acids (Met1-Gln173) from Gαi2 followed by a central part (Asp174-Ile266), including de switch region from Gα13QL, and the C-terminal (Leu267-Phe355) from Gαi2, as indicated. The reciprocal chimera, Gα13SWαi2QL, contains the first 194 amino acids (Met1-Gln194) from Gα13 followed by a central part (Asp195-Ile287), including the three switches, from Gαi2Q205L, and the C terminus (Leu288-Gln377) from Gα13. The activity of these chimeras was determined by their ability to stimulate a mutant SRE that responds to Rho (7Fromm C. Coso O.A. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (195) Google Scholar), and by their ability to interact with the RGL domain of PRG and to stimulate Rho. The different chimeric G proteins were detected by antibodies recognizing the N-terminal domain of either Gαi2 or Gα13 (Fig. 1C). As expected, a GTPase-deficient Gα13 strongly stimulated Rho-dependent pathways, while active Gαi2 was unable to do so. However, surprisingly, a chimeric Gαi2 containing what was expected to be the critical region for the interaction with RGL-RhoGEFs, did not gain the ability to stimulate Rho or to interact with PRG (Gαi2SWα13QL, Fig. 1C). Gα13, on the other hand, lost these properties when its switch region was replaced by that of Gαi2 (Gα13SWαi2QL, Fig. 1C). Direct evaluation of Rho-GTP content in transfected HEK-293T cells indicated that swapping the switches from Gα13 into Gαi2 provided to the resulting chimera only a very limited ability to stimulate Rho, while the reciprocal chimera was unable to do so (Fig. 1C, lower panel). The Structural Requirements for Rho Activation by Gα13 Extend toward the C-terminal Domain of Gα13—As swapping the switch region from Gα13QL into Gαi2QL was not sufficient to confer to Gαi2 the ability to stimulate Rho, we predicted that additional structural elements from Gα13 were required to support this effect. In order to test this possibility, we engineered additional chimeras where the contribution from Gα13QL into the Gαi2 backbone was extended from the switch region toward their N or C termini (Fig. 2, upper panel). Chimeras in which only the N- or C-terminal domains were swapped were also tested as controls. The expression of each chimera was confirmed by Western blotting (Fig. 2, lower panel). By this approach, we found that a chimeric G protein (Gαi2SW-Cα13QL), that includes the switch region and the C-terminal end, was able to stimulate Rho-dependent luciferase reporter gene comparable to that of the GTPase-deficient Gα13QL (Fig. 2, lower panel). The Active Gαi2SW-Cα13QL Chimera Requires the Integrity of the Switch Region and an Adjacent C-terminal Extension—To narrow down the minimal structural requirements for Gα13QL to stimulate Rho, the active chimera (Gαi2SW-Cα13QL) was further modified by replacing additional sequences for those from Gαi2 corresponding to each of the switch subregions, or the C-terminal domain, as indicated in Fig. 3, upper panel. The expression of this set of chimeras was demonstrated by Western blot (Fig. 3, lower panel). When either switch 1 or switch 1 and 2 in Gαi2SW-Cα13QL were substituted by those from Gαi2, the resulting chimeras, Gαi2SW2, 3-Cα13QL and Gαi2QLSW3-Cα13, respectively, were unable to stimulate Rho-dependent pathways (Fig. 3, lower panel). On the other hand, the contribution from Gα13 at the C-terminal end of Gαi2SW-Cα13QL was narrowed down by substituting it for sequences from Gαi2 as indicated in Fig. 3, upper panel. Based on this approach, we observed that the last 36 amino acids in Gαi2SW-Cα13QL (Pro320-Gln356) could be replaced with those from Gαi2 with no reduction in the Rho-stimulating activity of the resulting chimera, Gαi2SW-320α13QL, whereas a further reduction, replacing the last 61 amino-acids (Pro295-Gln356), resulted in a chimeric G protein, Gαi2SW-295α13QL, that was unable to activate Rho. Gαi2SW-320α13QL was therefore referred to herein as the “minimal active chimera” (Fig. 3, lower panel). Point Mutations on the Putative RGL Contact Sites within Switch 1 and 2 of the Minimal Active Chimera Gαi2SW-320α13QL Reduce Its Activity—Based on the structure shown in Fig. 1A (26Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar), amino acids from Gαi2 switch 1 and switch 2 region that were predicted to make contact with RGS4 were re-introduced by site-directed mutagenesis into the equivalent positions in Gαi2SW-320α13QL (Fig. 4, upper panel) (SW1 and SW2 mutants). The expression of these mutants was demonstrated by Western blot (Fig. 4, lower panel). Furthermore, as shown in Fig. 4, lower panel, Gαi2SW-320α13QL harboring mutations in the putative RGL contact sites in switch 1 or switch 2 exhibited a reduced ability to stimulate the SRE, which was nearly half of that of Gαi2SW-320α13QL, while the double mutant showed no activity. The ability of these chimeras to interact with PRG and to stimulate the increase in GTP-Rho was aligned" @default.
• W2040086538 created "2016-06-24" @default.
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• W2040086538 cites W2006473647 @default.
• W2040086538 cites W2009299953 @default.
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