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Mechanisms for Reversible Regulation between G13 and Rho Exchange Factors

2002; Elsevier BV; Volume: 277; Issue: 2 Linguagem: Inglês

10.1074/jbc.m105274200

1083-351X

Clark D. Wells, Mu-Ya Liu, M. T. Jackson, Stephen Gutowski, Pamela M. Sternweis, Jeffrey D. Rothstein, Tohru Kozasa, Paul C. Sternweis,

PI3K/AKT/mTOR signaling in cancer

The heterotrimeric G proteins, G12 and G13, mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Gα13 of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Gα12 and Gα13 is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for Gi and Gq. Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted “RGS box” region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Gα13 and activation of RhoA exchange activity by Gα13. In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Gα13 but was not stimulated by the α subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Gα13 also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Gα13 did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Gα13. These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Gα13 to both its RGS and DH domains. The heterotrimeric G proteins, G12 and G13, mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Gα13 of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Gα12 and Gα13 is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for Gi and Gq. Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted “RGS box” region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Gα13 and activation of RhoA exchange activity by Gα13. In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Gα13 but was not stimulated by the α subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Gα13 also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Gα13 did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Gα13. These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Gα13 to both its RGS and DH domains. guanine nucleotide exchange factor lysophosphatidic acid sphingosine 1-phosphate Dbl homology pleckstrin homology GTPase-activating protein glutathioneS-transferase Ras homology regulators of G protein signaling guanosine 5′-3-O-(thio)triphosphate cytomegalovirus amino acid(s) Heterotrimeric G proteins mediate signals from seven transmembrane receptors to a wide array of effectors, including adenylyl cyclase, ion channels, phospholipases, and the exchange factor p115 RhoGEF (1Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Google Scholar). Every G protein is composed of a heterotrimer made up of α, β, and γ subunits. The four G protein families, Gi, Gq, Gs, and G12, have been categorized by their sequence identity and the functional similarity of their α subunits (2Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Google Scholar, 3Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Google Scholar). The G12 family contains two members, α12 and α13 (4Strathmann M.P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5582-5586Google Scholar), which have been implicated in cellular transformation (5Jiang H. Wu D. Simon M.I. FEBS Lett. 1993; 330: 319-322Google Scholar), gastrulation ofDrosophila melanogaster (6Parks S. Wieschaus E. Cell. 1991; 64: 447-458Google Scholar), vascular development in mice (7Offermanns S. Mancino V. Revel J.P. Simon M.I. Science. 1997; 275: 533-536Google Scholar), and actin re-arrangement (8Buhl A.M. Johnson N.L. Dhanasekaran N. Johnson G.L. J. Biol. Chem. 1995; 270: 24631-24634Google Scholar, 9Gohla A. Harhammer R. Schultz G. J. Biol. Chem. 1998; 273: 4653-4659Google Scholar, 10Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Google Scholar). The cytoskeletal changes mediated by G13 have been shown in several studies to require the monomeric Rho GTPases (10Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Google Scholar). Both the heterotrimeric G proteins and monomeric Rho GTPases utilize the same basic cycle of regulation. The inactive proteins contain bound GDP. Activation is facilitated by guanine nucleotide exchange factors (GEFs)1 that promote dissociation of GDP and subsequent binding of GTP to the G protein. GTPase-activating proteins (GAPs) return the GTPase to the inactive state by accelerating hydrolysis of the terminal phosphate of the bound GTP (2Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Google Scholar, 3Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Google Scholar, 11Nobes C. Hall A. Curr. Opin. Genet. Dev. 1994; 4: 77-81Google Scholar). Members of the RGS (regulators of G protein signaling) family of proteins can act as GAPs for heterotrimeric G proteins (12Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Google Scholar, 13Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Google Scholar, 14Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Google Scholar). RGS proteins that have been characterized to date function by increasing the intrinsic rate of GTP hydrolysis of Gα subunits through allosteric binding (15Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Google Scholar). This mechanism was first worked out for RGS4, which acts upon Gαi and Gαq (16Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Google Scholar, 17Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Google Scholar). The smallest region capable of accelerating GTPase activity is called the RGS box. This domain is characterized by strong primary sequence identity and structural similarity among the four RGS family members for which structures are known: RGS4 (18Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Google Scholar), GAIP (19De de Alba E. Vries L. Farquhar M.G. Tjandra N. J. Mol. Biol. 1999; 291: 927-939Google Scholar), axin/conductin (20Spink K.E. Polakis P. Weis W.I. EMBO J. 2000; 19: 2270-2279Google Scholar), and RGS9 (21Slep K.C. Kercher M.A. He W. Cowan C.W. Wensel T.G. Sigler P.B. Nature. 2001; 409: 1071-1077Google Scholar). LARG (22Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Google Scholar), PDZ RhoGEF (23Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Google Scholar), p115 RhoGEF (24Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Google Scholar), and GTRAP48 (25Jackson M. Song W. Liu M.Y. Jin L. Dykes-Hoberg M. Lin C.I. Bowers W.J. Federoff H.J. Sternweis P.C. Rothstein J.D. Nature. 2001; 410: 89-93Google Scholar) are guanine nucleotide exchange factors for Rho; all share a highly conserved region that interacts specifically with Gα12and/or Gα13 (22Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Google Scholar, 23Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Google Scholar, 25Jackson M. Song W. Liu M.Y. Jin L. Dykes-Hoberg M. Lin C.I. Bowers W.J. Federoff H.J. Sternweis P.C. Rothstein J.D. Nature. 2001; 410: 89-93Google Scholar, 26Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Google Scholar). This region invariantly lies N-terminal to the tandem Dbl homology (DH) (27Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Google Scholar) and Pleckstrin homology (PH) (28Lemmon M.A. Ferguson K.M. Curr. Top. Microbiol. Immunol. 1998; 228: 39-74Google Scholar) domains found in all four proteins. Even though these N-terminal regions contain only weak sequence identity to a stereotypical RGS box, the structure of this region in p115 RhoGEF (aa 42–252) is similar to the folding pattern of other RGS boxes (29Chen Z. Wells C.D. Sternweis P.C. Sprang S.R. Nat. Struct. Biol. 2001; 8: 805-809Google Scholar). The high sequence identity among these regions in the four RhoGEFs suggests structural identity, and this clear subfamily of RGS domains is subsequently referred to as the rgRGS (RhoGEFRGS) domain. The best-studied member of this group, p115 RhoGEF, was initially isolated as a protein that tightly bound the nucleotide free form of RhoA and increased the nucleotide exchange rate of RhoA (24Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Google Scholar). The rgRGS domain within the N terminus of p115 RhoGEF was subsequently established as a GAP for Gα12 and Gα13(26Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Google Scholar). Most interestingly, p115 RhoGEF is also an effector of Gα13, which increases the activity of p115 RhoGEF as a guanine nucleotide exchange factor (GEF) for RhoA (1Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Google Scholar). Recently, GTRAP48 was found to be a GEF for RhoA that also binds Gα13, but the functional implications of this interaction are not known (25Jackson M. Song W. Liu M.Y. Jin L. Dykes-Hoberg M. Lin C.I. Bowers W.J. Federoff H.J. Sternweis P.C. Rothstein J.D. Nature. 2001; 410: 89-93Google Scholar). In this study, the interaction of p115 RhoGEF and GTRAP48 with the G12 family of heterotrimeric G proteins is more precisely defined. Deletion analysis of p115 RhoGEF provides evidence that regions outside of the apparent classic RGS box are required for accelerating the GTPase activity of Gα13. A mechanism for stimulation of p115 RhoGEF by Gα13 is suggested by the determination of a second binding site for Gα13 in the tandem DH/PH domains of p115 RhoGEF. Finally, GTRAP48 was found to bind Gα13 and act as a weak GAP on the α subunit, but its nucleotide exchange activity was not stimulated by Gα13. The cDNA encoding full-length p115 RhoGEF (24Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Google Scholar) was used for amplification of fragments of p115 RhoGEF by the polymerase chain reaction. All fragments were amplified with an N-terminal EcoRI site and a C-terminal HindIII site for cloning into pCMV5-myc (provided by Melanie Cobb), pGEX-KG (30Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Google Scholar), and a modified pTrc D expression vector as described previously (31Wells C.D. Gutowski S. Bollag G. Sternweis P.C. J. Biol. Chem. 2001; 276: 28897-28905Google Scholar). Briefly, the intervening sequence between the hexa-histidine tag (HIS) and the EcoRI restriction site of pTrc C (Invitrogen) was replaced with the amino acids Met-Gly-Ala. Fragments were transferred viaEcoRI/XbaI sites from pCMV5 into pVL1392-EE (31Wells C.D. Gutowski S. Bollag G. Sternweis P.C. J. Biol. Chem. 2001; 276: 28897-28905Google Scholar), which contains an N-terminal EE tag (EYMPME) (24Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Google Scholar). Baculoviruses were produced through co-transfection of SF9 cells with pVL1392-EE vectors and BakPak6 that was digested with Bsu36I (CLONTECH). The N-terminal DNA fragments of p115 RhoGEF were amplified by PCR and cloned into pGEX-KG and pTrc D. The different pieces of p115 RhoGEF are named by the primers used for their amplification. All cDNA constructs were sequenced to confirm correct amplification and construction. All GTRAP48 and p115 RhoGEF proteins were expressed via baculovirus in culturedSpodoptera frugiperda (SF9) cells or in the transformed BL21(DE3) strain of Escherichia coli. Recombinant EE-tagged proteins were expressed in SF9 cells after infection with baculovirus. The expressed proteins were purified from lysates by affinity chromatography with anti-EE coupled Sepharose (BAbCO) as described elsewhere (32Wells C. Jiang X. Gutowski S. Sternweis P.C. Methods Enzymol. 2002; 345: 371-372Google Scholar). N-terminal fragments of p115 RhoGEF and GTRAP48 were produced in E. coli as either HIS-tagged fusion proteins or as chimeras with GST (glutathioneS-transferase). GST-tagged proteins were purified by chromatography using glutathione-Sepharose (Amersham Biosciences, Inc.) and solution A (25 mm NaHEPES, pH 7.5, 1 mmdithiothreitol, 50 mm NaCl) containing the protease inhibitors (2.5 μg/ml leupeptin, 1 μg/ml pepstatin A, 21 μg/ml phenylmethylsulfonyl fluoride, 21 μg/mlNα-p-tosyl-l-lysine chloromethyl ketone, 21 μg/ml tosylphenylalanyl chloromethyl ketone, and 21 μg/mlNα-p-tosyl-l-arginine methyl ester). HIS-tagged proteins were purified by isolation with Ni2+-nitrilotriacetic acid resin (Qiagen) in 25 mm NaHEPES, pH 7.5, 2.5 mm β-mercaptoethanol, 50 mm NaCl, and the protease inhibitors. Gα13 was prepared as described by Singer et al(33Singer W.D. Miller R.T. Sternweis P.C. J. Biol. Chem. 1994; 269: 19796-19802Google Scholar). Prenyl RhoA was co-expressed with GST-tagged guanine nucleotide dissociation inhibitor via Baculovirus in SF9 cells (32Wells C. Jiang X. Gutowski S. Sternweis P.C. Methods Enzymol. 2002; 345: 371-372Google Scholar). Lysates were prepared by freeze-thawing cell pellets three times in Solution A containing protease inhibitors. After removing particulate material by centrifugation at 100,000 × g, the cytosol was passed over a glutathione-Sepharose column and eluted with the same solution containing 1% cholate to separate RhoA from GST-guanine nucleotide dissociation inhibitor. Proteins were concentrated, and cholate was removed by dilution of samples with solution A containing protease inhibitors and final concentration via pressure filtration through an Amicon PM10 membrane. Three purified protein pieces of p115 RhoGEF were used to generate polyclonal antisera (31Wells C.D. Gutowski S. Bollag G. Sternweis P.C. J. Biol. Chem. 2001; 276: 28897-28905Google Scholar). Antisera specific for fragments consisting of aa 1–252, 288–637, and 760–912 are termed U2760, U2762, and U2764, respectively. B860, an antiserum specific for Gα13 (33Singer W.D. Miller R.T. Sternweis P.C. J. Biol. Chem. 1994; 269: 19796-19802Google Scholar) and the antiserum specific for Gα12 (34Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Google Scholar) were described previously. A polyclonal antibody that specifically recognizes the myc tag (catalog number Sc-789) was purchased from Santa Cruz Biotechnology. Gα13 was activated by incubation with solution B (25 mm NaHEPES, pH 7.5, 100 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol) containing AMF (30 μm AlCl3, 5 mm MgCl2, and 10 mm NaF) and protease inhibitors for 10 min. Activated Gα13 (5 pmol) was mixed with the indicated concentrations of exchange factor, which had been pre-bound to anti-EE-Sepharose, and the final volume was adjusted to 200 μl with solution B and AMF if indicated. Samples were incubated for 1 h on a rocking platform at 4 °C. The beads were then pelleted in a microcentrifuge for 1 min and washed twice with solution B. The p115 RhoGEF proteins and Gα13 were eluted for 15 min at room temperature with 50 μl of solution B containing EE peptide (EYMPME) at a final concentration of 100 μg/ml. Gα13 in the supernatant was visualized by SDS-PAGE and by immunoblot analysis using the B860 antibody. The purified GST-tagged domains were bound to 20 μl of glutathione-Sepharose (packed beads) and washed in solution B to remove unbound protein. Activated Gα13 (5 pmol, activated as described above) was added to the indicated amount of immobilized rgRGS domain in 200 μl of solution B. After incubation on a rocking platform for 1 h at 4 °C, the samples were washed three times with solution B. The beads were then boiled in SDS sample buffer, and the eluted Gα13 was visualized by SDS-PAGE and by immunoblot analysis using the B860 antibody. COS cells were grown to 80% confluency in 60-mm dishes. The cells were then transfected using the FuGENE transfection reagent (Hoffman-LaRoche) with pCMV5-myc vectors that expressed the different exchange factors. Cells were also co-transfected with pCMV5 plasmids expressing either Gα13 or Gα12. Transfected cells were incubated at 37 °C in 5% CO2 for 24 h. Cells were then lysed in solution B containing 0.1% Triton X-100. Non-extracted material was removed from the lysates by centrifugation at 13,600 × g for 5 min. The cleared lysates were then incubated with protein G-Sepharose in solution B for 30 min at 4 °C to remove proteins that bound nonspecifically to the Sepharose resin. After removal of the resin, the samples were added to protein G-Sepharose coupled to a monoclonal antibody directed against the myc tag and further incubated for 1 h at 4 °C on a rocking platform. The Sepharose beads were pelleted and washed three times with solution B. Proteins were eluted by boiling in SDS sample buffer and visualized by immunoblot analysis with specific antibodies against either the myc-tag, Gα12, or Gα13. Gα13 (600 pmol) was loaded with [γ-32P]GTP for 15 min in 300 μl of 20 mm NaHEPES, pH 8.0, 1 mm dithiothreitol, 5 mm EDTA, 0.05% polyoxyethylene 10-laurelether, 5 μm GTP, and 50 cpm /fmol [γ-32P]GTP. Samples were then rapidly gel-filtered by centrifugation at 4 °C through 2 ml of Sephadex G50 resin that was previously equilibrated with buffer C (50 mm NaHEPES, pH 8.0, 1 mmdithiothreitol, 5 mm EDTA, and 0.05% polyoxyethylene 10-laurelether) to remove free [γ-32P]GTP and [32P]Pi. Hydrolysis of GTP was initiated by adding 60 pmol of the treated Gα13 (about 2 pmol of loaded Gα13) to buffer C containing 8 mmMgSO4, 1 mm GTP, and the proteins to be tested for GAP activity. After incubation for the indicated times at 4 °C, aliquots (50 μl) were quenched with 750 μl of 5% (w/v) NoritA in 50 mm NaH2PO4. The mixtures were then centrifuged at 2000 rpm for 5 min, and 500 μl of supernatant containing [32P]Pi was counted by liquid scintillation spectrometry. Binding of GTPγS to 2 μm RhoA was assayed at 30 °C in 20 μl of solution containing 20 μm GTPγS, [35S]GTPγS (200,000 cpm ), 50 mm NaHEPES, pH 7.5, 1 mmEDTA, 50 mm NaCl, 1 mm dithiothreitol, and 5 mm MgCl2. Gα13 was added to a final concentration of 100 nm after being activated for 10 min in binding buffer containing AMF. After mixing, assays were stopped at various times by the addition of 2 ml of filtration buffer (20 mm TrisCl, pH 8.0, 25 mm MgCl2, 100 mm NaCl), and the proteins were immediately collected by filtration through BA-85 filters (Intermountain Scientific). The amount of [35S]GTPγS bound to RhoA on each dried filter was determined by liquid scintillation spectrometry. The apparent rates of guanine nucleotide exchange for RhoA were examined at three to four concentrations to determine the average turnover rate for each p115 RhoGEF piece. Mutants of p115 RhoGEF and GTRAP48 were used to identify the domains responsible for binding to Gα13, stimulation of RhoA exchange activity by Gα13, and acceleration of GTPase activity of Gα12 and Gα13. Schematic representations of these mutants are shown in Figs.1A and 4A. Purified proteins derived from these constructs after expression via baculovirus infection of Sf9 cells or expression in E. coli are shown in Figs. 1B and 4B.Figure 4Constructs and expression of GTRAP48 and p115 RhoGEF.A, constructs derived from GTRAP48 and p115 RhoGEF are shown. The amino acids encoded in each construct are also listed. Nomenclature in parentheses is based on the oligomers used in their construction. B, representative preparations of purified proteins that were derived from GTRAP48 and p115 RhoGEF (see “Materials and Methods”) and used in this study were separated by SDS-PAGE and stained with Coomassie Blue R-250 dye.View Large Image Figure ViewerDownload (PPT) Stable expression of the N terminus of p115 RhoGEF (aa 1–248) as a fusion protein with GST has been shown previously, and aa 45–161 were predicted to form an RGS box (26Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Google Scholar). However, subsequent deletion mutants of this N terminus identified two areas outside of the predicted RGS box that were necessary for stable expression of protein. Because p115 RhoGEF was successively deleted from the N terminus by 7 or 13 amino acids, the level of expression remained stable. Further deletion up to aa 17 or 21 resulted in fragments that did not express intact protein. This N-terminal region, which apparently interferes with protein stability, ends at or before amino acid 25, because a fragment of p115 RhoGEF encoded by aa 25–252 can be expressed at 100–200 μg/gram ofE. coli (wet weight). The deletion of the first 41 amino acids resulted in a domain, p115 RhoGEF (aa 42–252), which was expressed at levels comparable to domains with intact N termini. Disruption of the region lying C-terminal to the predicted RGS box that lies between amino acids 161 and 252 also affected expression of protein. Protein fragments starting with aa 1 and ending at aa 248 or 252 express extremely well in E. coli (3–4 mg/g of packed wet cells). However, p115 RhoGEF (aa 1–215) could only be expressed poorly at 30–40 μg/g (wet weight), and this was only possible in SF9 cells. Fragments of p115 RhoGEF with shorter C termini, which encoded either aa 25–190 or aa 42–170, showed little to no detectable expression in either bacteria or SF9 cells (data not shown). The p115 rgRGS fragments consisting of aa 1–252, 25–252, and 42–252 were expressed and purified as fusion proteins with an N-terminal GST domain (Fig. 1A). All three bound preferentially to the activated form of Gα13 (Fig.2A). The relative binding affinity of p115 rgRGS (aa 1–252) and (aa 42–252) for Gα13 was then assessed by competitive binding (Fig.2B). HIS-tagged p115 rgRGS (aa 1–252) and (aa 42–252) proteins were added at increasing concentrations to compete with a fixed amount of the immobilized GST-tagged constructs for binding of Gα13. 60 pmol of HIS-tagged p115 rgRGS (aa 1–252) effectively reduced binding of 20 pmol of immobilized GST-tagged p115 rgRGS (aa 1–252) to Gα13. In contrast, 60 pmol of p115 rgRGS (aa 42–252) had little effect, and higher concentrations (180 and 540 pmol) were needed to substantially reduce binding of the Gα13 to 20 pmol of immobilized GST-tagged p115 rgRGS (aa 1–252). Similarly, HIS-tagged p115 rgRGS (aa 1–252) was a much more effective inhibitor of binding of Gα13 to immobilized GST-tagged p115 rgRGS (aa 42–252) than the HIS-tagged p115 rgRGS (aa 42–252). Both comparisons demonstrate that p115 rgRGS (aa 1–252) bound to Gα13 with 5- to 10-fold greater avidity than the truncated rgRGS of p115 RhoGEF (aa 42–252). Thus, the N-terminal residues of p115 rgRGS are not necessary for binding to Gα13 but do contribute significantly to the affinity of this interaction. Several N-terminal fragments of p115 RhoGEF, which included aa 1–252, 6–252, 13–252, 25–252, 42–252, and 1–215 (described in Figs. 1A and3A), were tested for their ability to stimulate the GTPase activity of Gα13. Single-turnover assays were utilized, which measure the release of [32P]Pi upon hydrolysis of [γ-32P]GTP that had been pre-bound to Gα13. Assays were performed as described by Singeret al (33Singer W.D. Miller R.T. Sternweis P.C. J. Biol. Chem. 1994; 269: 19796-19802Google Scholar), with the modifications outlined under “Materials and Methods.” As shown previously for a GST fusion protein containing the first 246 amino acids of p115 RhoGEF (26Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Google Scholar), the p115 rgRGS (aa 1–252) stimulated the GTPase activity of Gα13 as well as the full-length exchange factor (Fig.3B). However, a protein with further truncation at its C terminus, p115 rgRGS (aa 1–215), was a less effective stimulator of the GTPase activity of Gα13. Removal of additional C-terminal residues resulted in unstable proteins that had no detectable stimulation of the GTPase activity of Gα13 (data not shown). The effects of N-terminal deletion are shown in Fig. 3(C–F). Removal of the first 5 or 12 N-terminal amino acids, p115 rgRGS (aa 6–252) or p115 rgRGS (aa 13–252), respectively, did not alter GAP activities toward Gα13 (Fig.3C). Removal of the first 17 or 21 N-terminal amino acids did not allow expression of protein domains as discussed previously. P115 rgRGS (aa 25–252) was expressed modestly as a viable domain. Although an active GAP, the potency of this construct was only about 0.1% that of the full-length rgRGS domain encoded within aa 1–252 (Fig. 3, D–F). Finally, deletion of the N terminus up to the predicted RGS box, p115 RhoGEF (aa 42–252), produced a fragment that expressed well (see above). Despite binding strongly to Gα13 (about 10–20% as well as p115 rgRGS (aa 1–252), see Fig. 2B), this fragment had essentially no GAP activity toward Gα13 (Fig. 3, C–E). The hint of activity observed at 10 μm (Fig. 3E) is similar to nonspecific effects of adding other control proteins and is not increased at higher concentrations of p115 rgRGS (aa 42–252). GTRAP48 has been shown to bind to Gα13 (25Jackson M. Song W. Liu M.Y. Jin L. Dykes-Hoberg M. Lin C.I. Bowers W.J. Federoff H.J. Sternweis P.C. Rothstein J.D. Nature. 2001; 410: 89-93Google Scholar), but the functional consequences and potential interaction with Gα12 are unknown. The domain arrangements of GTRAP48 and p115 RhoGEF are compared in Fig.4A, and schematic descriptions of various constructs are shown. In a chimeric protein, N48C115, the N-terminal rgRGS region of p115 RhoGEF is replaced with the homologous region from GTRAP48. Examples of purified proteins that were expressed via these constructs with EE or GST tags are shown in Fig.4B. The ability of GTRAP48 to bind to Gα12 as well as Gα13 was assessed by immunoprecipitation after transient expression in COS cells. COS cells were transfected with either myc-tagged GTRAP48 or myc-tagged p115 RhoGEF and either constitutively active Gα13 (Q226L) or constitutively active Gα12 (Q229L). Both p115 RhoGEF and GTRAP48 bound to Gα13 in the presence of AlF 4−. Although p115 RhoGEF also bound well to Gα12, the interaction of this α subunit with GTRAP48 is apparently weaker and was hard to detect (Fig.5A, see “Discussion”). The dependence of association of these GTPase-deficient forms of Gα12 and Gα13 on AlF 4− may seem surprising. However, this reflects both the multiple states of the α subunits upon lysis of cells in GDP and slow conversion of Gα·GTP to Gα·GDP through slow hydrolysis or nucleotide exchange over the extensive timeframe required for the immunoprecipitation. To define the regions in GTRAP48 responsible for binding Gα13, two segments, which included sequences homologous to the rgRGS domain of p115 RhoGEF, were expressed and examined. The first segment encodes aa 1–539, which includes the PDZ-and proline-rich domains that precede the rgRGS domain (Fig. 4). The second construct encodes aa 285–495, which contains amino acids homologous to the N-terminal region of p115 RhoGEF (Fig. 4). Both constructs of GTRAP48 were found to preferentially bind the activated form of Gα13 (Fig. 5B). The relative affinities of GTRAP48, a fragment of GTRAP48 (aa 289–539), and p115 RhoGEF for Gα13 were assessed by competitive binding with immobilized GST-tagged p115 rgRGS aa (1) (Fig. 5C). Purified full-length p115 RhoGEF and HIS-tagged p115 rgRGS (aa 1–252) effectively attenuated binding of activated Gα13 to the immobilized p115 rgRGS at stoichiometries of 5- to 10-fold over the immobilized domain. In contrast, EE-tagged GTRAP48 or its HIS-tagged rgRGS domain, GTRAP48 (aa 289–539), at concentrations of 5- to 10-fold over the GST-tagged p115 rgRGS (aa 1–252) showed little competition for binding to Gα13. Thus, although the GTRAP48 rgRGS domain bound Gα13, it did so with a definitively lower affinity than the p115 rgRGS domain. The ability of GTRAP48 to stimulate the GTPase activity of Gα13 in a single-turnover assay is shown in Fig.6A. Both GTRAP48 and N48C115 (a chimera derived from GTRAP48 and p115 RhoGEF; Fig. 4A) displayed low but significant stimulation of the GTPase activity of Gα13. However, their activities were substantially less than the stimulation obtained with an equivalent amount of p115 RhoGEF. The N-terminal fragments of GTRAP48, which contain its rgRGS domain, failed to stimulate the GTPase activity of Gα13 at the concentrations tested (Fig. 6B). Thus, by this in vitro measure, the rgRGS domain is only a poor GAP for Gα13 at best. This contrasts with the robust GAP activity of p115 rgRGS. The N-terminal 248 amino acids in p115 RhoG