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• W2168142419 abstract "The activation of the Ras-related GTPase R-Ras, which has been implicated in the regulation of various cellular functions, by G protein-coupled receptors (GPCRs) was studied in HEK-293 cells stably expressing the M3 muscarinic acetylcholine receptor (mAChR), which can couple to several types of heterotrimeric G proteins. Activation of the receptor induced a very rapid and transient activation of R-Ras. Studies with inhibitors and activators of various signaling pathways indicated that R-Ras activation by the M3 mAChR is dependent on cyclic AMP formation but is independent of protein kinase A. Similar to the rather promiscuous M3 mAChR, two typical Gs-coupled receptors also induced R-Ras activation. The receptor actions were mimicked by an Epac-specific cyclic AMP analog and suppressed by depletion of endogenous Epac1 by small interfering RNAs, as well as expression of a cyclic AMP binding-deficient Epac1 mutant, but not by expression of dominant negative Rap GTPases. In vitro studies demonstrated that Epac1 directly interacts with R-Ras and catalyzes GDP/GTP exchange at this GTPase. Finally, it is shown that the cyclic AMP- and Epac-activated R-Ras plays a major role in the M3 mAChR-mediated stimulation of phospholipase D but not phospholipase C. Collectively, our data indicate that GPCRs rapidly activate R-Ras, that R-Ras activation by the GPCRs is apparently directly induced by cyclic AMP-regulated Epac proteins, and that activated R-Ras specifically controls GPCR-mediated phospholipase D stimulation. The activation of the Ras-related GTPase R-Ras, which has been implicated in the regulation of various cellular functions, by G protein-coupled receptors (GPCRs) was studied in HEK-293 cells stably expressing the M3 muscarinic acetylcholine receptor (mAChR), which can couple to several types of heterotrimeric G proteins. Activation of the receptor induced a very rapid and transient activation of R-Ras. Studies with inhibitors and activators of various signaling pathways indicated that R-Ras activation by the M3 mAChR is dependent on cyclic AMP formation but is independent of protein kinase A. Similar to the rather promiscuous M3 mAChR, two typical Gs-coupled receptors also induced R-Ras activation. The receptor actions were mimicked by an Epac-specific cyclic AMP analog and suppressed by depletion of endogenous Epac1 by small interfering RNAs, as well as expression of a cyclic AMP binding-deficient Epac1 mutant, but not by expression of dominant negative Rap GTPases. In vitro studies demonstrated that Epac1 directly interacts with R-Ras and catalyzes GDP/GTP exchange at this GTPase. Finally, it is shown that the cyclic AMP- and Epac-activated R-Ras plays a major role in the M3 mAChR-mediated stimulation of phospholipase D but not phospholipase C. Collectively, our data indicate that GPCRs rapidly activate R-Ras, that R-Ras activation by the GPCRs is apparently directly induced by cyclic AMP-regulated Epac proteins, and that activated R-Ras specifically controls GPCR-mediated phospholipase D stimulation. R-Ras, a member of the Ras superfamily of small GTPases, originally cloned through its homology to prototypic H-Ras, has been shown to interact in vitro with the three H-Ras downstream effectors, Raf-1, phosphatidylinositol (PI) 6The abbreviations used are: PI, phosphatidylinositol; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; GPCR, G protein-coupled receptor; mAChR, muscarinic acetylcholine receptor; PLC, phospholipase C; PLD, phospholipase D; PGE1, prostaglandin E1; 8-Br-cAMP, 8-bromo-cAMP; 8-pCPT-2Me-cAMP, 8-(4-chlorophenylthio)-2′-O-methyl-cAMP; Rp-CPT-cAMPS, 8-pCPT-adenosine-3′,5′-cyclic monophosphorothioate; VASP, vasodilator-stimulated phosphoprotein; PtdEtOH, phosphatidylethanol; IP3, inositol 1,4,5-trisphosphate; GST, glutathione S-transferase; siRNA, small interfering RNA; PKA, protein kinase A; GTPγS, guanosine 5′-[γ-thio]triphosphate; ddAdo, 2′, 5′-dideoxyadenosine; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); AMP-PNP, adenosine 5′-(β,γ-imido)triphosphate.6The abbreviations used are: PI, phosphatidylinositol; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; GPCR, G protein-coupled receptor; mAChR, muscarinic acetylcholine receptor; PLC, phospholipase C; PLD, phospholipase D; PGE1, prostaglandin E1; 8-Br-cAMP, 8-bromo-cAMP; 8-pCPT-2Me-cAMP, 8-(4-chlorophenylthio)-2′-O-methyl-cAMP; Rp-CPT-cAMPS, 8-pCPT-adenosine-3′,5′-cyclic monophosphorothioate; VASP, vasodilator-stimulated phosphoprotein; PtdEtOH, phosphatidylethanol; IP3, inositol 1,4,5-trisphosphate; GST, glutathione S-transferase; siRNA, small interfering RNA; PKA, protein kinase A; GTPγS, guanosine 5′-[γ-thio]triphosphate; ddAdo, 2′, 5′-dideoxyadenosine; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); AMP-PNP, adenosine 5′-(β,γ-imido)triphosphate. 3-kinase, and RalGDS (1Lowe D.G. Goeddel D.V. Mol. Cell. Biol. 1987; 7: 2845-2856Crossref PubMed Scopus (51) Google Scholar, 2Spaargaren M. Bischoff J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12609-12613Crossref PubMed Scopus (248) Google Scholar, 3Spaargaren M. Martin G.A. McCormick F. Fernandez-Sarabia M.J. Bischoff J.R. Biochem. J. 1994; 300: 303-307Crossref PubMed Scopus (55) Google Scholar, 4Marte B.M. Rodriguez-Viciana P. Wennström S. Warne P.H. Downward J. Curr. Biol. 1997; 7: 63-70Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, in vitro interaction of R-Ras with several Ras regulatory proteins, including various guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), has been reported (5Cullen P.J. Lockyer P.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 339-348Crossref PubMed Scopus (299) Google Scholar, 6Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar, 7Kinbara K. Goldfinger L.E. Hansen M. Chou F.-L. Ginsberg M.H. Nat. Rev. Mol. Cell Biol. 2003; 4: 767-776Crossref PubMed Google Scholar, 8Bernards A. Settleman J. Trends Cell Biol. 2004; 14: 377-385Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Despite these similarities, studies in different cell types demonstrated that R-Ras has biological functions distinct from classic H-Ras. Notably, although H-Ras inhibits cell adhesion in fibroblasts by reducing the affinity of integrins for their ligands, R-Ras has been shown to promote integrin activation (7Kinbara K. Goldfinger L.E. Hansen M. Chou F.-L. Ginsberg M.H. Nat. Rev. Mol. Cell Biol. 2003; 4: 767-776Crossref PubMed Google Scholar, 9Zhang Z. Vuori K. Wang H. Reed J.C. Ruoslahti E. Cell. 1996; 85: 61-69Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 10Sethi T. Ginsberg M.H. Downward J. Hughes P.E. Mol. Biol. Cell. 1999; 10: 1799-1809Crossref PubMed Scopus (87) Google Scholar, 11Self A.J. Caron E. Paterson H.F. Hall A. J. Cell Sci. 2001; 114: 1357-1366Crossref PubMed Google Scholar). Recently, it has been proposed that R-Ras might enhance integrin-mediated cell attachment and spreading through alterations in the cellular Ca2+ handling, by decreasing the Ca2+ content of the endoplasmic reticulum (12Koopman W.J.H. Bosch R.R. van Emst-de Vries S.E. Spaargaren M. De Pont J.J.H.H.M. Willems P.H.G.M. J. Biol. Chem. 2003; 278: 13672-13679Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The capacity of R-Ras to modulate cell adhesion by maintaining integrin activity can be regulated by phosphorylation of a tyrosine residue in its effector domain by an Eph receptor kinase (13Zou J.X. Wang B. Kalo M.S. Zisch A.H. Pasquale E.B. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13813-13818Crossref PubMed Scopus (172) Google Scholar). Meanwhile, it has been reported that Eph receptor signaling to R-Ras is controlled by tyrosine phosphorylation of R-Ras by the Src kinase on tyrosine 66, and by binding of R-Ras to the Src homology 2 domain-containing Eph receptor-binding protein SHEP1, which binds in addition to the Ras-related GTPase Rap1A (14Dodelet V.C. Pazzagli C. Zisch A.H. Hauser C.A. Pasquale E.B. J. Biol. Chem. 1999; 274: 31941-31946Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 15Zou J.X. Liu Y. Pasquale E.B. Ruoslahti E. J. Biol. Chem. 2002; 277: 1824-1827Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Notably, Rap1 has been shown to play a key role in the regulation of several aspects of cell adhesion, in particular in integrin-mediated cell adhesion (16Bos J.L. Curr. Opin. Cell Biol. 2005; 17: 123-128Crossref PubMed Scopus (392) Google Scholar), and thus R-Ras might modulate integrin signaling in concert with Rap GTPases. Other studies have shown that the semaphorin 4D receptor, plexin-B1, can down-regulate R-Ras activity by acting as an R-Ras-specific GAP in the presence of the Rho family member Rnd1, and thereby suppresses integrin activation and cell adhesion (17Oinuma I. Ishikawa Y. Katoh H. Negishi M. Science. 2004; 305: 862-865Crossref PubMed Scopus (315) Google Scholar). Furthermore, recent studies in different cell types demonstrated that R-Ras is involved in control of cell migration, apparently through spatio-temporal regulation of Rho and Rac activities (18Wozniak M.A. Kwong L. Chodniewicz D. Klemke R.L. Keely P.J. Mol. Biol. Cell. 2005; 16: 84-96Crossref PubMed Scopus (77) Google Scholar, 19Jeong H.-W. Nam J.-O. Kim I.-S. Cancer Res. 2005; 65: 507-515PubMed Google Scholar, 20Holly S.P. Larson M.K. Parise L.V. Mol. Biol. Cell. 2005; 16: 2458-2469Crossref PubMed Scopus (45) Google Scholar). In a recently generated R-Ras-null mouse, increased proliferation of vascular smooth muscle cells and enhanced angiogenesis was observed (21Komatsu M. Ruoslahti E. Nat. Med. 2005; 11: 1346-1350Crossref PubMed Scopus (98) Google Scholar).Despite the increasing evidence that R-Ras is obviously a key regulator of several cellular processes, our knowledge about the mechanisms leading to activation of R-Ras is very limited. In particular, it should be mentioned that in most studies the biological function of R-Ras was analyzed by ectopically expressing constitutively active or dominant negative R-Ras mutants. Activation of R-Ras, i.e. GTP loading, is catalyzed by several distinct GEFs, comprising about 20 distinct proteins (5Cullen P.J. Lockyer P.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 339-348Crossref PubMed Scopus (299) Google Scholar, 6Quilliam L.A. Rebhun J.F. Castro A.F. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 391-444Crossref PubMed Google Scholar, 7Kinbara K. Goldfinger L.E. Hansen M. Chou F.-L. Ginsberg M.H. Nat. Rev. Mol. Cell Biol. 2003; 4: 767-776Crossref PubMed Google Scholar). Of note, there is no report on a GEF that is specific for R-Ras. For example, the Ca2+/diacylglycerol-regulated GEFs, RasGRP1–3, have been reported to activate R-Ras, H-Ras, and Rap1 (22Ohba Y. Mochizuki N. Yamashita S. Chan A.M. Schrader J.W. Hattori S. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 20020-20026Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 23Yamashita S. Mochizuki N. Ohba Y. Tobiume M. Okada Y. Sawa H. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 25488-25493Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), although C3G and AND-34 have been demonstrated to promote GTP loading on R-Ras and Rap1 (22Ohba Y. Mochizuki N. Yamashita S. Chan A.M. Schrader J.W. Hattori S. Nagashima K. Matsuda M. J. Biol. Chem. 2000; 275: 20020-20026Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 24Mochizuki N. Ohba Y. Kobayashi S. Otsuka N. Graybiel A.M. Tanaka S. Matsuda M. J. Biol. Chem. 2000; 275: 12667-12671Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 25Gotoh T. Cai D. Tian X. Feig L.A. Lerner A. J. Biol. Chem. 2000; 275: 30118-30123Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). It should be emphasized, however, that none of these studies addressed the question whether activation of R-Ras by these GEFs is triggered by specific membrane receptors.The aim of this report was to study whether and by which mechanisms G protein-coupled receptors (GPCRs) activate R-Ras and to define a biological function of R-Ras in the GPCR actions. For this, we used the M3 muscarinic acetylcholine receptor (mAChR) stably expressed in HEK-293 cells. Numerous studies have demonstrated that this prototypical GPCR can couple to all major subtypes of heterotrimeric G proteins, not only to Gq proteins, as initially assumed (26Peralta E.G. Ashkenazi A. Winslow J.W. Ramachandran J. Capon D.J. Nature. 1988; 334: 434-437Crossref PubMed Scopus (547) Google Scholar, 27Offermanns S. Wieland T. Homann D. Sandmann J. Bombien E. Spicher K. Schultz G. Jakobs K.H. Mol. Pharmacol. 1994; 45: 890-898PubMed Google Scholar, 28Rümenapp U. Asmus M. Schablowski H. Woznicki M. Han L. Jakobs K.H. Fahami-Vahid M. Michalek C. Wieland T. Schmidt M. J. Biol. Chem. 2001; 276: 10168-10174Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), but also to G12, Gi, and Gs proteins. By this distinct G protein coupling, the M3 mAChR can lead to the regulation of various effector enzymes, including phospholipase C (PLC), phospholipase D (PLD), and adenylyl cyclase, and activation of small GTPases from distinct families (26Peralta E.G. Ashkenazi A. Winslow J.W. Ramachandran J. Capon D.J. Nature. 1988; 334: 434-437Crossref PubMed Scopus (547) Google Scholar, 28Rümenapp U. Asmus M. Schablowski H. Woznicki M. Han L. Jakobs K.H. Fahami-Vahid M. Michalek C. Wieland T. Schmidt M. J. Biol. Chem. 2001; 276: 10168-10174Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 29Evellin S. Nolte J. Tysack K. vom Dorp F. Thiel M. Oude Weernink P.A. Jakobs K.H. Webb E.J. Lomasney J.W. Schmidt M. J. Biol. Chem. 2002; 277: 16805-16813Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Here we report that the M3 mAChR strongly activates R-Ras, that this activation requires endogenously expressed cyclic AMP-activated Epac1 proteins as shown by silencing of cellular Epac1 using siRNAs, and that this cellular response is mimicked by typical Gs-coupled receptors. Evidence is provided that Epac-activated R-Ras controls the M3 mAChR-mediated stimulation of PLD.EXPERIMENTAL PROCEDURESMaterials, Expression Plasmids, and Transfection—Forskolin, prostaglandin E1, 2′,5′-dideoxyadenosine (dd-Ado), BAPTA/AM, and H-89 were from Calbiochem, Merck Biosciences, and adrenaline and wortmannin were from Sigma. 8-Br-cAMP, LY294002, and tyrphostin 23 were from Biomol, and 8-(4-chlorophenylthio)-2′-O-methyl cyclic AMP (8-pCPT-2Me-cAMP) and 8-pCPTadenosine-3′,5′-cyclic monophosphorothioate (Rp-CPT-cAMPS) were from BIOLOG Life Science Institute (Bremen, Germany). GTPγS was from Roche Applied Science. Antibodies against R-Ras, RhoA, and Epac1 were from Santa Cruz Biotechnology; the anti-phosphovasodilator-stimulated phosphoprotein (VASP, Ser-157) antibody was from Cell Signaling Technology; the anti-VASP antibody was from Calbiochem, Merck Biosciences, and the anti-cofilin antibody was from tebu-bio. The anti-GAP1 IP4BP antibody was kindly provided by D. Bouyoucef and P. Cullen and a mouse monoclonal antibody against Epac1 by J. Bos (30Kooistra M.R.H. Corada M. Decana E. Bos J.L. FEBS Lett. 2005; 579: 4966-4972Crossref PubMed Scopus (259) Google Scholar). [3H]-Oleic acid (5 Ci/mmol) was from PerkinElmer Life Sciences, and [3H]GDP (14.2 Ci/mmol) was from GE Healthcare. cDNAs encoding wild-type R-Ras, S43N R-Ras, G38V R-Ras, Epac1 (each subcloned into pMT2-HA), S17N Rap2B (subcloned in pRK5), the β2-adrenergic receptor (subcloned into pCDNA3), and GAP1 IP4BP (subcloned into pCI-neo) were kindly provided by Drs. M. Spaargaren, H. Rehmann, J. de Gunzburg, R. Jockers, D. Bouyoucef, and P. Cullen. M3 mAChR-expressing HEK-293 cells and N1E-115 neuroblastoma cells grown to near confluence on 145-mm culture dishes were transfected with an efficiency of at least 50% as reported before (31Schmidt M. Evellin S. Oude Weernink P.A. vom Dorp F. Rehmann H. Lomasney J.W. Jakobs K.H. Nat. Cell Biol. 2001; 3: 1020-1024Crossref PubMed Scopus (280) Google Scholar, 32von Dorp F. Sari A.Y. Sanders H. Keiper M. Oude Weernink P.A. Jakobs K.H. Schmidt M. Cell. Signal. 2004; 16: 921-928Crossref PubMed Scopus (15) Google Scholar), typically with 25 μg of DNA from each of the β2-adrenergic receptors and wild-type Epac1, 50 μg each of wild-type R-Ras and G38V R-Ras, 100 μg each of S17N Rap2B, S43N R-Ras, and R279K Epac1, or with the indicated amounts of plasmid DNA. For transfection of HEK-293 cells with siRNAs, cells on 35-mm dishes were transfected with 20 μl of Lipofectamine 2000 in 2 ml of Opti-MEM and 200 pmol of siRNA according to the manufacturer's instructions (Invitrogen). If vector DNA was co-transfected with siRNA, 4 μg of the individual vector DNA was used in a total amount of 12 μg. The specific siRNA (Eurogentec) was si-Epac1: 5′-CCAUCAUCCUGCGAGAAGA99-3′ (effective against human Epac1). To evaluate transfection efficiency, fluorescence-labeled siRNA (Alexa Fluor 488, Qiagen) was used, which also served as unspecific control siRNA. Expression of the encoded proteins was verified by immunoblotting of cell lysates with specific antibodies. Assays were performed 48 h after transfection.Measurement of PLD and PLC Activities—PLD activity was measured in HEK-293 cells prelabeled with [3H]oleic acid as formation of the specific PLD product, [3H]phosphatidylethanol ([3H]PtdEtOH), for 30 min at 37 °C in the presence of ethanol as reported previously (28Rümenapp U. Asmus M. Schablowski H. Woznicki M. Han L. Jakobs K.H. Fahami-Vahid M. Michalek C. Wieland T. Schmidt M. J. Biol. Chem. 2001; 276: 10168-10174Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Formation of [3H]PtdEtOH is expressed as percentage of total labeled phospholipids. PLC activity was measured for 1 min at 37 °C as accumulation of inositol 1,4,5-trisphosphate (IP3) as described before (29Evellin S. Nolte J. Tysack K. vom Dorp F. Thiel M. Oude Weernink P.A. Jakobs K.H. Webb E.J. Lomasney J.W. Schmidt M. J. Biol. Chem. 2002; 277: 16805-16813Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).Activation of R-Ras and VASP Phosphorylation—Serum-starved HEK-293 cells and N1E-115 neuroblastoma cells transfected with wild-type R-Ras were incubated for the indicated periods of time at 37 °C with the indicated agents. After cell lysis, activated R-Ras was extracted with glutathione S-transferase (GST)-tagged Raf1-RBD (Ras-binding domain of Raf-1) bound to glutathione-Sepharose beads and immunoblotting with the anti-R-Ras antibody as described before (29Evellin S. Nolte J. Tysack K. vom Dorp F. Thiel M. Oude Weernink P.A. Jakobs K.H. Webb E.J. Lomasney J.W. Schmidt M. J. Biol. Chem. 2002; 277: 16805-16813Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 33Stope M.B. vom Dorp F. Szatkowski D. Böhm A. Keiper M. Nolte J. Oude Weernink P.A. Rosskopf D. Evellin S. Jakobs K.H. Schmidt M. Mol. Cell. Biol. 2004; 24: 4664-4676Crossref PubMed Scopus (41) Google Scholar, 34Keiper M. Stope M.B. Szatkowski D. Böhm A. Tysack K. vom Dorp F. Saur O. Oude Weernink P.A. Evellin S. Jakobs K.H. Schmidt M. J. Biol. Chem. 2004; 279: 46497-46508Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Densitometric analysis of the bands was performed with ImageQuant software (Amersham Biosciences). For measurement of VASP phosphorylation, serum-starved HEK-293 cells were incubated for 5 min at 37 °C with 30 μm forskolin, followed by cell lysis in a buffer containing 1% SDS and 10 mm Tris/HCl, pH 7.4, and five passages through a 25-gauge needle. Thereafter, the lysates were clarified by centrifugation, followed by determination of protein concentration and incubation in Laemmli buffer for 10 min at 95 °C. After SDS-PAGE and transfer to nitrocellulose membranes, phosphorylated VASP (Ser-157) was detected with the anti-phospho-VASP antibody.Purification of Proteins—GST-tagged R-Ras, Rap2B, and ΔDEP Epac1 (Epac1-(149–881)) and His6-tagged C3G-(830–1078) (cDNAs kindly provided by Drs. M. Spaargaren, J. de Gunzburg, and H. Rehmann) were expressed in Escherichia coli and purified with glutathione-Sepharose or nickel-nitrilotriacetic acid superflow agarose beads as described before (31Schmidt M. Evellin S. Oude Weernink P.A. vom Dorp F. Rehmann H. Lomasney J.W. Jakobs K.H. Nat. Cell Biol. 2001; 3: 1020-1024Crossref PubMed Scopus (280) Google Scholar, 33Stope M.B. vom Dorp F. Szatkowski D. Böhm A. Keiper M. Nolte J. Oude Weernink P.A. Rosskopf D. Evellin S. Jakobs K.H. Schmidt M. Mol. Cell. Biol. 2004; 24: 4664-4676Crossref PubMed Scopus (41) Google Scholar, 35de Rooij J. Rehmann H. van Triest M. Cool R.H. Wittinghofer A. Bos J.L. J. Biol. Chem. 2000; 275: 20829-20836Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). To obtain the soluble and untagged proteins, the immobilized proteins were proteolytically digested by thrombin (Rap2B, ΔDEP Epac1), factor Xa (R-Ras) or eluted with 400 mm imidazole (C3G). For protease digestion, the washed beads were resuspended in thrombin buffer (50 mm Tris/HCl, pH 8.6, 150 mm NaCl, 2.5 mm CaCl2, 0.1% 2-mercaptoethanol) or factor Xa buffer (137 mm NaCl, 2.7 mm KCl, 6.5 mm Na2HPO4, 1.5 mm KH2PO4, 8.8 mm CaCl2, pH 7.2), respectively, and incubated for 2 h at room temperature with thrombin or factor Xa (followed by overnight incubation at 4 °C). Purification of the proteins was analyzed by SDS-PAGE. Expression and purification of GST-tagged ARF1 in Spodoptera frugiperda cells were performed as described before (36Schürmann A. Schmidt M. Asmus M. Bayer S. Fliegert F. Koling S. Maβmann S. Schilf C. Subauste M.C. Voβ M. Jakobs K.H. Joost H.-G. J. Biol. Chem. 1999; 274: 9744-9751Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), except that a different buffer (50 mm Tris/HCl, pH 7.5, 2 mm EDTA, 1 mm dithiothreitol, 250 mm sucrose, 10 μm phenylmethylsulfonyl fluoride, and 0.5 μg/ml leupeptin) was used.Measurement of the GEF Activities of Epac1 and C3G—Binding of GDP to R-Ras, Rap2B, and ARF1 was determined at room temperature by the filter binding method (37Ozaki N. Shibasaki T. Kashima Y. Miki T. Takahashi K. Ueno H. Sunaga Y. Yano H. Matsuura Y. Iwanaga T. Takai Y. Seino S. Nat. Cell Biol. 2000; 2: 805-811Crossref PubMed Scopus (394) Google Scholar). In brief, for GDP binding the recombinant GTPases were first made nucleotide-free by incubation for 5 min in an EDTA-containing loading buffer (20 mm Tris/HCl, pH 7.5, 100 mm NaCl, 2 mm EDTA, 1 mm AMP-PNP, and 1 mm dithiothreitol), supplemented with 3 μm [3H]GDP. Thereafter, MgCl2 was added to a final concentration of 5 mm, and the incubation was continued for a further 20 min. Finally, purified ΔDEP Epac1 or C3G-(830–1078) equilibrated for 15 min in exchange buffer, containing 20 mm Tris/HCl, pH 7.5, 80 mm NaCl, 5 mm MgCl2, 0.4 mg/ml bovine serum albumin, 1 mm dithiothreitol, 10 mm AMP-PNP, and 10 mm GTP, was added in the absence (C3G-(830–1078)) or presence of 1 mm 8-Br-cAMP(ΔDEP Epac1), and the reaction was continued for the indicated periods of time. Bound and free [3H]GDP were separated by adding 1.5 ml of washing buffer (20 mm Tris/HCl, pH 7.5, 8 mm NaCl, 10 mm MgCl2, 0.05% 2-mercaptoethanol) and filtration through nitrocellulose filters. The ratio of GEF protein to the GTPases in the exchange assays typically was 1:1, as assessed from the protein bands of Coomassie-stained gels.Protein Binding Assays—Serum-starved HEK-293 cells transfected with wild-type Epac1 were lysed in a buffer containing 50 mm Tris/HCl, pH 7.5, 200 mm NaCl, 2 mm MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml benzamidine, 10 μg/ml soybean trypsin inhibitor, 0.5 μg/ml aprotinin, and 0.5 μg/ml leupeptin. Nucleotide-free, GDP-bound, and GTPγS-bound forms of recombinant GST-tagged R-Ras, Rap2B, and ARF1, immobilized on glutathione-Sepharose beads, were prepared exactly as described (38Lutz S. Freichel-Blomquist A. Rümenapp U. Schmidt M. Jakobs K.H. Wieland T. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar). Thereafter, the beads (5–10 or 50 μg of protein) were incubated with lysates of the transfected cells (3–4 mg of protein) or purified ΔDEP Epac1 (50 μg of protein) overnight at 4 °C. Finally, after three washes, the beads were resolved in Laemmli buffer, subjected to SDS-PAGE, and transferred to nitrocellulose for Western blotting, using anti-Epac1 antibodies as indicated.Immunoblot Analysis—For detection of R-Ras, phospho-VASP, VASP (each at a dilution of 1:500), and Epac1 (dilution of 1:1000–1:10,000), equal amounts of protein were separated by SDS-PAGE on 10% acrylamide gels. After transfer to nitrocellulose membranes and a 1-h incubation with the antibodies at the above given dilution factors, the proteins were visualized by enhanced chemiluminescence.Generation of HEK-293 Cells Stably Expressing Retroviral Encoded siRNA Epac1—siRNA molecules targeted against Epac1 were delivered to HEK-293 cells by retroviral transfer as described (39Barton G.M. Medzhitov R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14943-14945Crossref PubMed Scopus (261) Google Scholar) with the following modifications. First, the human H1 promoter, suitable for expression of short RNA molecules, was cloned by PCR from genomic DNA using the primers 5′-CGCGGTCGACATGGAAATCGAACGCTGACGTCATCAACCCGCTC-3′ (forward) and 5′-CGCGGTCGACCTCGAGGCGCTTCGAAGTGGTCTCATACAGAACTTATAAGATTCC-3′ (reverse). This construct contains two artificial restriction sites for XhoI and Bsp119I for site-directed insertion of the respective siRNA constructs, including suitable transcription stop signals. The entire construct was sequenced and cloned into the SalI site within the 3′-long terminal repeat of the pQCXIH vector (Clontech). This vector contains a hygromycin resistance gene under the control of a cytomegalo-virus promoter followed by an internal ribosome entry site as well as enhanced green fluorescent protein sequence. siRNA sequences targeted against human Epac1 were designed according to current algorithms and consisted of the cDNA of the target transcript followed by nine nucleotides hairpin sequences and the reverse sequence. For human Epac1 (GenBank™ accession number NM_006105), the entire sequences were 5′-CGAACCCGGCACTTCGTGGTACATTATTCAAGAGATAATGTACCACGAAGTGCCTTTTTGGAAC-3′ (forward) and 5′-TCGAGTTCCAAAAAGGCACTTCGTGGTACATTATCTCTTGAATAATGTACCACGAAGTGCCGGGTT-3′ (reverse). Two oligonucleotides (0.06 μg/μl, final concentration) encoding these sequences were annealed in a stepwise process for 4 min at 90 °C, 10 min at 70 °C, 5 min at 62 °C, 5 min at 37 °C, 5 min at 25 °C, and 5 min at 10 °C in 100 mm NaCl, 50 mm Hepes buffer, pH 7.4. After hybridization the construct was ligated into the XhoI/Bsp119I site of the modified pQCXIH vector. All vector constructs used were sequence-verified. Packaging GP2–293 cells (Clontech) were co-transfected with the respective pQCXIH-siRNA vector and the pVSV-G plasmid (encoding the envelope protein of the vesicular stomatitis virus) by calcium-phosphate transfection. Supernatants were harvested and ultracentrifuged for enrichment of recombinant retroviruses. M3 mAChR-expressing HEK-293 cells were infected with the recombinant retroviruses for 24 h at 37 °C, and infection was monitored by fluorescent microscopy detection of enhanced green fluorescent protein. Stably infected cells were further selected by incubation with 200 μg/ml hygromycin. Epac1 transcripts were measured by quantitative real time PCR using the fluorescent dye SYBR Green. Total RNA samples were obtained using the NucleoSpin RNA II kit, including a DNA digestion step (Macherey-Nagel) according to the manufacturer's instructions. As control, cells infected with retroviruses lacking a siRNA insert were used. Reverse transcription was done with an oligo(dT)15 primer and 1 μg of RNA using SuperScript II reverse transcriptase (Invitrogen). Quantitative PCR was performed by monitoring the fluorescence of the SYBR Green dye (Platinum SYBR Green qPCR SuperMix UDG; Invitrogen) on a 7500 real time PCR system (Applied Biosystems). Applied primer pairs were specific for the sequences of Epac1 (5′-AGTTTCCCACCTCCACGAGGAC-3′; 5′-ACATAAGCCCAGGTGCTGGCTG-3′) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (5′-GGCGATGCTGGCGCTGAGT-3′; 5′-CATGGTTCACACCCATGACGA-3′), which was used for normalization. The relative transcription level of the EPAC1 gene in siRNA expressing cells compared with vector controls is expressed as ΔCT values (40Livak K.J. Schmittgen T.D. Methods (Orlando). 2001; 25: 402-408Crossref Scopus (119255) Google Scholar).Data Presentation—Data shown in the figures are either representative experiments or the mean ± S.E. of n independent experiments, each performed in triplicate. Comparisons between means were either with the Student's paired t test or one-way analysis of variance test, and a difference was regarded significant at p < 0.05.RESULTSActivation of R-Ras by the M3 mAChR—The M3 mAChR stably expressed in HEK-293 cells can couple to several types of heterotrimeric G proteins, thereby leading to stimulation of various effector enzymes and activation of small GTPases from different families (26Peralta E.G. Ashkenazi A. Winslow J.W. Ramachandran J. Capon D.J. Nature. 1988; 334: 434-437Crossref PubMed Scopus (547) Google Scholar, 27Offermanns S. Wieland T. Homann D. Sandmann J. Bombien E. Spicher K. Schultz G. Jakobs K.H. Mol. Pharmacol. 1994; 45: 890-898PubMed Google Scholar, 28Rümenapp U. Asmus M. Schablowski H. Woznicki M. Han L. Jakobs K.H. Fahami-Vahid M. Michalek C. Wieland T. Schmidt M. J. Biol. Chem. 2001; 276: 10168-10174Abstract Full Text Full Text PDF PubMed Scopus (75) Google Schola" @default.
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• W2168142419 title "Cyclic AMP-dependent and Epac-mediated Activation of R-Ras by G Protein-coupled Receptors Leads to Phospholipase D Stimulation" @default.
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