Hsp90 Interactions and Acylation Target the G Protein Gα12 but Not Gα13 to Lipid Rafts
2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês
10.1074/jbc.c200383200
ISSN1083-351X
AutoresAbdül Waheed, Teresa L.Z. Jones,
Tópico(s)Cell death mechanisms and regulation
ResumoThe heterotrimeric G proteins, G12and G13, are closely related in their sequences, signaling partners, and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet G12 and G13can act through different pathways, bind different proteins, and show opposing actions on some effectors. We investigated the compartmentalization of G12 and G13 at the membrane because other G proteins reside in lipid rafts, membrane microdomains enriched in cholesterol and sphingolipids. Lipid rafts were isolated after cold, nonionic detergent extraction of cells and gradient centrifugation. Gα12 was in the lipid raft fractions, whereas Gα13 was not associated with lipid rafts. Mutation of Cys-11 on Gα12, which prevents its palmitoylation, partially shifted Gα12 from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type Gα12 and a complete loss of the Cys-11 mutant from the lipid rafts and the appearance of a higher molecular weight form of Gα12 in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized Gα12 to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional Gα12 only in discrete membrane domains and thereby explain some of the nonoverlapping functions of G12 and G13 and control of these potent cell regulators. The heterotrimeric G proteins, G12and G13, are closely related in their sequences, signaling partners, and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet G12 and G13can act through different pathways, bind different proteins, and show opposing actions on some effectors. We investigated the compartmentalization of G12 and G13 at the membrane because other G proteins reside in lipid rafts, membrane microdomains enriched in cholesterol and sphingolipids. Lipid rafts were isolated after cold, nonionic detergent extraction of cells and gradient centrifugation. Gα12 was in the lipid raft fractions, whereas Gα13 was not associated with lipid rafts. Mutation of Cys-11 on Gα12, which prevents its palmitoylation, partially shifted Gα12 from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type Gα12 and a complete loss of the Cys-11 mutant from the lipid rafts and the appearance of a higher molecular weight form of Gα12 in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized Gα12 to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional Gα12 only in discrete membrane domains and thereby explain some of the nonoverlapping functions of G12 and G13 and control of these potent cell regulators. guanine nucleotide-binding protein heat shock protein of 90 kDa human dermal microvascular endothelial cells detergent-resistant membrane Heterotrimeric G proteins,1consisting of α, β, and γ subunits, act as molecular switches that transmit signals from heptahelical G protein-coupled receptors at the cell surface to intracellular effectors (1Hamm H.E. Gilchrist A. Curr. Opin. Cell Biol. 1996; 8: 189-196Crossref PubMed Scopus (204) Google Scholar). This family of proteins can be divided into four groups, Gs, Gi, Gq, and G12, based on sequence homology of their α subunits. The G12 proteins, Gα12 and Gα13, are ubiquitously expressed and share 67% amino acid sequence identity (2Dhanasekaran N. Dermott J.M. Cell. Signal. 1996; 8: 235-245Crossref PubMed Scopus (128) Google Scholar). Both G12and G13 can couple to the same receptors, bind to the same RGS (regulators of G protein signaling) proteins, regulate the same downstream pathways, and produce the same effects such as cytoskeletal rearrangements, oncogenic transformation, and apoptosis (2Dhanasekaran N. Dermott J.M. Cell. Signal. 1996; 8: 235-245Crossref PubMed Scopus (128) Google Scholar, 3Kozasa 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, 4Buhl 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, 5Fukuhara S. Chikumi H. Gutkind J.S. FEBS Lett. 2000; 485: 183-188Crossref PubMed Scopus (211) Google Scholar, 6Diviani D. Soderling J. Scott J.D. J. Biol. Chem. 2001; 276: 44247-44257Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 7Meigs T.E. Fields T.A. McKee D.D. Casey P.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 519-524PubMed Google Scholar, 8Althoefer H. Eversole-Cire P. Simon M.I. J. Biol. Chem. 1997; 272: 24380-24386Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). However, despite these appearances, the cellular and physiologic functions of Gα12 and Gα13 do not completely overlap. They act through different pathways to regulate Na+/H+ exchange (9Dhanasekaran N. Vara Prasad M.V.V.S. Wadsworth S.J. Dermott J.M. van Rossum G. J. Biol. Chem. 1994; 269: 11802-11806Abstract Full Text PDF PubMed Google Scholar), activate serum response factor (10Mao J. Yuan H. Xie W. Wu D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12973-12976Crossref PubMed Scopus (114) Google Scholar), and form Rho-dependent actin stress fibers (6Diviani D. Soderling J. Scott J.D. J. Biol. Chem. 2001; 276: 44247-44257Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar,11Gohla 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). Gα12 is more potent in inducing oncogenic transformation (12Vara Prasad M.V.V.S. Shore S.K. Dhanasekaran N. Oncogene. 1994; 9: 2425-2429PubMed Google Scholar), and Gα13 is more potent in inducing apoptosis (8Althoefer H. Eversole-Cire P. Simon M.I. J. Biol. Chem. 1997; 272: 24380-24386Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Between Gα12 and Gα13, Gα12 regulates paracellular permeability (13Meyer T.N. Schwesinger C. Denker B.M. J. Biol. Chem. 2002; 277: 24855-24858Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), and Gα13 activates depolarizing chloride channels (14Postma F.R. Jalink K. Hengeveld T. Offermanns S. Moolenaar W.H. Curr. Biol. 2001; 11: 121-124Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and stimulates p115 RhoGEF to catalyze nucleotide exchange on Rho (15Hart 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). The most striking difference is that Gα13-deficient mice die at midgestation with defects in angiogenesis (16Offermanns S. Mancino V. Revel J.-P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (294) Google Scholar), whereas Gα12-deficient mice are alive and without an obvious phenotype (17Offermanns S. Oncogene. 2001; 20: 1635-1642Crossref PubMed Scopus (75) Google Scholar). A recent report shows that Gα12 interacts with Hsp90 (heat shock protein of 90 kDa), and this interaction is required for Gα12 signaling (18Vaiskunaite R. Kozasa T. Voyno-Yasenetskaya T.A. J. Biol. Chem. 2001; 276: 46088-46093Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The Hsp90 interaction was specific for Gα12 because Gα13 did not bind or show functional interactions with Hsp90. Hsp90 is an abundant and ubiquitously expressed molecular chaperone with the unique property of binding and maintaining the activity of numerous signal transduction proteins (19Young J.C. Moarefi I. Hartl F.U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (719) Google Scholar, 20Richter K. Buchner J. J. Cell. Physiol. 2001; 188: 281-290Crossref PubMed Scopus (506) Google Scholar) including steroid hormone receptors, signaling kinases, nitric-oxide synthase, and thrombin receptors (21Pai K.S. Mahajan V.B. Lau A. Cunningham D.D. J. Biol. Chem. 2001; 276: 32642-32647Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Recently Hsp90 was discovered to serve as a scaffold to promote the interactions of the Akt kinase and its substrate, endothelial nitric-oxide synthase, through their binding to two of the multiple protein binding sites on Hsp90 (22Fontana J. Fulton D. Chen Y. Fairchild T.A. McCabe T.J. Fujita N. Tsuruo T. Sessa W.C. Circ. Res. 2002; 90: 866-873Crossref PubMed Scopus (301) Google Scholar). Compartmentalization of eukaryotic cell membranes into dynamic microdomains, referred to as lipid rafts, has been detected in numerous studies using different methodologies (23Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar, 24Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5164) Google Scholar, 25Shaul P.W. Anderson R.G. Am. J. Physiol. 1998; 275: L843-L851PubMed Google Scholar). These microdomains are enriched in cholesterol and sphingolipids and can be isolated by their resistance to cold, nonionic detergent extraction. Proteins modified with multiple acyl chains or glycosylphosphatidylinositol groups are targeted to lipid rafts (23Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar, 26Resh M.D. Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1079) Google Scholar). The Gα subunits of the Gs, Gi, and Gq classes undergo palmitoylation and/or myristoylation on their amino-terminal ends (27Mumby S.M. Curr. Opin. Cell Biol. 1997; 9: 148-154Crossref PubMed Scopus (238) Google Scholar) and localize to lipid rafts (28Miura Y. Hanada K. Jones T.L.Z. Biochemistry. 2001; 40: 15418-15423Crossref PubMed Scopus (40) Google Scholar, 29Pike L.J. Miller J.M. J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 30Moffett S. Brown D.A. Linder M.E. J. Biol. Chem. 2000; 275: 2191-2198Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 31Galbiati F. Volonte D. Meani D. Milligan G. Lublin D.M. Lisanti M.P. Parenti M. J. Biol. Chem. 1999; 274: 5843-5850Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Gα12 and Gα13 also undergo palmitoylation, the reversible addition of palmitate to cysteine residues through a thioester bond (32Jones T.L.Z. Gutkind J.S. Biochemistry. 1998; 37: 3196-3202Crossref PubMed Scopus (35) Google Scholar, 33Ponimaskin E. Harteneck C. Schultz G. Schmidt M.F.G. FEBS Lett. 1998; 429: 370-374Crossref PubMed Scopus (20) Google Scholar, 34Ponimaskin E. Behn H. Adarichev V. Voyno-Yasenetskaya T.A. Offermanns S. Schmidt M.F.G. FEBS Lett. 2000; 478: 173-177Crossref PubMed Scopus (20) Google Scholar, 35Bhattacharyya R. Wedegaertner P.B. J. Biol. Chem. 2000; 275: 14992-14999Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). A single cysteine residue (Cys-11) for Gα12 (32Jones T.L.Z. Gutkind J.S. Biochemistry. 1998; 37: 3196-3202Crossref PubMed Scopus (35) Google Scholar, 33Ponimaskin E. Harteneck C. Schultz G. Schmidt M.F.G. FEBS Lett. 1998; 429: 370-374Crossref PubMed Scopus (20) Google Scholar) and two or three cysteine residues (Cys-14, Cys-18, and Cys-37) for Gα13 are critical for this posttranslational modification (34Ponimaskin E. Behn H. Adarichev V. Voyno-Yasenetskaya T.A. Offermanns S. Schmidt M.F.G. FEBS Lett. 2000; 478: 173-177Crossref PubMed Scopus (20) Google Scholar, 35Bhattacharyya R. Wedegaertner P.B. J. Biol. Chem. 2000; 275: 14992-14999Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). We investigated the lipid raft localization of Gα12 and Gα13 to better understand the different signaling functions of these proteins. We found a distinct segregation of Gα12 and Gα13 at the membrane. Gα13 was not in lipid rafts, whereas Gα12was in lipid rafts with both acylation and Hsp90 interactions critical for this targeting. The QE antibody specific for Gα12, HD antibody specific for Gα13, AS antibody specific for Gαi, and SW antibody specific for Gβ were prepared by immunizing rabbits with the carboxyl-terminal peptide sequence of the respective subunits and affinity-purified. Antibodies against the following proteins were purchased: Gα12 and Gα13 against their amino-terminal sequences and Fyn (Santa Cruz Biotechnology), caveolin and Hsp90 (BD PharMingen), and Na+/K+-ATPase (Biomol Research Laboratories). OptiPrep was purchased from Invitrogen. Geldanamycin was obtained from Tocris Cookson Inc. MG132, a proteasome inhibitor, was from Calbiochem. Methyl-β-cyclodextrin, hydrocortisone, and epidermal growth factor were from Sigma. Simian kidney (COS-7) and human embryonic kidney (HEK293) cells were maintained in Dulbecco's modified Eagles medium with 10% fetal bovine serum. Human dermal microvascular endothelial cells that were transformed with SV-40 (HMEC1) (Biological Products Branch, Centers for Disease Control and Prevention, Atlanta, GA) were maintained in MCBD 131 medium supplemented with 5% fetal bovine serum, hydrocortisone (1 μg/ml), and epidermal growth factor (0.01 mg/ml). NIH 3T3 mouse fibroblasts that were stably transfected with wild-type and mutant plasmids of Gα12 were prepared and maintained as described previously (32Jones T.L.Z. Gutkind J.S. Biochemistry. 1998; 37: 3196-3202Crossref PubMed Scopus (35) Google Scholar). In geldanamycin experiments, cells were incubated in serum-free medium for 1 h and preincubated with 10 μm MG132 for 10 min, and then 1 μg/ml geldanamycin was added and incubated for 1 h. In cholesterol depletion experiments, COS cells were preincubated with serum-free medium for 2 h and then treated with 20 mm methyl-β-cyclodextrin in serum-free medium at 37 °C for 30 min. DRMs were prepared as described previously with some modifications (36Lindwasser O.W. Resh M.D. J. Virol. 2001; 75: 7913-7924Crossref PubMed Scopus (211) Google Scholar). Cells were washed twice and harvested in ice-cold phosphate-buffered saline (pH 7.4). Cell pellets were obtained by centrifugation at 1,000 × g for 10 min at 4 °C. The cell pellet was suspended in phosphate-buffered saline, and the protein concentration was determined (Bio-Rad). An aliquot of the cell suspension corresponding to 1 mg of protein was centrifuged at 5,000 ×g for 5 min at 4 °C. The cell pellet was resuspended in 0.5 ml of TNET buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 0.5% (v/v) Triton X-100) containing protease inhibitors, incubated on ice for 20 min, and then homogenized with 10 strokes in a Dounce homogenizer. The cell lysate was adjusted to 35% OptiPrep and overlaid with 3.0 ml of 30% OptiPrep in TNET and then with 200 μl of 5% OptiPrep in TNET in Beckman SW55 tubes. Samples were centrifuged for 4 h at 170,000 ×g at 4 °C and fractionated from the top (0.52 ml each for a total of nine fractions). The pellet was suspended in 0.52 ml of TNET buffer. SDS sample buffer containing 2-mercaptoethanol was added to aliquots of each fraction of the gradient. NIH 3T3 cells expressing the wild-type Gα12 or COS cells were homogenized in TNE buffer (TNET without Triton X-100) containing protease inhibitors by passing through a 25-gauge needle 12 times. The particulate and soluble fractions were separated by centrifugation at 125,000 ×g for 1 h. Aliquots of soluble and particulate fractions were treated with 200 μm MG132 for 10 min and then treated with 20 μg/ml of geldanamycin for 1 h. Triton X-100 was added to give a final concentration of 0.5% and incubated at 4 °C for 20 min, then diluted 3-fold in TNE buffer, and further incubated for 4 h. Detergent-soluble and -insoluble fractions were separated by centrifugation at 15,000 ×g and analyzed by immunoblotting. Equal volumes of gradient fractions were loaded on 12% polyacrylamide gels (Novex precast gels, Invitrogen) and subjected to SDS-PAGE and immunoblotting using Enhanced Chemiluminescence (Amersham Biosciences) for detection of the antibodies per the manufacturer's directions. Densitometry was performed using a UMAX scanner (model UTA-II) and Scion Image software. To determine whether Gα12 and Gα13 reside in lipid rafts, we prepared DRMs by treatment of COS cells with 0.5% Triton X-100 and centrifugation on an OptiPrep gradient. Caveolin, Fyn, and Gαi were found in fractions 1 and 2 consistent with their known partitioning to DRMs and lipid rafts (Fig. 1A) (25Shaul P.W. Anderson R.G. Am. J. Physiol. 1998; 275: L843-L851PubMed Google Scholar). Na+/K+-ATPase, an integral plasma membrane protein not found in lipid rafts (36Lindwasser O.W. Resh M.D. J. Virol. 2001; 75: 7913-7924Crossref PubMed Scopus (211) Google Scholar), was in the higher density fractions 6–8. Endogenous Gα12 was found in the DRM fraction 1; Gα13 was in the higher density fractions 7–9. We found the same pattern of partitioning after detergent solubilization and gradient centrifugation for the endogenous Gα12 and Gα13 proteins in two other cell lines, HMEC (Fig. 1B) and HEK293 (data not shown). In these cells, Gα12 and the lipid raft markers were in the DRM fractions, and Gα13 and Na+/K+-ATPase were in the higher density fractions. The partitioning of Gα12 and the lipid raft-associated proteins to the DRM fraction was abolished and decreased, respectively, by treatment with 20 mmmethyl-β-cyclodextrin, which depletes cellular cholesterol and disrupts lipid rafts (Fig. 1C). These results show that Gα12 segregates with proteins that prefer lipid rafts and Gα13 segregates with a plasma membrane protein that avoids lipid rafts. We investigated the role of the cysteine site of palmitoylation for targeting Gα12 to lipid rafts. Mutation of Cys-11 on Gα12 prevents [3H]palmitate incorporation, but the protein is still found in the particulate fraction (32Jones T.L.Z. Gutkind J.S. Biochemistry. 1998; 37: 3196-3202Crossref PubMed Scopus (35) Google Scholar). Both the wild-type Gα12 and a GTPase-deficient mutant of Gα12(Q229L) were in the DRM fractions in transfected NIH 3T3 cells (Fig.2A). Most of the nonpalmitoylated C11S mutant of Gα12 was shifted to the higher density fractions (Fig. 2A). This result is consistent with other studies showing that acylation can target proteins to lipid rafts (23Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2058) Google Scholar,26Resh M.D. Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1079) Google Scholar). Further mutation of the amino terminus of Gα12 at Ser-2 and Arg-6 changes the acylation to myristoylation (32Jones T.L.Z. Gutkind J.S. Biochemistry. 1998; 37: 3196-3202Crossref PubMed Scopus (35) Google Scholar) and restored a small amount of the DRM localization of Gα12(Fig. 2A). Therefore, the less hydrophobic myristate on the amino terminus could partially substitute for palmitate in targeting Gα12 to the DRM fractions. Given the importance of the cysteine site of acylation for the DRM partitioning of Gα12 and the lipid raft localization of other biacylated Gα subunits including Gαi in this study (29Pike L.J. Miller J.M. J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 30Moffett S. Brown D.A. Linder M.E. J. Biol. Chem. 2000; 275: 2191-2198Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 31Galbiati F. Volonte D. Meani D. Milligan G. Lublin D.M. Lisanti M.P. Parenti M. J. Biol. Chem. 1999; 274: 5843-5850Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), we were surprised that Gα12 with only one putative acylation site (32Jones T.L.Z. Gutkind J.S. Biochemistry. 1998; 37: 3196-3202Crossref PubMed Scopus (35) Google Scholar, 33Ponimaskin E. Harteneck C. Schultz G. Schmidt M.F.G. FEBS Lett. 1998; 429: 370-374Crossref PubMed Scopus (20) Google Scholar) was in the DRM fraction and that Gα13 with two or three putative acylation sites (34Ponimaskin E. Behn H. Adarichev V. Voyno-Yasenetskaya T.A. Offermanns S. Schmidt M.F.G. FEBS Lett. 2000; 478: 173-177Crossref PubMed Scopus (20) Google Scholar, 35Bhattacharyya R. Wedegaertner P.B. J. Biol. Chem. 2000; 275: 14992-14999Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) was in the higher density fractions. Not all palmitoylated proteins reside in lipid rafts (37Melkonian K.A. Ostermeyer A.G. Chen J.Z. Roth M.G. Brown D.A. J. Biol. Chem. 1999; 274: 3910-3917Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar), and the stoichiometry of palmitoylation on Gα13 is not known, but our present result suggests that Gα13 evades lipid rafts. Clearly further studies are required to understand the interaction that may prevent Gα13 targeting to lipid rafts. Hsp90 binds to Gα12 but not Gα13 (18Vaiskunaite R. Kozasa T. Voyno-Yasenetskaya T.A. J. Biol. Chem. 2001; 276: 46088-46093Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and Hsp90 can be part of a complex with caveolin (38Gratton J.-P. Fontana J. O'Connor D.S. Garcia-Cardena G. McCabe T.J. Sessa W.C. J. Biol. Chem. 2000; 275: 22268-22272Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Therefore, we investigated whether Hsp90 may be involved in the targeting of Gα12, but not G13, to the DRM fractions. To probe the interaction between Gα12 and Hsp90, we used geldanamycin, a fungal benzoquinone ansamycin, that specifically and tightly binds to the ATP binding site in Hsp90 and inhibits the ability of Hsp90 to form complexes with its substrate proteins (20Richter K. Buchner J. J. Cell. Physiol. 2001; 188: 281-290Crossref PubMed Scopus (506) Google Scholar). We included the proteasome inhibitor MG132 (39Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1249) Google Scholar) because geldanamycin can increase protein degradation through the ubiquitin/proteosomal pathway (19Young J.C. Moarefi I. Hartl F.U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (719) Google Scholar, 40Busconi L. Guan J. Denker B.M. J. Biol. Chem. 2000; 275: 1565-1569Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Treatment with MG132 alone did not change the DRM localization of Gα12 (data not shown). Geldanamycin and MG132 treatment of COS cells displaced Gα12 from the DRM fractions and led to the appearance of a 220-kDa band in fractions 6–9 (Fig.3A). Densitometric analysis showed a loss of Gα12 from the DRM fraction of about 60 ± 10% (mean ± S.E. for three experiments) for cells treated with geldanamycin plus MG132 compared with cells treated with vehicle alone. The lipid raft localization of caveolin, Gαi, Gβ, and Fyn was not changed by geldanamycin treatment (Fig.3B). Likewise, geldanamycin treatment did not alter the localization of Gα13 (Fig. 3B) and Na+/K+-ATPase (data not shown) to the higher density fractions. Only a trivial amount of Hsp90 was found in the DRM fraction, consistent with a previous report (41Triantafilou M. Miyake K. Golenbock D.T. Triantafilou K. J. Cell Sci. 2002; 115: 2603-2611Crossref PubMed Google Scholar) and with the cytosolic location of this abundant protein (Fig. 3B) (19Young J.C. Moarefi I. Hartl F.U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (719) Google Scholar, 20Richter K. Buchner J. J. Cell. Physiol. 2001; 188: 281-290Crossref PubMed Scopus (506) Google Scholar). Hsp90 was not found in the DRMs after geldanamycin treatment. Treatment with geldanamycin alone or with MG132 did not change the total amount of Gα12 (data not shown) in agreement with a previous report (18Vaiskunaite R. Kozasa T. Voyno-Yasenetskaya T.A. J. Biol. Chem. 2001; 276: 46088-46093Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). These results indicate that the loss of Gα12 from the DRM fractions after geldanamycin treatment was not due to a general disruption of lipid rafts or degradation of Gα12 but rather suggest it was due to a loss of Hsp90 binding. We also tested the effect of geldanamycin treatment on the DRM localization of the nonpalmitoylated C11S mutant of Gα12to determine the relationship between acylation and Hsp90 binding in the lipid raft localization of Gα12. Geldanamycin treatment of the transfected NIH 3T3 cells led to a further loss of the nonpalmitoylated C11S mutant from the DRM fraction so that the mutant Gα12 was only in the higher density fractions (Fig.4A). An increase in the intensity of the 220-kDa band was also seen after geldanamycin treatment in these cells. This result indicates that acylation and Hsp90 binding act independently to promote the DRM localization of Gα12. We investigated whether Hsp90 interactions maintain Gα12at lipid rafts. The crude membrane fraction of NIH 3T3 cells stably transfected with wild-type Gα12 was treated with geldanamycin and MG132 followed by solubilization with Triton X-100 under conditions similar to DRM preparation. Geldanamycin treatment increased the detergent solubility of Gα12 (Fig.4B, membrane) suggesting that Hsp90 interactions maintain Gα12 in the lipid rafts in addition to any possible role in directing Gα12 to these membrane microdomains. To our knowledge, this is the first report of a change in lipid raft localization after disruption of Hsp90 binding. Hsp90 is an abundant cytosolic protein that is not directly targeted to lipid rafts so its role in the targeting of Gα12 would be either 1) maintenance of Gα12 in a conformation that permits direct interaction of Gα12 with lipids and proteins in lipid rafts or 2) formation of a complex with proteins that have a high affinity for lipid rafts. In regard to the latter, Hsp90 has multiple protein binding sites (22Fontana J. Fulton D. Chen Y. Fairchild T.A. McCabe T.J. Fujita N. Tsuruo T. Sessa W.C. Circ. Res. 2002; 90: 866-873Crossref PubMed Scopus (301) Google Scholar) and readily forms complexes with other proteins including proteins that directly associate with lipid rafts (38Gratton J.-P. Fontana J. O'Connor D.S. Garcia-Cardena G. McCabe T.J. Sessa W.C. J. Biol. Chem. 2000; 275: 22268-22272Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar) or translocate to lipid rafts (41Triantafilou M. Miyake K. Golenbock D.T. Triantafilou K. J. Cell Sci. 2002; 115: 2603-2611Crossref PubMed Google Scholar). The presence of a 220-kDa band detected with an antibody to Gα12 in the high density fractions after geldanamycin and MG132 treatment is a novel finding. Higher molecular weight bands of Gα13 were not detected after geldanamycin treatment with antibodies to the amino and carboxyl terminus of Gα13(data not shown). The 220-kDa band was not due to immunoreactivity of another protein that is expressed after geldanamycin treatment because we saw an increase in the 220-kDa band after geldanamycin treatment of the cytosolic fractions of NIH 3T3 cells transfected with the wild-type Gα12 (Fig. 4B, cytosol) or COS cells (data not shown). The relative amounts of the 43- and 220-kDa bands could not be determined because the antibody to the amino terminus poorly detects the 43-kDa form probably because palmitoylation obscured the epitope (Fig. 2B), and the antibody to the carboxyl terminus did not detect the 220-kDa form (data not shown). The correlation of the loss of Gα12 immunodetection at 43 kDa in the DRMs (Figs. 3A and 4A) with an increase at 220 kDa suggests that this band is likely to contain the Gα12 protein, possibly as part of another protein or an SDS-resistant aggregate or oligomer. Hsp90 as a molecular chaperone maintains the proper folding of signal transduction proteins, which have an inherent instability because they undergo conformational changes as part of relaying signals (19Young J.C. Moarefi I. Hartl F.U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (719) Google Scholar). The presence of the 220-kDa band after geldanamycin treatment suggests that Hsp90 may be preventing aggregation of Gα12. Signal transduction requires not only affinity between molecules but also proximity. Some of the defects seen in G12 signaling after mutation of Cys-11, which prevents palmitoylation (32Jones T.L.Z. Gutkind J.S. Biochemistry. 1998; 37: 3196-3202Crossref PubMed Scopus (35) Google Scholar, 33Ponimaskin E. Harteneck C. Schultz G. Schmidt M.F.G. FEBS Lett. 1998; 429: 370-374Crossref PubMed Scopus (20) Google Scholar), or geldanamycin treatment, which blocks Hsp90 interactions (18Vaiskunaite R. Kozasa T. Voyno-Yasenetskaya T.A. J. Biol. Chem. 2001; 276: 46088-46093Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 21Pai K.S. Mahajan V.B. Lau A. Cunningham D.D. J. Biol. Chem. 2001; 276: 32642-32647Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), may be due to mistargeting of Gα12away from its signaling partners. Concentrating G12 in lipid rafts may significantly improve the kinetics of its signaling. Heterogeneity and compartmentalization within cellular membranes can also keep signaling partners apart to prevent interactions and increase the specificity of signaling. The differential membrane targeting of Gα12 and Gα13 may be responsible for increased specificity of receptor coupling seen for Gα12and Gα13 in intact cells under physiologic conditions compared with results using cell membranes (11Gohla 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). Specific control of the membrane location of G12 and G13 is especially important because they are potent inducers of cell growth, differentiation, shape change, and apoptosis. G12interactions with Hsp90 may explain the tight regulation of G12 signaling and the nonoverlapping functions of G12 and G13 because Hsp90 is both a scaffold and chaperone. If Gα12 remains bound directly or indirectly to Hsp90, it maintains a functional conformation to interact with its signaling partners. If it strays from Hsp90 and its membrane microdomain, Gα12 aggregates and loses activity. Gα12 would then only work when it is in the correct location and thus effectively prevent random collisions with signaling partners during diffusion in the membrane. The inability of Gα12 to compensate for the loss of Gα13 in the Gα13-deficient mice (16Offermanns S. Mancino V. Revel J.-P. Simon M.I. Science. 1997; 275: 533-536Crossref PubMed Scopus (294) Google Scholar) may be the result of its poor mobility. Besides Hsp90, Gα12 has also been found to interact with two other scaffold proteins in specialized cells (6Diviani D. Soderling J. Scott J.D. J. Biol. Chem. 2001; 276: 44247-44257Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar,13Meyer T.N. Schwesinger C. Denker B.M. J. Biol. Chem. 2002; 277: 24855-24858Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). G12 and G13 are like fraternal twins, similar but different. Our finding that they play their roles at different sites on the membrane gives a framework for understanding their regulation and the unfolding story of Hsp90 and G12signaling. More studies are needed to discover the ramifications of their interactions and membrane targeting for G protein signaling. We thank Fransisco Candal for the HMEC1 cells and Dr. Silvio Gutkind for valuable discussions.
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