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J. Biol. Chem., Vol. 277, Issue 36, 32409-32412, September 6, 2002

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ACCELERATED PUBLICATION
Hsp90 Interactions and Acylation Target the G Protein G₁₂ but Not G₁₃ to Lipid Rafts^*

Abdul A. Waheed and Teresa L. Z. Jones

From the Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 1, 2002, and in revised form, July 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The heterotrimeric G proteins, G₁₂ and G₁₃, are closely related in their sequences, signaling partners, and cellular effects such as oncogenic transformation and cytoskeletal reorganization. Yet G₁₂ and G₁₃ can 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. G₁₂ was in the lipid raft fractions, whereas G₁₃ was not associated with lipid rafts. Mutation of Cys-11 on G₁₂, which prevents its palmitoylation, partially shifted G₁₂ from the lipid rafts. Geldanamycin treatment, which specifically inhibits Hsp90, caused a partial loss of wild-type G₁₂ and a complete loss of the Cys-11 mutant from the lipid rafts and the appearance of a higher molecular weight form of G₁₂ in the soluble fractions. These results indicate that acylation and Hsp90 interactions localized G₁₂ to lipid rafts. Hsp90 may act as both a scaffold and chaperone to maintain a functional G₁₂ only in discrete membrane domains and thereby explain some of the nonoverlapping functions of G₁₂ and G₁₃ and control of these potent cell regulators.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Heterotrimeric G proteins,¹ consisting of , , and  subunits, act as molecular switches that transmit signals from heptahelical G protein-coupled receptors at the cell surface to intracellular effectors (1). This family of proteins can be divided into four groups, G_s, G_i, G_q, and G₁₂, based on sequence homology of their  subunits. The G₁₂ proteins, G₁₂ and G₁₃, are ubiquitously expressed and share 67% amino acid sequence identity (2). Both G₁₂ and 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 (2-8). However, despite these appearances, the cellular and physiologic functions of G₁₂ and G₁₃ do not completely overlap. They act through different pathways to regulate Na⁺/H⁺ exchange (9), activate serum response factor (10), and form Rho-dependent actin stress fibers (6, 11). G₁₂ is more potent in inducing oncogenic transformation (12), and G₁₃ is more potent in inducing apoptosis (8). Between G₁₂ and G₁₃, G₁₂ regulates paracellular permeability (13), and G₁₃ activates depolarizing chloride channels (14) and stimulates p115 RhoGEF to catalyze nucleotide exchange on Rho (15). The most striking difference is that G₁₃-deficient mice die at midgestation with defects in angiogenesis (16), whereas G₁₂-deficient mice are alive and without an obvious phenotype (17).

A recent report shows that G₁₂ interacts with Hsp90 (heat shock protein of 90 kDa), and this interaction is required for G₁₂ signaling (18). The Hsp90 interaction was specific for G₁₂ because G₁₃ 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 (19, 20) including steroid hormone receptors, signaling kinases, nitric-oxide synthase, and thrombin receptors (21). 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 (22).

Compartmentalization of eukaryotic cell membranes into dynamic microdomains, referred to as lipid rafts, has been detected in numerous studies using different methodologies (23-25). 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 (23, 26). The G subunits of the G_s, G_i, and G_q classes undergo palmitoylation and/or myristoylation on their amino-terminal ends (27) and localize to lipid rafts (28-31). G₁₂ and G₁₃ also undergo palmitoylation, the reversible addition of palmitate to cysteine residues through a thioester bond (32-35). A single cysteine residue (Cys-11) for G₁₂ (32, 33) and two or three cysteine residues (Cys-14, Cys-18, and Cys-37) for G₁₃ are critical for this posttranslational modification (34, 35).

We investigated the lipid raft localization of G₁₂ and G₁₃ to better understand the different signaling functions of these proteins. We found a distinct segregation of G₁₂ and G₁₃ at the membrane. G₁₃ was not in lipid rafts, whereas G₁₂ was in lipid rafts with both acylation and Hsp90 interactions critical for this targeting.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- The QE antibody specific for G₁₂, HD antibody specific for G₁₃, AS antibody specific for G_i, and SW antibody specific for G 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: G₁₂ and G₁₃ 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--cyclodextrin, hydrocortisone, and epidermal growth factor were from Sigma.

Cell Culture and Treatment with Methyl--cyclodextrin and Geldanamycin-- 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 G₁₂ were prepared and maintained as described previously (32). 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--cyclodextrin in serum-free medium at 37 °C for 30 min.

Preparation of Detergent-resistant Membranes (DRMs)-- DRMs were prepared as described previously with some modifications (36). 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.

Subcellular Fractionation and Triton X-100 Solubilization Experiments-- NIH 3T3 cells expressing the wild-type G₁₂ 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.

Immunoblotting and Miscellaneous Procedures-- 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.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

DRM Partitioning of G₁₂ but Not G₁₃-- To determine whether G₁₂ and G₁₃ 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 G_i were found in fractions 1 and 2 consistent with their known partitioning to DRMs and lipid rafts (Fig. 1A) (25). Na⁺/K⁺-ATPase, an integral plasma membrane protein not found in lipid rafts (36), was in the higher density fractions 6-8. Endogenous G₁₂ was found in the DRM fraction 1; G₁₃ was in the higher density fractions 7-9. We found the same pattern of partitioning after detergent solubilization and gradient centrifugation for the endogenous G₁₂ and G₁₃ proteins in two other cell lines, HMEC (Fig. 1B) and HEK293 (data not shown). In these cells, G₁₂ and the lipid raft markers were in the DRM fractions, and G₁₃ and Na⁺/K⁺-ATPase were in the higher density fractions. The partitioning of G₁₂ and the lipid raft-associated proteins to the DRM fraction was abolished and decreased, respectively, by treatment with 20 mM methyl--cyclodextrin, which depletes cellular cholesterol and disrupts lipid rafts (Fig. 1C). These results show that G₁₂ segregates with proteins that prefer lipid rafts and G₁₃ segregates with a plasma membrane protein that avoids lipid rafts.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   DRM localization and methyl--cyclodextrin treatment. COS-7 (A) and HMEC (B) cells were treated with 0.5% Triton X-100 on ice, homogenized, and subjected to floatation on an OptiPrep gradient and fractionated as described under "Experimental Procedures." Equal volumes from each fraction were subjected to SDS-PAGE followed by immunoblotting with the carboxyl-terminal antibody to G12 or the amino-terminal antibody to G13. Immunoblotting was also carried out with antibodies to the raft marker proteins, caveolin, Gi, and Fyn, and a non-raft marker protein, Na+/K+-ATPase. C, COS cells were incubated with 20 mM methyl--cyclodextrin at 37 °C for 30 min and subjected to Triton X-100 solubilization, OptiPrep density gradient centrifugation, and immunoblotting as described above.

Acylation and the DRM Localization of G₁₂ but Not G₁₃-- We investigated the role of the cysteine site of palmitoylation for targeting G₁₂ to lipid rafts. Mutation of Cys-11 on G₁₂ prevents [³H]palmitate incorporation, but the protein is still found in the particulate fraction (32). Both the wild-type G₁₂ and a GTPase-deficient mutant of G₁₂ (Q229L) were in the DRM fractions in transfected NIH 3T3 cells (Fig. 2A). Most of the nonpalmitoylated C11S mutant of G₁₂ 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 (23, 26). Further mutation of the amino terminus of G₁₂ at Ser-2 and Arg-6 changes the acylation to myristoylation (32) and restored a small amount of the DRM localization of G₁₂ (Fig. 2A). Therefore, the less hydrophobic myristate on the amino terminus could partially substitute for palmitate in targeting G₁₂ to the DRM fractions.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Lipid raft localization of wild-type and mutant G12 in stably transfected NIH 3T3 cells. A, NIH 3T3 cells expressing wild-type G12 and its mutants were homogenized in cold 0.5% Triton X-100 and separated into gradient fractions as described in the Fig. 1 legend. The antibody to the carboxyl terminus of G12 was used for all the G12-expressing cells except the C11S mutant for which the amino-terminal antibody was used. B, NIH 3T3 cells transfected with wild-type G12 were prepared as described above. Immunoblotting was performed with antibodies to G13 (amino-terminal antibody), caveolin, Gi, and Na+/K+-ATPase. C, NIH 3T3 cells stably transfected with vector alone (V) or containing the cDNA of the wild-type (WT) or mutant G12 proteins (C11S or S2G,C11S) were homogenized, and 40 µg of protein from the total cell lysate were separated by SDS-PAGE and analyzed by immunoblotting with either carboxyl- (C-term Ab) or amino-terminal (N-term Ab) antibodies to G12.

Given the importance of the cysteine site of acylation for the DRM partitioning of G₁₂ and the lipid raft localization of other biacylated G subunits including G_i in this study (29-31), we were surprised that G₁₂ with only one putative acylation site (32, 33) was in the DRM fraction and that G₁₃ with two or three putative acylation sites (34, 35) was in the higher density fractions. Not all palmitoylated proteins reside in lipid rafts (37), and the stoichiometry of palmitoylation on G₁₃ is not known, but our present result suggests that G₁₃ evades lipid rafts. Clearly further studies are required to understand the interaction that may prevent G₁₃ targeting to lipid rafts.

Hsp90 Interactions-- Hsp90 binds to G₁₂ but not G₁₃ (18), and Hsp90 can be part of a complex with caveolin (38). Therefore, we investigated whether Hsp90 may be involved in the targeting of G₁₂, but not G₁₃, to the DRM fractions. To probe the interaction between G₁₂ 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 (20). We included the proteasome inhibitor MG132 (39) because geldanamycin can increase protein degradation through the ubiquitin/proteosomal pathway (19, 40). Treatment with MG132 alone did not change the DRM localization of G₁₂ (data not shown). Geldanamycin and MG132 treatment of COS cells displaced G₁₂ 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 G₁₂ 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, G_i, G, and Fyn was not changed by geldanamycin treatment (Fig. 3B). Likewise, geldanamycin treatment did not alter the localization of G₁₃ (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 (41) and with the cytosolic location of this abundant protein (Fig. 3B) (19, 20). Hsp90 was not found in the DRMs after geldanamycin treatment. Treatment with geldanamycin alone or with MG132 did not change the total amount of G₁₂ (data not shown) in agreement with a previous report (18). These results indicate that the loss of G₁₂ from the DRM fractions after geldanamycin treatment was not due to a general disruption of lipid rafts or degradation of G₁₂ but rather suggest it was due to a loss of Hsp90 binding.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Geldanamycin treatment of COS cells. A, COS cells were preincubated with 10 µM MG132 for 10 min, and then treated with 1 µg/ml geldanamycin for 1 h. Cells underwent cold detergent extraction and OptiPrep gradient centrifugation as described in the Fig. 1 legend. Fractions were analyzed by SDS-PAGE and immunoblotting with a mixture of carboxyl- and amino-terminal antibodies against G12. B, immunoblotting was also carried out with antibodies to caveolin, Gi, G, Fyn, G13 (amino-terminal antibody), and Hsp90.

We also tested the effect of geldanamycin treatment on the DRM localization of the nonpalmitoylated C11S mutant of G₁₂ to determine the relationship between acylation and Hsp90 binding in the lipid raft localization of G₁₂. 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 G₁₂ 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 G₁₂.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Geldanamycin treatment of intact NIH 3T3 cells and its subcellular fractions. A, NIH 3T3 cells expressing C11S mutant were preincubated with 10 µM MG132 for 10 min and then treated with 1 µg/ml geldanamycin for 1 h. Cells underwent cold detergent extraction and were subjected to OptiPrep gradient centrifugation as in Fig. 1. A mixture of carboxyl- and amino-terminal antibodies was used to detect G12. B, NIH 3T3 cells expressing wild-type G12 were homogenized and separated into cytosol and crude membrane fractions by centrifugation. Aliquots of these fractions were treated with 200 µM MG132 for 10 min and then treated with 20 µg/ml geldanamycin (GA) for 1 h. Triton X-100 at a final concentration of 0.5% was added and incubated at 4 °C for 20 min, then diluted 3-fold with TNE buffer, and further incubated for 4 h. Triton X-100-soluble (S) and -insoluble (P) fractions were separated by centrifugation and analyzed by immunoblotting with a mixture of carboxyl- and amino-terminal antibodies to G12.

We investigated whether Hsp90 interactions maintain G₁₂ at lipid rafts. The crude membrane fraction of NIH 3T3 cells stably transfected with wild-type G₁₂ 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 G₁₂ (Fig. 4B, membrane) suggesting that Hsp90 interactions maintain G₁₂ in the lipid rafts in addition to any possible role in directing G₁₂ 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 G₁₂ would be either 1) maintenance of G₁₂ in a conformation that permits direct interaction of G₁₂ 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 (22) and readily forms complexes with other proteins including proteins that directly associate with lipid rafts (38) or translocate to lipid rafts (41).

The Higher Molecular Weight Form of G₁₂-- The presence of a 220-kDa band detected with an antibody to G₁₂ in the high density fractions after geldanamycin and MG132 treatment is a novel finding. Higher molecular weight bands of G₁₃ were not detected after geldanamycin treatment with antibodies to the amino and carboxyl terminus of G₁₃ (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 G₁₂ (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 G₁₂ 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 G₁₂ 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 (19). The presence of the 220-kDa band after geldanamycin treatment suggests that Hsp90 may be preventing aggregation of G₁₂.

Functional Implications-- 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 (32, 33), or geldanamycin treatment, which blocks Hsp90 interactions (18, 21), may be due to mistargeting of G₁₂ away 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 G₁₂ and G₁₃ may be responsible for increased specificity of receptor coupling seen for G₁₂ and G₁₃ in intact cells under physiologic conditions compared with results using cell membranes (11). 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. G₁₂ interactions 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 G₁₂ 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, G₁₂ aggregates and loses activity. G₁₂ 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 G₁₂ to compensate for the loss of G₁₃ in the G₁₃-deficient mice (16) may be the result of its poor mobility. Besides Hsp90, G₁₂ has also been found to interact with two other scaffold proteins in specialized cells (6, 13).

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 G₁₂ signaling. More studies are needed to discover the ramifications of their interactions and membrane targeting for G protein signaling.

	ACKNOWLEDGEMENTS

We thank Fransisco Candal for the HMEC1 cells and Dr. Silvio Gutkind for valuable discussions.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: National Institutes of Health, Bldg. 10/Rm. 9C101, Bethesda, MD 20892-1802. E-mail: tlzj@helix.nih.gov.

Published, JBC Papers in Press, July 12, 2002, DOI 10.1074/jbc.C200383200

	ABBREVIATIONS

The abbreviations used are: G protein, guanine nucleotide-binding protein; Hsp90, heat shock protein of 90 kDa; HMEC, human dermal microvascular endothelial cells; DRM, detergent-resistant membrane.

	REFERENCES

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