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• W1987674945 abstract "One mechanism utilized by cells to maintain signaling pathways is to regulate the levels of specific signal transduction proteins. The compound geldanamycin (GA) specifically interacts with heat shock protein 90 (hsp90) complexes and has been widely utilized to study the role of hsp90 in modulating the function of signaling proteins. In this study, we used GA to demonstrate that levels of heterotrimeric Gα subunits can be regulated through interactions with hsp90. In a dose-dependent manner, GA significantly reduced the steady state levels of endogenous Gαo expression in two cell lines (PC12 and GH3) and had a similar effect on Gαo transiently expressed in COS cells. Gαo synthesis and degradation was studied in PC12 cells and in transiently transfected COS cells. 35S labeling followed by immunoprecipitation demonstrated no effect of GA on the rate of Gαo synthesis, but GA accelerated degradation of Gαo in both PC12 cells and COS cells. The use of inhibitors, including lactacystin (a proteosome-specific inhibitor), suggests that Gαo is predominantly degraded through the proteosome pathway. In vitro translated35S-labeled Gαo could be detected in hsp90 immunoprecipitates, and this interaction did not require N-terminal myristoylation. Taken together, these results suggest that heterotrimeric Gαo subunits are protected from degradation by interaction with hsp90 and that the interaction of Gα subunits with heat shock proteins may be a general mechanism for regulating Gα levels in the cell. One mechanism utilized by cells to maintain signaling pathways is to regulate the levels of specific signal transduction proteins. The compound geldanamycin (GA) specifically interacts with heat shock protein 90 (hsp90) complexes and has been widely utilized to study the role of hsp90 in modulating the function of signaling proteins. In this study, we used GA to demonstrate that levels of heterotrimeric Gα subunits can be regulated through interactions with hsp90. In a dose-dependent manner, GA significantly reduced the steady state levels of endogenous Gαo expression in two cell lines (PC12 and GH3) and had a similar effect on Gαo transiently expressed in COS cells. Gαo synthesis and degradation was studied in PC12 cells and in transiently transfected COS cells. 35S labeling followed by immunoprecipitation demonstrated no effect of GA on the rate of Gαo synthesis, but GA accelerated degradation of Gαo in both PC12 cells and COS cells. The use of inhibitors, including lactacystin (a proteosome-specific inhibitor), suggests that Gαo is predominantly degraded through the proteosome pathway. In vitro translated35S-labeled Gαo could be detected in hsp90 immunoprecipitates, and this interaction did not require N-terminal myristoylation. Taken together, these results suggest that heterotrimeric Gαo subunits are protected from degradation by interaction with hsp90 and that the interaction of Gα subunits with heat shock proteins may be a general mechanism for regulating Gα levels in the cell. guanine nucleotide-binding protein 90-kDa heat shock protein geldanamycin polyacrylamide gel electrophoresis benzyloxycarbonyl-leucinyl-leucinyl-leucinyl-H lactacystin N-acetyl-leucinyl-leucinyl-methional-H hemagglutinin Cells respond to a wide range of physical, chemical, and hormonal stimuli through cell surface receptors that are coupled to heterotrimeric G proteins.1 G proteins are composed of Gα and Gβγ subunits and are attached to the plasma membrane through lipid modifications on Gα and Gγ subunits (reviewed in Ref. 1.Wedegaertner P.B. Biol. Signals Recept. 1998; 7: 125-135Crossref PubMed Scopus (69) Google Scholar). Activated receptors induce a conformational change in Gα that leads to GDP release and GTP binding. GTP-bound Gα dissociates from Gβγ, and both subunits can interact with a variety of intracellular effectors until the intrinsic GTPase activity of Gα hydrolyzes GTP to GDP. Many types of G protein-coupled receptors and G proteins are expressed within the same cell, and the mechanisms that generate specific cellular responses are not well understood. Some specificity lies at the interface of receptor-G protein and G protein-effector, but in reconstituted systems multiple G proteins can couple to the same sets of receptors and effectors (reviewed in Ref. 2.Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1289) Google Scholar). Furthermore, in a single cell type, a single Gα subunit can couple to at least three different effector pathways (3.Hunt T.W. Carroll R.C. Peralta E.G. J. Biol. Chem. 1994; 269: 29565-29570Abstract Full Text PDF PubMed Google Scholar). In the cell, multiple mechanisms are likely to be important for maintaining signaling specificity, and these include regulation by modulatory proteins such as receptor kinases (reviewed in Ref. 4.Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar) and RGS (regulators of G protein signaling) proteins (reviewed in Ref. 5.Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). An additional cellular mechanism to regulate signaling pathways is to control the degradation of individual signaling molecules (6.Madura K. Varshavsky A. Science. 1994; 265: 1454-1458Crossref PubMed Scopus (133) Google Scholar). Lipid modifications on the N terminus of Gα subunits are important for targeting and attachment to the plasma membrane, but the interaction of Gα subunits with cytosolic proteins prior to plasma membrane association have not been defined. The 90-kDa heat shock protein (hsp90) is a highly conserved protein chaperon representing up to 5% of total cell protein under non-stress conditions. Interestingly, hsp90 interacts with a diverse group of proteins involved in cellular signaling that include several families of tyrosine kinases and steroid hormone receptors. The functional implications for the interaction of signaling molecules with hsp90 depends upon the protein. Steroid hormone receptors are stabilized by interaction with hsp90, and this interaction is necessary for high-affinity ligand binding (7.Segnitz B. Gehring U. J. Biol. Chem. 1997; 272: 18694-18701Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). In contrast, the membrane-associated tyrosine kinase pp60v- svc interacts transiently with hsp90 following synthesis until the nascent protein is inserted in the membrane (8.Xu Y. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7074-7078Crossref PubMed Scopus (380) Google Scholar). The role of hsp90 in regulating the functions of some cellular proteins has been facilitated by the recognition that quinone ansamycin antibiotics, such as geldanamycin (GA), are highly specific inhibitors of hsp90-protein complexes (9.Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1337) Google Scholar). The GA-hsp90 complex has been crystallized and reveals GA binding to a highly conserved pocket of hsp90 (residues 9–232) (10.Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1257) Google Scholar). Because hsp90 interactions are important for the proper function of a variety of signaling molecules, we asked whether G protein α subunits could also interact with hsp90. We used the hsp90-specific compound geldanamycin to address this question, and we found that GA induced a decline in the level of endogenous Gαo in PC12 cells, GH3 cells, and transiently transfected COS cells without affecting the level of other cellular proteins. The enhanced degradation of Gαo occurred predominantly through the proteosome pathway. Furthermore, immunoprecipitates of hsp90 from in vitro translates of Gαo coprecipitated Gαo, and this interaction was independent of N-terminal myristoylation. GA was kindly provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute. Hemagglutinin (HA) antibody (clone SCP-12CA5-J) was purchased from Berkeley Antibody Co., and anti-heat shock protein 90 (mouse monoclonal, IgM) antibody was purchased from StressGen(Collegeville, PA). Protease inhibitors N-Ac-LLM, LCN, and Z-LLL were from Biomol Research Laboratories (Plymouth Meeting, PA). Chemicals were from Sigma, and chemiluminescence reagents were from Pierce. The buffers used were: A, 50 mmTris-HCl, pH 7.6, 6 mm MgCl2, 75 mmsucrose, 1 mm dithiothreitol, 1 mm EDTA, 3 mm benzamidine, 1 μg/ml leupeptin, soy and lima bean trypsin inhibitors; B, 10 mm Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate; C, 1% Triton X-100, 20 mm sodium molybdate. PC12 cells were kindly provided by Dr. Eva Neer (Brigham and Women's Hospital, Boston, MA) and cultured as described previously (11.Nusse O. Neer E.J. J. Cell Sci. 1996; 109: 221-228PubMed Google Scholar). GαocDNA in Bluescript (12.Denker B.M. Neer E.J. Schmidt C.J. J. Biol. Chem. 1992; 267: 6272-6277Abstract Full Text PDF PubMed Google Scholar) was amplified by polymerase chain reaction to generate a blunt C-terminal end that was then cloned into a modified Bluescript vector containing the HA epitope. The sequence was confirmed by dideoxynucleotide sequencing. HA Gαo was cloned into the eukaryotic expression vector pcDNA3 (Invitrogen) using theXbaI and ApaI sites. COS cells were cultured and transfected as described previously using LipofectAMINE (Life Technologies, Inc.) (13.Busconi L. Boutin P.M. Denker B.M. Biochem. J. 1997; 323: 239-244Crossref PubMed Scopus (10) Google Scholar), and 24–48 h after transfection cells were treated with GA dissolved in dimethyl sulfoxide or with dimethyl sulfoxide alone under various conditions. Levels of Gαo expression were determined 48–72 h after transfection of COS cells or from confluent monolayers of PC12 cells and GH3 cells after treatment with GA for specified conditions. Cells were washed with phosphate-buffered saline, suspended in 300 μl of cold Buffer A, frozen and thawed 3 times in liquid nitrogen, and passed 15 times through a 27-gauge needle. The homogenates were cleared by centrifuging at 1,500 rpm for 5 min at 4 °C. Total protein levels were determined by the Bradford method, and SDS-PAGE sample buffer was added to the supernatant. Equivalent amounts of total protein were analyzed by SDS-PAGE, and the level of Gαo expression was determined by Western blotting using the anti-HA monoclonal antibody at a dilution of 1:1000 or an anti-Gαo antibody (R4) at a dilution of 1:2000 (courtesy of E. Neer (14.Huff R.M. Axton J.M. Neer E.J. J. Biol. Chem. 1985; 260: 10864-10871Abstract Full Text PDF PubMed Google Scholar)). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies were used at a dilution of 1:10,000, and bands were visualized by chemiluminescence. PC12 cells and transfected COS cells (at 48 h) were treated with 2 μm GA for 18 h (COS cells) or 24 h (PC12 cells) and then incubated for 30 min in a methionine- and cysteine-free medium. Tran35S-labelTM (>1000 Ci/mmol, ICN Radiochemicals) was added to a final concentration of 200 μCi/ml for 60–90 min and chased with medium containing cold methionine/cysteine for various lengths of time. For synthesis studies, cells were labeled at t = 0 and immunoprecipitated at 30-min intervals. Cells were washed and scraped, and homogenates were prepared as described above. 35S-Labeled proteins were immunoprecipitated from homogenates in Buffer B after clearing with protein A-Sepharose. 12CA5 antibody (1/250 μl) for HA Gαo transiently expressed in COS cells (or R4 (1/100 μl) for PC12 cells) was added for 1–4 h at 4 °C followed by protein A-Sepharose for 1 h. Samples were centrifuged, and the pellets were washed three times with Buffer A. The immunoprecipitates were eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by autoradiography. cDNAs for wild-type Gαo (not HA-tagged) and myristoylation-minus mutant (G1A) were in vitro translated with [35S]methionine in a single-step rabbit reticulocyte lysate (Promega) as described previously (13.Busconi L. Boutin P.M. Denker B.M. Biochem. J. 1997; 323: 239-244Crossref PubMed Scopus (10) Google Scholar). The in vitro translation was mixed 1:1 with 2× Buffer C and then divided in half. One tube was incubated with the anti-hsp90 antibody coupled to protein A-Sepharose (15.Mimnaugh E.G. Worland P.J. Whitesell L. Neckers L.M. J. Biol. Chem. 1995; 270: 28654-28659Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and the other was a control of nonspecific IgM antibody similarly coupled to protein A-Sepharose. The mixtures were rocked at 4 °C for 1 h, centrifuged, and washed three times with cold Buffer C. Samples were eluted with SDS-PAGE sample buffer and analyzed as described above. Regulating the levels of signaling proteins is an important mechanism contributing to the accurate development and maintenance of signal transduction pathways. hsp90 interacts with several families of signal transduction proteins including receptor and non-receptor tyrosine kinases, serine threonine kinases, and mutated p53 (reviewed in Ref. 16.Buchner J. Trends Biochem. Sci. 1999; 24: 136-141Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). The compound GA destabilizes the interaction between hsp90 and its associated proteins leading to accelerated degradation and loss of function. Although Gβγ subunits have been shown to interact with hsp90 (17.Ianobe A. Takahashi K. Katada T. J. Biochem. (Tokyo). 1994; 115: 486-492Crossref PubMed Scopus (42) Google Scholar), little is known about the interaction of Gα subunits with heat shock proteins. To determine whether there is a role for hsp90 in regulating Gα subunits, we used GA to characterize Gαoprotein levels in two different systems: 1) endogenous Gαo in neuronal cell lines (PC12 and GH3) and 2) Gαo in transiently transfected COS cells. To facilitate future studies, we were interested in determining whether the mechanisms of Gαo degradation were similar in PC12 cells and in a transient expression system. When cells were treated with increasing concentrations of GA (Fig.1) for 18 h or with 2 μm GA for increasing lengths of time (not shown), there were dose- and time-dependent decreases in steady state Gαo levels for all three cell lines. Interestingly, 2 nm GA treatment caused a noticeable increase in steady state Gαo levels (Fig. 1) in all three cell lines. The explanation for this observation is not immediately apparent but may relate to compensatory changes in synthesis and/or degradation at low dose exposure to GA. C-terminal hemagglutinin epitope-tagged Gαo was used in the COS cell studies to facilitate subsequent experiments requiring immunoprecipitations. Transient expression of Gαo in COS cells results in over 75% of the protein separating into the particulate fraction (13.Busconi L. Boutin P.M. Denker B.M. Biochem. J. 1997; 323: 239-244Crossref PubMed Scopus (10) Google Scholar, 18.Busconi L. Denker B.M. Biochem. J. 1997; 328: 23-31Crossref PubMed Scopus (14) Google Scholar), an amount consistent with results in bovine brain (19.Denker B.M. Neer E.J. FEBS Lett. 1991; 279: 98-100Crossref PubMed Scopus (9) Google Scholar). In addition, placing the epitope on the C terminus does not affect separation into soluble and particulate fractions (not shown), and HA Gαoexchanges guanine nucleotides as determined by a tryptic conformation assay (not shown). Furthermore, the HA epitope on the C terminus of Gpa1, the yeast Gα subunit, did not affect the stability of the protein (6.Madura K. Varshavsky A. Science. 1994; 265: 1454-1458Crossref PubMed Scopus (133) Google Scholar). The closed arrows (Fig. 1) indicate Gαo; the open arrowhead indicates a nonspecific protein present in vector-transfected (PC) control cells (first lane) and in HA Gαo-transfected cells. The intensity of the nonspecific protein does not change with increasing GA doses, but there is a dose-dependent decrease in steady state Gαo levels (Fig. 1, closed arrows). The time-dependent effects of 2 μm GA were not apparent until after 3 h, and maximal effects were consistently seen with 18–24 h of exposure (not shown). Steady state Gαo levels were reduced by 78 ± 2% (n = 7) in COS cells and to a similar degree in PC12 and GH3 cells. The lower steady state protein levels of Gαo in the presence of GA could arise from effects on the rate of Gαo synthesis and/or degradation. To distinguish among these possibilities, the rate of Gαo synthesis in PC12 and COS cells was determined by comparing the amount of [35S]methionine/[35S]cysteine incorporated into Gαo at various times after labeling, and the Gαo degradation rate was measured by pulse-chase experiments. Fig. 2 A shows that the amount of label incorporated at 30, 60, 90, and 120 min was indistinguishable in PC12 cells in the presence or absence of GA. Similar results were obtained in transiently transfected COS cells (not shown). The rate of degradation was significantly faster in the presence of GA for endogenous Gαo in PC12 cells and in transfected COS cells (Fig. 2 B). Not surprisingly, the half-life of endogenous Gαo in PC12 cells is significantly longer (>24 h) than the half-life of Gαotransiently expressed in COS cells (∼6 h). Nevertheless, the effect of GA was similar in the two systems: a significant increase in the amount of degradation was evident at nearly every time point. Taken together, these results indicate that the predominant effect of GA on steady state Gαo levels is through accelerated degradation. Most intracellular protein degradation is catalyzed by calpains, lysosomal proteases, or by the ubiquitin-proteosome system (reviewed in Ref. 20.Mitch W.E. Goldberg A.L. N. Engl. J. Med. 1996; 335: 1897-1905Crossref PubMed Scopus (1010) Google Scholar). To test which of these pathways was responsible for the accelerated degradation of Gαo, a series of well characterized peptide aldehyde protease inhibitors were utilized (21.Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2206) Google Scholar). Two of these peptides, Z-LLL and N-Ac-LLM, exhibit similar inhibitory activity against calpains and lysosomal cathepsins, but Z-LLL is a more potent proteosome inhibitor than N-Ac-LLM (21.Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2206) Google Scholar, 22.Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1922) Google Scholar). Fig. 3 shows that when COS cells expressing Gαo are treated with GA plus an inhibitor of proteolysis (lanes 2–4) there are different effects on the levels of Gαo. In comparison with no inhibitor, treatment with N-Ac-LLM has no significant effect on Gαo levels (lane 3), and this is consistent with little degradation occurring through the calpain and lysosomal pathways. Z-LLL also blocks the proteosome pathway, and this inhibitor was partially capable of blocking degradation of Gαoinduced by GA (lane 2). This finding suggests that degradation of Gαo occurs through the proteosome pathway. To confirm this, we used lactacystin (LCN), the most specific inhibitor of the proteosome (inhibits all five proteolytic activities) (23.Mori S. Tanaka K. Omura S. Saito Y. J. Biol. Chem. 1995; 270: 29447-29452Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 24.Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1504) Google Scholar). As is seen in Fig. 3, lane 4, lactacystin significantly blocks degradation of Gαo (closed arrow). When the Western blots of total cellular extracts are exposed for increasing lengths of time, higher molecular mass species gradually become apparent (Fig. 3, bottom, lanes 2–4). Theopen arrows highlight the appearance of new bands initially between Gαo and the 50-kDa background band (lanes 2 and 3), whereas longer exposures show the development of new bands in the 50–110-kDa range (lane 4). Vector-transfected cells (lane 1) were maximally exposed to indicate the background bands. This pattern of higher molecular mass bands in the presence of LCN is characteristic of polyubiquitination, a covalent modification that targets proteins for degradation by the proteosome (reviewed in Ref. 20.Mitch W.E. Goldberg A.L. N. Engl. J. Med. 1996; 335: 1897-1905Crossref PubMed Scopus (1010) Google Scholar). This finding is consistent with degradation of Gαo through polyubiquitination and is similar to the yeast Gα subunit Gpa1, which has been shown to be degraded by this pathway (6.Madura K. Varshavsky A. Science. 1994; 265: 1454-1458Crossref PubMed Scopus (133) Google Scholar). The effects of GA on Gαo degradation are highly suggestive of an interaction between Gαo and hsp90. However, hsp90 complexes in cells are often of low affinity and disrupted by the detergents required for immunoprecipitations, and we were unable to detect this interaction in COS cells. As an alternative approach, we utilized the rabbit reticulocyte lysate system to look for this interaction. Rabbit reticulocyte lysate is rich in hsp90 and has been used to study the folding of several molecules (7.Segnitz B. Gehring U. J. Biol. Chem. 1997; 272: 18694-18701Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 9.Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1337) Google Scholar). Fig.4 shows that [35S]methionine-labeled Gαo can be detected in a complex with hsp90 although other control proteins are not detected (not shown). The control immunoprecipitations with nonspecific antibody contain almost no background (Fig. 4). Gαo was consistently detected in hsp90 immunoprecipitates (n = 4), but the fraction precipitated was low (<10% of starting material). The requirements for detergent and the low-affinity nature of the interaction between hsp90 and Gαo are the most likely explanations for this result. The hsp90 antibody can precipitate hsp90 alone or in a complex with other proteins, so we cannot exclude the possibility that the interaction of Gαo with hsp90 is indirect. The N terminus of Gαo is important for interactions with G protein Gβγ subunits and for interactions with the plasma membrane (12.Denker B.M. Neer E.J. Schmidt C.J. J. Biol. Chem. 1992; 267: 6272-6277Abstract Full Text PDF PubMed Google Scholar, 13.Busconi L. Boutin P.M. Denker B.M. Biochem. J. 1997; 323: 239-244Crossref PubMed Scopus (10) Google Scholar, 18.Busconi L. Denker B.M. Biochem. J. 1997; 328: 23-31Crossref PubMed Scopus (14) Google Scholar). Myristoylation of the N-terminal glycine (first amino acid after cleavage of methionine) and palmitoylation on cysteine 2 are also important to these functions. However, mutation of N-terminal glycine to alanine (G1A) did not disturb the interaction with hsp90 (Fig. 4), and likewise, this mutation in Gpa1 did not affect its degradation through the proteosome pathway (6.Madura K. Varshavsky A. Science. 1994; 265: 1454-1458Crossref PubMed Scopus (133) Google Scholar). Similar results were obtained with other Gαomutants previously characterized (13.Busconi L. Boutin P.M. Denker B.M. Biochem. J. 1997; 323: 239-244Crossref PubMed Scopus (10) Google Scholar, 18.Busconi L. Denker B.M. Biochem. J. 1997; 328: 23-31Crossref PubMed Scopus (14) Google Scholar), which are deleted up to 20 amino acids from the N terminus (not shown). These results indicate that myristoylation and an N-terminal amino acid sequence are not necessary for association of Gαo with the hsp90 complex. The identity of signaling molecules and their levels expressed in the membrane play an important role in signal transduction specificity. Critical to determining these levels are the mechanisms through which G protein α subunits are degraded. The results described above, and studies in yeast (6.Madura K. Varshavsky A. Science. 1994; 265: 1454-1458Crossref PubMed Scopus (133) Google Scholar), are consistent with degradation of Gα subunits through the ubiquitin-proteosome pathway. Several studies have demonstrated that activated Gα subunits (through cholera toxin (Gαs), by activating mutation, or by receptor stimulation) are more rapidly degraded, but the mechanisms of degradation are not addressed (25.Mitchell F.M. Buckley N.J. Milligan G. Biochem. J. 1993; 293: 495-499Crossref PubMed Scopus (55) Google Scholar, 26.Wise A. Lee T.W. MacEwan D.J. Milligan G. J. Biol. Chem. 1995; 270: 17196-17203Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 27.Levis M.J. Bourne H.R. J. Cell Biol. 1992; 119: 1297-1307Crossref PubMed Scopus (182) Google Scholar). Furthermore, Gα subunits have different half-lives depending upon the cell type. In GH4 cells the half-life of Gαo is about 28 h, but is greater than 72 h in cardiac myocytes (28.Silbert S. Michel T. Lee R. Neer E.J. J. Biol. Chem. 1990; 265: 3102-3105Abstract Full Text PDF PubMed Google Scholar). These different half-lives occur, in part, because of differences in rates of protein degradation (28.Silbert S. Michel T. Lee R. Neer E.J. J. Biol. Chem. 1990; 265: 3102-3105Abstract Full Text PDF PubMed Google Scholar). Our results suggest that steady state Gαo protein levels are regulated by interaction with an hsp90 complex that prevents degradation through the proteosome pathway and that N-terminal myristoylation is not required for this interaction. Although the time course and dose responses of GA treatment will vary among cell lines, the observation that this mechanism appears to be preserved in a transient expression system will make more detailed study of these mechanisms feasible. Other cytosolic proteins are likely to participate in this process and together provide an important mechanism for regulating Gα levels and function. We thank Dr. Eva Neer for continued support of this work." @default.
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• W1987674945 title "Degradation of Heterotrimeric Gαo Subunits via the Proteosome Pathway Is Induced by the hsp90-specific Compound Geldanamycin" @default.
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