Cholesterol Depletion Delocalizes Phosphatidylinositol Bisphosphate and Inhibits Hormone-stimulated Phosphatidylinositol Turnover

Caveolae and detergent-insoluble, glycosphingolipid-enriched domains (DIGs) are cholesterol-enriched membrane domains that have been implicated in signal transduction because a variety of signaling proteins as well as phosphatidylinositol bisphosphate (PtdInsP2) are compartmentalized in these domains. We report here that depletion of cellular cholesterol leads to the inhibition of epidermal growth factor- and bradykinin-stimulated PtdIns turnover in A431 cells. This is associated with the loss of compartmentalization of epidermal growth factor receptors, Gq, and PtdInsP2 in the low density membrane domains. Replacement of cellular cholesterol leads to the reorganization of signaling molecules in the low density domains and the reestablishment of hormone-stimulated PtdIns hydrolysis. Oxysterol derivatives show a variable ability to functionally replace the cholesterol in this system. These data are consistent with the hypothesis that localization of signaling proteins and lipids to cholesterol-enriched domains is required for the proper function of hormone-stimulated PtdIns turnover. Caveolae and detergent-insoluble, glycosphingolipid-enriched domains (DIGs) are cholesterol-enriched membrane domains that have been implicated in signal transduction because a variety of signaling proteins as well as phosphatidylinositol bisphosphate (PtdInsP2) are compartmentalized in these domains. We report here that depletion of cellular cholesterol leads to the inhibition of epidermal growth factor- and bradykinin-stimulated PtdIns turnover in A431 cells. This is associated with the loss of compartmentalization of epidermal growth factor receptors, Gq, and PtdInsP2 in the low density membrane domains. Replacement of cellular cholesterol leads to the reorganization of signaling molecules in the low density domains and the reestablishment of hormone-stimulated PtdIns hydrolysis. Oxysterol derivatives show a variable ability to functionally replace the cholesterol in this system. These data are consistent with the hypothesis that localization of signaling proteins and lipids to cholesterol-enriched domains is required for the proper function of hormone-stimulated PtdIns turnover. Caveolae are small, plasma membrane invaginations first identified by Palade and co-workers in the 1950s (1Palade G.E. J. Appl. Physiol. 1953; 24: 1424Google Scholar). Although caveolae are non-clathrin-coated invaginations, subsequent studies have shown that caveolae do possess a striated coat that appears to be comprised largely of caveolin (2Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.-S. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1864) Google Scholar). Caveolin is a 21-kDa integral membrane protein first described as a substrate for the tyrosine kinase, pp60src (3Glenney J.R. Zokas L. J. Cell Biol. 1989; 108: 2401-2408Crossref PubMed Scopus (360) Google Scholar, 4Glenney J.R. Soppet D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10517-10521Crossref PubMed Scopus (341) Google Scholar). It is a member of a family of three homologous proteins termed caveolin, caveolin-2, and caveolin-3 (5Tang Z. Scherer P.E. Okamoto T. Song K. Chu C. Kohtz D.S. Nishimoto I. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1996; 271: 2255-2261Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 6Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (492) Google Scholar). Caveolae appear to represent specialized lipid domains that contain high levels of glycosphingolipids and cholesterol (7Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2605) Google Scholar, 8Fiedler K. Kobayashi T. Kurzchalia T.V. Simons K. Biochemistry. 1993; 32: 6365-6373Crossref PubMed Scopus (225) Google Scholar). This unusual lipid composition renders caveolae and their constituent proteins resistant to solubilization with Triton X-100. Thus, caveolae can be isolated as a low density membrane fraction from cells lysed in the presence of Triton X-100 (7Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2605) Google Scholar). Because proteins might be redistributed during extraction of membranes with Triton X-100, detergent-free procedures have also been developed to isolate caveolae (9Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 10Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar). Cells that do not exhibit plasmalemmal caveolae nonetheless possess low density, Triton-resistant membrane domains (11Fra A.M. Williamson E. Simons K. Parton R.G. J. Biol. Chem. 1994; 269: 30745-30748Abstract Full Text PDF PubMed Google Scholar) referred to as detergent-insoluble, glycosphingolipid-enriched domains (DIGs) 1The abbreviations used are: DIGsdetergent-insoluble, glycosphingolipid-enriched domainsDMEMDulbecco's modified Eagle's mediumEGFepidermal growth factorMES4-morpholineethanesulfonic acidPDGFplatelet-derived growth factorPtdInsphosphatidylinositolPtdInsPphosphatidylinositol monophosphatePtdInsP2phosphatidylinositol bisphosphate.1The abbreviations used are: DIGsdetergent-insoluble, glycosphingolipid-enriched domainsDMEMDulbecco's modified Eagle's mediumEGFepidermal growth factorMES4-morpholineethanesulfonic acidPDGFplatelet-derived growth factorPtdInsphosphatidylinositolPtdInsPphosphatidylinositol monophosphatePtdInsP2phosphatidylinositol bisphosphate. (12Parton R.G. Simons K. Science. 1995; 269: 1398-1399Crossref PubMed Scopus (295) Google Scholar) or lipid rafts (13Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8054) Google Scholar). Cholesterol-enriched lipid rafts have been described in the Golgi and late endosomes suggesting that, among other things, these domains may participate in the apical sorting of proteins and glycolipids as well as in endocytic trafficking (13Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8054) Google Scholar). detergent-insoluble, glycosphingolipid-enriched domains Dulbecco's modified Eagle's medium epidermal growth factor 4-morpholineethanesulfonic acid platelet-derived growth factor phosphatidylinositol phosphatidylinositol monophosphate phosphatidylinositol bisphosphate. detergent-insoluble, glycosphingolipid-enriched domains Dulbecco's modified Eagle's medium epidermal growth factor 4-morpholineethanesulfonic acid platelet-derived growth factor phosphatidylinositol phosphatidylinositol monophosphate phosphatidylinositol bisphosphate. Substantial evidence also implicates caveolae/DIGs in signal transduction. Low density membrane fractions prepared in the presence or absence of Triton X-100 contain many proteins involved in signal transduction, including low molecular weight and heterotrimeric G proteins, src family kinases, EGF receptors, PDGF receptors, endothelin receptors, the phosphotyrosine phosphatase syp, Grb2, Shc, mitogen-activated protein kinase kinase, protein kinase C, and the p85 subunit of PtdIns 3-kinase (9Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 14Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (860) Google Scholar, 15Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z. Hermanowski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (811) Google Scholar, 16Chun M. Liyanage U.K. Lisanti M.P. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11728-11732Crossref PubMed Scopus (325) Google Scholar, 17Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 18Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). Treatment of fibroblasts with PDGF stimulates the tyrosine phosphorylation of proteins in the caveolin-enriched fraction but has little effect on the tyrosine phosphorylation of proteins in other membrane fractions (17Liu P. Ying Y. Ko Y.-G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). In addition, incubation of Rat 1 cells with EGF leads to the recruitment of Raf-1 to a caveolin-enriched fraction suggesting that the initial steps in the ras signaling pathway may originate in caveolae (18Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). Finally, caveolin has been shown to interact directly with G protein α subunits, src family kinases, EGF receptors, and protein kinase C, inhibiting their respective activities (19Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar, 20Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 21Oka N. Yamamoto Y. Schwencke C. Kawabe J. Ebina T. Ohno S. Couet J. Lisanti M.P. Ishikawa Y. J. Biol. Chem. 1997; 272: 33416-33421Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Together these results have been used to support the hypothesis that caveolae/DIGs provide a site within the plasma membrane at which signaling molecules are concentrated and through which initial signaling events proceed. Not only signaling proteins but also signaling lipids appear to be concentrated in low density membrane domains. Studies with Madin-Darby canine kidney, A431, and Neuro 2a cells indicate that as much as half of the cellular PtdIns 4,5-P2 in cells is present in low density, detergent-insoluble domains that are enriched in caveolin (22Hope H.R. Pike L.J. Mol. Biol. Cell. 1996; 7: 843-851Crossref PubMed Scopus (212) Google Scholar, 23Pike L.J. Casey L. J. Biol. Chem. 1996; 271: 26453-26456Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 24Liu Y. Casey L. Pike L.J. Biochem. Biophys. Res. Commun. 1998; 245: 684-690Crossref PubMed Scopus (94) Google Scholar).2 Stimulation of A431 cells with either EGF or bradykinin led to the time-dependent loss of PtdIns 4,5-P2 from the caveolin-enriched fraction with no change in the level of non-caveolar PtdIns 4,5-P2, suggesting that these low density domains are the primary source of PtdIns 4,5-P2 hydrolyzed in response to hormones (23Pike L.J. Casey L. J. Biol. Chem. 1996; 271: 26453-26456Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). If localization of PtdIns 4,5-P2 and other signaling proteins in low density membrane domains is required for hormone-stimulated PtdIns turnover, then disruption of these domains should result in the ablation of this biological response. We report here that treatment of A431 cells with the cholesterol-binding agent methyl-β-cyclodextrin leads to depletion of cellular cholesterol and results in the loss of compartmentalization of PtdIns 4,5-P2, EGF receptors, and Gq. This delocalization of signaling components is associated with a loss of EGF- and bradykinin-stimulated PtdIns turnover. Replacement of cellular cholesterol leads to the reorganization of the low density domains and the reestablishment of hormone-stimulated PtdIns hydrolysis. These data implicate cholesterol in the process of hormone-stimulated PtdIns turnover and are consistent with the hypothesis that the localization of signaling proteins and lipids to cholesterol-enriched domains is required for the function of this signaling pathway. Anti-caveolin-1 and anti-caveolin-2 antibodies were from Transduction Laboratories. Anti-Gq antibody was from Santa Cruz. The polyclonal antibody to the EGF receptor (DB-1) was prepared as described previously (25Schuh S.M. Newberry E.P. Dalton M.A. Pike L.J. Mol. Biol. Cell. 1994; 5: 739-746Crossref PubMed Scopus (10) Google Scholar). EGF was prepared according to the method of Savage and Cohen (26Savage R.C. Cohen S. J. Biol. Chem. 1972; 247: 7609-7611Abstract Full Text PDF PubMed Google Scholar). myo-3HInositol and the Enhanced Chemiluminescence kit were from Amersham Pharmacia Biotech. Methyl-β-cyclodextrin was from Aldrich. All other chemicals were from Sigma. A431 cells were maintained in DMEM containing 7% newborn calf serum and 3% fetal calf serum. For labeling, subconfluent cultures were switched to inositol-free DMEM containing 5% newborn calf serum and 1 μCi/mlmyo-3Hinositol and grown for a further 48 h, at which point the cultures were confluent. One 150-mm plate of A431 cells was washed in phosphate-buffered saline and scraped into 1 ml of ice-cold 150 mm Na2CO3, pH 11, 2 mm EDTA. The cells were passed through a 23-gauge needle 10 times and subsequently sonicated 3 times for 15 s on ice using a Branson 250 sonicator set at maximum power output for a microtip. The lysate was mixed with an equal volume of 80% sucrose in MES-buffered saline (25 mm MES, pH 6.5, 150 mmNaCl, 2 mm EDTA) and placed in the bottom of a centrifuge tube. 6 ml of 35% sucrose in MES-buffered saline and 4 ml of 5% sucrose in MES-buffered saline were layered on top of the lysate. The gradient was centrifuged for 3 h at 175,000 × gand fractionated into 10 1.2-ml fractions. The small pellet was resuspended in 1.2 ml of MES-buffered saline. Aliquots (800 μl) of each sucrose density gradient fraction were extracted in chloroform: methanol:HCl and inositol phospholipids analyzed as described previously (22Hope H.R. Pike L.J. Mol. Biol. Cell. 1996; 7: 843-851Crossref PubMed Scopus (212) Google Scholar). For analysis of cholesterol levels, lipids were extracted according to the method of Bligh-Dyer (27Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42411) Google Scholar), and cholesterol was assayed using the Cholesterol CII assay kit (Wako). Cells were plated in six-well dishes and labeled with myo-3Hinositol as described above. Before the assay, cells were switched to inositol-free DMEM containing 1 mg/ml bovine serum albumin. Vehicle, EGF (50 nm), or bradykinin (10 μm) was added to each well, and cells were incubated for 10 min at 37 °C in a CO2 incubator. Assays were terminated by aspiration of the medium, washing once in cold phosphate-buffered saline, and addition of 1 ml of 5% trichloroacetic acid. Inositol phosphates were isolated on Dowex columns as described previously (28Downes C.P. Michell R.H. Biochem. J. 1981; 198: 133-140Crossref PubMed Scopus (360) Google Scholar). Aliquots (100 μl) of sucrose density gradient fractions were subjected to SDS-polyacrylamide gel electrophoresis. were to were using and then incubated for 2 h with primary was using Enhanced Chemiluminescence according to the were as described by U. Biochemistry. 1995; PubMed Scopus Google Scholar). 6 of was in of Methyl-β-cyclodextrin was in ml of and to °C with in a The was added in small and the This a that mm For were into inositol-free DMEM containing 1 mg/ml bovine serum to a of We have previously that PtdInsP2 is compartmentalized in caveolae/DIGs and that is this compartmentalized PtdInsP2 that is in response to hormones (22Hope H.R. Pike L.J. Mol. Biol. Cell. 1996; 7: 843-851Crossref PubMed Scopus (212) Google Scholar, 23Pike L.J. Casey L. J. Biol. Chem. 1996; 271: 26453-26456Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). Because caveolae/DIGs are membrane domains that are enriched in effect cholesterol depletion have on the of these their ability to and the process of hormone-stimulated PtdIns turnover. Methyl-β-cyclodextrin is a cholesterol-binding agent that cholesterol from cells Y. T. K. K. J. J. Biochem. 1989; PubMed Scopus Google Scholar, W.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). A431 cells were with of methyl-β-cyclodextrin for min at 37 °C and then assayed for EGF- and bradykinin-stimulated PtdIns turnover. shown in inositol by The response to for inhibition of PtdIns turnover was for suggesting that the effect of was not but The effect of methyl-β-cyclodextrin on hormone-stimulated PtdIns turnover was and was associated with a in cellular cholesterol The data in that EGF- and bradykinin-stimulated PtdIns turnover were within 10 min the addition of inhibition was min of treatment with this this cellular cholesterol levels inhibition of hormone-stimulated PtdIns turnover half of the cellular cholesterol been inhibition was only of the cellular cholesterol been the effect of methyl-β-cyclodextrin on the of A431 cells were in the absence or presence of this for Low density membrane fractions were then and the of proteins enriched in caveolae/DIGs was by of the sucrose density 1 the top of the and fraction 10 is the bottom of the 4 the sucrose In the of the was in fraction this fraction as the treatment with this compartmentalization was and the of the was in the high density of the fractions and the was to fraction 4 in cells but was in the high density fractions in cells. Treatment of A431 cells with also led to the loss of compartmentalization of proteins in the low density fractions. the EGF receptor and were to fraction 4 in cells. treatment with of these proteins were from the low density of the gradient and in the high density of the the effect of cholesterol depletion on the compartmentalization of A431 cells were labeled with and subsequently with methyl-β-cyclodextrin for min Treatment with not the of PtdIns or in A431 cells and this treatment was associated with the loss of and PtdInsP2 from the low density, caveolin-enriched fraction of the sucrose and The and PtdInsP2 from fraction 4 were in the high density that was not a of but a of these lipids to other of the These data that depletion of cellular cholesterol results in the loss of hormone-stimulated PtdIns turnover and the delocalization of caveolin, signaling proteins and PtdInsP2 from the low density membrane methyl-β-cyclodextrin can cholesterol from of cholesterol and methyl-β-cyclodextrin can the of cholesterol into membranes U. Biochemistry. 1995; PubMed Scopus Google Scholar, J. J. J. Sci. Full Text PDF PubMed Scopus Google Scholar, U. Biochemistry. 1995; Scopus Google Scholar, K. Biochemistry. 1997; PubMed Scopus Google Scholar). the loss of hormone-stimulated PtdIns turnover and the loss of of caveolin-enriched membrane domains were to the depletion of A431 cells were first with for min at 37 the cells were washed and incubated for times with either bovine serum or in bovine serum and for bradykinin-stimulated PtdIns turnover. shown in treatment of cells with the loss of bradykinin-stimulated PtdIns turnover. was within min the addition of in the presence of bovine serum but was and that in the presence of the This may be to of cholesterol from membranes to the plasma Treatment with the also the of treatment on PtdIns turnover of the cholesterol levels of cells with that treatment with the led to the of cellular incubation in bovine serum medium not to an in cholesterol levels levels in cells with serum cells were or with for min at 37 the of the incubation the medium was of the were for the times with DMEM containing 1 mg/ml bovine serum and half of the were with the medium containing mm The medium was then and the washed with cold phosphate-buffered saline. were extracted and cholesterol levels as described The shown represent the of that the is at the level from the in a A431 cells were or with for min at 37 the of the incubation the medium was of the were for the times with DMEM containing 1 mg/ml bovine serum and half of the were with the medium containing mm The medium was then and the washed with cold phosphate-buffered saline. were extracted and cholesterol levels as described The shown represent the of that the is at the level from the Methyl-β-cyclodextrin of of cholesterol were also prepared to these cholesterol derivatives the of on PtdIns turnover. shown in was in the ability of the to hormone-stimulated PtdIns turnover. For EGF- and bradykinin-stimulated inositol and were to the of other derivatives were to for The hormone-stimulated PtdIns turnover, cholesterol was cholesterol in the of bradykinin-stimulated PtdIns turnover to levels to or only PtdIns turnover was in cells in cells. Thus, the of of the for PtdIns turnover was for the effect of was for the Treatment of A431 cells with also the loss of of the caveolin-enriched membrane The data in that was from the low density fraction a treatment with addition of led to the of the of the caveolin in the low density fraction of the EGF and from the low density domains treatment with but were to to the fraction incubation with The PtdInsP2 delocalization was also treatment with of the PtdInsP2 present in the low density was treatment of cells with of the and incubation with PtdInsP2 was to the low density and the level of this lipid in the caveolin-enriched fraction was to that in cells. treatment treatment with the fraction of PtdIns that was in the low density domains The data here that hormone-stimulated PtdIns turnover levels of cholesterol in cells. of as little as of the cellular cholesterol led to an loss in EGF- or bradykinin-stimulated PtdIns turnover. extraction of cholesterol from the cells in a further loss of hormone-stimulated inositol These data that this biological response is on the presence of cholesterol in The cholesterol of hormone-stimulated PtdIns may be by the localization of this signaling pathway to cholesterol-enriched membrane domains. We have shown previously that the of PtdInsP2 is in a caveolin-enriched membrane fraction that also EGF and (23Pike L.J. Casey L. J. Biol. Chem. 1996; 271: 26453-26456Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar).2 These domains contain high levels of cholesterol and glycosphingolipids (7Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2605) Google Scholar). We show here that the inhibition of PtdIns turnover that results from depletion of cholesterol is associated with a in the compartmentalization of PtdInsP2 as well as caveolin, EGF receptors, and Gq. Treatment with in the loss of of these components from the low density fraction and their in the high density of the sucrose These data that the of cholesterol the of the low density domains. This results in the delocalization of these molecules and the inhibition of hormone-stimulated PtdIns turnover. and PtdIns turnover was the of and a of inhibition at a of these data that treatment a that is to is not from these data the in hormone-stimulated PtdIns turnover is to the loss of compartmentalization of the delocalization of EGF and Gq, or a of these The and hormone-stimulated PtdIns turnover is further by the results of the in which cholesterol was in cells. cellular cholesterol in the of the caveolin-enriched membrane domains and the of EGF receptors, and to the low density membrane PtdIns turnover was with the of the low density domains. These data are consistent with the that the localization of PtdInsP2 and signaling proteins to cholesterol-enriched domains is required for hormone-stimulated PtdIns turnover. of the domains results in loss of of the domains is associated with the of Because the of can be by the of these results also that the effect of is to to cholesterol from cells by other of this Cholesterol in cells in a in the ability of EGF to PtdIns with cells. Although bradykinin-stimulated PtdIns turnover was also cholesterol the was that for PtdIns turnover. The ability of cholesterol and to with bradykinin-stimulated PtdIns turnover that may have on the EGF pathway other of low density Cholesterol has been shown to to the and by in membrane and molecular K. Biochemistry. 1997; PubMed Scopus Google Scholar). Thus, the of on hormone-stimulated PtdIns turnover may be not only by their to caveolae/DIGs but also to their ability to in membrane or interact directly with or other signaling The that but not are of the of is consistent with this If the of the were the only in their ability to PtdIns turnover, then many of the should have The of the and derivatives of cholesterol to the effect of on PtdIns turnover that these may PtdIns by a other of cholesterol in cholesterol-enriched domains. have of on signaling J. P. T. J.E. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) that the tyrosine phosphorylation of S.M. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) that the ability of PDGF to PtdIns 3-kinase and the of p85 with the PDGF treatment was shown to the of for and this was associated with a in the ability of this to inositol U. Biochemistry. 1995; PubMed Scopus Google Scholar, K. Biochemistry. 1997; PubMed Scopus Google Scholar). These that many of signaling are on cholesterol and may be through low density, cholesterol-enriched domains. In the data here that hormone-stimulated PtdIns turnover is the presence of cholesterol in the The in cellular cholesterol is associated with the loss of compartmentalization of Gq, and the EGF receptor in low density, caveolin-enriched membrane Replacement of cholesterol leads to the of these the of these signaling and the reestablishment of hormone-stimulated PtdIns turnover. These that hormone-stimulated PtdIns turnover the of cholesterol-enriched domains and that localization of protein and lipid components to these low density domains is for the proper function of this signaling system.