Immunopurification and Characterization of Rat Adipocyte Caveolae Suggest Their Dissociation from Insulin Signaling
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m211541200
ISSN1083-351X
AutoresRicardo Peres do Souto, Gino Vallega, Jonathan Wharton, Jørgen Vinten, Jørgen Tranum‐Jensen, Paul F. Pilch,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoAdipocytes play an important role in the insulin-dependent regulation of organismal fuel metabolism and express caveolae at levels as high or higher than any other cell type. Recently, a link between insulin signaling and caveolae has been suggested; nevertheless, adipocyte caveolae have been the subject of relatively few studies, and their contents have been minimally characterized. With the aid of a new monoclonal antibody, we developed a rapid procedure for the immunoisolation of caveolae derived from the plasma membrane of adipocytes, and we characterized their protein content. We find that immunopurified adipocyte caveolae have a relatively limited protein composition, and they lack the raft protein, flotillin, and insulin receptors. Immunogold labeling and electron microscopy of the adipocyte plasma membrane confirmed the lack of insulin receptors in caveolae. In addition to caveolins, the structural components of caveolae, their major protein constituents, are the semicarbazide-sensitive amine oxidase and the scavenger lipoprotein receptor CD36. The results are consistent with a role for caveolae in lipid flux in and of adipocytes. Adipocytes play an important role in the insulin-dependent regulation of organismal fuel metabolism and express caveolae at levels as high or higher than any other cell type. Recently, a link between insulin signaling and caveolae has been suggested; nevertheless, adipocyte caveolae have been the subject of relatively few studies, and their contents have been minimally characterized. With the aid of a new monoclonal antibody, we developed a rapid procedure for the immunoisolation of caveolae derived from the plasma membrane of adipocytes, and we characterized their protein content. We find that immunopurified adipocyte caveolae have a relatively limited protein composition, and they lack the raft protein, flotillin, and insulin receptors. Immunogold labeling and electron microscopy of the adipocyte plasma membrane confirmed the lack of insulin receptors in caveolae. In addition to caveolins, the structural components of caveolae, their major protein constituents, are the semicarbazide-sensitive amine oxidase and the scavenger lipoprotein receptor CD36. The results are consistent with a role for caveolae in lipid flux in and of adipocytes. plasma membrane(s) bovine serum albumin glucose transporter isoform 4 heavy microsome(s) light microsome(s) N-hydroxysuccinimide phosphate-buffered saline secretory compartment-associated membrane protein semicarbazide-sensitive amine oxidase vesicle-associated membrane protein Caveolae are 50–100-nm invaginations of the plasma membrane (PM)1 which are formed by the expression of one or more isoforms of caveolin, the protein that produces their distinct structure (1Galbiati F. Razani B. Lisanti M.P. Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar). The membrane lipid composition of caveolae is enriched in sphingolipids and cholesterol, and caveolae represent a subtype of membrane lipid rafts, that is, subdomains of the PM with specific lipid and protein compositions (2Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2065) Google Scholar). The physiological roles of caveolae remain uncertain (3Parton R.G. Nat. Rev. Mol. Cell. Biol. 2003; 4: 162-167Crossref PubMed Scopus (140) Google Scholar), but they have been suggested to participate in a large number of important cellular functions. These include the formation of transcytotic/endocytic vesicles in endothelial cells (4Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (628) Google Scholar, 5Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (557) Google Scholar), the organization/localization of numerous transmembrane signaling complexes in many cell types (6Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1727) Google Scholar, 7Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. 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Zhang X.L. Marks C.B. Macaluso F. Russell R.G. Li M. Pestell R.G. Di Vizio D. Hou Jr., H. Kneitz B. Lagaud G. Christ G.J. Edelmann W. Lisanti M.P. J. Biol. Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 14Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. Schedl A. Haller H. Kurzchalia T.V. Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1318) Google Scholar). On the other hand, these animals do have vascular abnormalities, particularly in the lung, and consequently, they have a reduced ability to exercise. Interestingly, with age they show abnormalities in lipid metabolism as a result of apparent adipocyte pathology (15Razani B. Combs T.P. Wang X.B. Frank P.G. Park D.S. Russell R.G. Li M. Tang B. Jelicks L.A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 2002; 277: 8635-8647Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). Indeed, normal adipocytes have perhaps the highest content of caveolae in any cell type. Estimates have been made that from 15 to 30% of the adipocyte PM are caveolae (15Razani B. Combs T.P. Wang X.B. Frank P.G. Park D.S. Russell R.G. Li M. Tang B. Jelicks L.A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 2002; 277: 8635-8647Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 16Carpentier J.L. Perrelet A. Orci L. J. Lipid Res. 1976; 17: 335-342Abstract Full Text PDF PubMed Google Scholar, 17Voldstedlund M. Tranum-Jensen J. Vinten J. J. Membr. Biol. 1993; 136: 63-73Crossref PubMed Scopus (38) Google Scholar), although why these structures are so abundant in adipocytes is unknown. It is now recognized that adipocytes play a complex and pivotal role in organismal fuel metabolism as both recipients and generators of endocrine/cytokine signals (18Morrison R.F. Farmer S.R. J. Nutr. 2000; 130: 3116S-3121SCrossref PubMed Google Scholar, 19Fruhbeck G. Gomez-Ambrosi J. Muruzabal F.J. Burrell M.A. Am. J. Physiol. 2001; 280: E827-E847Crossref PubMed Google Scholar). Indeed, Bergman (20Bergman R.N. Recent Prog. Horm. Res. 1997; 52: 359-385PubMed Google Scholar) has postulated that the actions of insulin on adipocytes are rate-limiting for the regulation of overall fuel homeostasis by this hormone. For this and other reasons, the possible role of lipid rafts and caveolae on insulin signaling and regulated GLUT4 trafficking has been studied extensively (for a recent review, see Ref. 21Bickel P.E. Am. J. Physiol. 2002; 282: E1-E10Crossref PubMed Google Scholar). As summarized by Bickel (22Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (564) Google Scholar), the published data are contradictory in that insulin receptors and GLUT4 are reported to be localized in adipocyte caveolae by some investigators and to be absent by others. Moreover and regardless of the presence of insulin receptors there, lipid rafts that may include caveolae have been suggested to be the locus of an important signaling complex downstream from the insulin receptor, linking it to GLUT4 translocation (22Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (564) Google Scholar, 23Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (148) Google Scholar, 24Chiang S.H. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Crossref PubMed Scopus (484) Google Scholar). Thus, the possible physiological role of caveolae in insulin action has generated considerable interest and activity. As with the study of caveolae in any cell, technical issues of isolation and purity may lie at the heart of the uncertainty regarding the composition and physiological function of adipocyte caveolae. Because caveolae are integral structures of the PM, methods needed to be devised to separate them from the bulk PM. As reviewed in Ref. 6Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1727) Google Scholar, these methods include mechanical disruption (sonication/shearing) and/or physicochemical treatment (brief extraction in Triton X-100 at 4 °C) followed by flotation in density gradients. Typically, these protocols are lengthy, and their specificity and effectiveness are questionable. Noncaveolar, detergent-resistant membrane rafts and cytoskeleton aggregates may copurify with caveolae under some of these conditions. An alternative approach is to coat the cell surface (of endothelial cells) with cationized silica to stabilize the PM and facilitate the detachment of caveolae (25Schnitzer J.E. Oh P. Jacobson B.S. Dvorak A.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1759-1763Crossref PubMed Scopus (231) Google Scholar). This procedure was improved further with the introduction of an immunoisolation step using anti-caveolin antibody (26Stan R.V. Roberts W.G. Predescu D. Ihida K. Saucan L. Ghitescu L. Palade G.E. Mol. Biol. Cell. 1997; 8: 595-605Crossref PubMed Scopus (177) Google Scholar, 27Oh P. Schnitzer J.E. J. Biol. Chem. 1999; 274: 23144-23154Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). As noted in the latter paper, the speed of caveolae isolation can be a critical parameter, and preparations obtained by immunoisolation show a more limited protein composition for caveolae than those involving detergent resistance. Here we describe a monoclonal antibody specific to caveolin-1 (7C8) which can be immobilized on acrylic beads and used to immunoisolate caveolae rapidly. We find that homogenization of adipocytes results in a certain amount of caveolae being pinched off from the PM, and these caveolae can be immunoisolated and characterized rapidly. We find caveolae purified by this method to be devoid of GLUT4, the insulin receptor, and flotillin. Indeed, and in agreement with previous freeze fracture studies (28Westermann M. Leutbecher H. Meyer H.W. Histochem. Cell Biol. 1999; 111: 71-81Crossref PubMed Scopus (43) Google Scholar), adipocyte caveolae have a relatively limited protein composition. In addition to the caveolins only two major protein components of caveolae were identified: (a) the semicarbazide-sensitive amine oxidase (SSAO), a very abundant adipocyte protein (29Morris N.J. Ducret A. Aebersold R. Ross S.A. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 9388-9392Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) of unknown physiological significance (30Jalkanen S. Salmi M. EMBO J. 2001; 20: 3893-3901Crossref PubMed Scopus (174) Google Scholar); and (b) the scavenger receptor CD36, which plays an important role in mammalian fatty acid/lipid metabolism (31Febbraio M. Abumrad N.A. Hajjar D.P. Sharma K. Cheng W. Pearce S.F. Silverstein R.L. J. Biol. Chem. 1999; 274: 19055-19062Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 32Febbraio M. Hajjar D.P. Silverstein R.L. J. Clin. Invest. 2001; 108: 785-791Crossref PubMed Scopus (937) Google Scholar). These results plus those from the caveolin-1 knockout (15Razani B. Combs T.P. Wang X.B. Frank P.G. Park D.S. Russell R.G. Li M. Tang B. Jelicks L.A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 2002; 277: 8635-8647Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar) are consistent with a major function of adipocyte caveolae in lipid trafficking into and out of these cells. Monoclonal anti-caveolin-1 antibody (clone 7C8) was raised in our laboratory following our published procedures used to obtain monoclonal antibodies against intracellular membrane proteins (33James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (472) Google Scholar, 34Thoidis G. Kotliar N. Pilch P.F. J. Biol. Chem. 1993; 268: 11691-11696Abstract Full Text PDF PubMed Google Scholar). The specificity of this reagent was confirmed by its recognition in Western blots of proteins corresponding in mass to caveolin-1α and -1β (see Fig.1A) and by immunoprecipitation of caveolin with a commercial antibody followed by Western blotting with 7C8 and by the reciprocal experiment of reversing the roles of the two antibodies. Monoclonal antibodies recognizing GLUT4 (33James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (472) Google Scholar, 34Thoidis G. Kotliar N. Pilch P.F. J. Biol. Chem. 1993; 268: 11691-11696Abstract Full Text PDF PubMed Google Scholar) and SCAMPs (33James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (472) Google Scholar, 34Thoidis G. Kotliar N. Pilch P.F. J. Biol. Chem. 1993; 268: 11691-11696Abstract Full Text PDF PubMed Google Scholar) have been described previously. Polyclonal rabbit anti-peptide antibodies against the insulin receptor were prepared as in Ref. 35Lee J. Pilch P.F. Shoelson S.E. Scarlata S.F. Biochemistry. 1997; 36: 2701-2708Crossref PubMed Scopus (44) Google Scholar. The following antibodies were commercially acquired: anti-caveolin-1 (C13630), anti-caveolin-2, anti-flotillin, and anti-TGN38 (from Transduction Laboratories); anti-VAMP2 (from Synaptic Systems); anti-actin (from Developmental Studies Hybridoma Bank, University of Iowa); anti-transferrin receptor (from Zymed Laboratories); anti-CD36 (Cascade Bioscience). Various researchers kindly provided sera against other proteins: SSAO (Dr. Antonio Zorzano, University of Barcelona, Spain); VAMP3 (Dr. Ronald Corley, Boston University School of Medicine); insulin receptor (Dr. Ken Siddle, Cambridge University, UK). Primary antibodies were detected in Western blots using secondary antibodies conjugated to horseradish peroxidase (Sigma) diluted 1:3,000 and chemiluminescent substrate (PerkinElmer Life Sciences). The protocol was adapted from Simpson et al. (36Simpson I.A. Yver D.R. Hissin P.J. Wardzala L.J. Karnieli E. Salans L.B. Cushman S.W. Biochim. Biophys. Acta. 1983; 763: 393-407Crossref PubMed Scopus (330) Google Scholar) as described previously (37Kandror K.V. Stephens J.M. Pilch P.F. J. Cell Biol. 1995; 129: 999-1006Crossref PubMed Scopus (93) Google Scholar). Briefly, epididymal fat pads were removed from male Sprague-Dawley rats (150–175 g) and transferred to KRP (12.5 mm HEPES, 120 mm NaCl, 6 mm KCl, 1.2 mm MgSO4, 1 mmCaCl2, 0.6 mm Na2HPO4, 0.4 mm NaH2PO4, 2.5 mmglucose, and 2% BSA (pH 7.4)) at 37 °C. Isolated adipocytes were obtained by collagenase B (Roche Applied Science) treatment at 37 °C for 45 min (38Rodbell M. J. Biol. Chem. 1964; 239: 375-385Abstract Full Text PDF PubMed Google Scholar). After recovery from digestion for 45 min, cells were stimulated or not with 20 nm insulin for 15 min. Hormonal action was stopped with 2 mm KCN. Cells were then transferred to HES (20 mm HEPES, 5 mm EDTA, 250 mm sucrose (pH 7.4)) and homogenized with a Teflon-glass tissue grinder. Subcellular fractions (PM, mitochondria and nuclei, heavy microsomes (HM), and light microsomes (LM)) were obtained by differential centrifugation and resuspended in HES. LM could be fractionated further (see Fig. 9) by sucrose velocity gradient (37Kandror K.V. Stephens J.M. Pilch P.F. J. Cell Biol. 1995; 129: 999-1006Crossref PubMed Scopus (93) Google Scholar). Microsomes (0.3 mg of total protein) were loaded over 4.6 ml of a 10–35% (w/v) sucrose gradient in 20 mm HEPES, 5 mm EDTA and spun for 55 min at 280,000 ×gmax. After centrifugation, fractions were collected from the bottom of the tube. All buffers used with subcellular fractions in this work contained a mixture of protease inhibitors consisting of 1 μm aprotinin, 10 μm leupeptin, 1 μm pepstatin (American Bioanalytical), and 5 mm benzamidine (Sigma). The procedure described in Souzaet al. (39Souza S.C. de Vargas L.M. Yamamoto M.T. Lien P. Franciosa M.D. Moss L.G. Greenberg A.S. J. Biol. Chem. 1998; 273: 24665-24669Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) was followed. Briefly, 3T3-L1 adipocytes at day 8 or 9 of differentiation were fixed in 2% paraformaldehyde for 10 min at 25 °C, washed, and treated with primary antibody (2–8 μg/ml) and respective secondary antibody labeled with Cy-3 or Cy-5 (Jackson Immunoresearch) diluted 1:250. Staining of lipid droplets was achieved with 1 μm Nile Red (Sigma). Fluorescence of dyes was assessed by confocal microscopy. The PM fraction (50–100 μg of total protein) suspended in PBS was solubilized with 60 mm octyl glucoside for 2 h at 4 °C with constant agitation. Insoluble material was removed by pelleting for 10 min in a microcentrifuge. Monoclonal and polyclonal anti-caveolin antibodies or nonspecific mouse and rabbit IgGs (5 μg) were incubated with the supernatant overnight at 4 °C, then 20 μl of protein A beads (Pierce) was added for 4 h. The supernatant with unbound proteins was collected, and the beads were washed four times with octyl glucoside in PBS buffer, rinsed once PBS, and eluted with SDS-PAGE loading buffer containing 2% SDS. Protein A-purified 7C8 antibody as well as nonspecific mouse IgG (Sigma) were immobilized to acrylic beads (Reacti-gel GF 2000, Pierce) at ∼1 mg of antibody/ml of resin, according to instructions from the manufacturer. Beads were blocked with 2% BSA in PBS for 2 h and washed with PBS. Microsomes resuspended in PBS (containing 0.1% BSA for biotinylated samples) were added at 5–20 μg of total protein/μl of resin for 16 h at 4 °C. The supernatant was recovered, and beads were washed with PBS. Bound vesicular proteins were eluted sequentially with 1% Triton X-100 in PBS and sample buffer for PAGE containing 2% SDS. LM were precleared with nonspecific IgG beads for 16 h at 4 °C. The unbound fraction was mixed with 7C8 beads for 16 h at 4 °C. Beads were washed extensively with PBS, and immunoadsorbed material was eluted with 100 μl of 0.2m NaHCO3 (pH 11.0) in the presence of 0.1% BSA for 30 min on ice. The supernatant was collected and the pH adjusted to 7.0 with 6 m HCl. The sample was fixed with 0.4% OsO4 for 1 h on ice, applied to carbon-coated 300 mesh copper grid, and incubated 30 min at room temperature for adsorption. The grid was rinsed sequentially with water and 1% sodium phosphotungstate (pH 7.4). Samples were analyzed on a Philips CM12 transmission electron microscope. Immunogold electron microscopy was performed in the following fashion. Adipocyte PM, adsorbed to EM grids with their cytoplasmic face exposed, were prepared and labeled as described previously (17Voldstedlund M. Tranum-Jensen J. Vinten J. J. Membr. Biol. 1993; 136: 63-73Crossref PubMed Scopus (38) Google Scholar). The primary monoclonal antibody against the C terminus of the insulin receptor β-subunit (CT-1) was kindly provided by Dr. K. Siddle, Cambridge, UK. The primary antibody against caveolin has been described earlier (40Vinten J. Voldstedlund M. Clausen H. Christiansen K. Carlsen J. Tranum-Jensen J. Cell Tissue Res. 2001; 305: 99-106Crossref PubMed Scopus (53) Google Scholar). The secondary antibody (rabbit anti-mouse immunoglobulins, DAKO, Denmark) was conjugated with 5-nm gold particles according to the protocol of Slot and Geuze (41Slot J.W. Geuze H.J. Eur. J. Cell Biol. 1985; 38: 87-93PubMed Google Scholar). The peptide used for control of the specificity of insulin receptor labeling was comprised of the 15 C-terminal amino acids of the human insulin receptor and was obtained from a commercial source. For cell surface labeling, some modifications of the procedure described in Ref.42Kandror K. Pilch P.F. J. Biol. Chem. 1994; 269: 138-142Abstract Full Text PDF PubMed Google Scholar were made. Primary amines of BSA from KRP buffer were blocked with acetic acid N-hydroxysuccinimide ester (Sigma) for 1 h at 37 °C and the reagent removed by dialysis at 4 °C. Isolation of adipocytes proceeded using KRP with blocked BSA until just before biotinylation, when cells were washed with KRP without BSA. Sulfosuccinimidobiotin (sulfo-NHS-biotin, Pierce) was added to adipocytes at a concentration of 0.5 mg/ml and incubated for 2–15 min in the presence or absence of insulin, according to specific experimental design. After labeling, cells were treated with 50 mm Tris (pH 7.4) to quench unreacted biotin and 2 mm KCN, then the fractionation followed the regular protocol. For biotinylation of isolated vesicles (either total LM or vesicles enriched by velocity gradient (43Kandror K.V. Coderre L. Pushkin A.V. Pilch P.F. Biochem. J. 1995; 307: 383-390Crossref PubMed Scopus (95) Google Scholar), samples were treated with 0.5 mg/ml sulfo-NHS-biotin for 30 min at 37 °C. The reaction was stopped with 100 mm Tris (pH 7.4) and vesicles recovered by centrifugation (230,000 × gmax, 120 min). Detection of biotinylated proteins transferred to Immun-Blot polyvinylidene difluoride membranes (Bio-Rad) was performed with 100 ng/ml streptavidin-horseradish peroxidase conjugate (Pierce) prepared in PBS-Tween with 2% BSA and chemiluminescent substrate. Sequence analysis was performed under the supervision of Dr. William Lane at the Harvard Microchemistry Facility by microcapillary reverse-phase high performance liquid chromatography nanoelectrospray tandem mass spectrometry on a Finnigan LCQ quadrupole ion trap mass spectrometer. Fig.1A shows that caveolin-1α and -1β are detected by Western blotting with 7C8, whereas a commercial polyclonal antibody (no longer available) recognizes only the α-form. These caveolin isoforms differ in the N terminus because of alternate initiation sites for translation, methionines 1 and 32, respectively, for α and β (44Scherer P.E. Tang Z. Chun M. Sargiacomo M. Lodish H.F. Lisanti M.P. J. Biol. Chem. 1995; 270: 16395-16401Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), and thus 7C8 must recognize an epitope between residue 32 and the C terminus of caveolin. Fig.1B shows by immunofluorescence that caveolin detected by 7C8 is almost exclusively localized in the cell surface, as expected. Fig.1C shows the labeling pattern in fractionated rat adipocytes of caveolin (by 7C8), as well as that for various other proteins of interest from resting and insulin-treated cells. As expected, insulin causes a redistribution of intracellular (LM and HM) GLUT4 to the cell surface (PM) (33James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (472) Google Scholar). As we demonstrated previously by Western blotting (45Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), the insulin receptor undergoes ligand-dependent endocytosis, whereas caveolin shows a small insulin-dependent decrease in the LM as a result of apparent redistribution to the PM (37Kandror K.V. Stephens J.M. Pilch P.F. J. Cell Biol. 1995; 129: 999-1006Crossref PubMed Scopus (93) Google Scholar). The change in PM caveolin cannot be accurately measured because of the large amount of caveolin already at the cell surface. Interestingly, flotillin, reported to be associated with caveolin by coimmunoprecipitation (46Volonte D. Galbiati F. Li S. Nishiyama K. Okamoto T. Lisanti M.P. J. Biol. Chem. 1999; 274: 12702-12709Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), has a different distribution than caveolin in the LM and HM membrane fractions (Fig.1C), does not coimmunoprecipitate with anti-caveolin antibodies (Fig. 2), and has a different sedimentation pattern than caveolin (see Fig. 9). Given that several groups have shown association of the insulin receptor with caveolin both in cell-free pulldowns (47Yamamoto M. Toya Y. Schwencke C. Lisanti M.P. Myers Jr., M.G. Ishikawa Y. J. Biol. Chem. 1998; 273: 26962-26968Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) and by biochemical and morphological methods in adipocytes (48Nystrom F.H. Chen H. Cong L.N. Li Y. Quon M.J. Mol. Endocrinol. 1999; 13: 2013-2024Crossref PubMed Google Scholar, 49Gustavsson J. Parpal S. Karlsson M. Ramsing C. Thorn H. Borg M. Lindroth M. Peterson K.H. Magnusson K.E. Stralfors P. FASEB J. 1999; 13: 1961-1971Crossref PubMed Scopus (317) Google Scholar, 50Kimura A. Mora S. Shigematsu S. Pessin J.E. Saltiel A.R. J. Biol. Chem. 2002; 277: 30153-30158Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), we wished to determine whether this was the case in rat adipocyte PM. We used monoclonal 7C8 and polyclonal anti-caveolin-1 antibodies as shown in Fig. 2 to immunoprecipitate proteins from the rat adipocyte PM and then Western blotted with the indicated antibodies. As has been shown in other cells (51Scherer P.E. Lewis R.Y. Volonte D. Engelman J.A. Galbiati F. Couet J. Kohtz D.S. van Donselaar E. Peters P. Lisanti M.P. J. Biol. Chem. 1997; 272: 29337-29346Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar), caveolins-1 and -2 form a stable complex that can be immunoprecipitated with both antibodies. None of these antibodies coimmunoprecipitate the insulin receptor under the described conditions. We verified this result with 7C8 using similar immunoprecipitation conditions except that Triton X-100 was the detergent used (data not shown). Interestingly, the caveolin complex immunoprecipitated with 7C8 or caveolin-1 antibodies excluded flotillin (also, see Figs. 7 and 9). Note that the lanes designated 7C8,IP, show nonspecific bands that bracket the location of flotillin. Although most of caveolin is in the PM fraction, ∼10% of the protein is found in internal membrane fractions (Fig.1C and Refs. 37Kandror K.V. Stephens J.M. Pilch P.F. J. Cell Biol. 1995; 129: 999-1006Crossref PubMed Scopus (93) Google Scholar and 52Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. Mastick C.C. Lodish H.F. J. Cell Biol. 1994; 127: 1233-1243Crossref PubMed Scopus (358) Google Scholar). Our original assumption was that this represented caveolin-rich vesicles, perhaps Golgi-derived (53Conrad P.A. Smart E.J. Ying Y.S. Anderson R.G. Bloom G.S. J. Cell Biol. 1995; 131: 1421-1433Crossref PubMed Scopus (219) Google Scholar), and we decided to immunoisolate and characterize these membranes. Antibody 7C8 was immobilized on acrylic beads and was used to immunoadsorb membranes from the LM fraction as shown in Fig.3. After binding of membranes, proteins were eluted sequentially in nondenaturing (1% Triton X-100) and denaturing (2% SDS) conditions. Surprisingly, of the panel of eight proteins tested that are postulated to be involved in aspects of endocytosis/exocytosis, only caveolins-1 and -2 were immunoadsorbed under these conditions. Proteins known to be markers of vesicular trafficking in adipocytes such as SCAMPs and VAMPs (54Kandror K.V. Pilch P.F. Am. J. Physiol. 1996; 271: E1-E14Crossref PubMed Google Scholar) were absent as was GLUT4. The reciprocal experiment of adsorbing GLUT4 was repeated and confirmed earlier results (37Kandror K.V. Stephens J.M. Pilch P.F. J. Cell Biol. 1995; 129: 999-1006Crossref PubMed Scopus (93) Google Scholar) that caveolin is absent from GLUT4 vesicles (data not shown). A trans-Golgi network marker (TGN38) and a recycling endosomal marker (transferrin receptor) also did not colocalize with immunoadsorbed caveolin-rich membranes. In more than 10 experiments, an average of 60% of caveolin-1 could be immunoadsorbed, and increasing the amount of immobilized antibody resulted in a maximum of 75% adsorption, presumably because of the topography of the caveolin complexes which prevented their complete immunoisolation. In any case, these results and those of the next two figures support the notion that caveolin-rich vesicles found in fractions enriched in intracellular vesicles represent caveolae pinched off from the PM. The immunoadsorbed material obtained as in the previous figure was treated with high pH buffer (0.2 m sodium bicarbonate (pH 11)) for elution of intact membranes. Analysis by electron microscopy of the structures recovered from 7C8 beads showed them to be vesicular (Fig. 4) and identical to caveolae isolated by other investigators using independent methodology (28Westermann M. Leutbecher H. Meyer H.W. Histochem. Cell Biol. 1999; 111: 71-81Crossref PubMed Scopus (43) Google Scholar). These structures have a thickening of the membrane which may correspond to caveolae coat, although the characteristic striations of caveolae which can be revealed by rapid freeze etching studies (55Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar) were absent. Moreover, it is possible to identify regions that might correspond to sealed openings of caveolae which would occur upon homogenization/disruption of cells (Fig. 4). The range
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