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The Membrane-spanning Domains of Caveolins-1 and -2 Mediate the Formation of Caveolin Hetero-oligomers

1999; Elsevier BV; Volume: 274; Issue: 26 Linguagem: Inglês

10.1074/jbc.274.26.18721

ISSN

1083-351X

Autores

Kallol Das, Renée Y. Lewis, Philipp E. Scherer, Michael P. Lisanti,

Tópico(s)

Signaling Pathways in Disease

Resumo

The mammalian caveolin gene family consists of caveolins-1, -2, and -3. The expression of caveolin-3 is muscle-specific. In contrast, caveolins-1 and -2 are co-expressed, and they form a hetero-oligomeric complex in many cell types, with particularly high levels in adipocytes, endothelial cells, and fibroblasts. These caveolin hetero-oligomers are thought to represent the functional assembly units that drive caveolae formation in vivo. Here, we investigate the mechanism by which caveolins-1 and -2 form hetero-oligomers. We reconstituted this reciprocal interactionin vivo and in vitro using a variety of complementary approaches, including the generation of glutathioneS-transferase fusion proteins and synthetic peptides. Taken together, our results indicate that the membrane-spanning domains of both caveolins-1 and -2 play a critical role in mediating their ability to interact with each other. This is the first demonstration that these unusual membrane-spanning regions found in the caveolin family play a specific role in protein-protein interactions. The mammalian caveolin gene family consists of caveolins-1, -2, and -3. The expression of caveolin-3 is muscle-specific. In contrast, caveolins-1 and -2 are co-expressed, and they form a hetero-oligomeric complex in many cell types, with particularly high levels in adipocytes, endothelial cells, and fibroblasts. These caveolin hetero-oligomers are thought to represent the functional assembly units that drive caveolae formation in vivo. Here, we investigate the mechanism by which caveolins-1 and -2 form hetero-oligomers. We reconstituted this reciprocal interactionin vivo and in vitro using a variety of complementary approaches, including the generation of glutathioneS-transferase fusion proteins and synthetic peptides. Taken together, our results indicate that the membrane-spanning domains of both caveolins-1 and -2 play a critical role in mediating their ability to interact with each other. This is the first demonstration that these unusual membrane-spanning regions found in the caveolin family play a specific role in protein-protein interactions. Caveolae, the "little caves" first described in electron micrographs of endothelial cells, have emerged in recent years as the site of the important dynamic regulatory events at the plasma membrane (1Severs N.J. J. Cell Sci. 1988; 90: 341-348Crossref PubMed Google Scholar, 2Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (590) Google Scholar, 3Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 4Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotgia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Both transcytosis and potocytosis occur within these cell surface organelles, as does the uptake of atherogenic oxidized low density lipoprotein particles via endothelial scavenger receptors, the uptake of cholera toxin and DNA tumor viruses, the processing of Alzheimer disease-related protein APP, and of the Scrapie prion protein PrP (1Severs N.J. J. Cell Sci. 1988; 90: 341-348Crossref PubMed Google Scholar, 2Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (590) Google Scholar, 3Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 4Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotgia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Caveolae have also been implicated in signal transduction, particularly by receptor tyrosine kinases and G proteins (2Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (590) Google Scholar, 3Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). Caveolins (Cav-1, -2, and -3) 1The abbreviations used are: Cav, caveolin; GST, glutathione S-transferase; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; Fmoc, N-(9-fluorenyl)methoxycarbonyl. are a family of cytoplasmic membrane-anchored scaffolding proteins that (i) help to sculpt caveolae membranes from the plasma membrane proper, and (ii) participate in the sequestration of inactive signaling molecules (2Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (590) Google Scholar,3Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). In the adult, caveolins-1 and -2 are co-expressed and are most abundant in type I pneumocytes, endothelia, fibroblastic cells and adipocytes, whereas the expression of caveolin-3 is restricted to striated muscle cells. Caveolae-like vesicles can be generated by expressing caveolin-1 or -3 in insect cells or in mammalian cell lines, providing an in vivo assay for caveolin-dependent vesicle formation (5Fra A.M. Williamson E. Simons K. Parton R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8655-8659Crossref PubMed Scopus (526) Google Scholar, 6Li S. Song K.S. Koh S.S. Kikuchi A. Lisanti M.P. J. Biol. Chem. 1996; 271: 28647-28654Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 7Li S. Galbiati F. Volonte D. Sargiacomo M. Engelman J.A. Das K. Scherer P.E. Lisanti M.P. FEBS Lett. 1998; 434: 127-134Crossref PubMed Scopus (114) Google Scholar, 8Engelman J.A. Wycoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. J. Biol. Chem. 1997; 272: 16374-16381Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). In addition, caveolin-induced vesicle formation appears to be isoform-specific. Expression of caveolin-2 alone under the same conditions failed to drive the formation of vesicles, either in insect cells or in mammalian cells (7Li S. Galbiati F. Volonte D. Sargiacomo M. Engelman J.A. Das K. Scherer P.E. Lisanti M.P. FEBS Lett. 1998; 434: 127-134Crossref PubMed Scopus (114) Google Scholar, 8Engelman J.A. Wycoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. J. Biol. Chem. 1997; 272: 16374-16381Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Thus, caveolin-2 may function as an accessory protein in conjunction with caveolin-1 (3Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 9Scherer 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 (472) Google Scholar). In support of this notion, caveolins-1 and -2 form a stable hetero-oligomeric complex of ∼200–400 kDa in cell types in which they are co-expressed (9Scherer 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 (472) Google Scholar). These caveolin hetero-oligomers are thought to represent the assembly units that drive the formation of caveolae membranes in nonmuscle cells (7Li S. Galbiati F. Volonte D. Sargiacomo M. Engelman J.A. Das K. Scherer P.E. Lisanti M.P. FEBS Lett. 1998; 434: 127-134Crossref PubMed Scopus (114) Google Scholar, 8Engelman J.A. Wycoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. J. Biol. Chem. 1997; 272: 16374-16381Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). However, caveolin-2 requires caveolin-1 to form high molecular mass oligomers; when caveolin-2 is expressed alone, it behaves as a mixture of monomers and dimers (7Li S. Galbiati F. Volonte D. Sargiacomo M. Engelman J.A. Das K. Scherer P.E. Lisanti M.P. FEBS Lett. 1998; 434: 127-134Crossref PubMed Scopus (114) Google Scholar, 8Engelman J.A. Wycoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. J. Biol. Chem. 1997; 272: 16374-16381Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). In contrast, caveolin-1 forms high molecular mass homo-oligomers of ∼350 kDa (10Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (478) Google Scholar, 11Monier S. Parton R.G. Vogel F. Behlke J. Henske A. Kurzchalia T. Mol. Biol. Cell. 1995; 6: 911-927Crossref PubMed Scopus (401) Google Scholar). Thus, it has been hypothesized that caveolin-2 molecules are embedded within or become tightly associated with high molecular mass homo-oligomers formed by caveolin-1 (9Scherer 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 (472) Google Scholar). The genes encoding murine caveolin-1 and caveolin-2 are co-localized within the A2 region of mouse chromosome 6 (6-A2) (12Engelman J.A. Zhang X.L. Galbiati F. Lisanti M.P. FEBS Lett. 1998; 429: 330-336Crossref PubMed Scopus (132) Google Scholar). HumanCAV1 and CAV2 co-map to 7q31 in a region of conserved synteny with murine 6-A2 (13Engelman J.A. Zhang X.L. Lisanti M.P. FEBS Lett. 1998; 436: 403-410Crossref PubMed Scopus (198) Google Scholar, 14Engelman J.A. Zhang X.L. Lisanti M.P. FEBS Lett. 1999; 448: 221-230Crossref PubMed Scopus (136) Google Scholar). Similarly, the muscle-specific CAV3 gene is conserved both at the level of sequence and chromosomal context between mouse and human (4Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotgia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). The humanCAV3 gene, which underlies an autosomal dominant form of limb-girdle muscular dystrophy (limb-girdle muscular dystrophy-1C), maps to 3p25, corresponding to the mouse region 6-E1 (12Engelman J.A. Zhang X.L. Galbiati F. Lisanti M.P. FEBS Lett. 1998; 429: 330-336Crossref PubMed Scopus (132) Google Scholar, 15Minetti C. Sotogia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco P. Egeo A. Donati M.A. Volonté D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (492) Google Scholar). Given that caveolins-1 and -2 are co-expressed, that they form a hetero-oligomeric complex in vivo, and even their genes are co-localized to the same chromosomal region in mouse and human, it is apparent that this interaction is of vital importance. For example, in cells transformed by activated oncogenes, caveolin-1 levels are selectively down-regulated, but caveolin-2 levels remain relatively constant (9Scherer 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 (472) Google Scholar). In these cells, caveolae are not formed. In addition, targeted down-regulation of caveolin-1 expression using an antisense approach prevents caveolae formation, induces cell transformation, and produces a tumorigenic phenotype in NIH 3T3 cells (16Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar). Thus, it is either the absence of caveolin-1 or the presence of unbound caveolin-2 within these cells that is responsible for their transformed phenotype (16Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar). Furthermore, once caveolin-1 expression is restored in these cells, reconstituting the interaction between caveolins-1 and -2, their transformed phenotype is ablated (16Galbiati F. Volonte D. Engelman J.A. Watanabe G. Burk R. Pestell R. Lisanti M.P. EMBO J. 1998; 17: 6633-6648Crossref PubMed Scopus (431) Google Scholar). 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Traverso G. Tsui L.C. Andrulis I.L. Jothy S. Park M. Oncogene. 1996; 13: 2001-2008PubMed Google Scholar). Given the usefulness of 7q31.1 andD7S522 loss of heterozygosity as markers for carcinogenesis, many laboratories are currently searching this chromosomal region for a novel tumor suppressor gene. Recently, we have shown that the human caveolin-1 and caveolin-2 genes map within ∼100 kilobases ofD7S522, in the middle of the 1 megabase smallest common deleted region for the presumed tumor suppressor gene (4Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotgia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 13Engelman J.A. Zhang X.L. Lisanti M.P. FEBS Lett. 1998; 436: 403-410Crossref PubMed Scopus (198) Google Scholar). Thus, we have proposed that the caveolin genes may represent the missing tumor suppressor genes at this locus (4Engelman J.A. Zhang X.L. Galbiati F. Volonte D. Sotgia F. Pestell R.G. Minetti C. Scherer P.E. Okamoto T. Lisanti M.P. Am. J. Hum. Genet. 1998; 63: 1578-1587Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 13Engelman J.A. Zhang X.L. Lisanti M.P. FEBS Lett. 1998; 436: 403-410Crossref PubMed Scopus (198) Google Scholar, 14Engelman J.A. Zhang X.L. Lisanti M.P. FEBS Lett. 1999; 448: 221-230Crossref PubMed Scopus (136) Google Scholar). Despite the emerging importance of caveolins-1 and -2, little is known about the mechanism by which they form a hetero-oligomeric complexin vivo. Here, we have addressed this issue using a variety of complementary approaches. Our results indicate that complex formation between caveolins-1 and -2 is mediated by interactions between their respective membrane-spanning domains. This is the first demonstration that these unusual membrane-spanning regions play a critical role in specific protein-protein interactions. Antibodies and their sources were as follows: anti-caveolin-1 IgG (mAb 2234 and mAb 2297 (34Scherer 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 (322) Google Scholar); gifts of Dr. Roberto Campos-Gonzalez, Transduction Laboratories); anti-caveolin-2 IgG (mAb 65 (9Scherer 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 (472) Google Scholar); gift of Dr. Roberto Campos-Gonzalez, Transduction Laboratories); anti-Myc epitope IgG (mAb 9E10; Santa Cruz Biotechnology); and anti-caveolin-1 (polyclonal antibody; rabbit anti-peptide antibody directed against caveolin-1 residues 2–21; Santa Cruz Biotechnology). Anti-fatty acyl CoA synthase IgG were the generous gift of Dr. Jean Schaffer (Washington University, St. Louis, MO). The blots containing caveolin-2-derived peptides ("peptides on paper") were custom synthesized by Research Genetics. Other reagents were purchased commercially: fetal bovine serum (JRH Biosciences); prestained protein markers (Life Technologies, Inc.). All other biochemicals used were of the highest purity available and obtained from regular commercial sources. An oligo(dT)-primed, random-primed mouse adipocyte cDNA library in the vector pcDNA1 (35Baldini G. Hohl T. Lin H. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5049-5052Crossref PubMed Scopus (195) Google Scholar) was screened using the deoxygenin-labeled human caveolin-2 cDNA as a probe. Library screening was performed as per the manufacturer's instructions (Roche Molecular Biochemicals). Positive clones were characterized by restriction digestion and sequencing. The clone with the longest 5′-end contained an ∼2.3-kilobase cDNA insert and was completely sequenced. This insert contained the complete coding sequence of murine caveolin-2. The murine caveolin-2 cDNA construct and vector alone were transiently expressed in Cos-7 cells using the DEAE-dextran method (9Scherer 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 (472) Google Scholar, 34Scherer 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 (322) Google Scholar). Forty-eight hours post-transfection, cells were scraped into lysis buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 60 mm octyl-glucoside). Cell extracts were analyzed by SDS-PAGE followed by Western blotting using a specific caveolin-2 mAb probe. All DNA manipulations, including ligations, bacterial transformation, and plasmid purification were carried out using standard procedures. Myc epitope-tagged forms of caveolins-1 and -3 in the mammalian expression vector pCB7 were as described previously (34Scherer 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 (322) Google Scholar,36Song K.S. Tang Z. Li S. Lisanti M.P. J. Biol. Chem. 1997; 272: 4398-4403Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 37Tang 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 (610) Google Scholar). Untagged murine caveolin-2 in the vector pcDNA1 was used for these co-transfection experiments. Briefly, Myc-tagged forms of caveolin-1 and untagged caveolin-2 (∼10–20 μg of DNA) were transiently transfected into Cos-7 cells using the DEAE-dextran method. Forty-eight hours post-transfection, cells were scraped into lysis buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 60 mm octyl-glucoside). Myc-tagged forms of caveolin-1 were immunoprecipitated with the monoclonal antibody, 9E10, that recognizes the Myc epitope (EQKLISEEDLN). Immunoprecipitates were washed with washing buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100) and were analyzed by SDS-PAGE (13% acrylamide) followed by Western blotting using antibodies directed against caveolin-2 to detect hetero-oligomer formation. 3T3-L1 murine fibroblasts (ATCC) were propagated in 10-cm dishes and differentiated to the adipoctye form according to conventional protocols (38Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. Corley-Mastick C. Lodish H.F. J. Cell Biol. 1994; 127: 1233-1243Crossref PubMed Scopus (356) Google Scholar). Day 6 adipocytes were subjected to metabolic labeling with35S-labeled methionine and cysteine in Dulbecco's modified Eagle's medium (methionine and cysteine-free) for a 10-min pulse. The cells were then chased for 0, 10, 30 or 60 min with Dulbecco's modified Eagle's medium in the presence of 300 μmcycloheximide, to prevent further protein synthesis. Cells were scraped into lysis buffer (20 mm Tris, pH 8.0, 150 mmNaCl, 1% Triton X-100, 60 mm octyl-glucoside) and subjected to immunoprecipitation. Extracts were first immunoprecipitated with anti-caveolin-1 IgG (mAb 2234) bound to protein A-Sepharose. After washing the beads with washing buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100), bound proteins were eluted with 1% SDS, 20 mmTris, pH 8.0, 150 mm NaCl, 1% Triton X-100. Half of the eluate was then diluted to a final concentration of 0.1% SDS with washing buffer and was re-immunoprecipitated with anti-caveolin-2 IgG. The resulting immunoprecipitates were analyzed by SDS-PAGE/autoradiography. Estimation of the molecular mass of caveolin-1 and -2 was performed essentially as described previously (9Scherer 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 (472) Google Scholar, 10Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (478) Google Scholar, 37Tang 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 (610) Google Scholar, 39Scherer 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). Briefly, cells were scraped into 20 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 60 mm octyl-glucoside, and the soluble extracts were loaded atop a 5–20% sucrose density gradient and subjected to centrifugation for 4 h at 100,000 rpm in a Beckman TLS-55 rotor. After centrifugation, gradient fractions were collected from the top. Molecular mass standards for velocity gradient centrifugation were as follows: carbonic anhydrase, 29 kDa; bovine serum albumin, 66 kDa; alcohol dehydrogenase, 150 kDa; β-amylase, 200 kDa; apoferritin, 443 kDa (Sigma). GST-caveolin-1 fusion proteins were constructed and purified as we described previously (10Sargiacomo M. Scherer P.E. Tang Z.-L. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (478) Google Scholar, 34Scherer 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 (322) Google Scholar, 40Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Crossref PubMed Scopus (559) Google Scholar). Briefly, regions of caveolin-1 were subcloned into the MCS of the vector pGEX-4T-1 (Amersham Pharmacia Biotech) and expressed in a suitableEscherichia coli strain (BL21, lacking lon and ompT proteases; Novagen, Inc.). GST-caveolin-1 fusion proteins were purified by affinity chromatography using glutathione-agarose (41Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (831) Google Scholar). The purified proteins bound to glutathione-agarose beads were incubated with 3T3-L1 adipocyte extracts. After binding, the beads were washed and analyzed by SDS-PAGE/Western blotting with anti-caveolin-2 IgG. Samples were separated by SDS-PAGE (12% acrylamide) and transferred to nitrocellulose. After transfer, nitrocellulose sheets were stained with Ponceau S to visualize protein bands and subjected to immunoblotting with anti-caveolin-1 IgG (mAb 2297; 1:400) or anti-caveolin-2 IgG (mAb 65; 1:1000). Incubation conditions were as described by the manufacturer (Promega; Amersham Pharmacia Biotech), except we supplemented our blocking solution with 1% bovine serum albumin and 1% nonfat dry milk (Carnation). Peptide blots (peptides on paper) containing a panel of peptides derived from the protein sequence of murine caveolin-2 were custom made by Research Genetics. Peptides were designed so that each peptide is 10 amino acids in length, and each pair of consecutive peptides overlaps by 5 amino acids. The caveolin-2 peptide blot was incubated with radioiodinated GST-caveolin-1 for ∼18 h at 4 °C, washed thoroughly with 20 mm Tris, pH 8.0, 150 mm NaCl, 0.2% Tween 20, and subjected to autoradiography. Purified GST-caveolin-1 fusion proteins were radioiodinated using the chloramine-T method (42Greenwood F.C. Hunter W.M. Glever J.S. Biochem. J. 1963; 89: 114-123Crossref PubMed Scopus (6739) Google Scholar). Prior to labeling, GST-caveolin-1 fusion proteins were dialyzed against 20 mmTris, pH 8.0, 150 mm NaCl, 0.2% Tween 20. Approximately 50–100 μg of protein was radioiodinated with 1 mCi of Na125I in presence of 20 μg of chloramine-T for 5 min at room temperature. After terminating the reaction with unlabeled NaI, the labeled proteins were separated from free 125I using a G-25 column. Caveolin-2-derived polypeptides were synthesized directly onto an activated polymeric membrane by Research Genetics. The peptide chemistry was standard Fmoc with coupling mediated through HOBt/DIC and Fmoc removal with 1:3 piperidine/DMF. For final peptide protecting group removal, the membrane was placed in a bath of 50:47.5:2.5:1.5:1 DCM/TFA/thioanisole/EDT/anisole for 1 h and finally washed and dried. These sheets were probed by immunoblotting with anti-caveolin-2 IgG or with radioiodinated GST-caveolin-1 as described above. The membrane-spanning domain of caveolin-1 (residues 102–134) fused to GST was amplified by polymerase chain reaction using a previously constructed GST fusion construct as the template. This polymerase chain reaction product then was subcloned into the pCB7 vector downstream of the cytomegalovirus promoter and termed pGST-Cav-1-MS. pGST-Cav-1-MS was co-transfected with (i) N-terminally Myc-tagged caveolin-1 and (ii) untagged caveolin-2 in Cos-7 cells using the Effectene transfection reagent (Qiagen, Inc), as per the manufacturer's instructions. Forty-eight hours posttransfection, cells were scraped into lysis buffer (20 mm Tris, pH 8.0, 150 mm NaCl, 1% Triton X-100, 60 mm octyl-glucoside). Myc-tagged caveolin-1 was immunoprecipitated with the monoclonal antibody 9E10, which recognizes the Myc epitope (EQKLISEEDLN). Immunoprecipitates were washed with washing buffer (20 mm Tris, pH 8.0, 150 mmNaCl, 1% Triton X-100) and analyzed by SDS-PAGE/Western blotting using anti-caveolin-2 IgG. The full-length cDNA for mouse caveolin-2 was cloned by screening a day 8 3T3-L1 adipocyte library. Twenty-two individual clones were analyzed and characterized by restriction digestion/DNA sequencing. The clone with the longest 5′-end contained a 2.3-kilobase cDNA fragment and was completely sequenced. This 2.3-kilobase insert contained the complete coding sequence of murine caveolin-2 and has been deposited in GenBankTM under accession number AF141322. Only the cDNAs for human and canine caveolin-2 have been previously cloned (9Scherer 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 (472) Google Scholar, 39Scherer 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, 43Scheiffele P. Verkade P. Fra A.M. Virta H. Simons K. Ikonen E. J. Cell Biol. 1998; 140: 795-806Crossref PubMed Scopus (264) Google Scholar). The open reading frame has an in frame stop codon preceding the first ATG, confirming the authenticity of the starting methionine. Fig.

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