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Multiple Functions of Caveolin-1

2002; Elsevier BV; Volume: 277; Issue: 44 Linguagem: Inglês

10.1074/jbc.r200020200

1083-351X

Pingsheng Liu, Michael J. Rudick, Richard G.W. Anderson,

Signaling Pathways in Disease

endoplasmic reticulum platelet-derived growth factor platelet-derived growth factor receptor autocrine motility factor transforming growth factor tyrosine kinase A/nerve growth factor receptor p75 neurotrophin receptor epidermal growth factor epidermal growth factor receptor glutathione S-transferase endothelial nitric-oxide synthase neuronal nitric-oxide synthase phospholipase C inositol triphosphate receptor Rous sarcoma virus transforming gene Src family tyrosine kinase C-terminal Src kinase G-protein-coupled receptor kinase 1 phospholipase D protein kinase C protein kinase A Src family tyrosine kinase urokinase-type plasminogen activator receptor heat shock protein 56 growth factor receptor bound protein S, G2 phase nuclear antigen GM1 ganglioside GD3 ganglioside immunoprecipitation phosphotyrosine high density lipoprotein The amino acid sequence of caveolin-1 predicts that it is an integral membrane protein, and there is strong experimental evidence that it has this property. For example, caveolin-1 is co-translationally inserted into the ER1 and shipped to the Golgi apparatus where it is incorporated into lipid domains that sort molecules for shipment to the cell surface (1Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (929) Google Scholar). The preferred location for caveolin-1 at the cell surface is the caveola, and it cannot be removed from these membranes without detergent (2Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1944) Google Scholar). Finally, the movements of green fluorescent protein-tagged caveolin-1 suggest that normally caveolin-1 moves with caveolae-derived vesicles to multiple interior compartments and then recycles back to the cell surface (3Thomsen P. Roepstorff K. Stahlhut M. van Deurs B. Mol. Biol. Cell. 2002; 13: 238-250Crossref PubMed Scopus (378) Google Scholar). By contrast, there is compelling evidence that caveolin-1 can be a soluble protein. Immunogold labeling first detected soluble caveolin-1 in the lumen of the ER after cells were exposed to cholesterol oxidase (4Smart E.J. Ying Y.S. Conrad P.A. Anderson R.G. J. Cell Biol. 1994; 127: 1185-1197Crossref PubMed Scopus (384) Google Scholar). Then a small pool of soluble caveolin-1 was found in fibroblast cytosol in a complex with chaperones (5Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). A routine survey of caveolin-1 distribution in different tissues identified cells that targeted caveolin-1 primarily to the cytosol (skeletal muscle cells and keratinocytes), to the lumen of secretory vesicles (serous cells of pancreas, fundic stomach, and salivary gland), and to mitochondria (airway epithelial cells and hepatocytes) (6Li W.P. Liu P. Pilcher B.K. Anderson R.G. J. Cell Sci. 2001; 114: 1397-1408Crossref PubMed Google Scholar, 7Liu P., Li, W.P. Machleidt T. Anderson R.G. Nat. Cell Biol. 1999; 1: 369-375Crossref PubMed Scopus (103) Google Scholar). Both the secreted and the cytosolic caveolin-1 appear to be embedded in lipoprotein-like particles, which may explain why they are soluble. Thus, caveolin-1 is an unusual protein that can be both an integral membrane protein and soluble in multiple cellular compartments. We believe this property is an important clue about its function. Caveolin-1 (VIP21) was first identified as a tyrosine-phosphorylated protein in Rous sarcoma transformed cells (8Glenney J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google Scholar) that was enriched in both caveolae (2Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1944) Google Scholar) and vesicles targeted to the apical surface of polarized epithelial cells (9Kurzchalia T.V. Dupree P. Parton R.G. Kellner R. Virta H. Lehnert M. Simons K. J. Cell Biol. 1992; 118: 1003-1014Crossref PubMed Scopus (467) Google Scholar). Caveolae (plasmalemmal vesicles) were first identified in 1953–1955 as endocytic structures that transport molecules across endothelial cells (10Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1741) Google Scholar). Typically caveolae are recognized in thin section EM images by their flask-shaped morphology. The cytosolic surface of each caveola, however, has a striated coat (2Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1944) Google Scholar) that is best seen in rapid-freeze deep-etch images. Caveolin-1 has been localized to the filaments that make up this coat. Caveolin-1 readily oligomerizes in vitro(1Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (929) Google Scholar), and recent structural studies indicate the N-terminal 101 amino acids assemble into heptameric subunits that appear to be the basic building block of each filament (11Fernandez I. Ying Y.-S. Albanesi J. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11193-11198Crossref PubMed Scopus (124) Google Scholar). The characteristics of other membrane coats, such as clathrin, COPI, and COPII, led to the expectation that caveolin was necessary for caveolae budding. Indeed, rapid-freeze deep-etch images showed caveolin-1 coats decorating membranes in different stages of membrane invagination, and cells lacking caveolin-1 appear not to have flask-shaped membranes (12Anderson R.G. Jacobson K. Science. 2002; 296: 1821-1825Crossref PubMed Scopus (1026) Google Scholar). Recent studies, however, call in to question whether caveolin-1 is needed for internalization and traffic of caveolae (see below). There are three caveolin genes expressed in mammals (designated caveolin-1, -2, and -3), and they code for five different isoforms of the protein (1Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (929) Google Scholar). Most tissues in the body express at least one of these isoforms. Notably lacking are cultured lymphocytes and certain neuronal cells. Caveolin-1 and -2 are usually co-expressed and assemble into hetero-oligomers in the ER and Golgi apparatus (13Scheiffele P. Verkade Fra M. Virta Simons Ikonen J. Cell Biol. 1998; 140: 795-806Crossref PubMed Scopus (265) Google Scholar). These oligomers mature into higher molecular weight complexes once they reach caveolae. Interestingly, caveolin-2 appears unable to exit the Golgi apparatus by itself (14Parolini I. Sargiacomo M. Galbiati F. Rizzo G. Grignani F. Engelman J.A. Okamoto T. Ikezu T. Scherer P.E. Mora R. Rodriguez-Boulan E. Peschle C. Lisanti M.P. J. Biol. Chem. 1999; 274: 25718-25725Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) and is rapidly degraded in cells not expressing caveolin-1. In skeletal and heart muscle cells, caveolin-3 replaces caveolin-1 in caveolae. Skeletal muscle cells selectively express the β isoform of caveolin-1, but it is targeted to the cytosol where it tends to collect along the Z-line (6Li W.P. Liu P. Pilcher B.K. Anderson R.G. J. Cell Sci. 2001; 114: 1397-1408Crossref PubMed Google Scholar). One way to understand the function of a protein is to identify its interacting partners. A variety of proteins have been identified that interact with either caveolin-1 or tyrosine-phosphorylated caveolin-1 (pY14). In addition, caveolin-1 interacts with both lipids and lipid anchors on proteins (Table I). These interactions predict that caveolin-1 functions in lipid traffic, membrane traffic, and signal transduction.Table IA partial list of proteins and lipids that interact with caveolin-1ProteinsInteracting region of Cav-1Detection methodRef.PDGFRα and -β82–101IP48Yamamoto M. Toya Y. Jensen R.A. Ishikawa Y. Exp. Cell Res. 1999; 247: 380-388Crossref PubMed Scopus (102) Google ScholarEGFR61–101IP49Couet J. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1997; 272: 30429-30438Abstract Full Text Full Text PDF PubMed Scopus (555) Google ScholarInsulin receptor81–101IP50Yamamoto 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 (254) Google ScholarTGFβRI61–101IP51Razani B. Zhang X.L. Bitzer M. von Gersdorff G. Bottinger E.P. Lisanti M.P. J. Biol. Chem. 2001; 276: 6727-6738Abstract Full Text Full Text PDF PubMed Scopus (293) Google ScholarTrkA/p75NTRWhole proteinIP52Bilderback T.R. Grigsby R.J. Dobrowsky R.T. J. Biol. Chem. 1997; 272: 10922-10927Abstract Full Text Full Text PDF PubMed Scopus (160) Google ScholarHedgehog receptor81–101IP53Karpen H.E. Bukowski J.T. Hughes T. Gratton J.P. Sessa W.C. Gailani M.R. J. Biol. Chem. 2001; 276: 19503-19511Abstract Full Text Full Text PDF PubMed Scopus (106) Google ScholarEstrogen receptor82–101IP54Schlegel A. Wang C. Pestell R.G. Lisanti M.P. Biochem. J. 2001; 359: 203-210Crossref PubMed Scopus (66) Google ScholarAndrogen receptorWhole proteinIP/two hybrid55Lu M.L. Schneider M.C. Zheng Y. Zhang X. Richie J.P. J. Biol. Chem. 2001; 276: 13442-13451Abstract Full Text Full Text PDF PubMed Scopus (212) Google ScholarH-Ras61–101GST56Song 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 (924) Google ScholarGαq/Gαo/GαsWhole proteinGST57Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Abstract Full Text Full Text PDF PubMed Scopus (560) Google ScholarAdenylyl cyclase/PLCβ282–101IP58Schreiber S. Fleischer J. Breer H. Boekhoff I. J. Biol. Chem. 2000; 275: 24115-24123Abstract Full Text Full Text PDF PubMed Scopus (44) Google ScholarTrp1/IP3R/Gq11Whole proteinIP59Lockwich T.P. Liu X. Singh B.B. Jadlowiec J. Weiland S. Ambudkar I.S. J. Biol. Chem. 2000; 275: 11934-11942Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholarc-Src61–101IP37Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (682) Google ScholarLyn81–101IP60Muller G. Jung C. Wied S. Welte S. Jordan H. Frick W. Mol. Cell. Biol. 2001; 21: 4553-4567Crossref PubMed Scopus (87) Google ScholarCskPY14Two hybrid/IP61Cao H. Courchesne W.E. Mastick C.C. J. Biol. Chem. 2002; 277: 8771-8774Abstract Full Text Full Text PDF PubMed Scopus (183) Google ScholarGRK1, -2, and -561–101IP62Carman C.V. Lisanti M.P. Benovic J.L. J. Biol. Chem. 1999; 274: 8858-8864Abstract Full Text Full Text PDF PubMed Scopus (157) Google ScholarCOX-2Whole proteinIP41Liou J.Y. Deng W.G. Gilroy D.W. Shyue S.K. Wu K.K. J. Biol. Chem. 2001; 276: 34975-34982Abstract Full Text Full Text PDF PubMed Scopus (87) Google ScholarPLD/PKCα82–101IP63Kim J.H. Han J.M. Lee S. Kim Y. Lee T.G. Park J.B. Lee S.D. Suh P.G. Ryu S.H. Biochemistry. 1999; 38: 3763-3769Crossref PubMed Scopus (59) Google ScholarPKA81–101IP64Razani B. Rubin C.S. Lisanti M.P. J. Biol. Chem. 1999; 274: 26353-26360Abstract Full Text Full Text PDF PubMed Scopus (155) Google ScholarPKCɛWhole proteinIP65Wu D. Foreman T.L. Gregory C.W. McJilton M.A. Wescott G.G. Ford O.H. Alvey R.F. Mohler J.L. Terrian D.M. Cancer Res. 2002; 62: 2423-2429PubMed Google ScholarIntegrin/cortactin/SrcWhole proteinIP66Wei Y. Yang X. Liu Q. Wilkins J.A. Chapman H.A. J. Cell Biol. 1999; 144: 1285-1294Crossref PubMed Scopus (372) Google ScholarIntegrin(α,β)/Shc/FynWhole proteinIP67Wary K.K. Mariotti A. Zurzolo C. Giancotti F.G. Cell. 1998; 94: 625-634Abstract Full Text Full Text PDF PubMed Scopus (622) Google ScholaruPAR/integrinβ1Whole proteinIP68Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (699) Google ScholareNOS82–101IP69Garcia-Cardena G. Fan R. Stern D.F. Liu J.W. Sessa W.C. J. Biol. Chem. 1996; 271: 27237-27240Abstract Full Text Full Text PDF PubMed Scopus (432) Google ScholarnNOSWhole proteinGST70Venema V.J., Ju, H. Zou R. Venema R.C. J. Biol. Chem. 1997; 272: 28187-28190Abstract Full Text Full Text PDF PubMed Scopus (221) Google ScholarFlotillin 1, 2/Cav-2Whole proteinIP71Volonte 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 (189) Google Scholar190-kDa pYWhole proteinIP72Liu P. Ying Y., Ko, Y.G. Anderson R.G. J. Biol. Chem. 1996; 271: 10299-10303Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar30-kDa pYWhole proteinIP73Mastick C.C. Saltiel A.R. J. Biol. Chem. 1997; 272: 20706-20714Abstract Full Text Full Text PDF PubMed Scopus (131) Google ScholarHSP56/cyc40/cycAWhole proteinIP5Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google ScholarFilamin1–101GST/2 hybrid74Stahlhut M. van Deurs B. Mol. Biol. Cell. 2000; 11: 325-337Crossref PubMed Scopus (268) Google ScholarGrb7PY 14GST75Lee H. Woodman S.E. Engelman J.A. Volonte D. Galbiati F. Kaufman H.L. Lublin D.M. Lisanti M.P. J. Biol. Chem. 2001; 276: 35150-35158Abstract Full Text Full Text PDF PubMed Scopus (105) Google ScholarStriatin/SG2NA/zinedinWhole proteinIP/GST76Gaillard S. Bartoli M. Castets F. Monneron A. FEBS Lett. 2001; 508: 49-52Crossref PubMed Scopus (58) Google ScholarConnexin 4382–101 and 135–178IP77Schubert A.L. Schubert W. Spray D.C. Lisanti M.P. Biochemistry. 2002; 41: 5754-5764Crossref PubMed Scopus (233) Google ScholarLipidsInteracting region of Cav-1Detection methodRef.CholesterolWhole proteinOverlay16Murata M. Peranen J. Schreiner R. Wieland F. Kurzchalia T.V. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (781) Google ScholarFatty acidsWhole proteinIP17Trigatti B.L. Anderson R.G. Gerber G.E. Biochem. Biophys. Res. Commun. 1999; 255: 34-39Crossref PubMed Scopus (187) Google ScholarGM1Whole proteinIP18Fra A.M. Masserini M. Palestini P. Sonnino S. Simons K. FEBS Lett. 1995; 375: 11-14Crossref PubMed Scopus (163) Google ScholarGD3Whole proteinIP78Kasahara K. Watanabe Y. Yamamoto T. Sanai Y. J. Biol. Chem. 1997; 272: 29947-29953Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar Open table in a new tab Cholesterol is required for the structure (2Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1944) Google Scholar) and function of caveolae (15Chang W.J. Rothberg K.G. Kamen B.A. Anderson R.G. J. Cell Biol. 1992; 118: 63-69Crossref PubMed Scopus (221) Google Scholar). In vitro assays have shown that caveolin-1 interacts with cholesterol (16Murata M. Peranen J. Schreiner R. Wieland F. Kurzchalia T.V. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (781) Google Scholar), suggesting that it may organize membrane cholesterol in caveolae. Caveolin-1 also binds with high affinity long chain unsaturated fatty acids (17Trigatti B.L. Anderson R.G. Gerber G.E. Biochem. Biophys. Res. Commun. 1999; 255: 34-39Crossref PubMed Scopus (187) Google Scholar), which may account for its ability to interact with the GM1 gangliosides (18Fra A.M. Masserini M. Palestini P. Sonnino S. Simons K. FEBS Lett. 1995; 375: 11-14Crossref PubMed Scopus (163) Google Scholar) that collect in caveolae. A strong argument can be made, however, that caveolin-1 plays a role in the import and export of cellular cholesterol by caveolae. The first indication caveolae were involved in cholesterol traffic came from the observation that in normal human fibroblasts cholesterol moves directly to surface caveolae within minutes after being synthesized in the ER (19Smart E.J. Ying Y. Donzell W.C. Anderson R.G. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). From caveolae the cholesterol then moves rapidly to other regions of the plasma membrane and to the extracellular space. The rapid transport of new cholesterol to the lymphocyte cell surface is dependent on the expression of caveolin-1 (19Smart E.J. Ying Y. Donzell W.C. Anderson R.G. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). Non-palmitoylated caveolin-1 is impaired in the transport of new cholesterol in these cells (20Uittenbogaard A. Smart E.J. J. Biol. Chem. 2000; 275: 25595-25599Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). ER to plasma membrane transport of new cholesterol is also inhibited by progesterone, and this steroid causes caveolin-1 to accumulate in internal membranes (1Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (929) Google Scholar). Finally, expression of a truncated caveolin-3 (CavDGV) dramatically lowers the cholesterol content of caveolae in CV1 cells, which can be reversed by the addition of exogenous cholesterol (21Roy S. Luetterforst R. Harding A. Apolloni A. Etheridge M. Stang E. Rolls B. Hancock J.F. Parton R.G. Nat. Cell Biol. 1999; 1: 98-105Crossref PubMed Scopus (127) Google Scholar). The mechanism of action of CavDGV is not known, although it is presumed that it interferes with caveolin-1 function. Plasma membrane cholesterol can move directly to the ER (22Lange Y. J. Biol. Chem. 1994; 269: 3411-3414Abstract Full Text PDF PubMed Google Scholar). Free cholesterol, cholesterol esters, and cholesterol ethers in HDL bound to SR-B1 can also move to intracellular sites from caveolae (23Graf G.A. Matveev S.V. Smart E.J. Trends Cardiovasc. Med. 1999; 9: 221-225Crossref PubMed Scopus (51) Google Scholar). Expression of caveolin has been linked to cholesterol import (24Fielding C.J. Fielding P.E. J. Lipid Res. 1997; 38: 1503-1521Abstract Full Text PDF PubMed Google Scholar), but the rapid migration of caveolin to the ER after the oxidation of caveolae cholesterol (4Smart E.J. Ying Y.S. Conrad P.A. Anderson R.G. J. Cell Biol. 1994; 127: 1185-1197Crossref PubMed Scopus (384) Google Scholar) suggests it has a direct role in cholesterol import. The mystery is how can caveolin-1, which is an integral membrane protein in caveolae, carry cholesterol to intracellular compartments like the ER. A solution to this puzzle may be cytosolic caveolin-1. Several laboratories have verified the initial discovery (5Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) that the cytosol of many cells contains a pool of soluble caveolin-1. This caveolin-1 is associated with cholesterol and behaves like a protein that is embedded in a particle with the size (25Ito J. Nagayasu Y. Kato K. Sato R. Yokoyama S. J. Biol. Chem. 2002; 277: 7929-7935Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and buoyant density (6Li W.P. Liu P. Pilcher B.K. Anderson R.G. J. Cell Sci. 2001; 114: 1397-1408Crossref PubMed Google Scholar) of HDL. Cholesterol esters that enter cells through caveolae appear to associate with cytosolic caveolin-1. Likewise, the same particle may also carry new cholesterol from the ER to the plasma membrane (23Graf G.A. Matveev S.V. Smart E.J. Trends Cardiovasc. Med. 1999; 9: 221-225Crossref PubMed Scopus (51) Google Scholar). Recently it has been found that apoA-1 binding to SR-B1 stimulates the formation of cytosolic lipid particles containing caveolin-1, cholesterol, and phospholipid (25Ito J. Nagayasu Y. Kato K. Sato R. Yokoyama S. J. Biol. Chem. 2002; 277: 7929-7935Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Importantly, caveolin-1 is secreted by exocrine secretory cells in HDL particles containing apoA-1, raising the possibility that caveolin-rich lipid particles in the cytoplasm are involved in the assembly of secreted lipoproteins. Soluble cytoplasmic caveolin particles may also be important for the biogenesis of cytoplasmic lipid droplets (26Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Crossref PubMed Scopus (278) Google Scholar). In summary, caveolin-1 has a function in intracellular and extracellular lipid transport. This function may account for the high level of caveolin-1 expression in adipocytes (6Li W.P. Liu P. Pilcher B.K. Anderson R.G. J. Cell Sci. 2001; 114: 1397-1408Crossref PubMed Google Scholar) as well as the apparent abnormalities in lipid metabolism that are seen in caveolin-1 null mice (27Razani 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 (479) Google Scholar). Caveolin-1 is not behaving like a simple lipid carrier protein but, instead, seems to be part of a novel intracellular lipid particle that transports lipids between organelles similar to the way that plasma lipoproteins move lipids between tissues. The precise lipid and protein composition of this particle remains to be determined. The principal site where one would expect caveolin-1 to function in membrane traffic is at the caveola. Caveolae are well known for their unique endocytic properties (reviewed in Ref. 10Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1741) Google Scholar). Caveolin-1 could attract proteins to caveolae the same way that clathrin adaptors attract transmembrane receptors to coated pits and/or function as a molecular motor that powers membrane invagination and budding. There is very little evidence, however, that caveolin-1 functions this way. Caveolin-1 has been reported to interact with several receptor tyrosine kinases (see Table I), including the EGF receptor (EGFR). The information for EGFR localization to caveolae/rafts, however, is in the extracellular domain of the receptor (28Yamabhai M. Anderson R.G. J. Biol. Chem. 2002; 277: 24843-24846Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), so an interaction with caveolin-1 probably has nothing to do with EGFR attraction to caveolae. A recent study also challenges the assumption that caveolin-1 is required for caveolae internalization. Receptor-mediated uptake of autocrine motility factor (AMF) normally occurs by both coated pit and caveolae pathways (29Le P.U. Guay G. Altschuler Y. Nabi I.R. J. Biol. Chem. 2002; 277: 3371-3379Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). The caveolae path delivers the factor directly to the ER whereas coated pits deliver it to endosomes. Delivery to the ER, however, appears to occur in cells deficient in caveolin-1. These cells lack visible, flask-shaped caveolae, which is in agreement with previous observations, but delivery of AMF to the ER is 5 times faster than in cells expressing caveolin-1. Identification of caveolae as the route of entry was made possible by transfecting these cells with a dominant-negative dynamin, which blocks vesicle budding from plasma membranes. Under these conditions the caveolin-deficient cells had numerous flask-shaped membranes that were morphologically indistinguishable from those found in caveolin-1-expressing cells, and they contained AMF receptors. In other words, without caveolin-1 caveolae internalization is so rapid that flask-shaped caveolae are difficult to find in thin section images unless internalization is blocked. These observations are in agreement with a recent report suggesting that internalization of a glycosylphosphatidylinositol -anchored protein through the caveolae pathway is the same regardless of whether the cells express caveolin-1 (30Nichols B.J. Nat. Cell Biol. 2002; 4: 374-378Crossref PubMed Scopus (218) Google Scholar). In contrast to these studies, endocytosis of albumin by endothelial cells derived from caveolin-1 null mice is markedly impaired (31Razani B. Engelman J.A. Wang X.B. Schubert W. 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). This study did not determine, however, whether the defect was because of malfunctioning caveolae or the absence of the albumin receptor GP60 at the cell surface. If the studies on AMF uptake are confirmed, then caveolin-1 must be a negative regulator of caveolae membrane internalization and traffic. How could this be? A regulatory rather than structural function for caveolin-1 actually agrees very nicely with several recent observations on the movement of membrane populations containing caveolin-green fluorescent protein (3Thomsen P. Roepstorff K. Stahlhut M. van Deurs B. Mol. Biol. Cell. 2002; 13: 238-250Crossref PubMed Scopus (378) Google Scholar). This probe has become an invaluable tool for studying the movement of caveolae and caveolae-derived membranes. Surprisingly, most caveolae on the surface of cells are immobile and not traveling to interior sites of the cell (3Thomsen P. Roepstorff K. Stahlhut M. van Deurs B. Mol. Biol. Cell. 2002; 13: 238-250Crossref PubMed Scopus (378) Google Scholar). Caveolae internalization is tightly regulated by PKC and tyrosine kinases (10Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1741) Google Scholar). Pathogens like SV40 virus can commandeer this regulatory system and stimulate their own internalization through caveolae by activating an unknown tyrosine kinase (32Pelkmans L. Kartenbeck J. Helenius A. Nat. Cell Biol. 2001; 3: 473-483Crossref PubMed Scopus (1065) Google Scholar). Caveolae tend to collect in actin-rich regions of the cell membrane, and they contain a number of actin-binding proteins. One is filamin, which may interact directly with caveolin-1 (Table I). Therefore, an important function of caveolin-1 may be to regulate the interaction of caveolae with the cortical actin cytoskeleton, thereby controlling whether caveolae are at the cell surface or traveling to interior sites. This interaction, in turn, may be controlled by tyrosine phosphorylation of caveolin-1. Immediately following the identification of caveolin-1 as a caveolae marker protein, procedures were developed for isolating caveolae from tissues as well as tissue culture cells (10Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1741) Google Scholar). An unanticipated outcome of using these isolation procedures was the discovery that caveolae are rich in multiple molecules that function in cellular signal transduction (10Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1741) Google Scholar). The ability to rapidly isolate these membrane domains led to the discovery that many signaling molecules are dynamically associated with caveolae. For example, quiescent fibroblasts have very little Raf-1 in caveolae but are enriched in EGFR. After EGF binds EGFR, Raf-1 is recruited to caveolae at the same time as EGFR moves to non-caveolae membranes (33Mineo C. James G.L. Smart E.J. Anderson R.G. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). Entire signaling modules like the PDGFR-Ras-ERK (extracellular signal-regulated kinase) module have been localized to caveolae, and they are fully functional even after caveolae are isolated away from the cell (34Liu P. Ying Y. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13666-13670Crossref PubMed Scopus (194) Google Scholar). In light of these discoveries, it was only natural to think that caveolin-1 modulated signal transduction by attracting signaling molecules to caveolae and regulating their activity. A scaffolding function for caveolin-1 arose out of a consideration of the propensity of this molecule to oligomerize (35Lisanti M.P. Scherer P. Tang Z.L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (602) Google Scholar). Purified caveolin-1 spontaneously forms oligomers in solution, and oligomerization maps to a region of the molecule between amino acids 80 and 101, which is on the N-terminal side of the putative membrane insertion region (36Sargiacomo M. Scherer P.E. Tang Z. Kubler E. Song K.S. Sanders M.C. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9407-9411Crossref PubMed Scopus (489) Google Scholar). An ability to polymerize fits well with the idea that caveolin-1 is a structural component of the filamentous caveolae coat. Li et al. (37Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar) were the first to show that a synthetic peptide matching the amino acid sequence between residues 80 and 101 in caveolin-1 inhibited Src kinase activity. Moreover, a GST fusion protein containing amino acids 61–101 of caveolin-1 preferentially interacts with inactive Src. The sequence between 80 and 101 was named the caveolin-1 scaffolding domain (37Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar). Subsequently a synthetic scaffolding domain peptide was used to isolate two binding motifs from a phage display library with the sequences φXφXXXφ and φXXXXφXφ, where φ is aromatic (38Couet J., Li, S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar). Many, but not all, caveolin-1-interacting proteins contain one of these sequences (reviewed in Ref. 1Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (929) Google Scholar). A key part of the caveolin-1 scaffolding domain hypothesis is the proposal that it functions to inactivate signaling molecules (39Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1362) Google Scholar). Many studies have documented an interaction between the caveolin-1 scaffolding domain and various signaling molecules (see Table I for a partial list). Although these interactions often suppress the signaling activity of the molecule as predicted (1Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (929) Google Scholar), in some cases the scaffolding domain stimulates activity (40Czarny M. Lavie Y. Fiucci G. Liscovitch M. J. Biol. Chem. 1999; 274: 2717-2724Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) or has no effect at all (41Liou J.Y. Deng W.G. Gilroy D.W. Shyue S.K. Wu K.K. J. Biol. Chem. 2001; 276: 34975-34982Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The most common assay used in these studies measures the effect of the scaffolding peptide (versus a scrambled peptide sequence) on signaling activity in vitro. Recently, however, this peptide has been found to inhibit eNOS activity after being introduced into living cells (42Bucci M. Gratton J.P. Rudic R.D. Acevedo L. Roviezzo F. Cirino G. Sessa W.C. Nat. Med. 2000; 6: 1362-1367Crossref PubMed Scopus (483) Google Scholar). The results of over 50 studies are in basic agreement with the caveolin-1 scaffolding domain hypothesis. Despite the agreement, other considerations do not fit the scaffolding domain hypothesis. Table I clearly demonstrates that caveolin-1 is a promiscuous molecule when it comes to interacting with proteins that collect in caveolae, but there is no way of knowing the true extent of its promiscuity. The scaffolding domain may simply be a particularly sticky region that binds non-specifically to many proteins in the cell. Recent studies on the structure of the 1–101 region of caveolin-1 suggest that amino acids 79–96 of the scaffolding domain have α-helix secondary structure (11Fernandez I. Ying Y.-S. Albanesi J. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11193-11198Crossref PubMed Scopus (124) Google Scholar). An interdigitating set of positively charged and aromatic amino acids forms one side of the helix whereas the other side is rich in polar, uncharged amino acids. Thus, the principal interacting surface of the scaffolding domain is a simple helix that, in addition, mediates lateral interactions between caveolins during caveolae coat filament assembly. It also has a tendency to interact non-specifically with membranes (43Schlegel A. Schwab R.B. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1999; 274: 22660-22667Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Another consideration is the lack of genetic evidence that the scaffolding domain functions as a repressor or activator of cell signaling. Introduction of mutations into this region of the molecule severely impairs traffic out of the Golgi apparatus (44Machleidt T., Li, W.P. Liu P. Anderson R.G. J. Cell Biol. 2000; 148: 17-28Crossref PubMed Scopus (98) Google Scholar), which makes it impossible to determine whether mutating this region of the molecule has an effect on signal transduction. Indeed, a naturally occurring mutation in this region of the caveolin-3 isoform is unstable and not found at the cell surface (45Minetti C. Sotgia F. Bruno C. Scartezzini P. Broda P. Bado M. Masetti E. Mazzocco M. Egeo A. Donati M.A. Volonte D. Galbiati F. Cordone G. Bricarelli F.D. Lisanti M.P. Zara F. Nat. Genet. 1998; 18: 365-368Crossref PubMed Scopus (504) Google Scholar). Finally, the reported phenotypes of mice lacking caveolin-1 are not in agreement with a strict requirement for the scaffolding domain in suppressing signal transduction. These animals certainly have defects in NO and calcium signaling, but their overall viability is inconsistent with an obligatory role for the scaffolding domain in regulating signal transduction. Caveolin-1 may modulate signal transduction through an interaction with lipids rather than proteins. As discussed above, there is good experimental and genetic evidence that caveolin-1 is involved in maintaining caveolae cholesterol levels. Caveolae cholesterol, in turn, is clearly required for the localization of certain signaling molecules to caveolae and for modulating the interaction between signaling molecules. One good example is the interaction between the PDGFR and multiple signaling molecules in caveolae. When cholestenone is substituted for cholesterol in caveolae, PDGF stimulates normal autophosphorylation of PDGFR, but none of the other PDGFR substrates become phosphorylated. This is not because the PDGFR has migrated out of caveolae nor is it because of a loss of the substrates from the domain (46Liu P. Wang P. Michaely P. Zhu M. Anderson R.G. J. Biol. Chem. 2000; 275: 31648-31654Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Rather, it appears that cholesterol is required for PDGFR to couple with its normal substrates. In other words, caveolae lipids themselves form a scaffold that organizes multiple signaling molecules and controls their interactions with PDGFR. The integrity of this “lipid scaffold” depends on cholesterol. A function for caveolin-1, therefore, is to maintain the caveolae lipid scaffold by removing oxidized cholesterol and replacing it with new cholesterol. Caveolin-1 appears to have principal functions in lipid transport, membrane traffic, and cell signaling. The mechanistic basis of these functions remains to be worked out but must be reflected in the complex and mysterious intracellular traffic of this protein. A model is proposed in Fig. 1 for how we think caveolin-1 might move from its site of synthesis to various compartments in the cell. Caveolin-1 appears to be inserted co-translationally into the ER membrane with its N- and C-terminal portions in the cytoplasm (Fig. 1, red path). It then is incorporated into vesicles (Fig. 1, 1) that move to the Golgi apparatus in a step that requires amino acids 66–70 (IDFED) (44Machleidt T., Li, W.P. Liu P. Anderson R.G. J. Cell Biol. 2000; 148: 17-28Crossref PubMed Scopus (98) Google Scholar). Within the Golgi apparatus caveolin-1 oligomerizes and becomes detergent-insoluble (47Lisanti M.P. Tang Z.L. Sargiacomo M. J. Cell Biol. 1993; 123: 595-604Crossref PubMed Scopus (172) Google Scholar). Oligomerization depends on amino acids 91–100 (TFTVTKYWFY) and 135–140 (KSFLIE). Vesicular transport to the cell surface (Fig. 1, 2) depends both on the ability of the molecule to oligomerize and on amino acids 71–80 (VIAEPEGTHS). Once it reaches the cell surface, presumably caveolin-1 becomes incorporated into functioning caveolae that internalize and recycle (Fig. 1,3). At some stage in the caveolae internalization cycle, we believe caveolin-1 can enter the cytoplasm of the cell as a soluble protein embedded in a lipid particle (Fig. 1, 4–6). The exact amino acids in the protein that control this step are not known but may depend on palmitoylation of cysteines 133, 143, and 156 (20Uittenbogaard A. Smart E.J. J. Biol. Chem. 2000; 275: 25595-25599Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). There are multiple targets for the soluble caveolin-1. It may go to the ER (Fig. 1, 4) and either pick up newly synthesized cholesterol for transport back to caveolae (return blue arrow) or enter the lumen of the ER. If the latter occurs, then the soluble caveolin-1 (Fig. 1, blue path) is incorporated into HDL-like particles that are secreted by the cell (7Liu P., Li, W.P. Machleidt T. Anderson R.G. Nat. Cell Biol. 1999; 1: 369-375Crossref PubMed Scopus (103) Google Scholar). Another possibility is that the soluble caveolin-1 remains in the cytosol (Fig. 1, green). Some of this caveolin-1 may be targeted to lipid droplets (26Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Crossref PubMed Scopus (278) Google Scholar). Finally, soluble caveolin-1 can go to mitochondria (Fig. 1, orange). Nothing is known about how the distribution of caveolin-1 is regulated or about the machinery responsible for converting caveolin-1 from a membrane to a soluble protein. We thank Brenda Pallares for administrative assistance.