Caveolin Isoforms Differ in Their N-terminal Protein Sequence and Subcellular Distribution. IDENTIFICATION AND EPITOPE MAPPING OF AN ISOFORM-SPECIFIC MONOCLONAL ANTIBODY PROBE

Caveolin, an integral membrane protein, is a principal component of caveolae membranes in vivo. Two isoforms of caveolin have been identified: a slower migrating 24-kDa species (α-isoform) and a faster migrating 21-kDa species (β-isoform). Little is known about how these isoforms differ, either structurally or functionally. Here we have begun to study the differences between these two isoforms. Microsequencing of caveolin reveals that both isoforms contain internal caveolin residues 47-77. In a second independent approach, we recombinantly expressed caveolin in a caveolin-negative cell line (FRT cells). Stable transfection of FRT cells with the full-length caveolin cDNA resulted in the expression of both caveolin isoforms, indicating that they can be derived from a single cDNA. Using extracts from caveolin-expressing FRT cells, we fortuitously identified a monoclonal antibody that recognizes only the α-isoform of caveolin. Epitope mapping of this monoclonal antibody reveals that it recognizes an epitope within the extreme N terminus of caveolin, specifically residues 1-21. These results suggest that α- and β-isoforms of caveolin differ in their N-terminal protein sequences. To independently evaluate this possibility, we placed an epitope tag at either the extreme N or C terminus of full-length caveolin. Results of these “tagging” experiments clearly demonstrate that (i) both isoforms of caveolin contain a complete C terminus and (ii) that the α-isoform contains a complete N terminus while the β-isoform lacks N-terminal-specific protein sequences. Mutational analysis reveals that these two isoforms apparently derive from the use of two alternate start sites: methionine at position 1 and an internal methionine at position 32. This would explain the ~3-kDa difference in their apparent migration in SDS-polyacrylamide electrophoresis gels. In addition, using isoform-specific antibody probes we show that caveolin isoforms may assume a distinct but overlapping subcellular distribution by confocal immunofluorescence microscopy. We discuss the possible implications of these differences between α- and β-caveolin. Caveolin, an integral membrane protein, is a principal component of caveolae membranes in vivo. Two isoforms of caveolin have been identified: a slower migrating 24-kDa species (α-isoform) and a faster migrating 21-kDa species (β-isoform). Little is known about how these isoforms differ, either structurally or functionally. Here we have begun to study the differences between these two isoforms. Microsequencing of caveolin reveals that both isoforms contain internal caveolin residues 47-77. In a second independent approach, we recombinantly expressed caveolin in a caveolin-negative cell line (FRT cells). Stable transfection of FRT cells with the full-length caveolin cDNA resulted in the expression of both caveolin isoforms, indicating that they can be derived from a single cDNA. Using extracts from caveolin-expressing FRT cells, we fortuitously identified a monoclonal antibody that recognizes only the α-isoform of caveolin. Epitope mapping of this monoclonal antibody reveals that it recognizes an epitope within the extreme N terminus of caveolin, specifically residues 1-21. These results suggest that α- and β-isoforms of caveolin differ in their N-terminal protein sequences. To independently evaluate this possibility, we placed an epitope tag at either the extreme N or C terminus of full-length caveolin. Results of these “tagging” experiments clearly demonstrate that (i) both isoforms of caveolin contain a complete C terminus and (ii) that the α-isoform contains a complete N terminus while the β-isoform lacks N-terminal-specific protein sequences. Mutational analysis reveals that these two isoforms apparently derive from the use of two alternate start sites: methionine at position 1 and an internal methionine at position 32. This would explain the ~3-kDa difference in their apparent migration in SDS-polyacrylamide electrophoresis gels. In addition, using isoform-specific antibody probes we show that caveolin isoforms may assume a distinct but overlapping subcellular distribution by confocal immunofluorescence microscopy. We discuss the possible implications of these differences between α- and β-caveolin. Caveolae are small flask-shaped invaginations located at or near the plasma membrane(1Severs N.J. J. Cell Sci. 1988; 90: 341-348Crossref PubMed Google Scholar, 2Anderson R.G.W. Curr. Opin. Cell Biol. 1993; 5: 647-652Crossref PubMed Scopus (171) Google Scholar). They are thought to exist in most cell types, although they are most abundant in endothelial cells, type I pneumocytes, adipocytes, fibroblasts, and smooth muscle cells (reviewed in Refs. 3Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (590) Google Scholar and 4Lisanti M.P. Scherer P.E. Tang Z.-L. Kubler E. Koleske A.J. Sargiacomo M.S. Semin. Dev. Biol. 1995; 6: 47-58Crossref Scopus (31) Google Scholar). Functionally, caveolae are involved in the uptake of small molecules such as folate (5Kamen B.A. Smith A.K. Anderson R.G.W. J. Clin. Invest. 1991; 87: 1442Crossref PubMed Scopus (106) Google Scholar) and the transport of macromolecules across capillary endothelial cells, including modified atherogenic low density lipoproteins(6Vasile E. Simionescu M. Simionescu N. J. Cell Biol. 1983; 96: 1677-1689Crossref PubMed Scopus (245) Google Scholar, 7Snelting-Havinga I. Mommaas M. Van Hinsbergh V. Daha M. Daems W. Vermeer B. Eur. J. Cell Biol. 1989; 1989: 27-36Google Scholar). Recently, we and others have proposed that caveolae may also participate in a subset of transmembrane signaling events, such as G-protein-coupled signaling(3Lisanti M.P. Scherer P. Tang Z.-L. Sargiacomo M. Trends Cell Biol. 1994; 4: 231-235Abstract Full Text PDF PubMed Scopus (590) Google Scholar, 8Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, 9Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar, 10Chang W.J. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. De Gunzburg J. Mumby S. Gilman A. Anderson R.G.W. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (311) Google Scholar, 11Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Crossref PubMed Scopus (343) Google Scholar). Caveolin, a 21-24-kDa integral membrane protein, has been identified as a principal component of caveolae membranes in vivo(12Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1868) Google Scholar). However, caveolin was first identified as a major v-src substrate in Rous sarcoma virus-transformed chick embryo fibroblasts (13Glenney J.R. Zokas L. J. Cell Biol. 1989; 108: 2401-2408Crossref PubMed Scopus (360) Google Scholar). Both cell transformation and tyrosine phosphorylation of caveolin are dependent on membrane attachment of v-src(14Glenney J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google Scholar), suggesting that caveolin may represent a critical substrate for cellular transformation. In support of this view, we have recently observed that both caveolin expression and caveolae are lost during cell transformation by activated oncogenes other than v-src (v-abl, bcr-abl, middle T antigen, and activated ras)(15Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (472) Google Scholar). These results support the hypothesis that caveolin may represent a candidate tumor suppressor protein(15Koleske A.J. Baltimore D. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1381-1385Crossref PubMed Scopus (472) Google Scholar). Indeed, Krev-1, a Ras-related transformation suppressor protein, is concentrated in purified caveolin-rich membrane domains (9Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar) and purified caveolae(10Chang W.J. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. De Gunzburg J. Mumby S. Gilman A. Anderson R.G.W. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (311) Google Scholar). Two major isoforms of caveolin are known to exist: a slower migrating 24-kDa species and a faster migrating 21-kDa species. For simplicity, we will designate them as α- and β-isoforms of caveolin, respectively. Little is known about how these isoforms differ. This may be important for understanding the role of caveolin and caveolae in normal and transformed cells. Caveolin is the product of a single gene(16Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar). Furthermore, as caveolin mRNA is a single species, it is unlikely that these two isoforms arise from differential mRNA splicing(9Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar, 16Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 17Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. J. Cell Biol. 1994; PubMed Scopus Google Scholar). both isoforms are caveolin it that is a between M.P. Tang Z.-L. Sargiacomo M. J. Cell Biol. 1993; PubMed Scopus Google Scholar, P. H. M. K. J. Cell Biol. 1992; PubMed Scopus Google Scholar). Caveolin is on M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google Scholar, M. Scherer P.E. Tang Z.-L. J.E. Lisanti M.P. 1994; Google Scholar). only the β-isoform is in while both are of phosphorylation in P.E. Lisanti M.P. Baldini G. Sargiacomo M. J. Cell Biol. 1994; PubMed Scopus Google Scholar). These to a difference that to be by a in vivo. these demonstrate that the β-isoform is an product of the as only the faster migrating β-isoform is in vivo. we have begun to study the differences between these two caveolin isoforms. monoclonal have been caveolin by Glenney and J.R. Zokas L. J. Cell Biol. 1989; 108: 2401-2408Crossref PubMed Scopus (360) Google Scholar, J.R. J. Biol. Chem. 1989; 264: 20163-20166Abstract Full Text PDF PubMed Google only a of these are with and caveolin, the that caveolin is from to J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google these we have fortuitously identified an monoclonal and epitope mapping of this monoclonal antibody has both and the differences between α- and β-caveolin. In addition, using this isoform-specific we show by confocal that may assume a distinct subcellular This is to be a for the of caveolin isoforms. full-length caveolin the J. Glenney monoclonal antibody was by of other protein normal and and antibody domains purified from as M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar, M.P. Tang Scherer P. Sargiacomo M. 1995; PubMed Scopus Google Scholar). To for caveolin, these domains on in to and using the type of density for single at the was with and by in the for at This other 21-24-kDa that to with caveolin. Caveolin isoforms and with caveolin these identified by Microsequencing was as we M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar). cells monoclonal antibody electrophoresis FRT and cells as M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, W. G. E. J. 1994; PubMed Scopus Google Scholar). to with and as M.P. A. Sargiacomo M. E. J. Cell Biol. 1989; PubMed Scopus Google Scholar). expression of a was by an with normal M.P. Tang Scherer P. Sargiacomo M. 1995; PubMed Scopus Google Scholar, M.P. E. J. Cell Biol. 1989; PubMed Scopus Google Scholar). type full-length caveolin and of caveolin the of the the of J. for expression in FRT or cells, respectively. In to recombinantly of caveolin in cells, we the epitope tag the N terminus or the C terminus of the caveolin cDNA using We placed as a between the epitope and the caveolin as has been P. H. M. K. J. Cell Biol. 1992; PubMed Scopus Google Scholar, 1991; PubMed Scopus Google Scholar). of the epitope tag and caveolin by FRT or cells using a of the M.P. Tang Scherer P. Sargiacomo M. 1995; PubMed Scopus Google Scholar, M.P. E. J. Cell Biol. 1989; PubMed Scopus Google Scholar). in with by using by immunofluorescence for expression of caveolin. type full-length caveolin expressed in FRT cells was using or of caveolin expressed in cells using monoclonal that recognizes the epitope residues at either position 1 or at position to by to to the caveolin cDNA by U. 96: PubMed Scopus Google Scholar). the the cells by the B. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). cells 1 was by at for and the was with an of and for of of a of cells was by by with caveolin caveolin terminus the transmembrane and the C terminus and of the N-terminal by and the of the to expressed in a and and caveolin by purified by using as 1993; PubMed Scopus Google Scholar). the caveolin was and to with a K. L. J. Cell Sci. 1992; Google Scholar). at and and for antibody by of purified using protein as by the FRT cells in at a with and for in and cells with and with in for to with for either at or on and with cells with (i) of normal and (ii) a of and and antibody and antibody first was while and antibody with between with and observed a confocal Two major isoforms of caveolin have been identified by or electrophoresis of cell including cells In to the difference between these two isoforms, we purified caveolin from a caveolin-rich and these isoforms to analysis N-terminal that the N terminus of is We internal with the of these in reveals that both caveolin isoforms contain internal caveolin residues 47-77. In addition, to contain a complete N-terminal residues and to contain a complete residues the faster migrating β-isoform a complete N-terminal of caveolin isoforms in a FRT cells to of caveolin mRNA or protein, although they contain M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, W. G. E. J. 1994; PubMed Scopus Google Scholar). a we expressed full-length caveolin in FRT cells to study results that both caveolin isoforms derive from a single cDNA. This is with a single species of caveolin mRNA (9Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar, 16Glenney J.R. FEBS Lett. 1992; 314: 45-48Crossref PubMed Scopus (189) Google Scholar, 17Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. J. Cell Biol. 1994; PubMed Scopus Google Scholar) and that in both P. H. M. K. J. Cell Biol. 1992; PubMed Scopus Google Scholar). However, these the that this is an of the in To this possibility, we derived cell FRT cells with the full-length caveolin cDNA. with monoclonal antibody reveals both α- and in FRT However, we that of the cell with monoclonal only These results suggest that monoclonal is and recognizes an epitope that is from β-caveolin. monoclonal antibody be as a to structurally and between α- and β-caveolin. We epitope mapping to monoclonal only the substrate for antibody we expressed full-length caveolin and of caveolin as to with either monoclonal antibody or on their differential recognizes an epitope within caveolin residues while recognizes an epitope within caveolin residues in both caveolin isoforms, these results are with the analysis that both isoforms contain caveolin residues 47-77. In addition, these results show that these two isoforms differ by a N-terminal This the of a that to the of N-terminal protein To the of the or of caveolin independently of we expressed of caveolin in cells. Using we placed a epitope tag at either the extreme N or C terminus of full-length caveolin Caveolin isoforms can be expression using a monoclonal antibody that is the with that both the and domains of caveolin the P. G. K. J. 1993; PubMed Scopus Google Scholar, D.J. Lublin D.M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), of either or caveolin with of caveolin a tag both caveolin isoforms with an antibody that recognizes the epitope and These results clearly demonstrate that both isoforms contain a complete C In caveolin only the α-isoform using an antibody that recognizes the However, both isoforms expressed in these cells with caveolin, as with and These results independently demonstrate that (i) caveolin isoforms derive from a single cDNA and (ii) that the α-isoform contains a complete N terminus while the β-isoform lacks N-terminal-specific protein as it to contain the These support results from epitope mapping of we have that caveolin isoforms differ in N-terminal protein it is how these two isoforms are is that or by a or an to β-caveolin. This would be a between α- and β-caveolin. we this possibility, it unlikely as both caveolin isoforms are caveolin M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, P. H. M. K. J. Cell Biol. 1992; PubMed Scopus Google Scholar). is alternate of Caveolin contains a second methionine at position that is in caveolin cDNA to for an This methionine as an internal start during of This would two caveolin isoforms that differ by and the would N-terminal protein such as residues as we of from a single mRNA is to two isoforms of other the and the B. D. W. B. PubMed Scopus Google Scholar, L. E. N. A. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). use of a methionine as a start is in by mRNA known as the M. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). analysis of methionine reveals that this as an internal start methionine at position in caveolin the for than methionine at position 1 at the two most critical that methionine as an internal start to caveolin isoforms. For these we caveolin and expression in cells. To evaluate the of methionine in caveolin isoforms, we the methionine at position 1 or at position to by to Caveolin only to the indicating that this can be derived from an internal start the normal start at position 1 is caveolin only the that an internal methionine at position is to the these that methionine can as an internal start to caveolin isoforms. Caveolin as distinct by M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1868) Google Scholar, P. H. M. K. J. Cell Biol. 1992; PubMed Scopus Google Scholar, J.R. D. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, M. U. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). In cells that contain caveolin or in cells caveolin is recombinantly these to of caveolae as by W.J. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. De Gunzburg J. Mumby S. Gilman A. Anderson R.G.W. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (311) Google Scholar, K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1868) Google Scholar, P. H. M. K. J. Cell Biol. 1992; PubMed Scopus Google Scholar, P. G. K. J. 1993; PubMed Scopus Google Scholar, E. Ying P. Anderson R.G.W. J. Cell Biol. 1994; PubMed Scopus Google Scholar). Using that both caveolin isoforms, and that than of both caveolin isoforms are to M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar, 10Chang W.J. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. De Gunzburg J. Mumby S. Gilman A. Anderson R.G.W. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (311) Google Scholar, K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1868) Google Scholar, P. G. K. J. 1993; PubMed Scopus Google Scholar, E. Ying P. Anderson R.G.W. J. Cell Biol. 1994; PubMed Scopus Google Scholar). However, independent of the and the plasma membrane has that at two distinct of caveolae may S. A. K. K. J. Cell Biol. 1993; Scopus Google Scholar, J. Cell Biol. 1993; PubMed Scopus (360) Google Scholar). differences between α- and distinct subcellular of these two isoforms. To this possibility, we with and using caveolin-expressing FRT cells assume a These two for experiments as they in species possible of the with was by confocal microscopy. with both was dependent on caveolin as was observed with FRT cells the of the the C terminus of caveolin and that this antibody specifically recognizes caveolin. observed with is in small are the cell and the cell However, is an of within the cell and the of the cell in with reveals a with are the In is an of the of the cell while within the of the cell However, a of that is within the of the cell in may represent the of the as caveolae are known to be concentrated at the K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1868) Google Scholar). In addition, as the α-isoform is from this this to the of the both caveolin isoforms contain a complete with the distribution of both isoforms. the antibody may the β-isoform by although it to both by of this antibody recognizes both isoforms or only the results demonstrate that these isoforms are within a single cell as is In addition, it be that caveolin-expressing FRT cells to using an M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar), than of caveolin was within membrane an that caveolin is expressed in FRT cells This is in with that FRT cells contain but to caveolin M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar) and that caveolin is to the or of E. Ying P. Anderson R.G.W. J. Cell Biol. 1994; PubMed Scopus Google Scholar). we have that caveolin isoforms differ in their extreme N-terminal protein but both contain a complete C This is on the of an monoclonal antibody epitope to residues of caveolin. This antibody to β-caveolin. most is that the β-isoform lacks an N-terminal-specific protein in the of this was by the N or C terminus of caveolin with the epitope tag to the of the N or C Mutational analysis reveals that these two isoforms apparently derive from the use of two alternate start sites: methionine at position 1 and an internal methionine at position 32. these for the ~3-kDa difference observed in the migration of the α- and of caveolin reveals or M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (863) Google Scholar, K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1868) Google Scholar, P. H. M. K. J. 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J. 1993; PubMed Scopus Google Scholar, E. Ying P. Anderson R.G.W. J. Cell Biol. 1994; PubMed Scopus Google Scholar). than of caveolin is within purified caveolae known as caveolin-rich membrane while caveolin is from these M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar, M.P. Tang Scherer P. Sargiacomo M. 1995; PubMed Scopus Google Scholar, E. Ying P. Anderson R.G.W. J. Cell Biol. 1994; PubMed Scopus Google Scholar). These and have also that both caveolin isoforms are within M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar, 10Chang W.J. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. De Gunzburg J. Mumby S. Gilman A. Anderson R.G.W. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (311) Google Scholar). However, we show by confocal immunofluorescence that these two isoforms may assume distinct but overlapping subcellular This is with independent suggesting the of at two distinct of S. A. K. K. J. Cell Biol. 1993; Scopus Google Scholar, J. Cell Biol. 1993; PubMed Scopus (360) Google Scholar). of from a single mRNA is to the two isoforms of other the and the B. D. W. B. PubMed Scopus Google Scholar, L. E. N. A. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). In the of these two isoforms are but are to subcellular is while the other L. E. N. A. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). is a for using alternate of to isoforms of a protein to within the both isoforms of caveolin are within M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar, 10Chang W.J. Ying Y. Rothberg K. Hooper N. Turner A. Gambliel H. De Gunzburg J. Mumby S. Gilman A. Anderson R.G.W. J. Cell Biol. 1994; 126: 127-138Crossref PubMed Scopus (311) Google Scholar), this N-terminal is for only phosphorylation in P.E. Lisanti M.P. Baldini G. Sargiacomo M. J. Cell Biol. 1994; PubMed Scopus Google Scholar), this N-terminal protein a by the of by an of this Caveolin contains for phosphorylation by protein C and M. Scherer P.E. Tang Z.-L. J.E. Lisanti M.P. 1994; Google Scholar, Z.-L. Scherer P.E. Lisanti M.P. 1994; PubMed Scopus Google Scholar). In addition, both protein and are concentrated in purified caveolin-rich membrane M.P. Scherer P.E. Vidugiriene J. Tang Z.-L. Hermanoski-Vosatka A. Tu Y.-H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (815) Google Scholar). phosphorylation is apparently important for the of caveolae as (i) protein C caveolae to and uptake of folate D. Ying B.A. Anderson R.G.W. J. Cell Biol. 1993; Scopus Google Scholar) and (ii) a the subcellular distribution of B. K. J. Cell Biol. 1994; PubMed Scopus Google Scholar). In support of differential of with the two isoforms of caveolin as an P. G. K. J. 1993; PubMed Scopus Google Scholar). These that differential the of a near the N terminus of caveolin an of protein sequences. However, they to support this they the of the N or C terminus of caveolin. Here we have these differences by and results demonstrate that contains a complete N and C while lacks N-terminal protein In addition, as is this will the use of this antibody as a for caveolin isoforms. We and for Cook for Glenney for monoclonal and and J. Koleske for in and caveolin.