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 ca veolae membranes in vivo. Two isoforms of caveolin have been identifie d: a slower migrating 24-kDa species (alpha-isoform) and a faster migr ating 21-kDa species (beta-isoform). Little is known about how these i soforms differ, either structurally or functionally. Here we have begu n to study the differences between these two isoforms. Microsequencing of caveolin reveals that both isoforms contain internal caveolin resi dues 47-77. In a second independent approach, we recombinantly express ed caveolin in a caveolin-negative cell line (FRT cells), Stable trans fection of FRT cells with the full-length caveolin cDNA resulted in th e expression of both caveolin isoforms, indicating that they can be de rived from a single cDNA. Using extracts from caveolin-expressing FRT cells, we fortuitously identified a monoclonal antibody that recognize s only the alpha-isoform of caveolin. Epitope mapping of this monoclon al antibody reveals that it recognizes an epitope within the extreme N terminus of caveolin, specifically residues 1-21. These results sugge st that alpha- and beta-isoforms of caveolin differ in their N-termina l protein sequences. To independently evaluate this possibility, we pl aced an epitope tag at either the extreme N or C terminus of full-leng th caveolin. Results of these ''tagging'' experiments clearly demonstr ate that (i) both isoforms of caveolin contain a complete C terminus a nd (ii) that the alpha-isoform contains a complete N terminus while th e beta-isoform lacks N-terminal-specific protein sequences. Mutational analysis reveals that these two isoforms apparently derive from the u se of two alternate start sites: methionine at position 1 and an inter nal methionine at position 32. This would explain the similar to 3-kDa difference in their apparent migration in SDS-polyacrylamide electrop horesis gels. In addition, using isoform-specific antibody probes we s how that caveolin isoforms may assume a distinct but overlapping subce llular distribution by confocal immunofluorescence microscopy. We disc uss the possible implications of these differences between alpha- and beta-caveolin.