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

Abstract

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 (alpha-isoform) and a faster migrating 21-kDa species (beta-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 alpha-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 alpha- and beta-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 alpha-isoform contains a complete N terminus while the beta-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 approximately 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 alpha- and beta-caveolin.

Extensive vascularization of developing mouse ovaries revealed by caveolin‐1 expression

“…2A-F). These observations are consistent with reports of a high density of caveolae on endothelial cells (Scherer et al, 1995;Glienke et al, 2000), of caveolin-1 protein expression in vasculature of the fetal lung (Ramirez et al, 2002), and of more widespread Cav-1 mRNA expression in the developing cardiovascular system in mice (Fig. 1A and Bullejos et al, unpublished data).…”

“…The function of caveolae remains controversial, but they are implicated in lipid regulation (Razani et al, 2002), endocytosis/transcytosis, and signal transduction (Okamoto et al, 1998;Drab et al, 2001;Matveev et al, 2001;Schlegel and Lisanti, 2001). Caveolae are particularly abundant in adipocytes and endothelial cells (Scherer et al, 1995). Caveolin-1 is known to cause invagination of caveolae from the plasma membrane (Fra et al, 1995), but no detailed expression profile has been reported for the Cav-1 gene.…”

Analysis of the CAVEOLIN-1 gene at human chromosome 7q31.1 in primary tumours and tumour-derived cell lines

“…In this case, the consequence of overexpressing Caveolin-1 in malignant breast epithelial cells, namely growth inhibition, may be less physiologically relevant than ®rst thought, since these cells do not represent an appropriate target cell type. Lee et al (1998) failed to satisfactorily explain how re-expression of CAVEOLIN-1 in carcinoma cells was at all consequential when it failed to result in de novo caveolae formation, as endocytosis/vesicular transport associated with the function of this protein is believed to be important for its e ects on growth (Koleske et al, 1995;Scherer et al, 1995). Perhaps an ability of Caveolin protein to modulate integrin-mediated growth signalling is more signi®cant than its ability to induce caveolae formation.…”

M‐caveolin, a muscle‐specific caveolin‐related protein

“…4). The internal initiation site for translation [21] and the conserved co asensus protein kinase C (PKC) phosphorylation site of Vca ceolin [26] are lacking in M-caveolin. The first region where th,: sequences show a high level of identity starts at the KEI (I_ :¢s-Glu-Ile) sequence (residue 20 of M-caveolin, residue 47 of V-.zaveolin) and extends beyond the putative intramembrane dvmain.…”

“…Rather than being different gene products the shorter form appears to be the result of translation from an inner initiation site [21]. We now describe the characterisation of a novel homologue of V-caveolin which is muscle specific.…”