Caveolae are plasma membrane specializations present in most cell types. Caveolin, a 22-kDa integral membrane protein, is a principal structural and regulatory component of caveolae membranes. Previous studies have demonstrated that caveolin co-purifies with lipid modified signaling molecules, including G ␣ subunits, HRas, c-Src, and other related Src family tyrosine kinases. In addition, it has been shown that caveolin interacts directly with G ␣ subunits and H-Ras, preferentially recognizing the inactive conformation of these molecules. However, it is not known whether caveolin interacts directly or indirectly with Src family tyrosine kinases. Here, we examine the structural and functional interaction of caveolin with Src family tyrosine kinases. Caveolin was recombinantly expressed as a glutathione S-transferase fusion. Using an established in vitro binding assay, we find that caveolin interacts with wild-type Src (c-Src) but does not form a stable complex with mutationally activated Src (v-Src). Thus, it appears that caveolin prefers the inactive conformation of Src. Deletion mutagenesis indicates that the Src-interacting domain of caveolin is located within residues 82-101, a cytosolic membrane-proximal region of caveolin. A caveolin peptide derived from this region (residues 82-101) functionally suppressed the auto-activation of purified recombinant c-Src tyrosine kinase and Fyn, a related Src family tyrosine kinase. We further analyzed the effect of caveolin on c-Src activity in vivo by transiently co-expressing full-length caveolin and c-Src tyrosine kinase in 293T cells. Co-expression with caveolin dramatically suppressed the tyrosine kinase activity of c-Src as measured via an immune complex kinase assay. Thus, it appears that caveolin structurally and functionally interacts with wild-type c-Src via caveolin residues 82-101. Besides interacting with Src family kinases, this cytosolic caveolin domain (residues 82-101) has the following unique features. First, it is required to form multivalent homo-oligomers of caveolin. Second, it interacts with G-protein ␣-subunits and down-regulates their GTPase activity. Third, it binds to wild-type H-Ras. Fourth, it is membrane-proximal, suggesting that it may be involved in other potential protein-protein interactions. Thus, we have termed this 20-amino acid stretch of caveolin residues the caveolin scaffolding domain.

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes. In this regard, caveolin co-purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS. Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions. For example, this domain interacts with G-protein ␣ subunits and Src-like kinases and can functionally suppress their activity. This caveolinderived protein domain has been termed the caveolinscaffolding domain. However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules.Here, we have used the caveolin-scaffolding domain as a receptor to select random peptide ligands from phage display libraries. These caveolin-selected peptide ligands are rich in aromatic amino acids and have a characteristic spacing in many cases. A known caveolin-interacting protein, G i2␣ , was used as a ligand to further investigate the nature of this interaction. G i2␣ and other G-protein ␣ subunits contain a single region that generally resembles the sequences derived from phage display. We show that this short peptide sequence derived from G i2␣ interacts directly with the caveolin-scaffolding domain and competitively inhibits the interaction of the caveolin-scaffolding domain with the appropriate region of G i2␣ . This interaction is strictly dependent on the presence of aromatic residues within the peptide ligand, as replacement of these residues with alanine or glycine prevents their interaction with the caveolinscaffolding domain. In addition, we have used this interaction to define which residues within the caveolin-scaffolding domain are critical for recognizing these peptide and protein ligands. Also, we find that the scaffolding domains of caveolins 1 and 3 both recognize the same peptide ligands, whereas the corresponding domain within caveolin-2 fails to recognize these ligands under the same conditions. These results serve to further demonstrate the specificity of this interaction. The implications of our current findings are discussed regarding other caveolin-and caveolae-associated proteins.Caveolae are plasma membrane-attached vesicular organelles that have a characteristic diameter in the range of 50 -100 nm (1, 2). Caveolae are present in most cell types but are especially abundant in adipocytes, endothelial cells, fibroblasts, and smooth muscle cells (3). In adipocytes and smooth muscle cells, they represent up to 20% of the total plasma membrane surface area. Endothelial cells contain ϳ5,000 -10,000 caveolae/cell. Although they were originally implicated in cellular transport processes (4), recent evidence suggests that they may participate in signal transduction-related events (5-11).Caveolin, a ...

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Caveolae are microdomains of the plasma membrane that have been implicated in organizing and compartmentalizing signal transducing molecules. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membrane in vivo. Recently, we and other laboratories have identified a family of caveolin-related proteins; caveolin has been retermed caveolin-1.Here, we examine the cell-type and tissue-specific expression of caveolin-2. For this purpose, we generated a novel mono-specific monoclonal antibody probe that recognizes only caveolin-2, but not caveolins-1 and -3. A survey of cell and tissue types demonstrates that the caveolin-2 protein is most abundantly expressed in endothelial cells, smooth muscle cells, skeletal myoblasts (L6, BC3H1, C2C12), fibroblasts, and 3T3-L1 cells differentiated to adipocytes. This pattern of caveolin-2 protein expression most closely resembles the cellular distribution of caveolin-1. In line with these observations, co-immunoprecipitation experiments with mono-specific antibodies directed against either caveolin-1 or caveolin-2 directly show that these molecules form a stable hetero-oligomeric complex. The in vivo relevance of this complex was further revealed by dual-labeling studies employing confocal laser scanning fluorescence microscopy. Our results indicate that caveolins 1 and 2 are strictly co-localized within the plasma membrane and other internal cellular membranes. Ultrastructurally, this pattern of caveolin-2 localization corresponds to caveolae membranes as seen by immunoelectron microscopy. Despite this strict co-localization, it appears that regulation of caveolin-2 expression occurs independently of the expression of either caveolin-1 or caveolin-3 as observed using two different model cell systems. Although caveolin-1 expression is down-regulated in response to oncogenic transformation of NIH 3T3 cells, caveolin-2 protein levels remain unchanged. Also, caveolin-2 protein levels remain unchanged during the differentiation of C2C12 cells from myoblasts to myotubes, while caveolin-3 levels are dramatically induced by this process. These results suggest that expression levels of caveolins 1, 2, and 3 can be independently up-regulated or down-regulated in response to a variety of distinct cellular cues.Caveolae are small omega-shaped indentations of the plasma membrane that have been implicated in signal transduction and vesicular transport processes (1, 2). Caveolae are found in most cell types but are extremely abundant in terminally differentiated cell types: adipocytes (3-5), simple squamous epithelia (type I pneumocytes and endothelial cells) (6), smooth muscle cells (7), and fibroblasts (8). Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo (9 -13).Several independent lines of evidence suggest that caveolin functions as a scaffolding protein within caveolae membranes. Caveolin forms a high molecular mass oligomeric complex (14, 15) that is thought to represent the assem...

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GSTcaveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61-101) and to a lesser extent the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.Caveolae are cholesterol-and sphingolipid-rich microdomains of the plasmalemma that have been implicated in a variety of cellular functions, including transcytosis of molecules and signal transduction events (1, 2). With respect to the latter function, structurally distinct dually acylated proteins involved in signal transduction (including G-protein ␣ subunits, Ha-Ras, Src family members, and endothelial nitric oxide synthase (eNOS) 1 ) reside in caveolae (3-6). In the case of certain Src members and eNOS, mutation of the cysteine palmitoylation sites prevents caveolae localization suggesting that palmitoylation is a "molecular zip code" for the trafficking of dually acylated proteins into glycolipid-rich microdomains of the plasmalemma (4, 6, 7).The major coat proteins of caveolae are the caveolin family of proteins (caveolin-1, -2 and -3 (1, 8)). Besides being intimately embedded within the lipid microdomain comprising caveolae, caveolins may regulate signaling via direct interaction with other resident proteins. For example, caveol...