IDENTIFICATION OF PEPTIDE AND PROTEIN LIGANDS FOR THE CAVEOLIN-SCAFFOLDING DOMAIN - IMPLICATIONS FOR THE INTERACTION OF CAVEOLIN WITH CAVEOLAE-ASSOCIATED PROTEINS

Caveolin, a 21-24-kDa integral membrane protein, is a principal compon ent of caveolae membranes, We have suggested that caveolin functions a s a scaffolding protein to organize and concentrate certain caveolin-i nteracting proteins within caveolae membranes, In this regard, caveoli n co purifies with a variety of lipid-modified signaling molecules, in cluding G-proteins, Src-like kinases, Ha-Ras, and eNOS, Using several independent approaches, it has been shown that a 20-amino acid membran e proximal region of the cytosolic amino-terminal domain of caveolin i s sufficient to mediate these interactions, For example, this domain i nteracts with G-protein cu subunits and Src-like kinases and can funct ionally suppress their activity, This caveolin-derived protein domain has been termed the caveolin scaffolding domain, However, it remains u nknown how the caveolin-scaffolding domain recognizes these molecules. Here, we have used the caveolin-scaffolding domain as a receptor to s elect random peptide ligands from phage display libraries, These caveo lin selected peptide ligands are rich in aromatic amino acids and have a characteristic spacing in many cases, A known caveolin-interacting protein, G(i2 alpha), was used as a ligand to further investigate the nature of this interaction. G(i2 alpha), and other G-protein alpha sub units contain a single region that generally resembles the sequences d erived from phage display. We show that this short peptide sequence de rived from G(i2 alpha) interacts directly with the caveolin-scaffoldin g domain and competitively inhibits the interaction of the caveolin-sc affolding domain with the appropriate region of G(i2 alpha). This inte raction is strictly dependent on the presence of aromatic residues wit hin the peptide ligand, as replacement of these residues with alanine or glycine prevents their interaction with the caveolin-scaffolding do main, In addition, we have used this interaction to define which resid ues within the caveolin-scaffolding domain are critical for recognizin g these peptide and protein ligands, Also, we find that the scaffoldin g domains of caveolins 1 and 3 both recognize the same peptide ligands , whereas the corresponding domain within caveolin-2 fails to recogniz e these ligands under the same conditions, These results serve to furt her demonstrate the specificity of this interaction, The implications of our current findings are discussed regarding other caveolin- and ca veolae-associated proteins.