A role for the caveolin scaffolding domain in mediating the membrane attachment of caveolin-1 - The caveolin scaffolding domain is both necessary andsufficient for membrane binding in vitro

Here, we have created a series of caveolin-1 (Cav-1) deletion mutants to ex amine whether the membrane spanning segment is required for membrane attach ment of caveolin-1 in vivo, One mutant, Cav-1-(1-101), contains only the cy toplasmic N-terminal domain and lacks the membrane spanning domain and the C-terminal domain, Interestingly, Cav-1-(1-101) still behaves as an integra l membrane protein but lacks any known signals for lipid modification. In s triking contrast, another deletion mutant, Cav-1-(1-81), behaved as a solub le protein, These results implicate caveolin-1 residues 82-101 (also known as the caveolin scaffolding domain) in membrane attachment. In accordance w ith the postulated role of the caveolin-1 scaffolding domain as an inhibito r of signal transduction, Cav-1-(1-101) retained the ability to functionall y inhibit signaling along the p42/44 mitogen-activated protein kinase casca de, whereas Cav-1-(1-81) was completely ineffective. To rule out the possib ility that membrane attachment mediated by the caveolin scaffolding domain was indirect, we reconstituted the membrane binding of caveolin-1 in vitro, By using purified glutathione S-transferase-caveolin-1 fusion proteins and reconstituted lipid vesicles, we show that the caveolin-1 scaffolding doma in and the C-terminal domain (residues 135-178) are both sufficient for mem brane attachment in vitro, However, the putative membrane spanning domain ( residues 102-134) did not show any physical association with membranes in t his in vitro system. Taken together, our results provide strong evidence th at the caveolin scaffolding domain contributes to the membrane attachment o f caveolin-1.