CAVEOLIN VERSUS CALMODULIN - COUNTERBALANCING ALLOSTERIC MODULATORS OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE

Nitric oxide is synthesized in diverse mammalian tissues by a family o f calmodulin-dependent nitric oxide synthases, The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal -transducing membrane domains termed plasmalemmal caveolae, Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-c almodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other s tructurally distinct signaling proteins via a specific region identifi ed within the caveolin sequence (amino acids 82-101) that appears to s ubserve the role of a ''scaffolding domain.'' We now report that the c o-immunoprecipitation of eNOS with caveolin is completely and specific ally blocked by an oligopeptide corresponding to the caveolin scaffold ing domain, Peptides corresponding to this domain markedly inhibit nit ric oxide synthase activity in endothelial membranes and interact dire ctly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli, The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reve rsed by Ca2+-calmodulin. These studies establish that caveolin, via it s scaffolding domain, directly forms an inhibitory complex with eNOS a nd suggest that caveolin inhibits eNOS by abrogating the enzyme's acti vation by calmodulin.