DIRECT INTERACTION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE AND CAVEOLIN-1 INHIBITS SYNTHASE ACTIVITY

Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated within endothelial plasmalemmal caveolae. It is not known, however, w hether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity. To answer these questions, we h ave cloned the bovine caveolin-1 cDNA and have investigated the eNOS-c aveolin-1 interaction in an in vitro binding assay system using glutat hione S-transferase (GST)-caveolin-1 fusion proteins and baculovirus-e xpressed bovine eNOS. We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system. Results obtained using both in vitro and in vivo protein interaction assays show that both Nand C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Interaction of eNOS with GST-caveolin -1 fusion proteins significantly inhibits enzyme catalytic activity. A synthetic peptide corresponding to caveolin-1 residues 82-101 also po tently and reversibly inhibits eNOS activity by interfering with the i nteraction of the enzyme with Ca2+/calmodulin (CaM). Regulation of eNO S in endothelial cells, therefore, may involve not only positive allos teric regulation by Ca2+/CaM, but also negative allosteric regulation by caveolin-1.