DYNAMIC REGULATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE - COMPLEMENTARY ROLES OF DUAL ACYLATION AND CAVEOLIN INTERACTIONS

N-Terminal myristoylation and thiopalmitoylation of the endothelial is oform of nitric oxide synthase (eNOS) are required for targeting the e nzyme to specialized signal-transducin microdomains of plasma membrane termed caveolae. We have previously documented that the subcellular l ocalization of eNOS is dynamically regulated by agonists such as brady kinin, which promotes enzyme depalmitoylation and translocation from c aveolae. More recently, we have shown that association of eNOS with ca veolin, the principal structural protein in caveolae, leads to enzyme inhibition, in a reversible process modulated by Ca2+-calmodulin (CaM) . We now report studies of the respective roles of acylation and caveo lin interaction for regulating eNOS activity. Using eNOS truncation an d deletion mutants expressed in COS-7 cells, we have identified an obl igatory role for the N-terminal half of eNOS in stabilizing its associ ation with caveolin. By exploring the differential effects of detergen ts (CHAPS vs octyl glucoside), we have shown that this direct interact ion between both proteins is facilitated by, but does not require, eNO S acylation, and, importantly, that treatment of intact aortic endothe lial cells with the calcium ionophore A23187 leads to the rapid disrup tion of the eNOS-caveolin complexes. Finally, using transiently transf ected COS-7 cells, we have observed that the myristoylation-deficient cytosol-restricted eNOS mutant (myr(-)) as well as the cytosolic fract ion of the palmitoylation-deficient eNOS mutant (palm(-)) may both int eract with caveolin; this association also leads to a marked inhibitio n of enzyme activity, which is completely reversed by addition of calm odulin. We conclude that the regulatory eNOS-caveolin association is i ndependent of the state of eNOS acylation, indicating that agonist-evo ked Ca2+/CaM-dependent disruption of the caveolin-eNOS complex, rather than agonist-promoted depalmitoylation of eNOS, relieves caveolin's t onic inhibition of enzyme activity. We therefore propose that caveolin may serve as an eNOS chaperone regulating NO production independently of the enzyme's residence within caveolae or its state of acylation.