Novel mechanisms of G protein-dependent regulation of endothelial nitric- oxide synthase

Endothelial nitric-oxide synthase (eNOS) plays a crucial role in the regulation of a variety of cardiovascular and pulmonary functions in both normal and pathological conditions. Multiple signaling inputs, including calcium, caveolin- 1, phosphorylation by several kinases, and binding to the 90-kDa heat shock protein (Hsp90), regulate eNOS activity. Here, we report a novel mechanism of G protein-dependent regulation of eNOS. We demonstrate that in mammalian cells, the alpha subunit of heterotrimeric G12 protein (G alpha(12)) can form a complex with eNOS in an activation- and Hsp90-independent manner. Our data show that G alpha(12) does not affect eNOS-specific activity, but it strongly enhances total eNOS activity by increasing cellular levels of eNOS. Experiments using inhibition of protein or mRNA synthesis show that G alpha(12) increases the expression of eNOS by increasing half-life of both eNOS protein and eNOS mRNA. Small interfering RNA-mediated depletion of endogenous G alpha(12) decreases eNOS levels. A quantitative correlation can be detected between the extent of down-regulation of G alpha(12) and eNOS in endothelial cells after prolonged treatment with thrombin. protein-dependent increase of eNOS expression represents a novel mechanism by which heterotrimeric G proteins can regulate the activity of downstream signaling molecules.