Deciphering the binding of caveolin-1 to client protein endothelial nitric oxide synthase (eNOS): scaffolding sub-domain identification, interaction modeling, and biological significance [Membrane Biology]

March 19th, 2014 by Trane, A. E., Pavlov, D., Sharma, A., Saqib, U., Lau, K., van Petegem, F., Minshall, R. D., Roman, L. J., Bernatchez, P. N.

Caveolin-1 (Cav-1) gene inactivation interferes with caveolae formation and causes a range of cardiovascular and pulmonary complications in vivo. Recent evidence suggest that blunted Cav-1 / endothelial nitric oxide synthase (eNOS) interaction, which occurs specifically in vascular endothelial cells, is responsible for the multiple phenotypes observed in Cav-1-null animals. Under basal conditions, Cav-1 binds eNOS and inhibits nitric oxide (NO) production via the Cav-1 scaffolding domain (CAV; aa 82-101). Although we have recently shown that CAV residue F92 is responsible for eNOS inhibition, the inactive F92A Cav-1 mutant unexpectedly retains its eNOS binding ability and can increase NO release, indicating the presence of a distinct eNOS-binding domain within CAV. Herein, we identified and characterized a small 10 aa CAV sub-sequence (90-99) that accounted for the majority of eNOS association with Cav-1 (Kd = 49nM), and computer modeling of CAV 90-99 docking to eNOS provides a rationale for the mechanism of eNOS inhibition by F92. Finally, using gene silencing and reconstituted cell systems, we show that intracellular delivery of a F92A CAV 90-99 peptide can promote NO bioavailability in eNOS- and Cav-1-dependent fashions. To our knowledge, these data provide the first detailed analysis of Cav-1 binding to one of its most significant client proteins, eNOS.
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