Deciphering the binding of caveolin-1 to client protein endothelial nitric-oxide synthase (eNOS): scaffolding subdomain identification, interaction modeling, and biological significance

TitleDeciphering the binding of caveolin-1 to client protein endothelial nitric-oxide synthase (eNOS): scaffolding subdomain identification, interaction modeling, and biological significance
Publication TypeJournal Article
Year of Publication2014
AuthorsTrane, AE, Pavlov, D, Sharma, A, Saqib, U, Lau, K, Van Petegem, F, Minshall, RD, Roman, LJ, Bernatchez, PN
JournalJournal of Biological Chemistry
Volume289
Issue19
Pagination13273-83
Date Published05/2014
ISSNPrint: 0021-9258; Online: 1083-351X
Abstract

Caveolin-1 (Cav-1) gene inactivation interferes with caveolae formation and causes a range of cardiovascular and pulmonary complications in vivo. Recent evidence suggests 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; amino acids 82-101). Although we have recently shown that CAV residue Phe-92 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-amino acid CAV subsequence (90-99) that accounted for the majority of eNOS association with Cav-1 (Kd = 49 nM), and computer modeling of CAV(90-99) docking to eNOS provides a rationale for the mechanism of eNOS inhibition by Phe-92. 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.

DOI10.1074/jbc.M113.528695