Deciphering the Binding of Caveolin-1 to Client Protein Endothelial Nitric-oxide Synthase (eNOS): SCAFFOLDING SUBDOMAIN IDENTIFICATION, INTERACTION MODELING, AND BIOLOGICAL SIGNIFICANCE

Background: One of the most significant client proteins of Cav-1 is the endothelial nitric-oxide synthase (eNOS), but their specific binding site is unknown. Results: We describe how Cav-1 binds to eNOS and how biologically active NO can be increased. Conclusion: We provide the most detailed characterization of eNOS binding to Cav-1. Significance: Our data provide a deeper understanding of Cav-1 signaling and NO generation in physiological processes.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 (K-d = 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.