title: Deciphering the binding of caveolin-1 to client protein endothelial nitric-oxide synthase (eNOS): scaffolding subdomain identification, interaction modeling, and biological significance.
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.
authors.affiliation: From the St. Paul's Hospital's Centre of Heart and Lung Innovation.
authors.forename: Andy E
authors.initials: AE
authors.lastname: Trane
authors.forename: Dmitri
authors.initials: D
authors.lastname: Pavlov
authors.forename: Arpeeta
authors.initials: A
authors.lastname: Sharma
authors.forename: Uzma
authors.initials: U
authors.lastname: Saqib
authors.forename: Kelvin
authors.initials: K
authors.lastname: Lau
authors.forename: Filip
authors.initials: F
authors.lastname: van Petegem
authors.forename: Richard D
authors.initials: RD
authors.lastname: Minshall
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Roman
authors.forename: Pascal N
authors.initials: PN
authors.lastname: Bernatchez
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D051242:Caveolin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003198:Computer Simulation; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D020125:Mutation, Missense; D009569:Nitric Oxide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: C495540:CAV1 protein, human; D051242:Caveolin 1; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III
keywords: Caveolae; Caveolin; Endothelium; Nitric Oxide; Nitric-oxide Synthase