TY - JOUR
T1 - Deciphering the binding of Caveolin-1 to client protein endothelial nitric-oxide synthase (eNOS)
T2 - Scaffolding subdomain identification, interaction modeling, and biological significance
AU - Trane, Andy E.
AU - Pavlov, Dmitri
AU - Sharma, Arpeeta
AU - Saqib, Uzma
AU - Lau, Kelvin
AU - Van Petegem, Filip
AU - Minshall, Richard D.
AU - Roman, Linda J.
AU - Bernatchez, Pascal N.
PY - 2014
Y1 - 2014
N2 - 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 increaseNOrelease, indicating the presence of a distincteNOSbinding 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.
AB - 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 increaseNOrelease, indicating the presence of a distincteNOSbinding 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.
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U2 - 10.1074/jbc.M113.528695
DO - 10.1074/jbc.M113.528695
M3 - Article
C2 - 24648521
AN - SCOPUS:84900447254
VL - 289
SP - 13273
EP - 13283
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 19
ER -