Interaction between caveolin-1 and the reductase domain of endothelial nitric-oxide synthase. Consequences for catalysis.
Endothelial nitric-oxide synthase (eNOS) is targeted to caveoli through interaction with caveolin-1 (cav-1). cav-1 binding to a consensus site in the eNOS oxygenase domain is proposed to antagonize calmodulin (CaM) binding and thereby inhibit eNOS nitric oxide (NO) synthesis. To study the mechanism, we examined how cav-1 scaffolding domain peptide (amino acids 82-101; cav-1P) would affect NO synthesis, NADPH oxidation, cytochrome c reduction, and ferricyanide reduction by full-length eNOS or its isolated oxygenase and reductase domains. Cav-1P equivalently inhibited NO synthesis and NADPH oxidation by full-length eNOS in a manner reversible by CaM but did not affect NADPH-independent NO synthesis by full-length eNOS or its oxygenase domain, indicating inhibition required the reductase domain. Similar concentrations of cav-1P inhibited cytochrome c reduction by full-length eNOS or the reductase domain (amino acids 492-1205) in a CaM-reversible manner, indicating cav-1P interaction with reductase or full-length eNOS are equivalent. Ferricyanide reduction was unaffected by cav-1P in all cases. Immunoblotting showed that full-length eNOS, eNOS oxygenase, and eNOS reductase all bound to an immobilized glutathione S-transferase-cav-1 fusion protein. Thus, cav-1 interacts independently with both oxygenase and reductase domains of eNOS. The reductase interaction occurs independent of a cav-1 binding motif, is CaM-reversible, and is of sufficient affinity to match cav-1P inhibition of NO synthesis by full-length eNOS. We propose that cav-1 binding to eNOS reductase compromises its ability to bind CaM and to donate electrons to the eNOS heme, thereby inhibiting NO synthesis.
Ghosh
S
S
Department of Immunology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio 44195, USA.
Gachhui
R
R
Crooks
C
C
Wu
C
C
Lisanti
M P
MP
Stuehr
D J
DJ
D000595:Amino Acid Sequence; D002384:Catalysis; D051242:Caveolin 1; D022461:Caveolins; D006418:Heme / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010088:Oxidoreductases / Q000378:metabolism*
D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D009569:Nitric Oxide; D006418:Heme; D010088:Oxidoreductases; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III