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.
Department of Immunology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio 44195, USA.
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S
Ghosh
R
R
Gachhui
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C
Crooks
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C
Wu
M P
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Lisanti
D J
DJ
Stuehr
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