Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity.
Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated within endothelial plasmalemmal caveolae. It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity. To answer these questions, we have cloned the bovine caveolin-1 cDNA and have investigated the eNOS-caveolin-1 interaction in an in vitro binding assay system using glutathione S-transferase (GST)-caveolin-1 fusion proteins and baculovirus-expressed bovine eNOS. We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system. Results obtained using both in vitro and in vivo protein interaction assays show that both N- and C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Interaction of eNOS with GST-caveolin-1 fusion proteins significantly inhibits enzyme catalytic activity. A synthetic peptide corresponding to caveolin-1 residues 82-101 also potently and reversibly inhibits eNOS activity by interfering with the interaction of the enzyme with Ca2+/calmodulin (CaM). Regulation of eNOS in endothelial cells, therefore, may involve not only positive allosteric regulation by Ca2+/CaM, but also negative allosteric regulation by caveolin-1.
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Vascular Biology Center, Department of Pediatrics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Zou
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Venema
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Venema
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D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins; D002478:Cells, Cultured; D002645:Chickens; D004285:Dogs; D004730:Endothelium, Vascular / Q000201:enzymology*; D004791:Enzyme Inhibitors; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
C495540:CAV1 protein, human; D002147:Calmodulin; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D019001:Nitric Oxide Synthase; D002118:Calcium