Interaction of neuronal nitric-oxide synthase with caveolin-3 in skeletal muscle. Identification of a novel caveolin scaffolding/inhibitory domain.
Neuronal nitric-oxide synthase (nNOS) has been shown previously to interact with alpha1-syntrophin in the dystrophin complex of skeletal muscle. In the present study, we have examined whether nNOS also interacts with caveolin-3 in skeletal muscle. nNOS and caveolin-3 are coimmunoprecipitated from rat skeletal muscle homogenates by antibodies directed against either of the two proteins. Synthetic peptides corresponding to the membrane-proximal caveolin-3 residues 65-84 and 109-130 and homologous caveolin-1 residues 82-101 and 135-156 potently inhibit the catalytic activity of purified, recombinant nNOS. Purified nNOS also binds to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. In vitro binding is completely abolished by preincubation of nNOS with either of the two caveolin-3 inhibitory peptides. Interactions between nNOS and caveolin-3, therefore, appear to be direct and to involve two distinct caveolin scaffolding/inhibitory domains. Other caveolin-interacting enzymes, including endothelial nitric-oxide synthase and the c-Src tyrosine kinase, are also potently inhibited by each of the four caveolin peptides. Inhibitory interactions mediated by two different caveolin domains may thus be a general feature of enzyme docking to caveolin proteins in plasmalemmal caveolae.
Venema
V J
VJ
Department of Pediatrics, Medical College of Georgia, Augusta, Georgia 30912, USA.rvenema@mail.mcg.edu
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Zou
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Venema
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism; D051244:Caveolin 3; D022461:Caveolins; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology*; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D051381:Rats
D002135:Calcium-Binding Proteins; C495549:Cav3 protein, rat; D051244:Caveolin 3; D022461:Caveolins; D008565:Membrane Proteins; D009124:Muscle Proteins; C091825:syntrophin alpha1; D019001:Nitric Oxide Synthase