Abstract

Endothelial nitric oxide synthase (eNOS) is a dually acylated peripheral membrane protein that targets to the Golgi region and caveolae of endothelial cells. Recent evidence has shown that eNOS can co-precipitate with caveolin-1, the resident coat protein of caveolae, suggesting a direct interaction between these two proteins. To test this idea, we examined the interactions of eNOS with caveolin-1 in vitro and in vivo. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Utilizing a series of GST-caveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with eNOS, the scaffolding domain (amino acids 61-101) and to a lesser extent the C-terminal tail (amino acids 135-178). Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities. These results suggest a common mechanism and site of inhibition. Utilizing GST-eNOS fusions, the site of caveolin binding was localized between amino acids 310 and 570. Site-directed mutagenesis of the predicted caveolin binding motif within eNOS blocked the ability of caveolin-1 to suppress NO release in co-transfection experiments. Thus, our data demonstrate a novel functional role for caveolin-1 in mammalian cells as a potential molecular chaperone that directly inactivates NOS. This suggests that the direct binding of eNOS to caveolin-1, per se, and the functional consequences of eNOS targeting to caveolae are likely temporally and spatially distinct events that regulate NO production in endothelial cells. Additionally, the inactivation of eNOS and nNOS by the scaffolding domain of caveolin-3 suggests that eNOS in cardiac myocytes and nNOS in skeletal muscle are likely subject to negative regulation by this muscle-specific caveolin isoform.

Highlights

  • From the ‡Department of Pharmacology, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut 06536, §Department of Biochemistry, University of Texas Health Sciences Center, San Antonio, Texas 782847760, and ʈDepartment of Molecular Pharmacology, The Albert Einstein College of Medicine, Bronx, New York 10461

  • Utilizing a series of GSTcaveolin-1 deletion mutants, we identified two cytoplasmic domains of caveolin-1 that interact with Endothelial nitric oxide synthase (eNOS), the scaffolding domain and to a lesser extent the C-terminal tail

  • Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS, and neuronal NOS activities

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Summary

Introduction

From the ‡Department of Pharmacology, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut 06536, §Department of Biochemistry, University of Texas Health Sciences Center, San Antonio, Texas 782847760, and ʈDepartment of Molecular Pharmacology, The Albert Einstein College of Medicine, Bronx, New York 10461. Incubation of endothelial cell lysates or purified eNOS with glutathione S-transferase (GST)-caveolin-1 resulted in the direct interaction of the two proteins. Incubation of pure eNOS with peptides derived from the scaffolding domains of caveolin-1 and -3, but not the analogous regions from caveolin-2, resulted in inhibition of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) activities.

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