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 baculovirusexpressed 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 Nand 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 Ca 2؉

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and Ser 635 regulates eNOS activity and contributes to the agonist-stimulated eNOS activation process. Endothelial nitric-oxide synthase (eNOS)1 is an important enzyme in the cardiovascular system producing nitric oxide (NO), a key regulator of blood pressure, platelet function, and vessel remodeling. Endothelial NOS is regulated by multiple mechanisms involving both protein-protein interactions with several different proteins, including caveolin-1 and Hsp90 (1), and post-translational modifications that include Nmyristoylation, cysteine palmitoylation, and multisite phosphorylation. The two most thoroughly studied phosphorylation sites have been the activation site, human Ser 1177

Neuronal nitric-oxide synthase (nNOS) has been shown previously to interact with ␣ 1 -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.

. Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase. Am J Physiol Heart Circ Physiol 285: H669-H678, 2003. First published April 3, 2003 10.1152/ajpheart.01025.2002 is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the ␣-or ␤-subunit of sGC show that the sGC ␤-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.smooth muscle cells; endothelium; vascular endothelial growth factor; bradykinin; cGMP accumulation SOLUBLE GUANYLATE CYCLASE (sGC), an ␣/␤-heterodimeric heme protein, catalyzes the conversion of GTP to cGMP in many cells including vascular endothelial cells (ECs) and vascular smooth muscle cells (SMCs). Activation of sGC is by direct binding of nitric oxide (NO) to the sGC heme prosthetic group. Formation of the nitrosyl heme adduct induces a conformational change in sGC that results in an increase in its enzymatic activity (21). The NO that activates sGC in various cells is the product of a reaction that is catalyzed by one of three distinct NO synthase (NOS) molecules, which catalyze the oxidation of L-arginine to produce L-citrulline and NO (1). In ECs, NO production is mediated by the constitutively expressed endothelial NOS (eNOS). Activation of eNOS is by Ca 2ϩ -calmodulin (CaM) after agonist-stimulated elevations in intracellular Ca 2ϩ concentrations. Two signaling pathways exist that involve eNOS and sGC. The first is an intercellular pathway whereby NO, which is produced by eNOS in ECs, diffuses to the underlying SMCs and promotes blood vessel relaxation (16). The second is an intracellular eNOS-sGC pathway that is essential for vascular endothelial growth factor (VEGF)-induced increases in EC permeability and proliferation (24,25,30).Initially, eNOS was thought to function as an isolated homodimer. It is now known, however, that eNOS exists in multiprotein complexes in which it interacts with other proteins. These pr...