Acylation Targets Endothelial Nitric-oxide Synthase to Plasmalemmal Caveolae

Abstract

Endothelial nitric-oxide synthase (eNOS) generates the key signaling molecule nitric oxide in response to intralumenal hormonal and mechanical stimuli. We designed studies to determine whether eNOS is localized to plasmalemmal microdomains implicated in signal transduction called caveolae. Using immunoblot analysis, eNOS protein was detected in caveolar membrane fractions isolated from endothelial cell plasma membranes by a newly developed detergent-free method; eNOS protein was not found in noncaveolar plasma membrane. Similarly, NOS enzymatic activity was 9.4-fold enriched in caveolar membrane versus whole plasma membrane, whereas it was undetectable in noncaveolar plasma membrane. 51-86% of total NOS activity in postnuclear supernatant was recovered in plasma membrane, and 57-100% of activity in plasma membrane was recovered in caveolae. Immunoelectron microscopy showed that eNOS heavily decorated endothelial caveolae, whereas coated pits and smooth plasma membrane were devoid of gold particles. Furthermore, eNOS was targeted to caveolae in COS-7 cells transfected with wild-type eNOS cDNA. Studies with eNOS mutants revealed that both myristoylation and palmitoylation are required to target the enzyme to caveolae and that each acylation process enhances targeting by 10-fold. Thus, acylation targets eNOS to plasmalemmal caveolae. Localization to this microdomain is likely to optimize eNOS activation and the extracellular release of nitric oxide.The endothelial isoform of nitric-oxide synthase (eNOS) 1 is one of three isoenzymes that converts L-arginine to L-citrulline plus the key signaling molecule nitric oxide (NO). eNOS is acutely activated by increases in endothelial intracellular calcium induced by the stimulation of diverse G-protein-coupled cell surface receptors and by physical stimuli such as hemodynamic shear stress and varying oxygenation. Endotheliumderived NO regulates blood pressure, platelet aggregation, and vascular smooth muscle mitogenesis. Diminished NO production has been implicated in the pathogenesis of a variety of vascular disorders including atherosclerosis and pulmonary hypertension (1-4).eNOS is a unique isoform of the enzyme in that it is primarily localized to the particulate subcellular fraction (1-4). Studies evaluating NOS enzymatic activity have demonstrated that functional eNOS is primarily associated with the plasma membrane (5). Since eNOS activity is acutely regulated by intralumenal factors and the NO produced is a labile, cytotoxic messenger molecule (1-4), the intracellular site of NO synthesis is likely to have a major influence on its biological activity in the vascular wall. We therefore sought to determine the specific subcellular location to which eNOS is targeted and designed studies to delineate whether the enzyme is localized to plasmalemmal caveolae. Caveolae are plasmalemmal microdomains that have been implicated in the transcytosis of macromolecules, the uptake of small molecules by potocytosis, and the compartmentalization of signaling molecules (6 -9). The ...

Dynamic Targeting of the Agonist-stimulated m2 Muscarinic Acetylcholine Receptor to Caveolae in Cardiac Myocytes

“…1A show that the majority of caveolin-3 and eNOS in ventricular myocytes appears in fractions 2 and 3, which correspond to the 5-15% sucrose equilibrium densities. This co-purification of eNOS and caveolin-3 is in agreement with our previous data on the co-immunoprecipitation of these two proteins from CHAPS-solubilized cardiac myocyte lysates (19) and on the co-isolation of eNOS and caveolin-1 in endothelial cells (36).…”

Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein

“…teins, such as the Src family tyrosine kinases [16], nitric oxide synthase [19] and heterotrimeric G-proteins [15,16]. DIGs isolated from epithelial tissue possess a characteristically high content of the 21-24 kDa integral membrane protein caveolin-1\VIP21 [20][21][22][23].…”

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

“…eNOS has been shown to be localized to small invaginations of the plasma membrane called caveolae (Feron et al, 1996;Garcia-Cardena et al, 1996;Shaul et al, 1996). Biochemically, caveolae represent a plasma membrane subdomain enriched with cholesterol, glycosphingolipids, and the structural proteins caveolins that interact, in vitro, with a variety of signal-transducing molecules.…”