Caveolin-1 detergent-solubility and association with endothelial nitric oxide synthase is modulated by agonist-stimulated tyrosine phosphorylation

Caveolae are small, bulb-shaped invaginations of the plasma membrane that are especially abundant in endothelial cells. A principal protein component of caveolae is caveolin-1. Caveolin-1 functions as a docking site for compartmentalizing certain signal transducing proteins in membrane microdomains at the cell surface. One of these signaling proteins is the endothelial nitric oxide synthase (eNOS). We have shown previously that eNOS is translocated to the detergent-insoluble, cytoskeletal fraction of bovine aortic endothelial cells (BAEC) in response to bradykinin (BK)-stimulation or tyrosine phosphatase inhibition. In the present study, we have examined whether caveolin-1 is translocated together with eNOS in response to these and other stimuli. Exposure of BAEC to BK, histamine, or ATP produces parallel, transient increases in the amounts of detergent-insoluble caveolin-1 and eNOS. Agonist-stimulated increases in insolubility are blocked by Src tyrosine kinase inhibitors and are potently mimicked by tyrosine phosphatase inhibitors. Solubility changes of caveolin-1 and eNOS are not due to direct tyrosine phosphorylation of either protein and do not represent a movement of either protein into or out of caveolae. Rather, increased detergent-insolubility reflects an increased association of the two proteins with each other and with caveolae cytoskeletal proteins. Agonist-activation of eNOS in endothelial cells thus appears to involve tyrosine phosphorylation-dependent changes in the interaction of eNOS with caveolin.