INHIBITORY INTERACTIONS OF THE BRADYKININ B2 RECEPTOR WITH ENDOTHELIAL NITRIC-OXIDE SYNTHASE

It has been shown previously that the endothelial nitric-oxide synthas e (eNOS) interacts reversibly with the plasmalemmal caveolae structura l protein, caveolin-1. The eNOS-caveolin-1 interaction inhibits eNOS c atalytic activity. In the present study, we show that eNOS also partic ipates in reversible inhibitory interactions with the G protein-couple d bradykinin B2 receptor, eNOS and the B2 receptor are coimmunoprecipi tated from endothelial cell lysates by antibodies directed against eit her of the two proteins. A glutathione S-transferase fusion protein co ntaining intracellular domain 4 of the receptor is bound by purified r ecombinant eNOS in in vitro binding assays. The fusion protein selecti vely inhibits the activity of purified eNOS. A synthetic peptide corre sponding to membrane-proximal residues 310-334 in intracellular domain 4 also potently inhibits eNOS activity (IC50 < 1 mu M). Treatment of cultured endothelial cells with bradykinin or Ca2+ ionophore promotes a rapid dissociation of the eNOS B2 receptor complex. These data demon strate that the bradykinin B2 receptor physically associates with eNOS in a ligand- and Ca2+ dependent manner. Reversible and inhibitory mem brane-docking interactions of eNOS, therefore, are not restricted to t hose with caveolin-1 but also occur with the bradykinin B2 receptor.