Inhibitory interactions of the bradykinin B2 receptor with endothelial nitric-oxide synthase

It has been shown previously that the endothelial nitric-oxide synthase (eNOS) interacts reversibly with the plasmalemmal caveolae structural protein, caveolin-1. The eNOS-caveolin-1 interaction inhibits eNOS catalytic activity. In the present study, we show that eNOS also participates in reversible inhibitory interactions with the G protein-coupled bradykinin B2 receptor, eNOS and the B2 receptor are coimmunoprecipitated from endothelial cell lysates by antibodies directed against either of the two proteins. A glutathione S-transferase fusion protein containing intracellular domain 4 of the receptor is bound by purified recombinant eNOS in in vitro binding assays. The fusion protein selectively inhibits the activity of purified eNOS. A synthetic peptide corresponding 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 demonstrate that the bradykinin B2 receptor physically associates with eNOS in a ligand- and Ca2+ dependent manner. Reversible and inhibitory membrane-docking interactions of eNOS, therefore, are not restricted to those with caveolin-1 but also occur with the bradykinin B2 receptor.