Abstract
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 microM). 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.
Highlights
Endothelial nitric-oxide synthase1 is targeted to endothelial plasmalemmal caveolae through direct interaction with the caveolae structural protein, caveolin-1
We have identified the functional consequences of receptor binding on Endothelial nitric-oxide synthase (eNOS) catalytic activity and have mapped the region in the receptor involved in interaction with the enzyme
To determine whether eNOS interacts with the B2 receptor in endothelial cells, we lysed bovine aortic endothelial cells (BAEC) in membrane-solubilizing buffer containing 1% Triton X-100 and subjected the lysates to immunoprecipitation with anti-eNOS and anti-B2 receptor antibodies
Summary
Endothelial nitric-oxide synthase (eNOS)1 is targeted to endothelial plasmalemmal caveolae through direct interaction with the caveolae structural protein, caveolin-1. One of the mechanisms for eNOS activation in endothelial cells appears to involve agonist-stimulated increases in intracellular Ca2ϩ and subsequent displacement by Ca2ϩ-CaM of caveolin-1 from its inhibitory interaction with eNOS.
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