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 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.
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H
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Department of Pediatrics, Medical College of Georgia, Augusta, Georgia 30912, USA.
Venema
V J
VJ
Marrero
M B
MB
Venema
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001011:Aorta; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D005982:Glutathione Transferase; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011993:Recombinant Fusion Proteins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D005982:Glutathione Transferase