Ju H, Venema V J, Marrero M B, Venema R C
Department of Pediatrics, Medical College of Georgia, Augusta, Georgia 30912, USA.
J Biol Chem. 1998 Sep 11;273(37):24025-9. doi: 10.1074/jbc.273.37.24025.
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.
先前的研究表明，内皮型一氧化氮合酶（eNOS）可与质膜小窝结构蛋白小窝蛋白-1发生可逆性相互作用。eNOS与小窝蛋白-1的相互作用会抑制eNOS的催化活性。在本研究中，我们发现eNOS还能与G蛋白偶联的缓激肽B2受体发生可逆性抑制性相互作用。用针对这两种蛋白中任一种的抗体，可从内皮细胞裂解物中共免疫沉淀出eNOS和B2受体。在体外结合试验中，含有该受体细胞内结构域4的谷胱甘肽S-转移酶融合蛋白可被纯化的重组eNOS结合。该融合蛋白能选择性抑制纯化eNOS的活性。一段与细胞内结构域4中膜近端残基310 - 334相对应的合成肽也能有效抑制eNOS活性（IC50 < 1 microM）。用缓激肽或Ca2+离子载体处理培养的内皮细胞，可促使eNOS - B2受体复合物快速解离。这些数据表明，缓激肽B2受体以配体和Ca2+依赖的方式与eNOS发生物理性结合。因此，eNOS的可逆性抑制性膜对接相互作用并不局限于与小窝蛋白-1的相互作用，也会与缓激肽B2受体发生。
