Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity
Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity
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DOI：
10.1074/jbc.272.30.18522
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发表时间：
1997-07-25
影响因子：
4.8
通讯作者：
Venema, RC
中科院分区：
文献类型：
--
作者：
Ju, H;Zou, R;Venema, RC
内皮一氧化氮合酶 (eNOS) 和 Caveolin-1 在内皮质膜小窝内相关。然而，目前尚不清楚 eNOS 和 Caveolin-1 是否直接或间接相互作用，或者该相互作用是否影响 eNOS 活性。为了回答这些问题，我们克隆了牛caveolin-1 cDNA，并使用谷胱甘肽S-转移酶（GST）-caveolin-1融合蛋白和杆状病毒表达的牛eNOS在体外结合测定系统中研究了eNOS-caveolin-1相互作用。我们还使用体内酵母双杂交系统绘制了参与相互作用的域。使用体外和体内蛋白质相互作用测定获得的结果表明，caveolin-1 的 N 和 C 末端胞质结构域均直接与 eNOS 加氧酶结构域相互作用。 eNOS 与 GST-caveolin-1 融合蛋白的相互作用显着抑制酶的催化活性。与 Caveolin-1 残基 82-101 相对应的合成肽还通过干扰酶与 Ca2+/钙调蛋白 (CaM) 的相互作用，有效且可逆地抑制 eNOS 活性。因此，内皮细胞中 eNOS 的调节可能不仅涉及 Ca2+/CaM 的正向变构调节，还涉及 Caveolin-1 的负向变构调节。
Endothelial nitric-oxide synthase (eNOS) and caveolin-1 are associated within endothelial plasmalemmal caveolae. It is not known, however, whether eNOS and caveolin-1 interact directly or indirectly or whether the interaction affects eNOS activity. To answer these questions, we have cloned the bovine caveolin-1 cDNA and have investigated the eNOS-caveolin-1 interaction in an in vitro binding assay system using glutathione S-transferase (GST)-caveolin-1 fusion proteins and baculovirus-expressed bovine eNOS. We have also mapped the domains involved in the interaction using an in vivo yeast two-hybrid system. Results obtained using both in vitro and in vivo protein interaction assays show that both Nand C-terminal cytosolic domains of caveolin-1 interact directly with the eNOS oxygenase domain. Interaction of eNOS with GST-caveolin-1 fusion proteins significantly inhibits enzyme catalytic activity. A synthetic peptide corresponding to caveolin-1 residues 82-101 also potently and reversibly inhibits eNOS activity by interfering with the interaction of the enzyme with Ca2+/calmodulin (CaM). Regulation of eNOS in endothelial cells, therefore, may involve not only positive allosteric regulation by Ca2+/CaM, but also negative allosteric regulation by caveolin-1.