Regulation of neuronal nitric-oxide synthase activity by somatostatin analogs following SST5 somatostatin receptor activation
Regulation of neuronal nitric-oxide synthase activity by somatostatin analogs following SST5 somatostatin receptor activation
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DOI：
10.1074/jbc.m602024200
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发表时间：
2006-07-14
影响因子：
4.8
通讯作者：
Buscail, Louis
中科院分区：
文献类型：
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作者：
Cordelier, Pierre;Esteve, Jean-Pierre;Buscail, Louis
生长抑素受体SST 5是一种抑制性G蛋白偶联受体，对各种细胞类型产生强烈的细胞生长抑制作用。我们以前报道过，SST 5的抗增殖作用导致抑制有丝分裂原诱导的细胞内cGMP水平和MAPK活性的增加。进行本研究以确定负责SST 5介导的抗增殖作用的早期分子事件。在这里，我们证明，在中国仓鼠卵巢细胞表达SST 5（CHO/SST 5细胞），生长抑素抑制细胞增殖诱导的一氧化氮供体和过度表达的神经元型一氧化氮合酶（nNOS）蛋白亚型。因此，nNOS活性和二聚化强烈抑制SST 5激活后，生长抑素类似物RC-160。在CHO/SST 5细胞中，nNOS被SST 5受体动态募集，并在RC-160处理后在酪氨酰残基处磷酸化。RC-160诱导SST 5-p60 src激酶复合物形成和随后的p60 src激酶活化。一个失活的p60 src激酶突变体与SST 5的共表达阻断了RC-160诱导的nNOS磷酸化和失活，并阻止了SST 5介导的抗增殖作用。在CHO/SST 5细胞中，RC-160处理后，p60（src）激酶与nNOS相关，通过酪氨酰残基磷酸化诱导其失活。使用重组蛋白，我们证明，这种磷酸化阻止nNOS同源二聚化。接下来，表面等离子体共振和突变分析揭示了p60（src）直接与nNOS磷酸化Tyr（604）相关。经证明，SST 5介导的nNOS活性抑制对于RC-160对胰腺内分泌肿瘤衍生细胞的抗增殖作用至关重要。因此，我们确定nNOS作为一种新的p60（src）激酶底物所必需的SST 5介导的抗增殖作用。
Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60src kinase complex formation and subsequent p60src kinase activation. Coexpression of an inactive p60src kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr(604). SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.