Mechanism of platelet inhibition by nitric oxide:: In vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase
Mechanism of platelet inhibition by nitric oxide:: In vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase
复制标题
DOI：
10.1073/pnas.95.9.4888
复制
发表时间：
1998-04-28
影响因子：
11.1
通讯作者：
Mendelsohn, ME
中科院分区：
文献类型：
--
作者：
Wang, GR;Zhu, Y;Mendelsohn, ME
一氧化氮（NO）是一种有效的血管扩张剂和血小板活化抑制剂。NO刺激cGMP的产生并激活cGMP依赖性蛋白激酶（G激酶），其通过未知的机制导致G α（q）-磷脂酶C-肌醇1，4，5-三磷酸信号传导的抑制和几种重要激动剂（包括血栓烷A（2）（TXA（2）的细胞内钙动员。为探讨NO抑制血小板生成的机制，研究了cGMP对血小板TXA 2受体的激活作用。非水解类似物8-溴环GMP（S-Br-cGMP）以浓度依赖性方式有效地抑制血小板膜中TXA（2）特异性GT3的活化，表明G激酶催化受体-G蛋白信号通路的某些近端组分的磷酸化。发现纳摩尔浓度的G激酶在体外催化血小板TXA（2）受体的磷酸化，但在这些实验中没有与TXA（2）受体共纯化的G α（q）。用免疫亲和法，从经8-Br-cGMP处理的P-32标记的细胞中证实了环鸟苷酸（cGMP）对TXA（2）受体的体内磷酸化。对体内磷酸化的TXA（2）受体的肽图研究证实cGMP介导TXA（2）受体羧基端的磷酸化。G激酶还催化与受体α和β形式的细胞质尾部相对应的肽的磷酸化，但不催化对照肽或与TXA（2）受体的第三胞浆内环相对应的肽的磷酸化。这些数据确定TXA（2）受体作为cGMP依赖性蛋白激酶底物，并支持NO抑制细胞功能的新机制，其中G激酶的激活通过催化G蛋白偶联受体的磷酸化抑制其信号传导。
Nitric oxide (NO) is a potent vasodilator and inhibitor of platelet activation. NO stimulates production of cGMP and activates cGMP-dependent protein kinase (G kinase), which by an unknown mechanism leads to inhibition of G alpha(q)-phospholipase C-inositol 1,4,5-triphosphate signaling and intracellular calcium mobilization for several important agonists, including thromboxane A(2) (TXA(2)). To explore the mechanism of platelet inhibition by NO, activation of platelet TXA(2) receptors in the presence of cGMP was studied. The nonhydrolyzable analog 8-bromo-cyclic GMP (S-Br-cGMP) potently inhibited activation of the TXA(2)-specific GTPase in platelet membranes in a concentration-dependent fashion, suggesting that G kinase catalyzes the phosphorylation of some proximal component of the receptor-G protein signaling pathway, Nanomolar concentrations of G kinase were found to catalyze the phosphorylation of platelet TXA(2) receptors in vitro, but not G alpha(q) copurifying with the TXA(2) receptors in these experiments. Using immunoaffinity methods, in vivo phosphorylation of TXA(2) receptors by cyclic GMP was demonstrated from P-32-labeled cells treated with 8-Br-cGMP, Peptide mapping studies of in vivo phosphorylated TXA(2) receptors demonstrated cGMP mediates phosphorylation of the carboxyl terminus of the TXA(2) receptor. G kinase also catalyzed the phosphorylation of peptides corresponding to the cytoplasmic tails of both alpha and beta forms of the receptor but not control peptide or a peptide corresponding to the third intracytoplasmic loop of the TXA(2) receptor. These data identify TXA(2) receptors as cGMP-dependent protein kinase substrates and support a novel mechanism for the inhibition of cell function by NO in which activation of G kinase inhibits signaling by G protein-coupled receptors by catalyzing their phosphorylation.