A highly effective dominant negative αs construct containing mutations that affect distinct functions inhibits multiple Gs-coupled receptor signaling pathways
A highly effective dominant negative αs construct containing mutations that affect distinct functions inhibits multiple Gs-coupled receptor signaling pathways
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DOI：
10.1074/jbc.m201330200
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发表时间：
2002-06-07
影响因子：
4.8
通讯作者：
Berlot, CH
中科院分区：
文献类型：
--
作者：
Berlot, CH
为了研究受体 G 蛋白信号通路的亚细胞组织，一个强大的显性失活 a。产生了含有改变不同功能的取代的突变体，并测试了其对 HEK-293 细胞中 G 偶联受体活性的影响。 α3β5环区的突变会增加受体亲和力，减少受体介导的激活，并损害腺苷酸环化酶的激活，与G226A（增加对βγ的亲和力）和A366S（减少对GDP的亲和力）结合。这个三重A。突变体可以抑制 97% 的黄体生成素受体向 G. 发出的信号，100% 抑制 100% 从降钙素受体向 G. 发出的信号。此外，这种α(s)突变体阻断从降钙素受体到G(q)的所有信号传导。这些结果得出关于受体 G 蛋白信号传导的两个结论。首先，各个受体可以接触 HEK-293 细胞膜中的多种类型的 G 蛋白。其次，不同的G蛋白a亚基可以相互竞争与相同受体的结合。这种显性阴性 a.构建体将有助于确定多种细胞和组织中不同受体-G蛋白相互作用之间的相互关系。
To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative a. mutant containing substitutions that alter distinct functions was produced and tested for its effects on G.-coupled receptor activity in HEK-293 cells. Mutations in the alpha3beta5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for betagamma, and A366S, which decreases affinity for GDP. This triple a. mutant can inhibit signaling to G. from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this alpha(s) mutant blocks all signaling from the calcitonin receptor to G(q). These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein a subunits can compete with each other for binding to the same receptor. This dominant negative a. construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues.