Targeted knockdown of G protein subunits selectively prevents receptor-mediated modulation of effectors and reveals complex changes in non-targeted signaling proteins
Targeted knockdown of G protein subunits selectively prevents receptor-mediated modulation of effectors and reveals complex changes in non-targeted signaling proteins
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DOI：
10.1074/jbc.m511551200
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发表时间：
2006-04-14
影响因子：
4.8
通讯作者：
Gilman, AG
中科院分区：
文献类型：
--
作者：
Krumins, AM;Gilman, AG
异源三聚体G蛋白信号传导特异性归因于G α、β和γ亚基的选择组合、它们与其他信号传导蛋白的相互作用以及它们在细胞中的定位。除了少数例外，体内存在的G蛋白亚基组合以及这些特定组合的意义在很大程度上是未知的。我们已经开始在HeLa细胞中解决这些问题：1）确定G α和G β亚基的浓度; 2）检查两个效应系统的受体依赖性活性（腺苷酸环化酶和磷脂酶C β）;和3）通过使用小干扰RNA系统地沉默G α和G β亚基中的每一个，同时定量效应子功能和其他相关的干扰RNA浓度的变化。网络中的蛋白质HeLa细胞表达等摩尔量的总G α和G β亚基。最常见的G α蛋白是每个G α亚家族的一个成员（G α（s）、G α（i3）、G α（11）和G α（13））。我们基本上废除了在这些细胞中表达的大多数G α和G β蛋白的表达，单独和一些组合。正如预期的那样，腺苷酸环化酶或磷脂酶C β的激动剂依赖性激活分别在G α（s）或G α（q/11）沉默后特异性消除。我们还证实，G β亚基是必要的稳定积累的G α蛋白在体内。G β亚基对效应子活性的受体依赖性调节表现出很小的亚型特异性。我们观察到个别基因沉默后G蛋白积累的补偿性变化，以及某些G α（q）和G α（i）亚家族成员表达之间的明显相互关系。这些发现为理解调节G蛋白信号网络的适应性和显著弹性的机制提供了基础。
Heterotrimeric G protein signaling specificity has been attributed to select combinations of G alpha, beta, and gamma subunits, their interactions with other signaling proteins, and their localization in the cell. With few exceptions, the G protein subunit combinations that exist in vivo and the significance of these specific combinations are largely unknown. We have begun to approach these problems in HeLa cells by: 1) determining the concentrations of G alpha and G beta subunits; 2) examining receptor-dependent activities of two effector systems (adenylyl cyclase and phospholipase C beta); and 3) systematically silencing each of the G alpha and G beta subunits by using small interfering RNA while quantifying resultant changes in effector function and the concentrations of other relevant proteins in the network. HeLa cells express equimolar amounts of total G alpha and G beta subunits. The most prevalent G alpha proteins were one member of each G alpha subfamily (G alpha(s), G alpha(i3), G alpha(11), and G alpha(13)). We substantially abrogated expression of most of the G alpha and G beta proteins expressed in these cells, singly and some in combinations. As expected, agonist-dependent activation of adenylyl cyclase or phospholipase C beta was specifically eliminated following the silencing of G alpha(s) or G alpha(q/11), respectively. We also confirmed that G beta subunits are necessary for stable accumulation of G alpha proteins in vivo. G beta subunits demonstrated little isoform specificity for receptor-dependent modulation of effector activity. We observed compensatory changes in G protein accumulation following silencing of individual genes, as well as an apparent reciprocal relationship between the expression of certain G alpha(q) and G alpha(i) subfamily members. These findings provide a foundation for understanding the mechanisms that regulate the adaptability and remarkable resilience of G protein signaling networks.