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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Silencing the expression of multiple G beta-subunits eliminates signaling mediated by all four families of G proteins
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Silencing the expression of multiple G beta-subunits eliminates signaling mediated by all four families of G proteins

机译：沉默多个Gβ亚基的表达消除了所有四个G蛋白家族介导的信号传导

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摘要

The G beta y-subunit complex derived from heterotrimeric G proteins can act to regulate the function of a variety of protein targets. We established lentiviral-based RNA interference in J774A.1 mouse macrophages to characterize the role of G beta in G protein-coupled receptor signaling. The expression of G beta 1 and G beta 2, the major subtypes present in J774A.1 cells, was eliminated by sequential treatment with small hairpin RNA expressing lentivirus. These beta y complex-deficient cells lost the ability to respond to G protein-mediated signals. Chemotaxis and the phosphorylation of Akt in response to C5a were both blocked. Similarly, C5a-mediated actin polymerization, C5a- and UTP-stimulated intracellular calcium mobilization, and the stimulation of cAMP formation by isoproterenol were all eliminated in the absence of the G beta-subunits. In addition, stabilization and membrane localization of several G alpha- and G gamma-subunit proteins was strongly effected. Furthermore, in DNA microarray analysis, regulation of gene expression stimulated by prostaglandin E2 and UTP was not observed in cells lacking G beta-subunits. In contrast, phagocytotic activity, serum-dependent cell growth and the patterns of gene expression induced by stimulating the Toll receptors with LPS were similar in wild-type cells and small hairpin RNA-containing cells. Thus, ablation of the G beta-subunits destabilized G alpha- and G gamma-subunits and effectively eliminated G protein-mediated signaling responses. Unrelated ligand regulated pathways remained intact. These cells provide a system that can be used to study signaling in the absence of most G protein-mediated functions.
机译：源自异源三聚体G蛋白的G beta y-亚基复合物可调节多种蛋白靶标的功能。我们在J774A.1小鼠巨噬细胞中建立了基于慢病毒的RNA干扰，以表征Gβ在G蛋白偶联受体信号传导中的作用。 J774A.1细胞中存在的主要亚型G beta 1和G beta 2的表达通过依次表达小发夹RNA的慢病毒进行处理而消除。这些缺乏βy复合体的细胞丧失了对G蛋白介导的信号作出反应的能力。趋化性和响应C5a的Akt磷酸化均被阻断。类似地，在不存在Gβ亚基的情况下，消除了C5a介导的肌动蛋白聚合，C5a和UTP刺激的细胞内钙动员以及异丙肾上腺素对cAMP形成的刺激。另外，强烈影响了几种Gα-和Gγ-亚基蛋白的稳定和膜定位。此外，在DNA芯片分析中，在缺乏Gβ亚基的细胞中未观察到前列腺素E2和UTP刺激的基因表达调控。相反，在野生型细胞和含有小发夹RNA的细胞中，吞噬活性，血清依赖性细胞生长以及通过用LPS刺激Toll受体诱导的基因表达模式相似。因此，消融的Gβ亚基使Gα亚基和Gγ亚基不稳定，并有效消除了G蛋白介导的信号传导反应。无关的配体调节途径保持完整。这些细胞提供了可用于在缺乏大多数G蛋白介导的功能的情况下研究信号转导的系统。

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