Analysis of Ga-mediated chemotaxis by lentiviral delivery of small interfering RNA
Analysis of Ga-mediated chemotaxis by lentiviral delivery of small interfering RNA
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DOI：
10.1073/pnas.0307549100
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发表时间：
2004-01-13
影响因子：
11.1
通讯作者：
Simon, MJ
中科院分区：
文献类型：
--
作者：
Hwang, JI;Fraser, IDC;Simon, MJ
免疫细胞通过 G 蛋白偶联受体对趋化信号做出反应。由于不同 G 蛋白亚型之间的冗余，试图阐明特定 G 蛋白家族成员在这些反应中的功能变得复杂。我们使用基于慢病毒的 RNA 干扰选择性消除 J774A.1 小鼠巨噬细胞中特定 G 蛋白亚基的表达。对补体因子 C5a 和 C3a 的趋化反应在缺乏 Gbeta(2) 的细胞中被消除，但在缺乏 Gbeta(1)、Galphai(2) 或 Galphai(3) 的细胞中不受影响。同样，C5a 介导的单细胞钙反应要么不存在，要么因 Gbeta(2) 敲低而显着延迟和减弱。对 Akt1 响应 C5a 的磷酸化水平的评估显示，野生型细胞和缺乏 Gbeta(1) 的细胞均出现快速且持续的磷酸化。缺乏 Gbeta(2) 的细胞保留快速反应，但无法维持磷酸化 Akt1 水平。缺乏 Gbeta(2) 的细胞的表型可以通过人 Gbeta(2) 或小鼠 Gbeta(1) 的过表达来逆转。这些数据证明了基于慢病毒的 RNA 干扰在信号通路系统分析中的有用性，并且表明在 J774A.1 细胞中，Gbeta(2) 衍生的 Gbetagamma 是 C5a 趋化性的最有效介质。
Immune cells respond to chemotactic signals by means of G protein-coupled receptors. Attempts to elucidate the function of specific G protein family members in these responses is complicated by redundancy among the different G protein isoforms. We have used lentiviral-based RNA interference to eliminate expression of specific G protein subunits selectively in J774A.1 mouse macrophages. The chemotactic response to the complement factors C5a and C3a is ablated in cells lacking Gbeta(2) but is unaffected in cells lacking Gbeta(1), Galphai(2), or Galphai(3). Similarly, the C5a-mediated calcium response of single cells is either absent or significantly delayed and weakened by Gbeta(2) knockdown. Assessment of Akt1 phosphorylation levels in response to C5a shows rapid and sustained phosphorylation in both wild-type cells and cells lacking Gbeta(1). Cells lacking Gbeta(2) retain the rapid response but cannot sustain phospho-Akt1 levels. The phenotype of cells lacking Gbeta(2) can be reversed by overexpression of either human Gbeta(2) or mouse Gbeta(1). These data demonstrate the usefulness of lentiviral-based RNA interference in the systematic analysis of a signaling pathway, and they suggest that in J774A.1 cells, Gbeta(2)-derived Gbetagamma is the most effective mediator of chemotaxis to C5a.