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首页> 外文期刊>American Journal of Physiology >RNA interference screen for RGS protein specificity at muscarinic and protease-activated receptors reveals bidirectional modulation of signaling
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RNA interference screen for RGS protein specificity at muscarinic and protease-activated receptors reveals bidirectional modulation of signaling

机译：RGS蛋白在毒蕈碱和蛋白酶激活受体上的RNA干扰筛选揭示了信号的双向调控

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摘要

Regulator of G protein signaling (RGS) proteins are considered key modulators of G protein-coupled receptor (GPCR)-mediated signal transduction. These proteins act directly on Gα subunits in vitro to increase their intrinsic rate of GTP hydrolysis; this activity is central to the prevailing view of RGS proteins as negative regulators of agonist-initiated GPCR signaling. However, the specificities of action of particular RGS proteins toward specific GPCRs in an integrated cellular context remain unclear. Here, we developed a medium-throughput assay to address this question in a wholly endogenous context using RNA interference. We performed medium-throughput calcium mobilization assays of agonist-stimulated muscarinic acetylcholine and protease-activated receptors in human embryonic kidney 293 (HEK293) cells transfected with individual members of a "pooled duplex" short interfering RNA library targeting all conventional human RGS transcripts. Only knockdown of RGS11 increased both carbachol-mediated calcium mobilization and inositol phosphate accumulation. Surprisingly, we found that knockdown of RGS8 and RGS9, but not other conventional RGS proteins, significantly decreased carbachol-mediated calcium mobilization, whereas only RGS8 knockdown decreased protease-activated receptor-1 (PAR-1)-mediated calcium mobilization. Loss of responsiveness toward carbachol and PAR-1 agonist peptide upon RGS8 knockdown appears due, at least in part, to a loss in respective receptor cell surface expression, although this is not the case for RGS9 knockdown. Our data suggest a cellular role for RGS8 in the stable surface expression of M3 muscarinic acetylcholine receptor and PAR-1, as well as a specific and opposing set of functions for RGS9 and RGS11 in modulating carbachol responsiveness similar to that seen in Caenorhabditis elegans.
机译：G蛋白信号转导（RGS）蛋白的调节剂被认为是G蛋白偶联受体（GPCR）介导的信号转导的关键调节剂。这些蛋白在体外直接作用于Gα亚基，以增加其GTP水解的固有速率。该活性对于RGS蛋白作为激动剂启动的GPCR信号转导的负调节剂的普遍观点至关重要。然而，在整合的细胞环境中，特定RGS蛋白对特定GPCR的作用特异性仍不清楚。在这里，我们开发了一种中等通量测定法，以利用RNA干扰在完全内源的情况下解决此问题。我们对人胚胎肾293（HEK293）细胞进行了中激动剂刺激的毒蕈碱乙酰胆碱和蛋白酶激活受体的中通量钙动员测定，该细胞被“池化双链”短干扰RNA文库的单个成员转染，靶向所有常规人RGS转录本。仅敲低RGS11会增加卡巴胆碱介导的钙动员和肌醇磷酸积累。出乎意料的是，我们发现敲低RGS8和RGS9，而不是其他常规RGS蛋白，可显着降低卡巴胆碱介导的钙动员，而只有RGS8敲低可降低蛋白酶激活受体1（PAR-1）介导的钙动员。 RGS8敲除后，对卡巴胆碱和PAR-1激动剂肽的反应性丧失似乎至少部分是由于各自受体细胞表面表达的丧失，尽管RGS9敲除并非如此。我们的数据表明，RGS8在M3毒蕈碱型乙酰胆碱受体和PAR-1的稳定表面表达中的细胞作用，以及RGS9和RGS11在调节卡巴胆碱反应性方面的一组特定且相反的功能，类似于秀丽隐杆线虫。

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