Probing the mutational landscape of regulators of G protein signaling proteins in cancer
Probing the mutational landscape of regulators of G protein signaling proteins in cancer
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DOI：
10.1126/scisignal.aax8620
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发表时间：
2020-02-04
影响因子：
7.3
通讯作者：
Garcia-Marcos, Mikel
中科院分区：
文献类型：
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作者：
DiGiacomo, Vincent;Maziarz, Marcin;Garcia-Marcos, Mikel
深度测序技术的出现揭示了癌症中G蛋白偶联受体(GPCR)信号通路的突变比以前所认识的更突出。一个迫在眉睫的主题是，癌症相关突变往往会导致GPCR途径激活增强，从而有利于致癌。G蛋白信号转导调节蛋白(RGS)是GPCR信号转导的关键调节物，通过GTP酶加速蛋白(GAP)活性抑制异源三聚体G蛋白的活性，GAP活性由一个被称为“RGS-box”的保守结构域赋予。在这里，我们开发了一条实验管道，系统地评估癌症中RGS缺口的突变情况。对具有GAP活性的20个RGS结构域进行的泛癌症生物信息学分析显示，数百个低频突变分布在保守的RGS结构域结构中，与G蛋白结合的位置略有丰富。我们经验性地测试了代表所有RGS GAP亚家族的多个突变，并使用可扩展的基于酵母的分析方法对G蛋白界面和非界面位置进行了采样。最后，在哺乳动物细胞中使用G蛋白活性生物传感器验证了突变体的子集。我们的发现表明，相当大一部分RGS蛋白突变通过不同的机制导致功能丧失，包括G蛋白结合界面的破坏，蛋白质稳定性的丧失，或变构对G蛋白偶联的影响。此外，我们的结果还验证了一种可扩展的管道，用于快速表征RGS蛋白中与癌症相关的突变。
The advent of deep-sequencing techniques has revealed that mutations in G protein-coupled receptor (GPCR) signaling pathways in cancer are more prominent than was previously appreciated. An emergent theme is that cancer-associated mutations tend to cause enhanced GPCR pathway activation to favor oncogenicity. Regulators of G protein signaling (RGS) proteins are critical modulators of GPCR signaling that dampen the activity of heterotrimeric G proteins through their GTPase-accelerating protein (GAP) activity, which is conferred by a conserved domain dubbed the "RGS-box." Here, we developed an experimental pipeline to systematically assess the mutational landscape of RGS GAPs in cancer. A pan-cancer bioinformatics analysis of the 20 RGS domains with GAP activity revealed hundreds of low-frequency mutations spread throughout the conserved RGS domain structure with a slight enrichment at positions that interface with G proteins. We empirically tested multiple mutations representing all RGS GAP subfamilies and sampling both G protein interface and noninterface positions with a scalable, yeast-based assay. Last, a subset of mutants was validated using G protein activity biosensors in mammalian cells. Our findings reveal that a sizable fraction of RGS protein mutations leads to a loss of function through various mechanisms, including disruption of the G protein-binding interface, loss of protein stability, or allosteric effects on G protein coupling. Moreover, our results also validate a scalable pipeline for the rapid characterization of cancer-associated mutations in RGS proteins.