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.

中文翻译：

---

探索癌症中G蛋白信号蛋白调节因子的突变态势。

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认识的更为突出。一个新兴的主题是与癌症相关的突变倾向于引起增强的GPCR途径活化，从而有利于致癌性。G蛋白信号转导（RGS）蛋白的调节剂是GPCR信号转导的关键调节剂，可通过其GTPase加速蛋白（GAP）活性抑制异源三聚体G蛋白的活性，该活性由称为“ RGS-box”的保守结构域赋予。在这里，我们开发了一条实验管道，可以系统地评估RGS GAP在癌症中的突变情况。对具有GAP活性的20个RGS结构域的全癌生物信息学分析显示，数百个低频突变分布在整个RGS结构域保守结构中，在与G蛋白接触的位置略有富集。我们根据经验测试了代表所有RGS GAP亚家族的多个突变，并使用基于酵母的可扩展分析方法对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，RGS蛋白突变的相当大一部分会通过各种机制导致功能丧失，包括G蛋白结合界面的破坏，蛋白稳定性的丧失或对G蛋白偶联的变构作用。此外，