G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and are a common drug target. They can be stabilised in different conformational states by ligands to activate multiple transducers and effectors leading to a variety of cellular responses. The potential of agonists to activate select pathways has important implications for drug discovery. Thus, there is a clear need to profile the initial GPCR signal transduction event, activation of G proteins, to enhance understanding of receptor coupling and guide drug design. The BRET-based biosensor suite, TRUPATH, was recently developed to enable quantification of the activation profiles of all non-visual G proteins (excluding G_olf and G₁₄) and has since been utilised in numerous studies. However, it fails to detect G_q/11 activation for a number of GPCRs previously reported to display promiscuous secondary coupling to G_q/11. Here we report modifications to the Gα_q and Gα₁₁ biosensors in the switch I region that prevent intrinsic GTPase activity (R183C/Q). Except for the PAC1R, substitution with cancer-associated mutations, Cys or Gln, significantly increased sensitivity to allow detection of robust, reliable, and representative G_q/11 responses to Class B1 GPCRs. We also demonstrate the utility of these modified biosensors for promiscuously coupled class A GPCR that have primary G_s-coupling. Thus, we propose that modification to Gα_q/11 may also be necessary in other biosensor systems to enable detection of G_q/11 activation.

中文翻译：

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癌症相关突变增强了 Trupath GαQ/11 系统的敏感性

G 蛋白偶联受体 (GPCRs) 是最大的细胞表面受体家族，是常见的药物靶点。它们可以通过配体稳定在不同的构象状态，以激活多个传感器和效应器，从而导致各种细胞反应。激动剂激活选择途径的潜力对药物发现具有重要意义。因此，显然需要分析初始 GPCR 信号转导事件、G 蛋白的激活，以增强对受体偶联的理解并指导药物设计。最近开发了基于 BRET 的生物传感器套件 TRUPATH，以实现对所有非视觉 G 蛋白（不包括 G _olf和 G ₁₄) 并已被用于许多研究。然而，它未能检测到先前报道的许多 GPCR 的 G _q/11激活，这些 GPCR 显示出与 G _q/11的混杂二次耦合。在这里，我们报告了对开关 I 区域中 Gα _q和 Gα ₁₁生物传感器的修改，这些修改阻止了内在的 GTPase 活性 (R183C/Q)。除 PAC1R 外，癌症相关突变 Cys 或 Gln 的替代显着提高了灵敏度，以允许检测对 B1 类 GPCR 的稳健、可靠和有代表性的 G _q/11反应。我们还展示了这些修改后的生物传感器对于具有初级 G _{s的混杂耦合的 A 类 GPCR 的效用}-耦合。因此，我们建议在其他生物传感器系统中也可能需要对 Gα _{q/11进行修改，以检测 G q/11}激活。