Heterotrimeric G proteins constitute the primary transducers of G protein–coupled receptor (GPCR) signaling. In addition to mediating ligand-induced GPCR activation, G proteins transduce basal signaling in various physiological and pathophysiological settings evoked by constitutively active, native GPCRs or disease-related receptor mutants. Optical biosensors have been developed and optimized to monitor GPCR ligand–induced activation of G proteins, but these biosensors cannot be used to detect constitutively active GPCRs. Here, we designed and validated eight bioluminescence resonance energy transfer (BRET)–based G protein sensors that can measure the activity of all four major families of G proteins. We also established a protocol to identify constitutive GPCR or G protein signaling in live cells. These G protein–based, tricistronic activity sensors (G-CASE) rely on the encoding of all three G protein subunits by a single plasmid, enabling their expression at the desired relative amounts and resulting in reduced signal variability in mammalian cells. We also present an experimental protocol to use the G-CASE sensor toolbox to quantify constitutive signaling of native and mutated GPCRs through these heterotrimeric transducers. This approach will help to characterize constitutively active GPCRs and their role in health and disease.

中文翻译：

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使用基于 BRET 的 G 蛋白生物传感器定量评估组成型 G 蛋白偶联受体活性

异三聚体 G 蛋白构成 G 蛋白偶联受体 (GPCR) 信号传导的主要转导子。除了介导配体诱导的 GPCR 激活外，G 蛋白还在由组成型活性天然 GPCR 或疾病相关受体突变体引起的各种生理和病理生理环境中转导基础信号传导。已经开发和优化了光学生物传感器以监测 GPCR 配体诱导的 G 蛋白活化，但这些生物传感器不能用于检测组成型活性 GPCR。在这里，我们设计并验证了八种基于生物发光共振能量转移 (BRET) 的 G 蛋白传感器，它们可以测量所有四个主要 G 蛋白家族的活性。我们还建立了一个协议来识别活细胞中的组成型 GPCR 或 G 蛋白信号传导。这些基于 G 蛋白，三顺反子活性传感器 (G-CASE) 依赖于单个质粒对所有三个 G 蛋白亚基的编码，使其能够以所需的相对量表达，并导致哺乳动物细胞中信号变异性的降低。我们还提出了一个实验协议，使用 G-CASE 传感器工具箱通过这些异源三聚体传感器量化原生和突变 GPCR 的组成信号。这种方法将有助于表征组成型活性 GPCR 及其在健康和疾病中的作用。我们还提出了一个实验协议，使用 G-CASE 传感器工具箱通过这些异源三聚体传感器量化原生和突变 GPCR 的组成信号。这种方法将有助于表征组成型活性 GPCR 及其在健康和疾病中的作用。我们还提出了一个实验协议，使用 G-CASE 传感器工具箱通过这些异源三聚体传感器量化原生和突变 GPCR 的组成信号。这种方法将有助于表征组成型活性 GPCR 及其在健康和疾病中的作用。