Schihada Hannes, Shekhani Rawan, Schulte Gunnar
Section for Receptor Biology and Signaling, Department of Physiology and Pharmacology, Karolinska Institutet, Biomedicum, Solnavägen 9, SE-17165 Stockholm, Sweden.
Sci Signal. 2021 Sep 7;14(699):eabf1653. doi: 10.1126/scisignal.abf1653.
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.
异源三聚体G蛋白是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发的各种生理和病理生理环境中传导基础信号。光学生物传感器已被开发和优化，用于监测GPCR配体诱导的G蛋白激活，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。我们还建立了一种在活细胞中鉴定组成型GPCR或G蛋白信号传导的方案。这些基于G蛋白的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，并减少哺乳动物细胞中的信号变异性。我们还提出了一个实验方案，使用G-CASE传感器工具箱通过这些异源三聚体转导子量化天然和突变GPCR的组成型信号传导。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。
