G protein–coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes—namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)–based effector membrane translocation assays, we dissected the mechanisms underlying endosomal G_q trafficking and activity following activation of G_q-coupled receptors, including the angiotensin II type 1, bradykinin B₂, oxytocin, thromboxane A₂ alpha isoform, and muscarinic acetylcholine M₃ receptors. Our data reveal that GPCR-promoted activation of G_q at the plasma membrane induces its translocation to endosomes independently of β-arrestin engagement and receptor endocytosis. In contrast, G_q activity at endosomes was found to rely on both receptor endocytosis-dependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular G_q signaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.

G 蛋白偶联受体 (GPCR) 是细胞稳态的看门人，也是大部分药物的靶点。除了它们在质膜上的信号活动外，有人提出它们的作用可能是由于内膜（即内体）中 G 蛋白的易位和激活所致。这可能会对我们理解 GPCR 靶向药物的信号如何在细胞内传播产生重大影响。然而，关于在亚细胞区室中驱动 G 蛋白运动和激活的机制知之甚少。使用生物发光共振能量转移（BRET）系效应膜易位测定，我们解剖底层内体G中的机制_q下列G的活化贩卖和活动_q-偶联受体，包括血管紧张素 II 1 型、缓激肽 B ₂、催产素、血栓素 A ₂ α 异构体和毒蕈碱乙酰胆碱 M ₃受体。我们的数据显示，GPCR 促进的质膜_Gq激活诱导其易位至内体，而与 β-抑制蛋白参与和受体内吞作用无关。相比之下，发现内体的G _q活性依赖于受体内吞作用依赖性和非依赖性机制。除了阐明控制亚细胞 G _q的分子过程 信号，我们的研究提供了一套工具，这些工具通常适用于研究内体和其他亚细胞细胞器的 G 蛋白易位和激活，以及信号传播对药物作用的贡献。