BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated G activation at early endosomes.

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 trafficking and activity following activation of G-coupled receptors, including the angiotensin II type 1, bradykinin B, oxytocin, thromboxane A alpha isoform, and muscarinic acetylcholine M receptors. Our data reveal that GPCR-promoted activation of G at the plasma membrane induces its translocation to endosomes independently of β-arrestin engagement and receptor endocytosis. In contrast, G 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 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.