Opioid receptors reveal a discrete cellular mechanism of endosomal G protein activation

Many GPCRs initiate a second phase of G protein-mediated signaling from endosomes, which inherently requires an increase in G protein activity on the endosome surface. G _s -coupled GPCRs are thought to achieve this by internalizing and allosterically activating cognate G proteins again on the endosome membrane. Here we demonstrate that the μ-opioid receptor (MOR), a G _i -coupled GPCR, increases endosomal G protein activity in a different way. Leveraging conformational biosensors, we resolve the subcellular activation dynamics of endogenously expressed MOR and G _i/o -subclass G proteins. We show that MOR activation triggers a transient increase of active-state G _i/o on the plasma membrane that is followed by a prolonged increase on endosomes. Contrary to the G _s -coupled GPCR paradigm, however, we show that the MOR-induced increase of active-state G _i/o on endosomes requires neither internalization of MOR nor activation of MOR in the endosome membrane. We propose a distinct and additional cellular mechanism for GPCR-triggered elevation of G protein activity on endosomes that is mediated by regulated trafficking of the activated G protein rather than its activating GPCR.