Conformational biosensors delineate endosomal G protein regulation by GPCRs.

Many GPCRs trigger a second phase of G protein-coupled signaling from endosomes after signaling from the plasma membrane, necessitating GPCRs to increase the concentration of active-state G proteins on the endosome membrane. How this is achieved remains unclear. Here, we show that three G(s)-coupled GPCRs-the β2-adrenergic receptor, VIP-1 receptor, and adenosine 2B receptor-each trigger a net redistribution of Gα(s) from the plasma membrane to endosomes at native expression levels and without requiring receptor internalization. We then show that active-state Gα(s) production on endosomes, in contrast, is GPCR internalization-dependent. We further identify location bias in the selectivity of GPCR coupling between G(s) and G(q) on endosomes relative to the plasma membrane. We propose that endosomal G(s) regulation involves discrete GPCR-G protein coupling reactions, one at the plasma membrane controlling G(s) concentration and another at endosomes controlling G(s) activity, and that GPCR endocytosis can switch signaling selectivity between G protein classes.