Location-biased activation of the proton-sensor GPR65 is uncoupled from receptor trafficking.

The canonical view of G protein-coupled receptor (GPCR) function is that receptor trafficking is tightly coupled to signaling. GPCRs remain on the plasma membrane (PM) at the cell surface until they are activated, after which they are desensitized and internalized into endosomal compartments. This canonical view presents an interesting context for proton-sensing GPCRs because they are more likely to be activated in acidic endosomal compartments than at the PM. Here, we show that the trafficking of the prototypical proton-sensor GPR65 is fully uncoupled from signaling, unlike that of other known mammalian GPCRs. GPR65 internalizes and localizes to early and late endosomes, from where they signal at steady state, irrespective of extracellular pH. Acidic extracellular environments stimulate receptor signaling at the PM in a dose-dependent manner, although endosomal GPR65 is still required for a full signaling response. Receptor mutants that were incapable of activating cAMP trafficked normally, internalize and localize to endosomal compartments. Our results show that GPR65 is constitutively active in endosomes, and suggest a model where changes in extracellular pH reprograms the spatial pattern of receptor signaling and biases the location of signaling to the cell surface.