Location-Biased Signaling of Proton-Sensing Receptor GPR65 Within the Endocytic Pathway

Abstract

G protein-coupled receptors (GPCRs) transduce distinct extracellular signals, including light, hormones, neurotransmitters, and ions, into diverse cellular signaling responses. These cellular responses underlie an array of physiological processes, ranging from the control of blood pressure, immune response, and neurological diseases to the progression of cancer. Considering the implications of GPCR signaling, understanding how GPCRs are regulated in human physiology and disease is very important.

The location of GPCRs within the cell is an increasingly recognized variable shaping signaling diversity at a cellular level. GPCR location at endosomes shapes downstream transcriptional responses. Endosomal signaling of prototypical GPCRs induces gene transcriptional responses with distinct cellular functions than surface receptor signaling.

The subcellular location of GPCR signaling presents an interesting context for proton-sensing GPCRs because they are more likely to be activated at acidic endosomal compartments than at the plasma membrane on the cell surface. In this dissertation, I have used the proton-sensing receptor GPR65 as a prototype to study how acidic environments at distinct cellular compartments, with an elevated proton concentration, change the signaling patterns of proton-sensing GPCRs. GPR65 is highly overexpressed in many solid tumors and is emerging as an attractive target to treat cancer since its response to acidic environments is implicated in tumor signaling and immune function. Because GPR65 is a physiologically relevant but understudied receptor, GPR65 is an ideal candidate to study how receptor location and acidic environments shape the signaling of proton-sensing GPCRs.

I first investigated whether GPR65 follows the tight coupling of receptor signaling and trafficking and whether it can be selectively activated in endosomal compartments. Through confocal microscopy, receptor mutagenesis, and biochemical assays, I 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 it can signal at steady state, irrespective of extracellular pH. Receptor mutants that were incapable of signaling trafficked normally, internalize, and localize to endosomal compartments. These findings show that GPR65 is constitutively active in endosomes and suggest a model where changes in extracellular pH reprogram the spatial pattern of receptor signaling and bias the location of signaling to the cell surface.

Next, I determined the effect of spatial organization on GPR65 signaling using a biosensor of the second messenger signaling molecule cAMP together with inhibitors of intracellular signaling proteins and effectors. I show that GPR65 increases intracellular second messenger cAMP presumably via two distinct signaling pathways which require soluble adenylyl cyclase activation. cAMP production by surface GPR65 requires EPAC and PLC, while cAMP generated by internalized receptors requires PLC activation dependent on the release of Gβγ subunits. These results suggest a model where activation of GPR65 elicits diverse and distinct signaling pathways at different cellular locations.

This work adds to our understanding of how receptor location inside the cell is intricately linked to receptor signaling and lays the groundwork for one day targeting receptor location to influence cellular responses, with greater efficacy and fewer adverse effects for patients suffering from life-threatening diseases such as cancer. Defining factors that modulate the spatial organization of GPR65 signaling together with the identification of compounds with functional selectivity, may result in clinically valuable tools for diseases involving proton-sensing receptor GPR65.