Proton‐Sensing Receptor GPR65 Dynamically Traffics To and Signals From Multiple Cellular Compartments

Abstract

Proton‐sensing G protein‐coupled receptors (GPCRs) allow cells to sense and respond to changes in physiological pH. Over the last 20 years, proton‐sensing membrane proteins have been implicated in a variety of biological functions and disease states, including inflammation, and stimulation and inhibition of oncogenic signaling pathways. Proton‐sensing GPCRs are unique in that, upon internalization and trafficking to acidic intracellular compartments, they are exposed to environments that are more likely to activate the receptor. However, little is known about whether and how proton‐sensing receptors signal from intracellular compartments. Characterizing proton‐sensing receptor trafficking and signaling is critical to understand how these receptors integrate responses from multiple locations in the cell, and how these responses change during the progression of diseases associated with acidic microenvironments. Here we use proton‐sensing receptor GPR65 as a prototype to study how receptor location influences proton‐sensing receptor signaling. We use live imaging of GPR65‐expressing cells to show that GPR65 internalizes from the plasma membrane at both neutral and acidic pH. Further, GPR65 localizes to early and late endosomes irrespective of extracellular pH. However, GPR65 increases second messenger cAMP by coupling to Gs proteins upon exposure to acidic extracellular pH. To determine whether GPR65 at internal compartments contributes to cAMP production, we pre‐treated GPR65‐expressing cells with Dyngo4a, an endocytosis inhibitor. GPR65 internalization was required for a full cAMP response after acidic extracellular pH. Together, our findings show that GPR65 dynamically traffics and signals from multiple cellular compartments irrespective of extracellular pH. Importantly, our results suggest that, unlike for most known GPCRs, activation of GPR65 is uncoupled from receptor trafficking.