Site-Directed Mutagenesis and Time-Resolved Fluorescence Spectroscopy Reveal That Two Hydrogen-Bonded Carboxylic Side Chains Are Essential for GPR68 Proton Sensing and G Protein Binding

GPR68 is a pH-sensing G protein-coupled receptor widely expressed throughout the body. It has been implicated in various diseases, including neurodegeneration, chronic inflammation, and cancer, emphasizing its role in pathophysiology. The identities of the structural elements essential for pH sensing have been controversial, as experiments and sequence analyses have been interpreted to suggest that an extracellular histidine cluster or, by contrast, an internal carboxylic triad, senses the extracellular pH. Recent molecular simulations and hydrogen-bond network analyses suggested instead a unifying mechanism whereby the extracellular histidine residues and the internal carboxylic triad couple to each other via a protonation-sensitive hydrogen-bond network that includes a fourth internal carboxylic group, E103^3.34. However, without experimental verification, there remains a gap in our understanding of the mechanism by which GPR68 is activated by protons. To this aim, here we have studied 16 GPR68 mutations, which we selected based on the hydrogen-bond network analyses and conservation of key residues. We implemented a cell-based homogeneous time-resolved fluorescence assay to monitor the Gα_q/11 and Gα_s coupled signaling pathways. We used this assay to study how each mutation alters the basal activity levels, half-maximal activation values, and reactivation at lower pH values. Our data identify E103^3.34Q as a gain-of-function mutant essential for the proton-sensing mechanism of GPR68. We further discovered that E149^4.53Q, a constitutively active mutant, prefers Gα_s coupling over Gα_q/11 and that wild-type GPR68 is more sensitive toward Gα_q/11 coupling over Gα_s.