The evolution and mechanism of GPCR proton sensing

Of the 800 G protein-coupled receptors (GPCRs) in humans, only three (GPR4, GPR65, and GPR68) regulate signaling in acidified microenvironments by sensing protons (H⁺). How these receptors have uniquely obtained this ability is unknown. Here, we show these receptors evolved the capability to sense H⁺ signals by acquiring buried acidic residues. Using our informatics platform pHinder, we identified a triad of buried acidic residues shared by all three receptors, a feature distinct from all other human GPCRs. Phylogenetic analysis shows the triad emerged in GPR65, the immediate ancestor of GPR4 and GPR68. To understand the evolutionary and mechanistic importance of these triad residues, we developed deep variant profiling, a yeast-based technology that utilizes high-throughput CRISPR to build and profile large libraries of GPCR variants. Using deep variant profiling and GPCR assays in HEK293 cells, we assessed the pH-sensing contributions of each triad residue in all three receptors. As predicted by our calculations, most triad mutations had profound effects consistent with direct regulation of receptor pH sensing. In addition, we found that an allosteric modulator of many class A GPCRs, Na⁺, synergistically regulated pH sensing by maintaining the pK_a values of triad residues within the physiologically relevant pH range. As such, we show that all three receptors function as coincidence detectors of H⁺ and Na⁺. Taken together, these findings elucidate the molecular evolution and long-sought mechanism of GPR4, GPR65, and GPR68 pH sensing and provide pH-insensitive variants that should be valuable for assessing the therapeutic potential and (patho)physiological importance of GPCR pH sensing.