Abstract
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 pKa 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.
Highlights
Maintain their status as understudied and “pharmacologically dark” receptors [28, 29]
We show that 3 human G protein–coupled receptor (GPCR) evolved the ability to sense pH by acquiring buried acidic residues that emerged in GPR65 and were later inherited by GPR4 and GPR68
GPR4, GPR65, and GPR68 share a unique triad of buried acidic residues pHinder is an informatics program that uses computational geometry to identify structural features, such as buried ionizable residues, that are predictive of pH sensing [40]
Summary
Isom1,2,3,* From 1The Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA; 2The Department of Tumor Biology, University of Miami Sylvester Comprehensive Cancer Center, Miami, Florida, USA; and 3The Institute for Data Science Computing, University of Miami, Coral Gables, Florida, USA
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