The Molecular Evolution and Mechanism of Proton‐sensing GPCRs

Abstract

Membrane receptors regulate many aspects of cell biology by enabling cells to sense and respond to their external environment. This is especially true for G protein-coupled receptors (GPCRs), capable of activating various intracellular signaling pathways upon stimulation by extracellular molecules ranging from small metabolites to large protein ligands. Three human GPCRs (GPR4, GPR65, and GPR68) are uniquely known for their proton (H+)-sensing capabilities, enabling the regulation of cell biology and physiology merely in response to acidosis. However, the mechanism underlying this proton sensing is poorly understood. In this study, we show these receptors evolved the ability to sense H+ by acquiring a triad of buried acidic residues. Using our informatics platform, pHinder, we initially identified this buried acidic triad as a distinct structural feature of proton-sensing GPCRs. Phylogenetics revealed the buried acidic triad emerged in GPR65, the ancestral proton-sensing receptor, and was maintained during the evolution of GPR4 and GPR68. To experimentally test the mechanistic importance of these triad residues, we developed Deep Variant Profiling (DVP), a method combining high-throughput CRISPR gene editing and GPCR signaling assays in yeast to profile large libraries of GPCR variants. Our results validated that most triad residues are the primary source of H+ sensing, and also show that Na+, an allosteric modulator of many other GPCRs, synergistically regulates H+ sensing by tuning the pKa values of triad residues within the physiological pH range. These findings reveal an evolutionarily supported mechanism of proton sensing by GPR4, GPR65, and GPR68, and also provide pH-insensitive variants for understanding how GPCR-based proton sensing regulates cell biology and physiology.