The evolution and mechanism of GPCR proton sensing

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 p K 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.

Proton-gated coincidence detection is a common feature of GPCR signaling

“…Furthermore, as reported in Fig. 5E, these pH titrations confirmed that the adenosine receptor A2a (ADORA2A) is activated by acidic pH in a manner strikingly similar to the known acid-activated proton sensor GPR68 (8). Furthermore, these pH profiles established that ADORA2A senses protons through the G i/o , G q , and G 12/13 families of Gα subunits but did not indicate proton-sensing through the canonical ADORA2A Gα subunit G α,s .…”

“…As shown in Fig. 1D, we previously engineered a high-throughput platform for studying human GPCRs that uses the isolated Ste2 pathway: Dynamic Cyan Induction by Functional Integrated Receptors, DCyFIR (8,17,18,21). In the DCyFIR platform, a human GPCR is coupled to a MAP kinase signaling cascade via a human/ yeast C-terminal Gα chimera in which the last five residues of the native yeast Gα subunit, Gpa1, are replaced with the last five residues of a human Gα (Fig.…”

“…S3), appears unaffected by pH. As it stands, discriminating such receptors from their pH-sensitive counterparts a priori remains a formidable challenge, although we are beginning to make progress on this front (8,12).…”

“…Recently, we have shown that acid-sensing GPCRs GPR4, GPR65, and GPR68 are molecular coincidence detectors of H + and Na + ions. In this case, H + is an activator, and Na + is a negative allosteric modulator of GPR4, GPR65, and GPR68 signaling (8). Additionally, GPR68 has recently been shown to integrate H + and mechanical inputs in response to simultaneous extracellular acidification and membrane stretch (9).…”

Proton-Sensing GPCRs in Health and Disease

“…Indeed, the G2A receptor seems to be less sensitive to pH changes than the other three members of the proton-sensing GPCR family, probably due to the lack of crucial histidines at the respective positions [8]. Moreover, a recent publication suggested a triad of acidic amino acids for proton detection that are conserved in GPR4, OGR1, and TDAG8, but not in G2A, which may additionally explain the weak proton activation of G2A, and which provides molecular evidence for the evolution of protonsensing GPCRs allowing phylogenetic classification of the receptors [15].…”