Mechanistic Insights into pH‐sensing GPCRs

Many aspects of protein function, cell biology, human physiology, and disease are influenced by pH changes. However, our mechanistic understanding of pH effects in most of these areas is limited. This is especially true in the case of cell signaling, for which very few pH‐sensing receptors and signaling cascades have been identified. To address this issue, we are studying how pH regulates G protein‐coupled receptors (GPCRs), the largest and most therapeutically targeted family of cell‐surface receptors in humans. Unlike most cellular proteins, GPCRs are regularly exposed to a wide range of dynamic pH values (from pH 7.4 on the cell surface, to pH 5.0 in endosomes) and can be chronically exposed to acidic microenvironments caused by cancer and processes such as inflammation. While there are more than 800 GPCR family members, only three receptors (i.e. GPR4, GPR65, and GPR68) are known to be activated directly by pH. In this study, we used structural informatics and cell‐based signaling assays to predict and validate residues responsible for pH sensing by these receptors. As part of these efforts, we engineered and interrogated 44 GPR4, GPR65, and GPR68 point mutations in a library of 440 cell strains that covered all possible GPCR‐Gα subunit signaling combinations. This comprehensive strain library included 14 mutations predicted by our structural informatics software, pHinder, and the full set of 30 mutations for these receptors available in the literature. Our results for the known mutants agree with previous reports and expand our understanding of the relationship between Gα‐signaling specificity and pH sensing. More importantly, several of our newly predicted mutations appear to have a greater influence on the pH‐sensing capabilities of these receptors than any of the previously reported GPR4, GPR65, and GPR68 mutations to date. Most of the pH‐sensitive residues predicted by pHinder were found within the protein core, a region that has been largely overlooked when considering pH‐sensing properties of GPCRs. Our cell‐based experiments confirmed that these residues are critical for pH sensing and provide a more thorough knowledge‐base for understanding GPCR pH sensing. We anticipate that the outcome of these studies will enable us to more comprehensively quantify the extent of pH sensing across the GPCRome and rationally engineer new pH‐sensing GPCRs for use in industry, synthetic biology, and medicine. Support or Funding Information R35GM119518R01NS103906R03TR002908