Modulating proton sensing by the adenosine receptor A2

For years, it was believed that G-protein-coupled receptors (GPCRs) activated or modulated by changes in physiological pH were mechanistically regulated by pH-sensitive histidine residues on the receptor’s exterior. More recent studies have shown that traditional acid-sensing GPCRs (GPR4, GPR65, GPR68) contain buried acidic amino acids in their 7-transmembrane regions and can be activated by pH-dependent mechanisms. The focus of our research has been the adenosine A2A receptor (A2AR) and its ability to activate at low pH levels without having the typical acidic triad structure found in previously studied acid-sensing GPCRs. We used a combination of bioinformatics and a humanized yeast-based platform, DCyFIR, to identify potential structures involved in the proton-sensing mechanism of A2AR. We also validated some of our A2AR variant yeast phenotypes using mammalian cells. Our data suggest that certain mutations eliminate the pH sensitivity of the A2AR while preserving agonist-induced signaling. Additionally, we demonstrated that one of our mutations (N284D) eliminates pH sensing but increases agonist potency, thereby providing a structural explanation for how pH-sensing and sodium binding occur that differs from previously established models. Overall, these data indicate that G-protein-coupled receptors can sense pH in several ways and demonstrate the potential of a novel, pH-insensitive A2AR in acid-related contexts.