Proton perception and activation of a proton-sensing GPCR.

Maintaining pH at cellular, tissular, and systemic levels is essential for human health. Proton-sensing GPCRs regulate physiological and pathological processes by sensing the extracellular acidity. However, the molecular mechanism of proton sensing and activation of these receptors remains elusive. Here, we present cryoelectron microscopy (cryo-EM) structures of human GPR4, a prototypical proton-sensing GPCR, in its inactive and active states. Our studies reveal that three extracellular histidine residues are crucial for proton sensing of human GPR4. The binding of protons induces substantial conformational changes in GPR4's ECLs, particularly in ECL2, which transforms from a helix-loop to a β-turn-β configuration. This transformation leads to the rearrangements of H-bond network and hydrophobic packing, relayed by non-canonical motifs to accommodate G proteins. Furthermore, the antagonist NE52-QQ57 hinders human GPR4 activation by preventing hydrophobic stacking rearrangement. Our findings provide a molecular framework for understanding the activation mechanism of a human proton-sensing GPCR, aiding future drug discovery.