Structural basis and biased signaling of proton sensation by GPCRs mediated by extracellular histidine rearrangement

Proton sensing by membrane G protein-coupled receptors (GPCRs) is crucial in controlling the breathing rate, bone formation, and cardiovascular and kidney functions. However, the structural basis and mechanistic insights of proton sensing-induced GPCR activation remains unclear. Here, we report seven cryo-EM structures of GPR4 and GPR68, two well-known proton-sensing receptors, at different pH values and in complex with Gs or Gq trimers. Structural inspection, structure-based pKa calculations, and mutational and computational analyses revealed that the protonation of two conserved extracellular histidines in ECL2 and TM6, H ^ECL2-45.47 and H ^7.36 , induced polar network formation and other conformational changes to tether TM6 to ECL2, and these changes constitute the central common mechanisms of the proton-induced activation of both GPR4 and GPR68. Unexpectedly, proton sensation by specific extracellular histidine determined biased G protein subtype coupling of GPR4. Moreover, GPR68 has an additional pH-sensing residue, H84 ^2.67 , whose protonation facilitates tighter interactions between TM2, ECL1 and ECL2, which may contribute to the more acidic optimal pH range for GPR68 activation than for GPR4 activation. The propagation path connecting proton-sensing histidines to the toggle switch and the structural basis of the Gs and Gq coupling of GPR4 and GPR68 were characterized. In summary, our study provides structural and mechanistic insights into the proton sensing, activation and downstream effector coupling mechanisms of two proton-sensing GPCRs.