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

Mol Cell. 2025 Apr 17;85(8):1658-1673.e7. doi: 10.1016/j.molcel.2025.03.018. Epub 2025 Apr 10.

Abstract

Proton sensing by G protein-coupled receptors (GPCRs) is crucial in many life activities. However, its underlying mechanism remains unclear. Here, we report 8 cryoelectron microscopy (cryo-EM) structures of human GPR4 and GPR68 at different pH values and in complex with Gs or Gq trimers or in apo state. Structural inspection, structure-based pKa calculations, and mutational and computational analyses revealed that protonation of two conserved extracellular histidines induced polar network formation and other conformational changes to tether 7-transmembrane (TM7) to second extracellular loop (ECL2), and these changes constitute the central mechanisms of proton-induced activation of GPR4 and GPR68. Unexpectedly, proton sensation by specific extracellular histidine determined biased G protein coupling of GPR4. Moreover, GPR68's additional pH-sensing H842.67 enhances its function in a more acidic optimal pH range. The propagation path connecting proton-sensing histidines to the toggle switch was characterized. Collectively, we provide structural insights into the proton sensing, activation, and downstream effector coupling mechanisms of proton-sensing GPCRs.

Keywords: GPCR; cryo-EM structure; human GPR4; proton sensation; sensory biology.

MeSH terms

  • Cryoelectron Microscopy
  • GTP-Binding Protein alpha Subunits, Gq-G11 / chemistry
  • GTP-Binding Protein alpha Subunits, Gq-G11 / genetics
  • GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism
  • GTP-Binding Protein alpha Subunits, Gs / chemistry
  • GTP-Binding Protein alpha Subunits, Gs / genetics
  • GTP-Binding Protein alpha Subunits, Gs / metabolism
  • HEK293 Cells
  • Histidine* / chemistry
  • Histidine* / genetics
  • Histidine* / metabolism
  • Humans
  • Hydrogen-Ion Concentration
  • Protein Binding
  • Protein Conformation
  • Protons*
  • Receptors, G-Protein-Coupled* / chemistry
  • Receptors, G-Protein-Coupled* / genetics
  • Receptors, G-Protein-Coupled* / metabolism
  • Receptors, G-Protein-Coupled* / ultrastructure
  • Signal Transduction

Substances

  • Histidine
  • Receptors, G-Protein-Coupled
  • Protons
  • GPR68 protein, human
  • GPR4 protein, human
  • GTP-Binding Protein alpha Subunits, Gq-G11
  • GTP-Binding Protein alpha Subunits, Gs