Published 6 February 2025 in Cell (doi 10.1016/j.cell.2024.11.036):
Molecular basis of proton sensing by G protein-coupled receptors
Matthew K Howard,* Nicholas Hoppe,* Xi-Ping Huang,° Darko Mitrovic,° Christian B Billesbølle, Christian B Macdonald, Eshan Mehrotra, Patrick Rockefeller Grimes, Donovan D Trinidad, Lucie Delemotte, Justin G English, Willow Coyote-Maestas,’ Aashish Manglik’
Three proton-sensing G protein-coupled receptors (GPCRs)-GPR4, GPR65, and GPR68-respond to extracellular pH to regulate diverse physiology. How protons activate these receptors is poorly understood. We determined cryogenic-electron microscopy (cryo-EM) structures of each receptor to understand the spatial arrangement of proton-sensing residues. Using deep mutational scanning (DMS), we determined the functional importance of every residue in GPR68 activation by generating ∼9,500 mutants and measuring their effects on signaling and surface expression. Constant-pH molecular dynamics simulations provided insights into the conformational landscape and protonation patterns of key residues. This unbiased approach revealed that, unlike other proton-sensitive channels and receptors, no single site is critical for proton recognition. Instead, a network of titratable residues extends from the extracellular surface to the transmembrane region, converging on canonical motifs to activate proton-sensing GPCRs. Our approach integrating structure, simulations, and unbiased functional interrogation provides a framework for understanding GPCR signaling complexity.
*Equal contributions; °equal contributions; ‘co-corresponding authors
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