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

doi:10.1016/j.cell.2024.11.036

Molecular basis of proton sensing by G protein-coupled receptors

Cell. 2024 Dec 26:S0092-8674(24)01373-4. doi: 10.1016/j.cell.2024.11.036. Online ahead of print.

Affiliations

¹ Tetrad graduate program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Bioengineering and Therapeutic Science, University of California, San Francisco, San Francisco, CA 94143, USA.
² Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA; Biophysics graduate program, University of California, San Francisco, San Francisco, CA 94143, USA.
³ Department of Pharmacology and the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH PDSP), The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁴ Science for Life Laboratory, Department of Applied Physics, KTH Royal Institute of Technology, 12121 Solna, Stockholm, Stockholm County 114 28, Sweden.
⁵ Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Department of Bioengineering and Therapeutic Science, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Tetrad graduate program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94143, USA.
⁸ Department of Medicine, Division of Infectious Disease, University of California, San Francisco, San Francisco, CA 94143, USA.
⁹ Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.
¹⁰ Department of Bioengineering and Therapeutic Science, University of California, San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub, San Francisco, CA 94148, USA; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: [email protected].
¹¹ Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub, San Francisco, CA 94148, USA; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, CA 94115, USA. Electronic address: [email protected].

PMID: 39753132
DOI: 10.1016/j.cell.2024.11.036

Abstract

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.

Keywords: GPCR; cryo-EM; proton; proton sensing; signaling; structure.