Elucidating the Activation Mechanism of the Proton-sensing GPR68 Receptor

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• GPR68 is a proton-sensing G-protein Coupled Receptor (GPCR) implicated in physiological processes and diseases, including cancer.
• The precise molecular mechanism underlying GPR68 activation by extracellular pH changes remains largely undetermined.

Purpose of the Study:

• To elucidate the activation mechanism of GPR68 using a multidisciplinary computational and in vitro approach.
• To identify key residues and structural features involved in GPR68 proton-sensing and activation.

Main Methods:

• Molecular Dynamics (MD) simulations of GPR68 to model pH-induced changes.
• Analysis of residue interactions, motions, and pKa values during simulations.
• Sequence analysis and site-directed mutagenesis to investigate residue pairings and structural elements.

Main Results:

• MD simulations revealed global and local rearrangements in GPR68 consistent with partial activation upon protonation.
• Extracellular histidine and transmembrane acidic residues exhibited upshifted pKa values, supporting their role in activation.
• A novel histidine-acidic residue pairing in the extracellular region was identified and validated.
• A previously unrecognized hydrophobic lock in the extracellular region was found to stabilize the inactive GPR68 conformation.

Conclusions:

• Protonation of specific residues triggers partial activation of GPR68.
• The identified extracellular residue pairing and hydrophobic lock are critical for GPR68 conformational stability and activation regulation.
• These findings provide new insights into the molecular basis of GPR68 function in physiological and pathological contexts.