GPR68 : A Proton Sensing G Protein-Coupled Receptor

G protein-coupled receptor 68 (GPR68) is an orphan receptor that is activated by extracellular acidity, plays a crucial role in various physiological processes, and is highly expressed in various types of cancer. Lack of a publicly available 3D structure of the receptor hinders the current understanding of how GPR68 senses extracellular protons. Theories suggest that extracellular histidines or a buried carboxylic triad play a role in its proton sensing function.

In my doctoral thesis, I utilized protein engineering to express and purify human GPR68 in the presence of a proprietary antagonist. However, the protein quality was unsuitable for determining the structure of the receptor via X-ray crystallography and single particle cryogenic electron microscopy (cryo-EM). I then attempted to determine the structure of the zebrafish GPR68 via cryo-EM.

I established a protocol for validating structural models of GPR68 based on various strategies and used the best compatible model for classical molecular dynamics to identify crucial proton sensing regions of the receptor. I found that GPR68 has a robust and highly dynamic water-mediated hydrogen-bond network that connects the extracellular histidine and carboxylate clusters to the buried carboxylates via a putative allosteric pocket of GPR68. This hydrogen bond network is sensitive to changes in the protonation states of specific buried carboxylates and may induce sufficient structural changes to allow further connection of the buried acidic residues to the intracellular G protein binding site.

These observations and hypotheses were experimentally validated by establishing a homogeneous time-resolved fluorescence (HTRF) based functional assay for GPR68. Using GPR68 mutants in this assay, I have shown that specific surface carboxylates and a buried acidic residue that had no prior indication to be functional play a substantial role in the proton sensing ability of GPR68.

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Kapur, B., 2024. GPR68: A Proton Sensing G Protein-Coupled Receptor.
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