Deep mutational scanning of GPR68 pH activation and surface expression

Homeostatic control of acid-base balance is a vital aspect of human physiology. GPR68 is a representative member of the proton-sensing family of G Protein-Coupled Receptors (GPCRs) which enable precise sensing of pH in humans. The mechanism by which GPR68 is activated by protons, and the specific residues involved, have remained elusive. Comprehensive mapping of all possible mutations at each position in GPR68 will enable determination of which residues are responsible for coordinating protons and hydrogen bonding networks in GPR68. This information will be critical for understanding the activation mechanism of this unique class of receptors and provide critical information for future therapeutic development. Here, we perform a parallel deep mutational scan on the entirety of GRP68 to measure the effect each mutation has on both receptor activation in response to a drop in extracellular pH and the effect each mutation has on surface expression of the receptor. With this multi-parametric dataset, we have identified which positions and interactions are key to GPR68 activation and expression. Further, this provides a broadly-applicable framework for the study of GPCR mutational effects on molecular recognition, allostery, and cell biology.