Abstract
G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in humans. More than 800 unique receptors bind diverse ligands such as hormones, peptides, and protons to guide cellular decision making. Although GPCRs are the most common target of FDA approved drugs, many receptors still lack well-defined physiological roles (e.g. “orphan” receptors) and biological tools for preclinical drug discovery. This is especially true for biological scenarios where receptors are exposed to dynamic pH environments such as when internalized into endosomes or in the acidic tumor microenvironment. In this dissertation, I report my efforts to expand our fundamental understanding of GPCR biology and pharmacology by developing a cell-based model system optimal for high-throughput ligand/drug discovery and pH studies called Dynamic Cyan induction by Functional Integrated Receptors (DCyFIR). Using CRISPR genome editing, I helped create a suite of 300 DCyFIR strains, each barcoded with a unique GPCR-Ga coupling combination. This feature motivated me to develop and optimize a multiplexed assay, known as DCyFIRplex, that enables large compound libraries to be screened against hundreds of GPCR-Ga pairs simultaneously in a single tube. I used the DCyFIRplex method to screen established GPCR ligands and a large library of human metabolites against the 300 DCyFIR strains and discovered unexpected interactions for both well-studied and orphan GPCRs. Furthermore, I utilized the DCyFIR technology to comprehensively assess the effects of pH on GPCR signaling for 280 GPCR-Ga coupling combinations. Through this effort, I discovered that many GPCRs function as molecular coincidence detectors that couple proton (H+) binding to receptor activity. My results demonstrate that the majority of human GPCRs exhibit signaling that is negatively regulated by acidic pH, and that all modes of GPCR pharmacology (efficacy, potency, cooperativity, and antagonism) can be modulated by pH. Together, these results establish a new paradigm for GPCR biology and pharmacology in acidic environments.