PROJECT SUMMARY Coincident signals are an essential feature of cellular communication. By detecting simultaneous inputs, cells can filter signal from noise in complex chemical environments to mount proper responses. In many biological and pathological contexts, coincident pH signals regulate the activity of individual proteins and signaling networks. Examples include acidotic signals in maturing endosomes, inflammatory zones, synapses, and tumor microenvironments. Our long-term objective is to understand how these coincident pH signals regulate biology through different classes of cell surface receptors. We have developed a comprehensive and ambitious research program for studying proton-gated (H+-gated) coincidence detection by G protein-coupled receptors (GPCRs), the largest and most therapeutically targeted class of transmembrane receptors in humans. More than 800 GPCRs detect a rich diversity of inputs, including H+. Although a few pH-sensing GPCRs are activated by protons alone, we have shown that H+-gated coincidence detection is a far more common feature of GPCR regulation. In this mode of proton sensing, GPCR agonism and/or inhibition is concurrently modulated by pH. Our efforts to illuminate this context-dependent mechanism for controlling GPCR activity have led us to establish a new frontier in cell signaling biology that is likely relevant to all receptor classes. The broad objective of our proposed research program is to pursue an in-depth understanding of H+-gated signaling and pharmacology for a wide variety of GPCRs. By creating innovative wet-lab and computational technologies, developing cutting-edge cell models, and building large libraries of GPCRs in cell-based assay systems, our lab can extensively study the effects of pH on GPCR signaling. As such, we can profile ambitious numbers of GPCRs and ligands as a function of pH using our yeast based DCyFIR platform and human cell models. Our proposed program of research comprises three project areas that synergistically utilize these unique capabilities: H+-gated GPCR coincidence detection of metabolites and drugs, pH regulation of secreted peptide and protein sensing by GPCRs, and pH- intelligent nanobody research tools and therapeutic leads for GPCRs. Over the next five years, our goal is to illuminate the mechanisms by which H+-gated coincidence detection regulates the selectivity of endogenous and artificial agonists, inhibitors, modulators, approved drugs, and conformationally-selective nanobody probes for a sizeable fraction of the human GPCRome. We anticipate these efforts will enable us to both design and repurpose an array of therapeutic leads, exploratory probes, and pharmacological tools for selectively targeting, controlling, and studying GPCR signaling mechanisms at discrete physiologic pH values. As such, we anticipate our ambitious research program will establish a new paradigm for GPCR biology and pharmacology in acidotic scenarios.