Illuminating Druggable Targets via Interrogation of Direct GPCR-kinase Interactions G protein-coupled receptors (GPCRs) and kinases represent two of the most highly druggable classes of membrane proteins, serving as targets for over one-third of FDA-approved drugs. Nevertheless, both of these protein superfamilies remain therapeutically underexploited, as many GPCRs and kinases still lack clinically useful ligands. A significant number of these understudied GPCRs and kinases may communicate with one another via noncanonical mechanisms not captured by existing functional assays. Our goal is to identify these noncanonical mechanisms, which will not only teach us how understudied GPCRs affect cell physiology, but also help us develop new assays to identify therapeutically beneficial ligands. We have discovered a new GPCR- kinase communication mechanism that does not conform to traditional signaling paradigms in the GPCR field. Instead, active GPCRs directly bind to and sequesters protein kinase A catalytic ɑ (PKA-Cɑ) subunits at the membrane, blocking phosphorylation of soluble PKA substrates to ultimately affect a plethora of cellular signaling processes. We hypothesize that a significant number of understudied GPCRs in the IDG portfolio control their respective intracellular signaling mechanisms via sequestration of PKA-C. This new idea likely applies not just to the heavily studied PKA-C isoform, but also to the understudied PKA-C and PKA-C isoforms that are designated as IDG targets and implicated in cancer and metabolic disorders. In this R03 grant we critically evaluate the above hypothesis, in the process developing concepts and generating reagents to interrogate understudied GPCRs and kinases more generally. Our strategy is to design a high-throughput assay for GPCR / PKA-C interactions, and then use this assay to systematically evaluate interactions between the GPCRs and/or PKA-C isoforms in IDG’s portfolio. By identifying and characterizing GPCR / PKA-C interactions on a broad scale, our work will reveal the extent to which GPCR / PKA-C interactions represent a general theme in receptor biology. The proposed studies will provide a foundation for at least one future R01 grant focusing on the specific GPCR / PKA-C interactions we identify, particularly those involved in developmental and cancer biology, nervous system function, and the biology of motile and primary cilia. The proposed research capitalizes on existing IDG- generated resources, including the PRESTO-Tango system and nanoBRET kinase target engagement assays, both of which we can use following relatively minor modifications, along with the AMIS database. The availability of these key reagents, combined with our expertise in cultured cell functional assays, will allow us to complete the project within one year. Our work will establish a new paradigm to understand and therapeutically target understudied GPCRs and kinases. This will provide major insights into these proteins’ biological functions and regulatory mechanisms and enable efforts to identify drugs that modulate these interactions. Given the ubiquity of GPCRs and PKA in biology, our studies are likely to be relevant to many aspects of human health and disease.

PROJECT NARRATIVE: Many of the drugs in the modern pharmacopeia target G protein-coupled receptors (GPCRs) or kinases, yet a large number of these proteins have yet to be targeted successfully for therapeutic purposes. By testing a new hypothesis that many such “dark” GPCRs and kinases communicate directly with one another, our research will increase our understanding of how GPCRs and kinases function within cells, and provide new concepts and tools to identify drugs that target these proteins. We expect our findings to have implications for cancer, metabolic disorders, and a range of other human diseases.