SUMMARY/ABSTRACT ADGRB3 (a.k.a. BAI3) is a G protein-coupled receptor (GPCR) that is predominantly expressed in brain and is a member of a poorly understood class of GPCRs known as adhesion GPCRs. Mutations and SNPs in ADGRB3 are linked to both neuropsychiatric diseases and cancers, suggesting this gene is a high value therapeutic target. However, no mechanistic explanations regarding ADGRB3 function have been proposed to explain these genetic associations. Previous work has identified the C1QL secreted family of proteins as the high-affinity extracellular ligands for ADGRB3. The signals induced by this ligand-GPCR binding are unknown. The long-term goal is to discover the functions of ADGRB3-C1QL interactions in the brain, then to use this knowledge for therapeutic gain. Although gene expression patterns of Adgrb3 and the C1ql family have been described in mice, their protein subcellular localizations are almost entirely unknown. In order to generate testable hypotheses regarding their function, the exact locations of ADGRB3-C1QL interaction need to be determined. Therefore, the objective of this application is to determine the subcellular localization of ADGRB3 and an associated C1QL ligand. Preliminary studies identify two possible and completely novel cellular contexts where ADGRB3 and a C1QL may co-localize, which will be investigated in greater detail in this proposal. The hypothesis is that ADGRB3 will be expressed by neurons and localized to the synaptic cleft, and will bind to C1QL1 expressed from distinct adjacent cells. The proposed research is innovative because it will 1) use cutting edge CRISPR/Cas9 technology to facilitate immunolocalization and 2) be the first to thoroughly characterize the subcellular locations of these proteins. Each cellular context investigated will feature virus-mediated introduction of CRISPR components resulting in the knockin of epitope tags allowing precise tracking of subcellular localization of ADGRB3 and its C1QL1 ligand, which will reveal critical spatiotemporal details about ADGRB3 and establish tools and a pathway for further functional study. This research is significant because it will form the etiological and biochemical foundation for subsequent research on disorders of ADGRB3 signaling, and demonstrate which cellular contexts/diseases might benefit from a small molecule drug targeting the ADGRB3-C1QL1 binding interface. It will also provide prerequisite data for future grant applications. Future research will focus on demonstrating the functional consequences of these receptor-ligand interactions in their respective cellular contexts, and fully elucidating ADGRB3 signaling mechanisms (which is expected to be generalizable). The longer-term goal is to design agonists or antagonists to target the ADGRB3-C1QL1 binding interface as a potential therapy related to at least one of the cellular contexts where ADGRB3 is present.

NARRATIVE The proposed research is relevant to the Common Fund Program "Illuminating the Druggable Genome" because it focuses on revealing the function of the understudied G protein-coupled receptor (GPCR) called adhesion GPCR B3 (ADGRB3), and its endogenous ligands. The proposed research is relevant to public health because it will identify novel ligand/receptor interactions as potential drug targets for therapeutic treatment of diseases of brain function.