PROJECT SUMMARY Adhesion G protein-coupled receptors (aGPCR) are important regulators of conserved developmental processes associated with various diseases - especially cancers and represent potential targets for drug discovery. The aGPCR B3 (ADGRB3) is one of the listed targets eligible for this FOA. Identified by the Pharos database as a high-value therapeutic target with broad applicability, the multi-domain architecture of ADGRB3, its expression in multiple tissues and cell types, and its interaction partners strongly suggest a pleiotropy of function. However, as an understudied receptor, ADRGB3’s activation mechanism, impact on cell physiology, and signaling pathway remains largely unknown. More specifically, it is unknown whether ADGRB3 is allosterically regulated via ligand or interdomain interactions in extracellular N-terminal fragment (NTF), leading to ADGRB3 activation via the Stachel tethered agonist. Central to our research program is ADGRB3 in the brain, where it is known to change neuron morphology and synapse health. Our laboratory has studied ADGRB3 and its ligands, the synapse organizing C1QL proteins, in the context of synapse adhesion and neuronal morphological changes. We published the first crystal structures of C1QLs and expanded the synaptic interaction network of ADGRB3 by showing that C1QL3 mediates a quaternary interaction between ADGRB3 and neuronal pentraxins. Our rationale is that the biophysical understanding of ligand C1QL and NTF ADGRB3 interactions offers a unique opportunity to visualize their chemistry and conformation, providing first insights into the ADGRB3 allosteric mechanism of activation. This proposal aims to determine complex structures between C1QL ligands and various NTF ADGRB3 constructs, decipher their oligomeric states, examine NTF interdomain interactions, and test ADGRB3 impact on cell morphology. Our strategy is innovative because it will provide a deep biophysical understanding that can be directly probed in a cellular context. The proposed research is significant because it gives a solid foundation for our longer-term goal of designing agonists or antagonists to target the NTF ADGRB3 binding interface. The results will have an immediate positive impact as they directly address critical gaps in our understanding of ADGRB3 and provide a generalizable molecular approach. Our results and techniques will apply to developing therapeutics for diseases linked to ADBRB3. Thus, it has enormous potential to generally advance biophysical investigations and pharmaceutical manipulation of a class of signaling proteins necessary for human health.

PROJECT NARRATIVE This proposal is relevant to the Common Fund Program "Illuminating the Druggable Genome" through its focus on revealing the structure and function of the understudied G protein-coupled receptor (GPCR) adhesion GPCR B3 (ADGRB3) and its endogenous ligands. ADGRB3 is linked to multiple diseases, including cancer and psychiatric conditions, but its mechanisms of action are not sufficiently understood. The proposed set of experiments will therefore provide essential information for future work by us and others to develop therapeutics for the many diseases linked to ADGRB3.