Structural and functional characterization of synaptic adhesion GPCR ADGRB3 binding interactions

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ABSTRACT Adhesion GPCRs (aGPCRs) represent a major class of GPCRs and are characterized by the presence of extracellular adhesive regions and membrane proximal GAIN domains that are linked to typical GPCR 7TM and cytoplasmic regions. aGPCRs have important roles including cell adhesion and migration, synaptogenesis and immunity. Recent structural studies have yielded considerable insights into their activation at the level of the 7TM and cytoplasmic regions, showing that insertion of a stachel peptide present in the stalk region into the transmembrane domains drives conformational changes during activation. In contrast, adhesive interactions of the membrane distal adhesive regions remain poorly characterized and, in particular, the structural mechanisms by which these interactions cause release of the stachel sequence for activation are unknown. This proposal aims to structurally characterize the adhesive interactions and activation mechanism of ADGRB3, a member of an aGPCR family that is found at neuronal synapses and in heart muscle that is critical for synaptogenesis, synaptic plasticity, synapse elimination and myoblast fusion. We will employ structure-based approaches including cryo-EM structure determination of ADGRB3 and its adhesive complexes complemented by functional assays to relate structural insights to receptor activation. Aim 1 focuses on the structure of the complete ADGRB3 ectodomain and its adhesive fragments using a combination of structural approaches including cryo-EM, x-ray crystallography, and SAXS. Successful outcome of this aim would provide insights into the organization of the ADGRB3 adhesive region and its coupling to the membrane proximal activation regions. Aim 2 focuses on characterization of ADGRB3 adhesive binding interactions with previously identified and candidate ligands using biophysical and in vitro binding assays to delineate binding regions and complex compositions and stoichiometries. Structures of defined adhesive complexes will be determined using cryo-EM and x-ray crystallography and complexes formed between lipid membranes will be visualized by cryo-ET to examine their organization and assembly in a near-native state. Activation of ADGRB3 by adhesive ligand binding will be assessed in functional assays and effects of mutations targeting identified interfaces and key structural residues will be tested. Successful completion of this aim will provide direct insights into ADGRB3 adhesion and activation of aGPCR signaling. Overall, the proposal aims to provide fundamental mechanistic insights that could identify new targets for therapeutics.