Structural and functional characterization of synaptic adhesion GPCR ADGRB3 binding interactions

摘要 粘附GPCR（aGPCR）代表了GPCR的主要类别，并且特征在于存在粘附分子。 与典型的GPCR 7 TM连接的细胞外粘附区和膜近端GAIN结构域 和细胞质区域。aGPCR在细胞粘附和迁移、突触发生等方面具有重要作用 和豁免权最近的结构研究对它们在细胞水平上的激活产生了相当多的见解。 7 TM和细胞质区域，显示存在于茎区stachel肽插入到 跨膜结构域在活化过程中驱动构象变化。相反，粘合剂 膜远端粘合剂区域的相互作用的特征仍然很差，特别是， 这些相互作用引起Stachel序列释放以激活的结构机制是 未知该建议旨在从结构上表征粘合剂相互作用和活化机制 ADGRB 3是aGPCR家族的一员，在神经元突触和心肌中发现， 对于突触发生、突触可塑性、突触消除和成肌细胞融合至关重要。我们会委聘 基于结构的方法，包括ADGRB 3及其粘合剂的cryo-EM结构测定 通过功能测定补充复合物，以将结构见解与受体活化联系起来。要求1 集中于完整ADGRB 3胞外域及其粘附片段的结构，使用组合 包括冷冻电镜、X射线晶体学和小角X射线散射。这一目标的成功实现 将提供对ADGRB 3粘附区域的组织及其与膜的偶联的深入了解 近端激活区。目的2集中于表征ADGRB 3粘附性结合相互作用， 使用生物物理和体外结合测定来描述结合 区域和复杂的组成和化学计量。定义的粘合剂复合物的结构将是 使用冷冻EM和X射线晶体学确定，脂质膜之间形成的复合物将 通过cryo-ET可视化，以检查它们在接近天然状态下的组织和组装。激活 通过粘附配体结合的ADGRB 3将在功能测定和突变靶向的影响中进行评估。 将测试已确定的界面和关键结构残留物。成功实现这一目标将为 直接洞察ADGRB 3粘附和aGPCR信号传导的激活。总体而言，该提案旨在 提供了基本的机制见解，可以确定新的治疗目标。

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.