Structural and functional characterization of synaptic adhesion GPCR ADGRB3 binding interactions

摘要 粘附性GPCRs(AGPCRs)代表GPCRs的一大类，其特征是存在 与典型的GPCR7TM连锁的细胞外粘附区和膜近端增益域 和细胞质区域。AGPCRs在细胞黏附和迁移、突触形成等方面具有重要作用 和豁免权。最近的结构研究已经对它们在水平上的激活产生了相当大的见解 7TM和细胞质区域，显示存在于茎区域Stachel肽插入到 跨膜结构域在激活过程中驱动构象变化。相比之下，粘合剂 膜远端粘附区的相互作用仍未得到很好的描述，尤其是 这些相互作用导致Stachel序列释放以供激活的结构机制是 未知。这项建议旨在从结构上表征粘结剂的相互作用和激活机制 ADGRB3，aGPCR3家族的成员，发现于神经元突触和心肌中， 对突触发生、突触可塑性、突触消除和成肌细胞融合至关重要。我们将聘用 基于结构的确定ADGRB3及其胶粘剂结构的低温电子显微镜方法 与功能分析相补充的复合体，将结构洞察力与受体激活联系起来。目标1 重点研究了完整的ADGRB3胞外区的结构及其结合使用的粘附性片段 结构方法包括低温电子显微镜、X射线结晶学和小角X射线光电子能谱。这一目标的成功实现 将提供对ADGRB3粘附区的组织及其与膜的偶联的深入了解 近端激活区。目标2重点描述ADGRB3胶粘剂结合与 使用生物物理和体外结合试验来描绘结合的先前确定的和候选的配体 区域和复杂的成分和化学计量。定义的粘合剂复合体的结构如下 用低温电子显微镜和X射线结晶学测定，脂膜之间形成的络合物将是 由CRYO-ET可视化，以检查它们在接近自然状态下的组织和组装。激活 通过粘合配体结合的ADGRB3将在功能分析和突变靶向的效果中进行评估 将测试已确定的界面和关键结构残留物。成功完成这一目标将提供 直接洞察ADGRB3黏附和激活aGPCR信号。总体而言，该提案旨在 提供基本的机械洞察，可以确定治疗的新靶点。

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.