Molecular basis of activation of the prototypic class B G protein-coupled secretin receptor

项目摘要/摘要 我们的总体目标是阐明B类GPCRs激活的分子基础，使用 以原型分泌素受体(Secr)为模型。洞察力将填补知识中的关键空白，并促进最终 开发具有不同活性特征的药物。B类GPCR包括下列既定目标 治疗糖尿病、肥胖症、骨质疏松症、偏头痛、焦虑和抑郁。然而，治疗学仍然存在 次优，而且受体类对小分子、口服活性药物的开发一直难以奏效， 至少部分是由于缺乏对受体所需的结构和功能动力学的了解 激活。我们现在对这两个方面都有了新的、独特的见解，包括 相关的、全长的、G蛋白偶联的全息受体，强调了 受体N端胞外区(ECD)和跨膜糖蛋白结构域核心在配体结合和 受体激活。组件的目的是为了理解这一构象动力学 用于激动剂结合和受体激活的接口，并利用我们在使用单粒子冷冻方面的突破- EM为这项工作提供一个结构框架。目的1，阐明与分泌素有关的分子事件 多肽与其受体核心的结合，以及其活性的关键决定因素，检验了以下假设 ECD和核心域的定向(和相互作用)在指导和定位 作用部位附近的正构体激动剂药效团。我们将使用半胱氨酸捕获来探索这个基因座 比较同样应用于野生型受体的类似非活性和活性探针的空间近似性 作为二聚化缺陷的受体构建。合理的结合构效分析和广泛的应用范围 还将对激动剂药效团的生物活性进行评估，结果用于提供 对激活和效应器专一性的决定因素的洞察。目的2，研究相对定向和相对定向 Secr ECD和CORE之间的交互，探索这种相互关系的功能含义，测试 假设这些区域可以以不同的方式相互作用，从而影响静止和激活状态。这 将通过受体突变来改变结构域相互作用，建立结构域-结构域 通过将半胱氨酸结合在受体核心的顶部和ECD的底部以及 使用受体氨基末端预测表面的免疫探针来确定通路和被 用于共振转移技术。目的3，阐明Secr非活性和活性/全息结构 粒子冷冻-EM，测试将生化和功能数据映射到高分辨率的假设 三维结构，包括激动剂-受体-异三聚体G蛋白和非活性拮抗剂占据的受体， 将有助于阐明受体信号传导的分子基础。总之，这项工作将提供基本的 了解B类gpr结构和功能的进展，对药物有很高的帮助 针对这些受体的开发。

PROJECT SUMMARY/ABSTRACT Our OVERALL OBJECTIVE is to elucidate the molecular basis of activation of class B GPCRs, using the prototypic secretin receptor (SecR) as a model. Insights will fill key gaps in knowledge and facilitate ultimate development of drugs exhibiting various activity profiles. Class B GPCRs include established targets for treatment of diabetes, obesity, osteoporosis, migraine, anxiety, and depression. However, therapeutics remain suboptimal, and the receptor class has been refractory to development of small molecule, orally active drugs, at least in part, due to lack of understanding of the structure and functional dynamics required for receptor activation. We now have novel, unique insights into both of these, including high-resolution structures of related, full-length, G protein-coupled holoreceptors, that highlight the importance of the interface between the receptor N-terminal extracellular domain (ECD) and the transmembane domain core in ligand binding and receptor activation. Component aims are directed toward understanding the conformational dynamics of this interface for agonist binding and receptor activation, and using our breakthroughs in use of single particle cryo- EM to provide a structural framework for this work. Aim 1, elucidates molecular events involved in secretin peptide engagement with its receptor core, and key determinants for its activity, testing the hypothesis that orientation (and interaction) of ECD and core domains plays a critical role in directing and positioning the orthosteric agonist pharmacophore near its site of action. We will explore this locus using cysteine trapping to compare spatial approximations for analogous inactive and active probes, applied to wild type receptor, as well as dimerization-deficient receptor constructs. Rational structure-activity analysis for binding and a broad range of biological activities of the agonist pharmacophore will also be performed, with results used to provide insights into determinants of activation and effector specificity. Aim 2, investigates the relative orientations and interactions between SecR ECD and core, exploring functional implications of this interrelationship, testing the hypothesis that these domains can interact in various ways that affect states of quiescence and activation. This will be approached by receptor mutagenesis to modify domain interactions, establishment of domain-domain disulfide bonds by incorporating cysteines at the top of the receptor core and bottom of the ECD, as well as using immunologic probes of predicted surfaces of the receptor amino terminus to determine access and to be used in resonance transfer techniques. Aim 3, elucidates SecR inactive and active/holostructure using single particle cryo-EM, testing the hypothesis that mapping of biochemical and functional data onto high resolution 3D structures, including agonist-receptor-heterotrimeric G protein and inactive antagonist-occupied receptor, will help to elucidate the molecular basis for receptor signaling. Together, this work will provide fundamental advances in understanding of class B GPCR structure and function, with insights highly useful in drug development targeting these receptors.