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

项目总结/摘要 我们的总体目标是阐明B类GPCR激活的分子基础，使用 原型分泌素受体（SecR）作为模型。洞察力将填补知识的关键空白， 开发具有各种活性特征的药物。B类GPCR包括以下目标的确定目标： 治疗糖尿病、肥胖症、骨质疏松症、偏头痛、焦虑症和抑郁症。然而，治疗方法仍然存在 次优的，并且受体类对于小分子口服活性药物的开发是难治的， 至少部分是由于缺乏对受体所需的结构和功能动力学的理解， activation.我们现在对这两个方面都有了新颖独特的见解，包括高分辨率的结构， 相关的，全长，G蛋白偶联全息受体，突出了界面之间的重要性， 受体N-末端胞外结构域（ECD）和跨膜结构域核心在配体结合中的作用， 受体激活组件的目的是为了理解这一构象动力学 激动剂结合和受体活化的界面，并利用我们在单颗粒冷冻- EM为这项工作提供了一个结构框架。目的1，阐明促胰液素参与的分子事件 肽与其受体核心的接合，以及其活性的关键决定因素，检验了以下假设： ECD和核心结构域的方向（和相互作用）在引导和定位细胞中起着关键作用。 正构激动剂药效团靠近其作用位点。我们将使用半胱氨酸捕获来探索这个位点， 比较类似的非活性和活性探针的空间近似值，也适用于野生型受体 作为二聚化缺陷型受体构建体。合理的结合结构-活性分析和广泛的 还将进行激动剂药效团的生物活性的测定，结果用于提供 深入了解激活和效应特异性的决定因素。目的2，研究相对取向和 SecR ECD和核心之间的相互作用，探索这种相互关系的功能含义，测试 假设这些域可以以各种方式相互作用，影响静止和激活状态。这 将通过受体诱变来修饰结构域相互作用，建立结构域-结构域 通过在受体核心的顶部和ECD的底部掺入半胱氨酸，以及 使用受体氨基末端的预测表面的免疫学探针来确定通路， 用于共振转移技术。目的3，利用单克隆抗体阐明SecR的非活性和活性/全结构 粒子冷冻EM，测试的假设，映射的生化和功能数据到高分辨率 3D结构，包括激动剂-受体-异源三聚体G蛋白和无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 对B类GPCR结构和功能的理解进展，对药物治疗非常有用 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.