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

原型 B 类 G 蛋白偶联促胰液素受体激活的分子基础

• 批准号: 10238892
• 负责人: LAURENCE J MILLER
• 金额: $ 37.23万
• 依托单位: MAYO CLINIC ARIZONA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10238892
• 关键词: Address; Affect; Affinity; Agonist; Antibodies; Anxiety; Binding; Biochemical; Biological; Biological Assay; Cardiovascular Diseases; Cardiovascular Physiology; Cholestasis; Complement; Complex; Coupled; Cryoelectron Microscopy; Cyclic AMP; Cysteine; Data; Dependence; Development; Diabetes Mellitus; Dimerization; Distal; Docking; Epitopes; Event; Exhibits; Extracellular Domain; Family member; Fluorescence Resonance Energy Transfer; Funding Mechanisms; G-Protein-Coupled Receptors; GTP-Binding Proteins; Global Change; Goals; Grant; Heterotrimeric GTP-Binding Proteins; Immunologics; Investigation; Knowledge; Length; Ligand Binding; Ligands; Maps; Mental Depression; Metabolic; Metabolic syndrome; Methodology; Migraine; Modeling; Molecular; Molecular Conformation; Mutagenesis; N-terminal; Nature; Obesity; Oral; Osteoporosis; Pathway interactions; Peptides; Pharmaceutical Preparations; Photoaffinity Labels; Play; Positioning Attribute; Production; Proteins; Pruritus; Receptor Activation; Receptor Signaling; Refractory; Resolution; Role; Secretin; Series; Signal Pathway; Signal Transduction; Site; Specificity; Structure; Surface; Techniques; Testing; Therapeutic; Variant; Work; analog; design; disulfide bond; drug development; experience; gastrointestinal; insight; novel; particle; pharmacophore; receptor; receptor function; receptor structure function; secretin receptor; small molecule; three dimensional structure; tool

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.

项目概要/摘要 我们的总体目标是利用以下方法阐明 B 类 GPCR 激活的分子基础： 原型促胰液素受体（SecR）作为模型。见解将填补知识方面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 包括已建立的目标 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 次优，并且受体类别难以开发小分子、口服活性药物， 至少部分是由于缺乏对受体所需结构和功能动力学的了解 激活。我们现在对这两个问题有了新颖、独特的见解，包括高分辨率的结构 相关的全长 G 蛋白偶联全感受器，强调了 受体 N 末端胞外结构域 (ECD) 和配体结合中的跨膜结构域核心 受体激活。组件的目标是了解该组件的构象动力学 激动剂结合和受体激活的界面，并利用我们在使用单颗粒冷冻技术方面的突破 EM 为这项工作提供了一个结构框架。目标 1，阐明促胰液素涉及的分子事件 肽与其受体核心的结合，以及其活性的关键决定因素，检验以下假设： ECD 和核心领域的定位（和相互作用）在指导和定位 正位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，也应用于野生型受体 作为二聚化缺陷的受体构建体。结合和广泛范围的合理结构-活性分析 还将进行激动剂药效团的生物活性分析，结果用于提供 深入了解激活和效应器特异性的决定因素。目标 2，调查相对方向和 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 将通过受体诱变来改变结构域相互作用，建立结构域-结构域 通过在受体核心顶部和 ECD 底部掺入半胱氨酸来形成二硫键，以及 使用受体氨基末端预测表面的免疫探针来确定接近和 用于共振转移技术。目标 3，使用单一的方法阐明 SecR 非活性和活性/全结构 粒子冷冻电镜，测试将生化和功能数据映射到高分辨率的假设 3D结构，包括激动剂-受体-异三聚体G蛋白和失活的拮抗剂占据的受体， 将有助于阐明受体信号传导的分子基础。总之，这项工作将为 对 B 类 GPCR 结构和功能的理解取得进展，其见解在药物中非常有用 针对这些受体的开发。