Secretin Receptor Structure, Function and Regulation

描述(申请人提供)：分泌素受体是B家族G蛋白偶联受体的原型，这组受体包含几个重要的潜在药物靶点。这项工作的长期目标是更好地了解这些受体的结构、功能和调节，获得有助于开发新的治疗策略和新药的见解，这些新药可以作用于这些分子中的各种靶点。建议的项目旨在测试、扩展和完善我们最近提出的天然激动剂占据的分泌素受体的分子模型，并阐明受体激活和调节的分子基础。这项提议有三个广泛的目标。第一个目标是获得完整的天然激动剂占据的分泌素受体的全球结构的详细结构洞察，为配体对接和从氨基末端结构域到核心螺旋束结构域的构象变化的传播提供分子基础。这将使用定向光亲和标记，包括新的双可光敏探针，以及使用可访问表面的免疫学探针，并测试具有靶向受体突变的进化分子模型来进行研究。第二个目的是研究促胰液素受体的胞浆表面，重点是与G蛋白偶联的分子决定因素，G蛋白偶联到该受体的生理四聚体结构的构型，以及这对受体功能的影响。这将通过受体突变选择性地破坏G蛋白偶联来进行研究，通过与相关区域的多肽竞争来确认结构决定因素。参与激动剂对接和G蛋白偶联的原型分子的光亲和标记将被用来探索与受体同源二聚体相关的亲和状态的四级结构。第三个目标是探索开发受体活性药物的潜在策略，包括激动剂、拮抗剂和变构调节剂。这将利用生化、分子生物学和药理学方法进行研究，以深入了解正构激动剂和拮抗剂的详细结构决定因素，并确定内源性激动剂序列在受体氨基末端可能的变构作用部位。总之，这些努力应该提供最好的分子细节，以了解该受体家族中任何受体的配体结合、激活和调节的结构和机制。与公共卫生相关：该项目旨在阐明分泌素受体的天然配体结合、激活、信号启动和调节的结构基础，分泌素受体是非常重要的G蛋白偶联受体B家族的原型成员。这包括生化、分子生物学、生物物理学和细胞生物学技术。这些见解将被用来启动合理的方法来开发作用于这些受体的激动剂、拮抗剂和变构调节剂。

DESCRIPTION (provided by applicant): The secretin receptor is prototypic of Family B G protein-coupled receptors, a group containing several important potential drug targets. The long-term goal of this work is to better understand the structure, function, and regulation of these receptors, gaining insights that will facilitate the development of new therapeutic strategies and new drugs that can act at a variety of targets within these molecules. The proposed projects are designed to test, extend, and refine our recently-proposed molecular model of the natural agonist-occupied secretin receptor and to elucidate the molecular basis of receptor activation and regulation. There are three broad aims for this proposal. The first aim is designed to acquire detailed structural insights into the global structure of the intact natural agonist-occupied secretin receptor, providing insights into the molecular basis for ligand docking and propagation of conformational changes from the amino-terminal domain to the core helical bundle domain. This will be investigated using directed photoaffinity labeling, including novel dual photolabile probes, as well as using immunological probes of accessible surfaces, and testing the evolving molecular models with targeted receptor mutagenesis. The second aim is designed to examine the cytosolic face of the secretin receptor, focusing on molecular determinants of coupling with G proteins, the configuration of G protein coupling to the physiologic quaternary dimeric structure of this receptor, and the impact of this on receptor function. This will be investigated using receptor mutagenesis to selectively disrupt G protein coupling, confirming the structural determinants by competition with peptides from the relevant regions. Photoaffinity labeling of the protomers involved in agonist docking and the G protein coupling will be used to explore the quaternary structure of affinity states associated with receptor homo-dimers. The third aim is designed to explore potential strategies for the development of receptor-active drugs, including agonists, antagonists, and allosteric modulators. This will be investigated using biochemical, molecular biological and pharmacological approaches to gain insights into the detailed structural determinants for orthosteric agonists and antagonists, as well as to define the possible allosteric site of action of the endogenous agonist sequence within the receptor amino terminus. Together, these efforts should provide the finest level of molecular detail available for understanding the structure and mechanisms of ligand binding, activation, and regulation of any receptor in this receptor family. PUBLIC HEALTH RELEVANCE: This project is directed to elucidate the structural basis for natural ligand binding, activation, initiation of signaling, and regulation of the secretin receptor, a prototypic member of the very important Family B group of G protein-coupled receptors. This includes biochemical, molecular biological, biophysical, and cell biological techniques. These insights will be utilized to initiate rational approaches toward the development of agonists, antagonists, and allosteric modulators acting at these receptors.