Secretin Receptor Structure, Function, and Regulation

描述(申请人提供)：分泌素受体是重要的B类G蛋白偶联受体家族的原型。这项工作的长期目标是更好地了解这组受体的结构、功能和调节，获得有助于开发能够作用于这些靶点的新治疗策略和新药的见解。这些项目旨在测试、扩展和完善最近提出的天然激动剂占据的分泌素受体的分子模型，并阐明通过质膜内的寡聚作用激活和调节受体的分子基础。这项提议有三个广泛的目标。第一个目的是探索这样的假设，即分泌素受体的氨基末端区域提供了一个关键的配体结合口袋，该口袋在与天然多肽激动剂结合时经历构象重排。这将通过使用一系列激动剂和拮抗剂探针对受体内的特定位置进行光亲和标记，通过在激动剂和拮抗剂探针中开发和应用荧光指示剂，以及通过应用荧光共振能量转移技术来进行研究。第二个目的是探索激活信号从受体氨基末端传递到受体体内的分子机制，检验新的假设，即受体序列中存在内源性激动剂，该受体序列与激动剂结合后暴露出来。这将通过电荷修饰配体的光亲和标记、定点受体突变和使用合成候选分子的生物活性研究来检验。第三个目的是研究分泌素受体寡聚的分子基础和功能重要性，以此作为调节健康和疾病中促胰液素受体的一种机制。这将用生物发光共振能量转移和改良的受体结构来检验，研究对功能和受体关联的影响。除了野生型受体，最近在各种肿瘤中描述的具有显性负抑制活性的促胰液素受体的错配变体也将被研究。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。

DESCRIPTION (provided by applicant): The secretin receptor is prototypic of the important Class B family of G protein-coupled receptors. The long-term goal of this work is to better understand the structure, function, and regulation of this group of receptors, gaining insights that will facilitate the development of new therapeutic strategies and new drugs that can act at these targets. The projects are designed to test, extend, and refine the recently proposed molecular model of the natural agonist-occupied secretin receptor and to elucidate the molecular basis of receptor activation and receptor regulation by oligomerization within the plasma membrane. There are three broad aims for this proposal. The first aim is designed to explore the hypothesis that the amino-terminal domain of the secretin receptor provides a critical ligand-binding pocket that undergoes a conformational rearrangement upon binding the natural peptide agonist. This will be investigated by photoaffinity labeling specific sites within the receptor using series of agonist and antagonist probes, by developing and applying fluorescent indicators within agonist and antagonist probes, and by the application of fluorescence resonance energy transfer techniques. The second aim is designed to explore the molecular mechanism of transduction of the activation signal from the receptor amino terminus to the receptor body, examining the novel hypothesis predicting the presence of an endogenous agonist within the receptor sequence that is exposed upon agonist binding. This will be examined by photoaffinity labeling with charge-modified ligands, site-directed receptor mutagenesis, and biological activity studies using synthetic candidate molecules. The third aim is designed to examine the molecular basis and functional importance of secretin receptor oligomerization as a mechanism to regulate the secretin receptor in health and disease. This will be examined using bioluminescence resonance energy transfer with modified receptor constructs, studying impact on function and receptor association. In addition to wild type receptor, a misspliced variant of the secretin receptor recently described in various neoplasms that has dominant negative inhibitory activity will also be studied. 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.