Secretin Receptor Structure, Function, and Regulation

描述（由申请人提供）：促胰液素受体是G蛋白偶联受体的重要B类家族的原型。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，获得有助于开发新的治疗策略和可以作用于这些靶点的新药的见解。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促胰液素受体的分子模型，并阐明受体激活和受体调节的分子基础，通过质膜内的寡聚化。这项建议有三大目标。第一个目的是探索的假设，分泌素受体的氨基末端结构域提供了一个关键的配体结合口袋，在结合天然肽激动剂进行构象重排。这将通过使用一系列激动剂和拮抗剂探针光亲和标记受体内的特定位点，通过开发和应用激动剂和拮抗剂探针内的荧光指示剂，以及通过应用荧光共振能量转移技术来研究。第二个目的是探索从受体氨基末端到受体体的活化信号转导的分子机制，检查预测在激动剂结合后暴露的受体序列内存在内源性激动剂的新假设。这将通过电荷修饰配体的光亲和标记、定点受体诱变和使用合成候选分子的生物活性研究来检查。第三个目的是研究分泌素受体寡聚化的分子基础和功能重要性，作为一种机制，以调节分泌素受体在健康和疾病。这将使用生物发光共振能量转移与修饰的受体结构进行检查，研究对功能和受体缔合的影响。除野生型受体外，还将研究最近在各种肿瘤中描述的分泌素受体的错误剪接变体，其具有显性负抑制活性。总之，这些努力应该提供最精细的分子细节水平，可用于理解配体结合，激活和调节该受体家族中任何受体的结构和机制。

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.