SECRETIN RECEPTOR STRUCTURE, FUNCTION, AND REGULATION

描述（改编自申请人的摘要）： 工作是扩大对促胰液素家族受体的理解， 作用于这些受体的药物的发展。该提案的重点是 促胰液素受体的结构表征， 针对VIP受体。指导这项工作的一般假设是 详细了解了原型分泌素受体的结构， 并进入其配体结合和激活的分子机制， 为合理发展战略提供重要线索， 受体活性药物目的1是为了探讨假设， 该受体的氨基末端结构域，具有保守的半胱氨酸残基 和二硫键，折叠成独特的稳定结构。这将是 使用生物化学、分子生物学和细胞生物学方法进行研究， 导致定义可用作特定键的方法 分子建模的约束条件。目标2旨在检验假设 分泌素受体的氨基末端结构域提供了一个关键的 天然肽配体的结合口袋。研究将使用 互补的亲和标记方法，具有一系列独特的 光不稳定探针和受体诱变以确定分子近似 在配体和受体内的残基之间。Aim 3旨在测试 分泌素家族受体被拴系的 转导机制，在结构和功能上独立 受体结构域以独特的方式相互作用。本系列的实验重点 的研究将转移到受体的体内。突变将是 在家族内保守的残基上进行， 建筑的支持作用，以及对残留物，可以与 受体结合和激活的选择性。最小 将确定与组成型活性一致的受体结构 使用一系列截短突变体。此外，一种新颖的反向模式 用特异性结合到所述抗体中的光不稳定探针进行亲和标记。 接收器将执行。这将直接探索分子接近 在受体内不同的区域之间。

DESCRIPTION (Adapted from the applicant's abstract): The long-term goal of the work is to expand understanding of secretin family receptors and to facilitate the development of drugs acting at these receptors. This proposal focuses on structural characterization of the secretin receptor with selected studies also directed toward the VIP receptor. The general hypothesis guiding this work is that detailed insights into the structure of the prototypic secretin receptor and into the molecular mechanisms of its ligand-binding and activation will provide important leads to strategies for the rational development of receptor-active drugs. Aim 1 is designed to explore the hypothesis that the amino-terminal domain of this receptor, with its conserved cysteine residues and disulfide bonds, folds into a unique stable structure. This will be investigated using biochemical, molecular biological, and cell biological approaches leading to the definition of specific bonds that can be used as constraints for molecular modeling. Aim 2 is designed to test the hypothesis that the amino-terminal domain of the secretin receptor provides a critical binding pocket for the natural peptide ligand. Studies will use the complementary approaches of affinity labeling with a series of unique photolabile probes and receptor mutagenesis to define molecular approximations between residues within the ligand and the receptor. Aim 3 is designed to test the hypothesis that secretin family receptors are activated by a tethered transduction mechanism in which structurally and functionally independent receptor domains interact in a unique way. Experimental focus for this series of studies will be shifted to the body of the receptor. Mutagenesis will be performed on residues that are conserved within the family and likely play an architectural support role, as well as on residues that can be correlated with the selectivity of binding and activation of the receptors. The minimal receptor structure consistent with constitutive activity will be determined using a series of truncation mutants. Additionally, a novel mode of reverse affinity labeling with a photolabile probe incorporated specifically into the receptor will be performed. This will directly explore molecular proximity between distinct domains within the receptor.