SECRETIN RECEPTOR STRUCTURE, FUNCTION, AND REGULATION

描述(改编自申请者的摘要)： 工作是扩大对分泌素家族受体的理解，并促进 作用于这些受体的药物的发展。这项提案的重点是 促胰液素受体的结构特征及其精选研究 指向VIP受体。指导这项工作的一般假设是 对原型分泌素受体结构的详细洞察 并探讨了其配体结合和激活的分子机制 为合理发展战略提供重要指导 受体活性药物。目标1旨在探索这样一个假设，即 该受体的氨基末端结构域及其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 使用生化、分子生物学和细胞生物学进行研究 导致定义可用作以下用途的特定债券的方法 分子建模的约束条件。《目标2》旨在检验这一假设 促胰液素受体的氨基末端区域提供了一个关键的 天然多肽配体的结合袋。研究将使用 一系列独特的亲和标记互补方法 光敏探针和受体突变来确定分子近似 在配体和受体内的残基之间。AIM 3的设计目的是测试 促胰液素家族受体被拴系激活的假说 结构和功能独立的转导机制 受体结构域以一种独特的方式相互作用。本系列的实验重点 研究的重点将转移到受体的体内。诱变将是 在家族内保守的残基上执行，并可能发挥作用 架构支持角色，以及可以关联的残留物 受体结合和激活的选择性。最小的 将确定与构效活性一致的受体结构 使用一系列截断突变体。此外，一种新的反转模式 亲和标记与亲和标记的光敏探针 将进行受体检查。这将直接探索分子的近似性。 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.