SECRETIN RECEPTOR STRUCTURE, FUNCTION, AND REGULATION

为了更好地理解 胃肠肽激素分泌素在健康和疾病， 目前的建议侧重于结构，功能和监管， 胰泌素受体。分泌素很重要 在生理学上并且在发病机制中具有潜在的重要性， 胰腺疾病的诊断和治疗。我们的一般假设 结构和调节主题是否对分泌物起作用 受体将与大多数G蛋白的受体不同， 偶联受体，并将提供深入了解分子基础 外分泌胰腺的综合功能。我们建议遵循 分泌素受体分子的互补途径 以及调节受体功能的细胞机制。在 制备，胰腺分泌素受体cDNA已被克隆， 测序和表达;融合蛋白，其掺入了用于 重组受体的有效亲和纯化已经被 构建和验证;并且已经开发了新的放射性配体。 分泌素受体的cDNA序列已经建立了其封闭的 与VIP、PACAP、PTH和GHRF受体的关系，以及 缺乏大多数G蛋白的典型结构基序- 偶联受体预测的大而复杂的，富含半胱氨酸的 这些受体的胞外域有趣地补充了大的 其天然配体的药效学结构域。在这项工作中，我们将 表征该受体结构域的结构，假设 其在激动剂结合中的关键作用。这包括分析模式 二硫键和糖基化的功能影响， 每个.激动剂结合结构域将直接使用 用掺入光不稳定残基的新型探针进行亲和标记 “内在”和“跨越”药效团，并间接使用 包括缺失、受体嵌合体和 定点突变体受体磷酸化的影响将是 在模型重组受体携带细胞系统中研究，和 诱变方法将用于定义关键结构域， 残基最后，胰腺腺泡细胞上的分泌素受体， 一个完善的刺激活动耦合模型，将是 研究以确定原型天然细胞中的受体调节过程， 细胞环境理解细胞的结构和调节 促胰液素受体的主题应该具有广泛的意义， 这个新的重要受体家族，以及提供基础， 探索这种受体在正常生理学中的作用， 发展，以及疾病的发病机制。

With the long-term goal of better understanding the roles of the gastrointestinal peptide hormone secretin in health and disease, the current proposal focuses on structure, function, and regulation of the secretin receptor in the exocrine pancreas. Secretin is important physiologically and is potentially important in the pathogenesis, diagnosis, and treatment of pancreatic disease. Our general hypothesis is that structural and regulatory themes active for the secretin receptor will be distinct from those of the majority of G protein- coupled receptors, and will provide Insights into the molecular basis for integrated function of the exocrine pancreas. We propose to follow complementary pathways directed toward the secretin receptor molecule and the cellular mechanisms for regulating receptor function. In preparation, the pancreatic secretin receptor cDNA has been cloned, sequenced, and expressed; fusion proteins which incorporate tags for efficient affinity purification of the recombinant receptor have been constructed and validated; and novel radioligands have been developed. The secretin receptor cDNA sequence has established its close relationship to receptors for VIP, PACAP, PTH, and GHRF, and the absence of structural motifs typical of the majority of G protein- coupled receptors. The predicted large and complex, cysteine-rich ectodomains of these receptors interestingly complement the large pharmacophoric domains of their natural ligands. In this work, we will characterize the structure of this domain of the receptor, postulating its key role in agonist binding. This includes analysis of the patterns of disulfide bonding and glycosylation, and the functional impact of each. The agonist-binding domain will be defined both directly using affinity labeling with novel probes incorporating photolabile residues "intrinsic to" and "spanning" the pharmacophore, and indirectly using molecular constructs which include deletions, receptor chimeras, and site-specific mutants. The effects of receptor phosphorylation will be studied in model recombinant receptor-bearing cell systems, and mutagenesis approaches will be utilized to define key domains and residues. Finally, the secretin receptor on the pancreatic acinar cell, a well-established model for stimulus-activity coupling, will be studied to define receptor regulatory processes in a prototypic native cellular environment An understanding of structural and regulatory themes for the secretin receptor should have broad implications for this new important receptor family, as well as providing the basis for exploring the roles of this receptor in normal physiology, in development, and in the pathogenesis of disease.