SECRETIN RECEPTOR STRUCTURE, FUNCTION, AND REGULATION

其长期目标是更好地理解 胃肠肽类激素分泌素与健康和疾病 目前的提案集中在结构、功能和监管方面 胰腺外分泌物中的分泌素受体。促胰液素很重要 在生理上和在发病机制中潜在的重要作用， 胰腺疾病的诊断和治疗。我们的一般假设 对于分泌素来说，结构和调控主题是活跃的吗 受体将与大多数G蛋白的受体不同- 偶联受体，并将提供对分子基础的洞察 用于胰腺外分泌的综合功能。我们建议遵循以下原则 针对促胰液素受体分子的互补途径 以及调节受体功能的细胞机制。在……里面 制备胰腺分泌素受体基因已被克隆， 对融合蛋白进行测序和表达，该融合蛋白包括标签 重组受体的高效亲和纯化 构建和验证；并开发了新的放射性配体。 分泌素受体的cdna序列已经建立了它的近似性 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，富含半胱氨酸 有趣的是，这些受体的胞外区与大的 它们的天然配基的药理作用域。在这项工作中，我们将 描述受体的这个区域的结构，假设 它在激动剂结合中的关键作用。这包括对图案的分析 对二硫键和糖基化的影响以及对功能的影响 每个人。激动剂结合结构域将直接使用 亲和标记光敏残基的新型探针 “内在的”和“跨越”的药效团，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 在模型重组受体承载细胞系统中进行研究，以及 将利用诱变方法来定义关键结构域和 残留物。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 研究确定原型原生体中的受体调节过程 蜂窝环境对结构和调节的理解 分泌素受体的主题应该具有广泛的意义 这一新的重要受体家族，以及为 探索这种受体在正常生理中的作用 发展，并在疾病的发病机制中。

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.