RECEPTORS FOR CCK AND OTHER GI HORMONES

CCKA、蛙皮素（Bn）和m3胆碱能（m3 Ach）受体是主要的 胰腺外分泌的调节器。 从表面上看， 这些感受器看起来是一样的。然而，各种研究表明， 表明腺泡细胞对这三种受体的反应不同。 这些受体之间不同的特征包括， 配体结合亲和力状态和受体特性 调节，包括脱敏，内化和下调- 调控目前建议的重点是确定 这些受体差异的结构和功能基础， 从而了解受体的作用。这三个基因的cDNA 受体最近被克隆。利用这些克隆体， 分子和细胞生物学技术，我们将确定具体的 受体结构域和细胞组分，其：1）决定受体 结合亲和力状态及其伴随的信号级联; 2） 参与受体的内化和下调;以及3） 与受体的快速脱敏有关。校长 方法之一是通过转移， 三种受体之间的同源结构域。使用这种方法， 这三种受体应该会产生新的重要信息， 通过单一的标准缺失和诱变研究无法获得 受体。我们将特别关注受体G蛋白 相互作用和受体磷酸化，因为它们可能 影响受体功能的几个方面。我们将确定具体 能够与受体相互作用的G蛋白α亚基 通过受体和G蛋白α亚基在组织中的共表达 培养细胞。确认生理相关相互作用受体 和来自大鼠腺泡细胞的G蛋白将被免疫共沉淀。 受体中的磷酸化残基将使用抗- 受体抗体和/或标记有由 克隆抗体然后将使用定点诱变来 研究受体上特异性磷酸化位点的作用 功能和调节。一般来说，分析潜在的机制和 相互作用将在转染的细胞系中进行， 技术，如受体嵌合体和定点突变体。 然而，在这方面， 尽可能验证和分析生理机制 将在正常大鼠胰腺腺泡细胞中进行。这些研究 将提供对相似性基础的理解， 它们之间的结合亲和力和受体调节的差异 三个重要的受体，将被发现。 参与其他受体调节的机制和相互作用 并最终进入胰腺外分泌的调节。

CCKA, bombesin (Bn) and m3 cholinergic (m3Ach) receptors are major regulators of the exocrine pancreas. At a superficial level the actions of these receptors appear identical. However, a variety of studies indicate that acinar cells respond differently to these three receptors. Characteristics which vary between these receptors include, the number of ligand binding affinity states, and characteristics of receptor regulation, including desensitization, internalization, and down- regulation. The focus of the current proposal is to identify the structural and functional basis of the differences in these receptors and thereby, to learn how the receptors act. The cDNAs for these three receptors have recently been cloned. Utilizing these clones and techniques of molecular and cell biology we will determine the specific receptor domains and cellular components which: l) determine receptor binding affinity states and their attendent signal cascades; 2) are involved in internalization and down-regulation of the receptors; and 3) are involved in rapid desensitization of the receptors. The principal approach will be one of constructing chimeric receptors by transferring homologous domains among the three receptors. Using this approach with these three receptors should yield novel and important information unavailable through standard deletion and mutagenesis studies of single receptors. We will focus particular attention on receptor G protein interactions and receptor phosphorylation because they are likely to influence several aspects of receptor function. We will identify specific G protein alpha-subunits which are able to interact with the receptors by co-expression of receptors and G protein alpha-subunits in tissue culture cells. To confirm physiologically relevant interactions receptors and G proteins from rat acinar cells will be co-immunoprecipitated. Phosphorylated residues in the receptors will be identified using anti- receptor antibodies and/or receptors tagged with epitopes recognized by monoclonal antibodies. Site-directed mutagenesis will then be used to investigate the roles of specific phosphorylation sites on receptor function and regulation. In general, analysis o potential mechanisms and interactions will be conducted in transfected cell lines using molecular techniques such as receptor chimeras and site-directed mutants. However, whenever possible, verification and analysis of physiological mechanisms will be conducted in normal rat pancreatic acinar cells. These studies will provide an understanding of the basis of the similarities and differences in binding affinities and receptor regulation between these three important receptors. Thereby, insight will be gained into the mechanisms and interactions involved in the regulation of other receptors and ultimately into the regulation of the exocrine pancreas.