STRUCTURE AND FUNCTION OF CHOLECYSTOKININ RECEPTORS

胆囊收缩素受体的结构和功能

• 批准号: 3232711
• 负责人: STEVEN Alan ROSENZWEIG
• 金额: $ 14.86万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-30 至 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3232711
• 关键词: Anura; affinity chromatography; affinity labeling; antibody; antireceptor antibody; brain; calcium transporting ATPase; cell membrane; cholecystokinin; complementary DNA; density gradient ultracentrifugation; gel electrophoresis; gel filtration chromatography; high performance liquid chromatography; hormone receptor; immunoelectroadsorption; laboratory rabbit; laboratory rat; membrane proteins; pancreas; phorbols; phosphatidylinositols; phosphorylation; protein biosynthesis; protein kinase C; protein structure function; retina; tyrosine

The long-range objective of this proposal is to characterize the structural and functional properties of the cholecystokinin receptor on pancreatic acinar cells and retinal neuronal cells. Comparative studies will be directed at examining the affinities and molecular weights of the CCK receptors on rat pancreas and toad retina by competition binding analyses and affinity labeling techniques. Functional comparisons of these receptors will be obtained by monitoring the relative effectiveness of CCK-8 to stimulate 45Ca2+ efflux and polyphosphoinositide turnover in intact pancreatic lobules and retinal monolayers, respectively. We will also compare the pancreatic and retinal CCK receptors using specific antibody probes. To this end, a major emphasis has been placed on obtaining specific polyclonal antibodies to the Mr 81 K CCK recognition subunit of the receptor isolated from rat pancreatic plasma membranes. Rat pancreatic plasma membranes will be solubilized with Nonidet P-40 and the Mr 81 K protein will be purified by a number of sequential chromatographic steps followed by SDS gel electrophoresis. It will then be electroeluted from gel slices and either immobilized onto nitrocellulose paper and used to immunize rabbits or electroblotted onto activated glass for NH2-terminal microsequence analysis on a gas-phase sequenator. In the latter case, we will obtain the sequence of the first 10-20 NH2- terminal amino acid residues of this protein and with this data, have mg quantities of the corresponding peptide synthesized. Synthetic peptide will then be used to immunize rabbits and obtain site-specific antibodies to the CCK receptor. Using the specific antibody probes generated, we will complement our comparisons of the pancreatic and retinal CCK receptors by Western blotting analysis and immunoprecipitation of biosynthetically labeled receptor. We will also determine whether the CCK receptor in pancreas undergoes phosphorylation on tyrosyl residues in response to CCK binding or whether it undergoes regulatory phosphorylation by agents such as protein kinase C. These studies will provide considerable information, at the molecular level, of CCK receptor structure and function and will greatly enhance our understanding of the role CCK play in the regulation of pancreatic and retinal function.

该提案的长期目标是描述 胆囊收缩素的结构和功能特性 胰腺腺泡细胞和视网膜神经元细胞上的受体。 比较研究将旨在检查相似性 大鼠胰腺上 CCK 受体的分子量和分子量 蟾蜍视网膜竞争结合分析和亲和力标记 技术。 这些受体的功能比较将是 通过监测 CCK-8 的相对有效性获得 刺激 45Ca2+ 外流和多磷酸肌醇周转 分别是完整的胰腺小叶和视网膜单层。 我们还将比较胰腺和视网膜 CCK 受体 使用特异性抗体探针。 为此，重点强调 已致力于获得针对该疾病的特异性多克隆抗体 Mr 81 K CCK 受体识别亚基分离 大鼠胰腺质膜。 大鼠胰血浆 膜将用 Nonidet P-40 和 Mr 81 溶解 K蛋白将通过一系列连续的纯化 色谱步骤，然后进行 SDS 凝胶电泳。 它 然后将从凝胶切片中电洗脱并固定 涂在硝化纤维纸上并用于免疫兔子或 电印迹到活性玻璃上的 NH2 末端 气相测序仪上的微序列分析。 在后者中 这种情况下，我们将得到前10-20个NH2-的序列 该蛋白质的末端氨基酸残基以及该数据， 合成了毫克量的相应肽。 然后合成肽将用于免疫兔子 获得 CCK 受体的位点特异性抗体。 使用 生成特异性抗体探针，我们将补充我们的 胰腺和视网膜CCK受体的比较 蛋白质印迹分析和免疫沉淀 生物合成标记受体。 我们还将确定 胰腺中的CCK受体是否经历 酪氨酰残基磷酸化响应 CCK 结合 或者它是否经历了药物的调节性磷酸化 例如蛋白激酶C。这些研究将提供相当多的信息 CCK 受体结构的分子水平信息 和功能，并将大大增强我们对 CCK在胰腺和视网膜调节中的作用 功能。