STRUCTURE AND FUNCTION OF CHOLECYSTOKININ RECEPTORS

胆囊收缩素受体的结构和功能

基本信息

批准号：
3232713
负责人：
STEVEN Alan ROSENZWEIG
金额：
$ 14.35万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-09-30 至 1993-08-31
项目状态：
已结题

项目摘要

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.

该提议的远程目标是表征胆囊动蛋白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。比较研究将致力于检查亲和力大鼠胰腺上CCK受体的分子量和分子量通过竞争约束分析和亲和力标签的蟾蜍视网膜技术。这些受体的功能比较将是通过监视CCK-8的相对有效性获得刺激45CA2+外排和polyphosphoinsitide在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受体使用特定的抗体探针。为此，重点已被放置在获得特定的多克隆抗体从从中分离的受体的MR 81 K CCK识别亚基大鼠胰质膜。大鼠胰腺等离子体膜将用非IDET P-40和MR 81溶解 K蛋白将通过许多顺序纯化色谱步骤，然后是SDS凝胶电泳。它然后将从凝胶切片中电解并固定在硝酸纤维素纸上，用于免疫兔子或电脑到活化玻璃的NH2-末端对气相序列的微序列分析。在后者情况，我们将获得第一个10-20 NH2-的序列该蛋白质的末端氨基酸残基以及此数据，具有合成的相应肽的毫克量。然后，合成肽将用于免疫兔子和获得对CCK受体的位点特异性抗体。使用生成的特定抗体探针，我们将补充我们的比较胰腺和视网膜CCK受体蛋白质印迹分析和免疫沉淀生物合成标记的受体。我们还将确定胰腺中的CCK受体是否经历 CCK结合响应于CCK的酪酶残基上的磷酸化还是它是否经过药物的调节磷酸化例如蛋白激酶C.这些研究将提供相当大的 CCK受体结构的分子水平的信息和功能，并将大大增强我们对 CCK在胰腺和视网膜的调节中扮演角色功能。