BIOSYNTHESIS AND DEGRADATION OF IP3 RECEPTORS

IP3 受体的生物合成和降解

• 批准号: 6343038
• 负责人: SURESH K JOSEPH
• 金额: $ 28.02万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6343038
• 关键词: SDS polyacrylamide gel electrophoresis; autoradiography; calcium ion; cell line; endoplasmic reticulum; enzyme biosynthesis; gel filtration chromatography; immunoprecipitation; inositol phosphates; membrane channels; membrane proteins; molecular chaperones; proteasome; protein degradation; protein isoforms; protein structure; receptor expression; tissue /cell culture; transfection; ubiquitin; western blottings

Ca2+ mobilization from internal stores is important in the acute regulation of cell metabolism and in regulating key steps in cell growth, cell division and programmed cell death. Binding of the intracellular messenger D-myo-Inositol 1,4,5-trisphosphate (IP3) by a specific receptor/Ca2+ ion channel (IP3R) mediates the mobilization of Ca2+ from intracellular stores. Although the structural and functional aspects of this receptor are under active investigation, there has been no systematic study of the biosynthesis and degradation of this class of important intracellular ion-channels and this represents the focus of this grant proposal. Our initial studies have shown that chronic treatment of WB cells with the Ca2+ mobilizing agonist Angiotensin II causes down-regulation of IP3R isoforms by activating the ubiquitin/proteasome pathway. We have also identified selective interactions of nascent IP3R isoforms with the Ca2+-binding molecular chaperones calnexin and calreticulin. These studies will be extended using several experimental models including cultured cells, cell-free translation systems and expression of specific IP3R cDNAs in transfected cells. The specific aims of this proposal are: [1]. To characterize early steps in the assembly of IP3Rs - The kinetics and mechanism of oligomerization will be studied. [2]. To study the association of IP3Rs with molecular chaperones - The consequences of chaperone association for IP3R assembly and function will be assessed. [3].To characterize the structural features of IP3R isoforms that are important for regulated degradation - Mutant IP3Rs will be tested as degradation substrates in various model systems. [4]. To define the role of the ubiquitin/proteasome pathway in the regulated turnover of IP3R - The regulation of proteasomal activity in ER membranes will be investigated. Our long-term goal is to understand the factors that regulate the levels of individual IP3R isoforms. The proposed studies are also intended to provide fundamental insights into the mechanisms of ion-channel assembly and the regulated degradation of polytopic membrane proteins in the endoplasmic reticulum.

内部储存的 Ca2+ 动员对于细胞代谢的急性调节以及细胞生长、细胞分裂和程序性细胞死亡的关键步骤的调节非常重要。 细胞内信使 D-肌醇 1,4,5-三磷酸 (IP3) 通过特定受体/Ca2+ 离子通道 (IP3R) 的结合介导细胞内储存的 Ca2+ 的动员。 尽管该受体的结构和功能方面正在积极研究中，但尚未对此类重要的细胞内离子通道的生物合成和降解进行系统研究，这是本拨款提案的重点。 我们的初步研究表明，用 Ca2+ 动员激动剂血管紧张素 II 长期治疗 WB 细胞，会通过激活泛素/蛋白酶体途径导致 IP3R 亚型下调。 我们还确定了新生 IP3R 亚型与 Ca2+ 结合分子伴侣钙联蛋白和钙网蛋白的选择性相互作用。 这些研究将使用多种实验模型进行扩展，包括培养细胞、无细胞翻译系统和转染细胞中特定 IP3R cDNA 的表达。 该提案的具体目标是：[1]。为了表征 IP3R 组装的早期步骤 - 将研究寡聚的动力学和机制。 [2]。研究 IP3R 与分子伴侣的关联 - 将评估伴侣关联对 IP3R 组装和功能的影响。 [3]. 为了表征对于调节降解很重要的 IP3R 亚型的结构特征 - 将在各种模型系统中测试突变的 IP3R 作为降解底物。 [4]。为了确定泛素/蛋白酶体途径在 IP3R 调节周转中的作用 - 将研究 ER 膜中蛋白酶体活性的调节。 我们的长期目标是了解调节各个 IP3R 同工型水平的因素。 拟议的研究还旨在为离子通道组装机制和内质网中多面体膜蛋白的调节降解提供基本见解。