BIOSYNTHESIS AND DEGRADATION OF IP3 RECEPTORS

从内部存储的Ca 2+动员是重要的细胞代谢的急性调节和调节细胞生长，细胞分裂和程序性细胞死亡的关键步骤。 细胞内信使D-myo-Inositol 1，4，5-trisphosphate（IP 3）通过特异性受体/Ca 2+离子通道（IP 3R）的结合介导Ca 2+从细胞内储存的动员。 虽然这种受体的结构和功能方面正在积极的调查，一直没有系统的研究，这类重要的细胞内离子通道的生物合成和降解，这代表了重点，这项拨款建议。 我们的初步研究表明，长期治疗WB细胞与Ca 2+动员激动剂血管紧张素II引起下调IP 3R亚型通过激活泛素/蛋白酶体途径。 我们还确定了新生的IP 3R亚型与钙结合分子伴侣钙连接蛋白和钙网蛋白的选择性相互作用。 这些研究将使用几种实验模型进行扩展，包括培养细胞，无细胞翻译系统和在转染细胞中表达特异性IP 3R cDNA。 该提案的具体目标是：[1]。为了表征IP 3Rs组装的早期步骤-将研究低聚反应的动力学和机理。[2]的文件。为了研究IP 3R与分子伴侣的关联-将评估IP 3R组装和功能的伴侣关联的后果。[3].为了表征对调节降解重要的IP 3R同种型的结构特征-将在各种模型系统中测试突变IP 3R作为降解底物。[4]的文件。为了确定泛素/蛋白酶体途径在IP 3R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个体IP 3R亚型水平的因素。 拟议的研究也旨在提供基本的见解离子通道的组装和调节内质网中的多位膜蛋白的降解机制。

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.