BIOSYNTHESIS AND DEGRADATION OF IP3 RECEPTORS

Ca2+从内部储存的动员在细胞代谢的急性调节和细胞生长、细胞分裂和程序性细胞死亡的关键步骤是重要的。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）结合介导细胞内Ca2+的动员。尽管该受体的结构和功能方面正在积极研究中，但还没有对这类重要的细胞内离子通道的生物合成和降解进行系统的研究，这是本基金申请的重点。我们的初步研究表明，用Ca2+动员激动剂血管紧张素II慢性治疗WB细胞通过激活泛素/蛋白酶体途径导致IP3R亚型下调。我们还发现了新生IP3R亚型与Ca2+结合分子伴侣钙连联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、无细胞翻译系统和转染细胞中特异性IP3R cdna的表达。本提案的具体目标是：为了描述IP3Rs组装的早期步骤-寡聚化的动力学和机制将被研究。[2]。为了研究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]。为了描述IP3R异构体的结构特征，这对调节降解很重要-突变IP3R将作为降解底物在各种模型系统中进行测试。[4]。为了确定泛素/蛋白酶体途径在IP3R的调节转换中的作用，将研究内质网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IP3R亚型水平的因素。提出的研究还旨在为内质网中离子通道组装和多聚膜蛋白的调节降解机制提供基本见解。

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.