MECHANISM OF INOSITOL TRISPHOSPHATE ACTION

描述：体内游离钙浓度的升高。 细胞质隔室是该机制的一个组成部分，通过 哪些细胞对激素、生长因子和某些 神经递质。D-肌醇-1，4，5-三磷酸(IP3)是一种 细胞内信使介导的激素性钙动员 细胞内存储。这种分子与特定的受体相互作用。 (IP3R)，已被纯化并被证明是配体门控的钙离子 频道。这项建议的中心主题是研究结构， IP3受体的功能和调节。所有的研究都建议 利用重组受体片段或小脑膜， 肝细胞或WB细胞(一种大鼠肝上皮细胞系)。这个 该提案的具体目标是：1)确定 跨膜结构域及其膜插入机制。 这将使用无细胞翻译/易位试验进行研究 用编码假定的跨膜结构域的cRNA编程。这个 该系统将用于对有争议的型号进行实验测试 基于亲水性分析的受体跨膜组织 并鉴定对同源和同源的跨膜结构域 2)研究钙离子对IP3R的调控机制 和磷酸化。重组融合蛋白及其蛋白水解性研究 受体的裂解结构域将被用来识别钙结合 受体上的位置以及与钙调节蛋白的相互作用。 调节IP3R功能的蛋白激酶和磷酸酶将是 已确认身份。神经元和神经细胞的差异调节的基础 外周IP3R亚型通过钙离子和磷酸化 调查过了。3)研究受体与细胞因子的相互作用 细胞骨架。I型IP3R与锚定蛋白的相互作用 进一步分析了WB-IP3R与 将对细胞骨架基质进行表征。4)研究相互作用 在受体的不同结构域之间和不同受体之间 异构体。重组融合蛋白及其蛋白水解性切割 受体的结构域将被用来进一步定位配体- 结合结构域并研究其与C末端通道的相互作用 域。第I类和第I类异齐聚的初步观察 III将对IP3R进行进一步调查。这项提案的重点是 获取有关IP3R蛋白的基本信息。长期目标是 了解这些蛋白质在单个细胞中是如何发挥作用的 钙离子瞬变过程中复杂的时空模式及其原因 这样的信号被解码以改变生理反应。

DESCRIPTION: An elevation of the free calcium concentration in the cytoplasmic compartment is an integral component of the mechanism by which cells respond to hormones, growth-factors and certain neurotransmitters. D- myo-Inositol 1,4,5-trisphosphate (IP3) is an intracellular messenger mediating the hormonal mobilization of Ca2+ from intracellular stores. This molecule interacts with a specific receptor (IP3R) that has been purified and shown to be a ligand-gated Ca2+ channel. The central theme of this proposal is to study the structure, function and regulation of IP3 receptors. All the studies proposed utilize recombinant receptor fragments or cerebellum membranes, hepatocytes or WB-cells (a rat liver epithelial cell line). The specific aims of the proposal are: 1) Determination of the topology of the transmembrane domains and mechanism of their membrane insertion. This will be studied using a cell-free translation/translocation assay programmed with cRNA encoding the putative transmembrane domains. The system will be used to experimentally test controversial models of transmembrane organization of the receptor based on hydropathy analysis and to identify transmembrane domains important for homo- and heteroligomerization; 2) Study the mechanism of IP3R regulation by Ca2+ and phosphorylation. Recombinant fusion proteins and proteolytically cleaved domains of the receptor will be used to identify Ca2+ binding sites on the receptor and interactions with Ca2+ regulatory proteins. The protein kinases and phosphatases that regulate IP3R function will be identified. The basis for the differential regulation of neuronal and peripheral IP3R isoforms by Ca2+ and phosphorylation will be investigated. 3) To study interaction of the receptor with the cytoskeleton. Interaction of the Type-I IP3R with ankyrin will be further analyzed and the interaction of the WB-IP3R with the cytoskeletal matrix will be characterized. 4) To study interactions between different domains of the receptor and between different receptor isoforms. Recombinant fusion proteins and proteolytically cleaved domains of the receptor will be used to further localize the ligand- binding domain and to study its interaction with the C-terminal channel domain. Initial observations on heteroligomerization of type-I and type- III IP3R will be further investigated. This proposal is focused on obtaining basic information on IP3R proteins. The long-term goal is to understand how these proteins function in individual cells to generate complex spatial and temporal patterns in their Ca2+ transients and how such signals are decoded to alter physiological responses.