MECHANISM OF INOSITOL TRISPHOSPHATE ACTION

描述：心肌细胞中游离钙浓度升高， 细胞质区室是该机制的组成部分， 哪些细胞会对激素、生长因子和某些 神经传递素D-肌-肌醇1，4，5-三磷酸（IP 3）是一种 细胞内信使介导的激素动员的Ca 2+从 细胞内储存。这种分子与特定的受体相互作用 （IP 3R），其已被纯化并显示为配体门控的Ca 2 + 频道这项建议的中心主题是研究结构， IP 3受体的功能和调节。所有提出的研究 利用重组受体片段或小脑膜， 肝细胞或WB细胞（大鼠肝上皮细胞系）。的 具体目标的建议是：1）确定的拓扑结构， 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 用编码推定的跨膜结构域的cRNA编程。的 系统将用于实验测试有争议的模型， 基于亲水性分析的受体的跨膜组织 并确定跨膜结构域的重要性， 研究Ca ~（2+）对IP_3受体的调节机制 和磷酸化。重组融合蛋白和蛋白水解 受体的切割结构域将用于鉴定Ca 2+结合 受体上的位点和与Ca 2+调节蛋白的相互作用。 调节IP 3R功能的蛋白激酶和磷酸酶将被 鉴定神经元和神经元的差异调节的基础 通过Ca 2+和磷酸化的外周IP 3R同种型将是 研究了3)为了研究受体与 细胞骨架I型IP 3R与锚蛋白的相互作用将是 进一步分析了WB-IP 3R与 将表征细胞骨架基质。4)来研究 在受体的不同结构域之间以及在不同受体之间 同种型。重组融合蛋白和蛋白水解切割的 受体的结构域将用于进一步定位配体- 结合结构域，并研究其与C-末端通道的相互作用 域对I型和I型的异低聚化的初步观察 三是IP 3R将进一步研究。该提案的重点是 获得关于IP 3R蛋白的基本信息。长期目标是 了解这些蛋白质如何在单个细胞中发挥作用， 复杂的空间和时间模式在他们的Ca 2+瞬变和如何 这些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.