ADP-ribose dependent calcium entry in the immune system

描述（申请人的摘要）： 对钙进入免疫系统细胞的调节知之甚少。我 初步数据描述了一种新的钙的分子特征， 由ADP-核糖（ADPR）控制的进入途径。这一特征包括 脊椎动物ADP-核糖水解酶鉴定 （ADPRH-1），以及先前鉴定的基因 编码ADPR门控钙通道的蛋白产物。基于 这些数据，我建议探讨ADPR代谢和机制， 调节ADPR介导的钙进入免疫系统通过三个 具体目标：具体目标1：在大鼠中游离ADPR代谢的表征 免疫系统该目标将表征细胞生物学、结构/功能 ADPRH-1在ADPR调节中的生理作用 新陈代谢.这些分析将包括生产ADPRH- 1缺陷型 细胞系及其使用各种形式的ADPRH-1的互补。具体目标 2：调节游离ADPR水平的免疫系统机制。这一目标 将重点分析ADPR代谢与免疫细胞的关系 操作合理地与游离ADPR的产生有关。它将利用 测量总细胞游离ADPR的经典方法和 细胞溶质游离ADPR的电生理学测定。具体目标3： 细胞生物学特性和结构/功能关系的表征 ADPR门控阳离子通道：游离ADPR可能的关键效应子。这些 分析将包括筛选细胞系和原代细胞类型， 通道表达和ADPR介导的电流，以及结构/功能 ADPR门控通道的ADPRH- 1同源区的作用分析 在门控过程中。这些目标的实验将为我们提供 关于游离ADPR的代谢和 ADPR依赖性钙离子内流在免疫系统功能中的作用。

DESCRIPTION (Applicant's Abstract): The molecular mechanisms involved in the regulation of calcium entry into immune system cells are poorly understood. My preliminary data describe the molecular characterization of a novel calcium entry pathway controlled by ADP-ribose (ADPR). This characterization includes the identification of a novel highly specific vertebrate ADP-Ribose Hydrolase (ADPRH-1), and the functional demonstration that a previously identified gene encodes a protein product which is an ADPR gated calcium channel. Based on these data, I propose to explore ADPR metabolism and the mechanisms which regulate ADPR-mediated calcium entry within the immune system through three specific aims: Specific Aim 1: Characterization of free ADPR metabolism in the immune system. This aim will characterize the cell biology, structure/function relationships, and physiologic roles of ADPRH-1 in the regulation of ADPR metabolism. These analyses will include the production of an ADPRH- 1 deficient cell line and its complementation using various forms of ADPRH-1. Specific Aim 2: Immune system mechanisms for the regulation of free ADPR levels. This aim will focus on analyses of ADPR metabolism in relation to immune cell manipulations plausibly linked to the production of free ADPR. It will utilize both classic methods for measurement of total cellular free ADPR and a novel electrophysiologic assay for cytosolic free ADPR. Specific Aim 3: Characterization of the cell biology and structure/function relationships of the ADPR gated cation channel: a possible key effector of free ADPR. These analyses will include screening of cell lines and primary cell types for channel expression and ADPR-mediated currents, and a structure/function analysis of the role of an ADPRH- 1 homology region of the ADPR gated channel in the gating process. Together, the experiments in these aims will provide us with substantial new information regarding the metabolism of free ADPR and the role of ADPR dependent calcium entry in immune system function.