ADP-ribose dependent calcium entry in the immune system

描述(申请人摘要)：涉及的分子机制 对钙离子进入免疫系统细胞的调节知之甚少。我的 初步数据描述了一种新型钙的分子特征 腺苷二磷酸核糖(ADPR)控制的进入途径。这一特征包括 一种新的高度特异的脊椎动物ADP-核糖水解酶的鉴定 (ADPRH-1)，以及先前发现的一个基因的功能证明 编码一种蛋白质产物，它是一种ADPR门控钙通道。基于 这些数据，我建议探索ADPR的代谢及其机制 通过三种途径调节ADPR介导的钙离子进入免疫系统 特定目标：特定目标1：确定人体内游离ADPR代谢的特征 免疫系统。这一目标将表征细胞的生物学、结构/功能 ADPRH-1在ADPR调控中的关系和生理作用 新陈代谢。这些分析将包括ADPRH-1缺陷的生产 不同形式的ADPRH-1对细胞株及其互补作用的研究特定目标 2：免疫系统调节游离ADPR水平的机制。这一目标 将重点分析ADPR代谢与免疫细胞的关系 操纵似乎与生产免费的ADPR有关。它将利用 两种测量细胞总游离ADPR的经典方法和一种新的 细胞内游离ADPR的电生理测定。具体目标3： 细胞生物学和结构/功能关系的表征 ADPR门控阳离子通道：可能是游离ADPR的关键效应因子。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ADPRH-1同源区域的作用分析 在浇注过程中。总而言之，这些目标的实验将为我们提供 提供了大量关于游离ADPR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.