REGULATION OF THE PLASMA MEMBRANE CALCIUM ATPASE

质膜钙ATP酶的调节

• 批准号: 6498787
• 负责人: EMANUEL Ernst STREHLER
• 金额: $ 23.39万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498787
• 关键词: calcium flux; calcium transporting ATPase; calmodulin; laboratory rat; molecular cloning; protein isoforms; protein localization; protein protein interaction; protein structure function; tissue /cell culture; transfection; yeast two hybrid system

Temporal and spatial control of intracellular Ca2+ fluctuations are of critical importance for eukaryotic cell function. The long-term research goal underlying this proposal is to understand the regulation of Ca2+ extrusion by the plasma membrane Ca2+ ATPase (PMCA) as the primary high affinity calcium efflux system of eukaryotic cells. Over twenty PMCA isoforms are generated from a multigene family and via alternative RNA splicing. The complex pattern of tissue and cellular distribution of these isoforms suggests their functional adaptation to the specific needs of a given cell. This project will explore the hypothesis that specific plasma membrane Ca2+ ATPase isoforms are targeted to multiprotein complexes where they associate with other receptors, transporters, channels, and signaling molecules to form "units of local Ca2+ regulation" at specific membrane sites. This concept has recently gained experimental support by the demonstration that some PMCA isoforms interact via their C-terminal tails with PDZ (PSD-95/Dlg/ZO-1) protein-protein interaction domains present in a growing number of multi-modular proteins thought to cluster and anchor membrane transporters and signaling proteins at the plasma membrane. The specific aims are (1) to determine the effect of PDZ domain interaction on the function of PMCAs; (2) to identify and characterize bona fide PDZ domain proteins that interact with different PMCA isoforms; and (3) to determine the cellular localization and possible co-localization of PMCA isoforms with candidate interacting PDZ domain proteins. Methods will include functional Ca2+ uptake assays, yeast two-hybrid screenings and biochemical, molecular and immunological techniques to identify and characterize specific protein-protein interactions; as well as confocal light microscopy combined with protein overexpression and fluorescent labeling methods to determine specific protein localizations. These experiments will open a new area for investigations of the physiological role of Ca2+ ATPase isoforms in the local control of Ca2+ regulation at the plasma membrane. For the plasma membrane Ca2+ ATPases, this represents a paradigm shift, changing their static image as housekeeping system responsible for the global maintenance of intracellular Ca2+ levels to one of a dynamic component involved in local Ca2+ control and Ca2+ signal transduction.

细胞内Ca ~（2+）波动的时间和空间控制 对真核细胞功能至关重要。 长期 这项建议的研究目标是了解监管 质膜Ca ~（2+）ATP酶（PMCA）对Ca ~（2+）的挤出作用 真核细胞的初级高亲和力钙外排系统。 超过 二十种PMCA同种型由多基因家族产生， 选择性RNA剪接。 组织和细胞的复杂模式 这些异构体的分布表明它们的功能适应于 特定细胞的特定需求。 本项目将探讨 假设特异性质膜Ca 2 + ATP酶同工型 靶向多蛋白复合物， 受体、转运蛋白、通道和信号分子， 在特定的膜位点的“局部Ca 2+调节单位”。这 这一概念最近得到了实验的支持， 一些PMCA亚型通过它们的C末端尾部与PDZ相互作用， （PSD-95/Dlg/ZO-1）蛋白质-蛋白质相互作用结构域存在于 越来越多的多模块蛋白被认为是聚集和锚 膜转运蛋白和质膜上的信号蛋白。 具体目的是：（1）确定PDZ结构域的作用 PMCAs功能的相互作用;（2）识别和表征 真正的PDZ结构域蛋白与不同的PMCA相互作用 同种型;和（3）确定细胞定位和可能的 PMCA同种型与候选相互作用PDZ结构域的共定位 proteins. 方法将包括功能性Ca 2+摄取测定、酵母 双杂交筛选和生化、分子和免疫学 鉴定和表征特定蛋白质-蛋白质的技术 相互作用;以及共聚焦光学显微镜结合蛋白质 过表达和荧光标记方法来确定特异性 蛋白质定位这些实验将为 Ca 2 + ATP酶同工酶的生理作用的调查， 局部控制质膜上的Ca 2+调节。 用于等离子体 膜Ca 2 + ATP酶，这代表了一种范式转变，改变了它们的 静态图像作为内务系统负责全局 维持细胞内Ca 2+水平的动态成分之一 参与局部Ca ~（2+）调控和Ca ~（2+）信号转导。