REGULATION OF THE PLASMA MEMBRANE CALCIUM ATPASE

质膜钙ATP酶的调节

• 批准号: 6351275
• 负责人: EMANUEL Ernst STREHLER
• 金额: $ 22.72万
• 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 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351275
• 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.

细胞内钙波动的时间和空间控制是 对真核细胞功能至关重要。长期的 这项建议背后的研究目标是理解监管 质膜Ca~(2+)-ATPase(PMCA)对Ca~(2+)的排泄 真核细胞的初级高亲和力钙外流系统。完毕 从一个多基因家族中产生了20个PMCA亚型 可供选择的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在功能上与 特定单元格的特定需求。本项目将探索 质膜钙ATPase亚型特异性假说 靶向多蛋白复合体，在那里它们与其他 受体、转运体、通道和信号分子形成 在特定的膜位置的“局部钙调节单位”。这 概念最近通过演示获得了实验支持 一些PMCA亚型通过其C-末端与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质被认为是聚集和锚定的 质膜上的膜转运蛋白和信号蛋白。 具体目的是(1)确定PDZ结构域的影响 对PMCA功能的相互作用；(2)识别和表征 与不同PMCA相互作用的真PDZ结构域蛋白 异构体；以及(3)确定细胞定位和可能 PMCA亚型与候选相互作用PDZ结构域的共定位 蛋白质。方法包括功能性钙摄取试验、酵母菌 双杂交筛选与生化、分子和免疫学 鉴定和鉴定特定蛋白质-蛋白质的技术 相互作用；以及结合蛋白质的共聚焦光学显微镜 过表达和荧光标记方法确定特异性 蛋白质定位。这些实验将开辟一个新的领域 Ca~(2+)-ATPase异构体在心肌梗死中的生理作用 质膜上钙离子调节的局部调控。对于血浆来说 膜Ca~(2+)-ATPase，这代表着范式的转变，改变了它们的 静态形象作为负责全球事务的内务系统 维持细胞内钙离子水平为一种动态成分 参与局部钙调控和钙信号转导。