Signaling in Living Cells

活细胞中的信号传导

• 批准号: 6874900
• 负责人: NARASIMHAN GAUTAM
• 金额: $ 32.13万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6874900
• 关键词: CHO cells; G protein; bioimaging /biomedical imaging; biological signal transduction; biosensor device; biotechnology; cell surface receptors; chimeric proteins; fluid flow; fluorescence; fluorescence resonance energy transfer; guanine nucleotide binding protein; imaging /visualization /scanning; method development; posttranslational modifications; protein localization; protein structure function; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): The molecular mechanisms underlying G protein signaling have so far been elucidated using purified proteins and lysed cells. Little information is available about the behavior of G protein pathways in a living mammalian cell. The broad aim is to visualize the functioning of a G protein signaling pathway and its cross-talk with other signaling pathways both spatially and temporally in live cells. G protein based biosensors have been designed containing signaling proteins fused to cyan and yellow fluorescent proteins (CFP & YFP). The sensors respond to receptor stimulation or inactivation with a corresponding fluorescence resonance energy transfer (FRET) signal change. Images of cells expressing the sensors acquired during signal processing provide direct information in real time on the properties of the pathway in different parts of the cell. The specific aims will be (i) to develop methods for imaging G protein signaling activity in mammalian cells. (ii) To test models about (a) the spatial distribution of signaling machinery and (b) the spatio-temporal progression of localized G protein activation. (iii) To test the hypotheses that a G protein pathway affects the spatio-temporal dynamics of (a) another G protein signaling activity and (b) ras and rap activity. Experiments will be performed with transfected and endogenous receptors. A vascular smooth muscle cell line will be used to examine other endogenous pathways. The majority of commercially available drugs are targeted at G protein coupled receptors. The biosensors developed here can be of considerable value pharmacologically. They can be used to screen for novel drugs and identify ligands for orphan receptors in non-invasive assays. In the longterm, this approach can help observe networks of signaling pathways functioning in a cell as it senses, processes and responds to a signal.

描述（由申请人提供）：迄今已使用纯化的蛋白质和裂解细胞阐明了G蛋白信号传导的分子机制。关于活哺乳动物细胞中G蛋白途径的行为的信息很少。广泛的目的是可视化G蛋白信号通路的功能及其与其他信号通路在活细胞中均与其他信号通路的串扰。基于G蛋白的生物传感器已设计为含有融合青色和黄色荧光蛋白（CFP和YFP）的信号蛋白。传感器以相应的荧光共振能量转移（FRET）信号变化对受体刺激或失活反应。表达信号处理过程中获得的传感器的单元格的图像可实时直接信息，以了解细胞不同部分的途径性质。 具体目的是（i）开发用于成像哺乳动物细胞中G蛋白信号活性的方法。 （ii）测试有关（a）信号机制的空间分布以及（b）局部G蛋白激活的时空进程。 （iii）测试G蛋白途径的假设会影响（a）另一种G蛋白信号活性和（b）RAS和RAP活性的时空动力学。实验将使用转染和内源性受体进行。血管平滑肌细胞系将用于检查其他内源性途径。 大多数市售药物针对G蛋白偶联受体。这里开发的生物传感器在药理学上具有相当大的价值。它们可用于筛查新药物，并在非侵入性测定中鉴定孤儿受体的配体。从长远来看，这种方法可以帮助观察信号通路的网络在感知，过程和对信号响应时在单元格中起作用。