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MOLECULAR DYNAMICS OF RECEPTORS OF LIPID SOLUBLE SIGNAL MOLECULES

脂溶性信号分子受体的分子动力学

基本信息

批准号：
11470006
负责人：
KAWATA Mitsuhiro
金额：
$ 9.41万
依托单位：
KYOTO PREFECTURAL UNIVERSITY OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

GFP-GR chimera construct was transfected into the cultured cells. By using luciferase assay GFP-GR chimera system was confirmed to be functional. DEX-inducible mouse mammary tumor virus promoter (MMTV)-Luc reporter was induced by DEX.Real-time imaging study has shown that in the absence of ligand GFP-GR was present in the cytoplasm of cells. Exposure of DEX to cultured cells induced the nuclear accumulation of GFP-GR and its translocation from cytoplasm-to-nucleus was time-dependent manner. Thirty min after DEX treatment GFP-GR was completely translocated into the nucleus. No green fluorescence was observed in the nucleolus. The same results were observed in the primary cultured hippocampal neurons and cortical glia. GFP-GR from the small cytoplasm of the perikarya to the nucleus was observed after 15 to 30 min after the DEX as well as corticosterone treatment, indicating that there was no conspicuous difference of trafficking manner of GR in the presence of the ligands among cell types. GFP-MR chimera construct was also transfected into COS-1 cells, hippocampal neurons, and cortical glia. As in the case of GFP-GR, GFP-MR chimera construct should be verified to be transcriptionally active before observing the real-time imaging. GFP-MR was observed in the cytoplasm of most of the COS-1 cells without the ligand. Aldosterone, an agonist for MR, induced the translocation of GFP-MR from the cytoplasm to the nucleus in a time-dependent manner. Within 30 min after the treatment green fluorescence was completely present in the nucleus and nucleolus was devoid of GFP-MR.Corticosterone treatment caused the similar accumulation of GFP-MR in the nucleus of the COS-1 cells where no fluorescence was observed at the nucleolus. Cells that were transfected by GFP-ERa construct and were not treated by estrogen showed the GFP-ERa in the nucleus. Nucleolus was devoid of GFP-ERa.

将GFP-GR嵌合体构建体转染到培养的细胞中。荧光素酶检测证实GFP-GR嵌合体系统具有功能。DEX诱导的小鼠乳腺肿瘤病毒启动子（MMTV）-Luc报告基因在DEX诱导下表达，实时荧光成像研究表明，在缺乏配体的情况下，GFP-GR表达于细胞质中。DEX诱导GFP-GR在细胞核内聚集，并呈时间依赖性地从细胞质向细胞核移位。DEX处理30 min后，GFP-GR完全转位入核。核仁内未见绿色荧光。在原代培养的海马神经元和皮层胶质细胞中也观察到同样的结果。DEX和皮质酮处理后15 ~ 30 min，GFP-GR从胞体的小胞质进入细胞核，表明GR在配体存在下的运输方式在不同细胞类型间无明显差异。GFP-MR嵌合体构建体也转染到COS-1细胞、海马神经元和皮质胶质细胞中。与GFP-GR的情况一样，在观察实时成像之前，GFP-MR嵌合体构建体应被验证为具有转录活性。GFP-MR在大多数没有配体的COS-1细胞的细胞质中观察到。醛固酮，一种激动剂的MR，诱导GFP-MR从细胞质易位到细胞核中的时间依赖性的方式。在处理后的30分钟内，绿色荧光完全存在于细胞核中，并且核仁中没有GFP-MR。皮质酮处理引起类似的GFP-MR在COS-1细胞的细胞核中的积累，而在核仁中没有观察到荧光。用GFP-ER α构建体转染且未用雌激素处理的细胞在细胞核中显示GFP-ER α。核仁缺乏GFP-ER α。