DYNAMICS AND REGULATION OF NUCLEAR RECEPTORS OF LIPID SOLUBLE MOLECULE IDENTIFIED BY USING GFPS

GFPS鉴定脂溶性分子核受体的动力学和调控

基本信息

批准号：
13470005
负责人：
KAWATA Mitsuhiro
金额：
$ 10.3万
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：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Nuclear receptors which include glucocorticoid receptor (GR), mineralocorticoid receptor (MR), estrogen receptor (ER) α and β, and androgen receptor (AR) are ligand-dependent transcriptional factor. With the advent of green fluorescent protein (GFP) and its color variants, the subcellular distribution of many nuclear receptors has been found to be much more dynamic than previously thought. GFP studies showed that microtubules are not involved in the translocation of GR and MR from cytoplasm to nucleus, but that HSP90 and importin system, at least in part, are strongly relevant to this translocation. Hormonal stimulation induced intranuclear receptor distribution from a homogeneous pattern to a heterogeneous dot-like image. GR and MR showed a partial colocalization at 30 min and nearly complete colocalization at 60 min after addition of corticosterone. The occurrence of FRET between GR and MR was observed in the nucleus. FRAP analysis showed that cytoplasmic GR was dynamic and nuclear GR was also mobile, but its mobility was different. Upon estradiol treatment ER α and β in the same cell were relocalized to show discrete pattern, and they were localized at the same discrete cluster, suggesting that both subtypes of Ers were bound to the same nuclear sites. In the absence of the ligand, any significant relationship between the diffuse distribution of Ers and the discrete distribution of Brg-1 was not observed. In the presence of the estradiol, however, the discrete staining pattern of ER α and β were mostly overlapped with Brg-1, indicating that most of the Ers clusters are involved in the chromatin remodeling machinery.

随着绿色荧光蛋白（GFP）及其出现，核受体包括糖皮质激素受体（GR）、盐皮质激素受体（MR）、雌激素受体（ER）α和β以及雄激素受体（AR）。研究发现，许多核受体的亚细胞分布比以前认为的更加动态，表明微管不参与 GR 和 MR 从细胞质到细胞核的易位。 HSP90 和 importin 系统至少部分与这种易位密切相关，激素刺激诱导的核内受体分布从均匀模式到异质点状图像，GR 和 MR 在 30 分钟时显示出部分共定位，在 30 分钟时显示出几乎完全共定位。添加皮质酮后60分钟，FRAP分析观察到细胞核中GR和MR之间发生FRET，细胞质GR是动态的，核GR也是动态的。雌二醇处理后，同一细胞中的 ER α 和 β 重新定位以显示离散模式，并且它们位于相同的离散簇，这表明 Ers 的两种亚型都结合到相同的核位点。在没有配体的情况下，没有观察到 Ers 的弥散分布和 Brg-1 的离散分布之间的任何显着关系。然而，在雌二醇存在的情况下，ER α 和 β 的离散染色模式大部分是重叠的。与 Brg-1 一起，表明大多数 Ers 簇参与染色质重塑机制。