Environmental Effects on the Dynamism of Receptors of Lipophilic Signal Molecules in the Brain and its Functional Significance

环境对脑内亲脂信号分子受体动态的影响及其功能意义

基本信息

批准号：
16200026
负责人：
KAWATA Mitsuhiro
金额：
$ 31.62万
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 (A)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

The present research project was undertaken to investigate two types of steroid hormone receptors (glucocorticoids and sex steroids) dynamism by using real-time imaging with fluorescence recovery after photobleaching (FRAP) and fluorescent resonance energy transfer (FRET.) Glucocorticod receptor (GR) and mineralocorticoid receptor (MR) were localized in the cytoplasm in the absence of the ligand and they translocate to the nucleus after ligand binding. FRET demonstrated that importin a was involved in GR/MR translocation from the cytoplasm to the nucleus and GR and MR dimerize within the nucleus. FRAP showed that the movement of ligand-binded GR/MR was restricted in the nucleus. Upon estradiol treatment ERa and b were relocalized to show discrete pattern, and they were localized at the same discrete cluster. FRET clearly showed the interaction of ERa and ERb. In the presence of the estradiol, however, the discrete staining pattern of ERa and b were mostly overlapped with Brg-1, indicating that most of the ERs clusters are involved in the chromatin remodeling machinery. FRAP showed that nuclear ERa and b were dynamic and mobile in the absence of the ligand, but its mobility was slightly decreased after the ligand treatment. Nuclear matrix which was scaffolding sites within the nucleus was composed of actin and actin-related peptides. Treatment by detergent caused complete loss of ERa in the unliganded condition, but liganded ERa stick to the nuclear matrix. Nuclear matrix was an important factor to determine the dynamism of unliganded and liganded ERa.

本研究项目旨在通过使用光漂白后荧光恢复 (FRAP) 和荧光共振能量转移 (FRET) 实时成像技术来研究两种类型的类固醇激素受体（糖皮质激素和性类固醇）的动态性。在没有配体的情况下，盐皮质激素受体（MR）定位于细胞质中，并在配体结合后转位至细胞核。 FRET证明importin a参与GR/MR从细胞质到细胞核的易位以及GR和MR在细胞核内二聚化。 FRAP表明配体结合的GR/MR的运动在细胞核内受到限制。雌二醇治疗后，ERa 和 b 重新定位以显示离散模式，并且它们位于相同的离散簇。 FRET 清楚地显示了 ERa 和 ERb 的相互作用。然而，在雌二醇存在的情况下，ERa 和 b 的离散染色模式大部分与 Brg-1 重叠，表明大多数 ER 簇参与染色质重塑机制。 FRAP表明，在没有配体的情况下，核ERa和b是动态的和可移动的，但在配体处理后其移动性略有下降。核基质是细胞核内的支架位点，由肌动蛋白和肌动蛋白相关肽组成。去污剂处理导致未配体条件下的 ERa 完全丧失，但配体的 ERa 粘附在核基质上。核基质是决定未配体和配体ERa活力的重要因素。