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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.

目前的研究项目进行调查两种类型的类固醇激素受体（糖皮质激素和性类固醇）的动力学通过使用实时成像与荧光漂白后恢复（FRAP）和荧光共振能量转移（FRET）。糖皮质激素受体（GR）和盐皮质激素受体（MR）在无配体时定位于细胞质，配体结合后移位至细胞核。FRET结果表明，importin a参与GR/MR从胞质向胞核的转位，并参与GR和MR在胞核内的二聚化。FRAP显示配体结合的GR/MR在细胞核内的运动受到限制。雌二醇处理后，ER a和B重新定位，显示离散模式，它们定位在同一离散簇。FRET清楚地显示了ERa和ERb的相互作用。然而，在雌二醇的存在下，ER a和B的离散染色模式大多与Brg-1重叠，表明大多数ER簇参与染色质重塑机制。FRAP结果显示，在无配体存在时，细胞核ER a和B是动态的，具有移动的特性，但经配体处理后，其移动性略有下降。核基质是细胞核内的支架部位，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状态下ER a完全丢失，但配体ER a粘附于核基质。核基质是决定配体和未配体ER α活力的重要因素。