Mechanisms of Steroid Receptor Activation

类固醇受体激活机制

• 批准号: 6432193
• 负责人: JULIANNA BARSONY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432193
• 关键词: DNA binding protein; cell line; cell nucleus; chimeric proteins; chromatin; corticosteroid receptors; cytoplasm; dexamethasone; glucocorticoids; green fluorescent proteins; hormone regulation /control mechanism; intermolecular interaction; intracellular transport; mifepristone; plasmids; protein localization; protein transport; receptor binding; receptor expression; receptor sensitivity

Members of the ligand-induced nuclear receptor superfamily are medically important regulators of cellular activity. Hormones initiate a receptor activation process leading to receptor binding to specific DNA recognition sites in the promoter regions of their target genes. Using microscopy and fluorescent protein chimeras of nuclear receptors, we pioneered studies establishing that ligand binding regulates the subcellular targeting of glucocorticoid (GR), vitamin D (VDR), and retinoid X receptors (RXR). We have continued to use advanced microscopy techniques to address the roles of receptor trafficking in hormone actions.We demonstrated that heterodimerization with RXR has a profound effect on VDR subcellular distribution and that this effect is physiologically important. When expressed separately, the steady-state distribution of the YFP-RXR was more nuclear than the GFP-VDR. Experiments measuring fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that both VDR and RXR constantly shuttle between the cytoplasm and the nucleus and that RXR shuttles at a faster rate than VDR. Coexpression of RXR-BFP with GFP-VDR promoted nuclear accumulation of the later by influencing both nuclear import and retention. RXR-BFP also promoted hormone-dependent nuclear accumulation of a nuclear localization signal mutant receptor (nlsGFP-VDR), and rescued its transcriptional activity. Heterodimerization mutant RXR failed to alter GFP-VDR and nlsGFP-VDR distribution or activity. This finding on the ability of RXR to facilitate the nuclear import of VDR is most likely applicable for other heterodimerization partners of RXR, such as the thyroid hormone receptor and the retinoic acid receptor. We and others have observed that ligand binding induces formation of multiple nuclear foci of GFP-GR, GFP-VDR, YFP-RXR, GFP-ER but the physiological importance of these foci remained to be elucidated. Studies on a cell line harboring a large array of MMTV-LTR allowed direct observation of human GR binding to this array. Mutational analysis demonstrated a correlation between hormone-dependent nuclear foci formation and DNA binding for both the VDR and the GR. In addition, fluorescence energy transfer experiments (FRET) revealed that calcitriol induces intranuclear foci formation of VDR/RXR heterodimers. Because VDR and RXR bind to DNA as heterodimers, this finding also suggest a correlation between focal receptor accumulation and DNA-binding. Current efforts are directed towards characterizing this and other subnuclear domains involved in nuclear receptor targeting.

配体诱导的核受体超家族成员在医学上是细胞活动的重要调节因子。激素启动受体激活过程，导致受体与其靶基因启动子区域的特定DNA识别位点结合。利用核受体的显微镜和荧光蛋白嵌合体，我们率先研究了配体结合调节糖皮质激素(GR)、维生素D(VDR)和维甲酸X受体(RXR)的亚细胞靶向。我们继续使用先进的显微镜技术来研究受体转运在激素活动中的作用。我们证明了与RXR的异源二聚对VDR亚细胞分布有深远的影响，并且这种影响在生理上是重要的。当单独表达时，YFP-RXR的稳态分布比GFP-VDR更具核性。测量光漂白后荧光恢复(FRAP)和光漂白中的荧光损失(FLIP)的实验表明，VDR和RXR都在细胞质和细胞核之间持续穿梭，并且RXR的穿梭速度比VDR快。RXR-BFP和GFP-VDR的共表达通过影响核的输入和保留促进了后者的核积累。RXR-BFP还促进激素依赖的核定位信号突变受体(nlsGFP-VDR)的核积累，并挽救其转录活性。异二聚化突变体RXR未能改变GFP-VDR和nlsGFP-VDR的分布或活性。这一关于RXR促进VDR核进口能力的发现很可能适用于RXR的其他异二聚化伙伴，如甲状腺激素受体和维甲酸受体。我们和其他人已经观察到配体结合诱导GFP-GR、GFP-VDR、YFP-RXR、GFP-ER的多个核焦点的形成，但这些焦点的生理意义仍未阐明。对含有大量MMTV-LTR的细胞系的研究允许直接观察到人GR与该阵列的结合。突变分析表明，VDR和GR的激素依赖核灶形成和DNA结合之间存在相关性。此外，荧光能量转移实验(FRET)显示骨化三醇可诱导VDR/RXR异源二聚体形成核内焦点。由于VDR和RXR以异源二聚体的形式与DNA结合，这一发现也表明焦点受体积累和DNA结合之间存在相关性。目前的研究方向是确定核受体靶向所涉及的这一结构域和其他亚核结构域。