Mechanisms of Nuclear Receptor Activation

核受体激活机制

• 批准号: 6984041
• 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/6984041
• 关键词: 1,25 dihydroxycholecalciferol; DNA binding protein; cell nucleus; chimeric proteins; corticosteroid receptors; cytoplasm; endocytosis; fluorescence recovery after photobleaching; fluorescence resonance energy transfer; glucocorticoids; green fluorescent proteins; guanosinetriphosphatases; hormone regulation /control mechanism; intermolecular interaction; intracellular transport; microscopy; molecular chaperones; nuclear receptors; protein localization; protein transport; receptor binding; receptor expression; receptor sensitivity; retinoid binding proteins; vitamin D receptors

Members of the ligand-induced nuclear receptor superfamily are medically important regulators of cellular activity. Hormones initiate a receptor activation process leading to receptor redistribution and 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). Our studies demonstrated that ligand binding induces formation of multiple nuclear foci of GFP-GR, GFP-VDR, YFP-RXR, and GFP-ER. Mutational analysis demonstrated a correlation between hormone-dependent nuclear foci formation and DNA binding for both the GFP-VDR and the GFP-GR. This notion was further supported by our co-localization and fluorescence energy transfer experiments (FRET). After calcitriol treatment, GFP-VDR and RXR-BFP co-localized in the nuclear foci and FRET demonstrated formation of VDR/RXR dimers at these foci. Dimerization incompetent GFP-VDR and RXR-BFP failed to co-localize and failed to generate FRET signal at the foci. Because VDR and RXR bind to DNA as heterodimer, this finding suggested a correlation between focal receptor accumulation and DNA-binding. Furthermore, dynamic microscopy experiments such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) revealed that both VDR and RXR move rapidly within the nucleus and that hormone binding slowes this intranuclear movement. Our ongoing studies are aimed to identify the protein responsible for keeping nuclear receptors in motion within the nucleus. The second aim of our studies is to elucidate the roles of receptor trafficking between the cytoplasm and the nucleus in the regulation of hormone actions. Our dynamic microscopy and cell permeabilization experiments demonstrated that liganded and unliganded YFP RXR and GFP-VDR both shuttle rapidly between the cytoplasm and the nucleus. Using mutational analysis, we identified two nuclear localization signals (NLSs) in the DNA-binding region of the RXR and clarified the contribution of several putative NLSs in the nucleocytoplasmic shuttling of VDR. We also identified regions of VDR and RXR significant for export. In addition, export of the unliganded GFP-VDR was sensitive to treatment with leptomycin B (LMB), an inhibitor of cargo protein binding to the Crm-1 export receptor. Experiments with NLS and NES mutants of VDR and RXR demonstrated that redirection of VDR and RXR signaling with either nuclear exclusion or retention results in the development of abnormal hormone-induced transcriptional regulation. Our results indicated that both VDR and RXR import contributes to calcitriol induced transcriptional activities. Moreover, our results provided the first demonstration of the importance of export for transcriptional activity of a nuclear receptor. Our studies have changed the paradigm of VDR and RXR localization within the cell and introduced a dynamic model for VDR activation. Ongoing studies are aimed to understand the roles of receptor export in the mechanism of hormone actions.

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，导致受体重新分布，并与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。我们的研究表明，配体结合诱导了GFP-GR、GFP-VDR、YFP-RXR和GFP-ER多个核焦点的形成。突变分析表明，GFP-VDR和GFP-GR的激素依赖核灶形成和DNA结合之间存在相关性。我们的共定位和荧光能量转移实验(FRET)进一步支持了这一观点。骨化三醇治疗后，GFP-VDR和RXR-BFP共定位于核灶，FRET显示这些病灶形成VDR/RXR二聚体。二聚体失活的GFP-VDR和RXR-BFP不能共定位，不能在病灶处产生FRET信号。由于VDR和RXR以异源二聚体的形式与DNA结合，这一发现表明焦点受体积累和DNA结合之间存在相关性。此外，光漂白后荧光恢复(FRAP)和光漂白中的荧光损失(FLIP)等动态显微镜实验表明，VDR和RXR都在细胞核内快速移动，激素结合减缓了这种核内移动。我们正在进行的研究旨在确定负责在细胞核内保持核受体运动的蛋白质。 我们研究的第二个目的是阐明受体在细胞质和细胞核之间的运输在激素作用调节中的作用。我们的动态显微镜和细胞通透性实验表明，连接和未连接的YFP RXR和GFP-VDR都能在细胞质和细胞核之间快速穿梭。通过突变分析，我们在RXR的DNA结合区确定了两个核定位信号(NLSS)，并阐明了几个可能的NLSS在VDR核质穿梭中的作用。我们还确定了VDR和RXR的重要出口地区。此外，未连接的GFP-VDR的出口对软霉素B(LMB)敏感，LMB是一种与CRM-1出口受体结合的货物蛋白的抑制剂。用VDR和RXR的NLS和NES突变体进行的实验表明，VDR和RXR信号的重定向与核排斥或保留导致了激素诱导的异常转录调节的发展。我们的结果表明，VDR和RXR的导入都有助于骨化三醇诱导的转录活性。此外，我们的结果首次证明了出口对于核受体转录活性的重要性。我们的研究改变了VDR和RXR在细胞内定位的范式，并引入了VDR激活的动态模型。正在进行的研究旨在了解受体输出在激素作用机制中的作用。