Mechanisms of Nuclear Receptor Activation

核受体激活机制

• 批准号: 6810576
• 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/6810576
• 关键词: 1,25 dihydroxycholecalciferol; DNA binding protein; cell line; cell nucleus; chimeric proteins; corticosteroid receptors; cytoplasm; endocytosis; glucocorticoids; 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的成员是医学上重要的细胞活性调节剂。激素启动受体活化过程，导致受体重新分布并结合到其靶基因启动子区的特异性DNA识别位点。使用显微镜和荧光蛋白嵌合体的核受体，我们开创性的研究建立，配体结合调节糖皮质激素（GR），维生素D（VDR），和类维生素A X受体（RXR）的亚细胞靶向。我们的研究表明，配体结合诱导GFP-GR、GFP-VDR、YFP-RXR和GFP-ER的多个核灶的形成。突变分析表明，GFP-VDR和GFP-GR的核灶形成与DNA结合之间存在相关性，这一观点得到了我们的共定位和荧光能量转移实验（FRET）的进一步支持。在骨化三醇处理后，GFP-VDR和RXR-BFP共定位于核灶中，并且FRET证明在这些灶处形成VDR/RXR二聚体。不能二聚化的GFP-VDR和RXR-BFP不能共定位并且不能在病灶处产生FRET信号。由于VDR和RXR以异源二聚体的形式与DNA结合，这一发现表明局灶性受体蓄积与DNA结合之间存在相关性。此外，动态显微镜实验，如荧光恢复后的光漂白（FRAP）和荧光损失的光漂白（FLIP）显示，VDR和RXR迅速移动的细胞核内，激素结合减慢这种核内运动。我们正在进行的研究旨在确定负责保持核受体在细胞核内运动的蛋白质。 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调节激素作用中的作用。我们的动态显微镜和细胞透化实验表明，配体和未配体的YFP RXR和GFP-VDR都在细胞质和细胞核之间快速穿梭。使用突变分析，我们确定了两个核定位信号（NLS）的RXR的DNA结合区，并澄清了几个假定的NLS的VDR的核质穿梭的贡献。我们还确定了VDR和RXR的重要出口地区。此外，未配体的GFP-VDR的输出对用来普霉素B（LMB）（一种与Crm-1输出受体结合的货物蛋白的抑制剂）处理敏感。用VDR和RXR的NLS和内斯突变体进行的实验表明，用核排斥或保留重定向VDR和RXR信号传导导致异常的核转录诱导的转录调控的发展。我们的研究结果表明，VDR和RXR的输入有助于骨化三醇诱导的转录活性。此外，我们的研究结果提供了第一次证明的重要性，出口的转录活性的核受体。我们的研究改变了细胞内VDR和RXR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。