Mechanisms of Nuclear Receptor Activation

核受体激活机制

• 批准号: 7337523
• 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/7337523
• 关键词: Mechanisms; Nuclear; Receptor; Activation

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 slows this intranuclear movement. Our ongoing studies are aimed to identify the protein responsible for keeping nuclear receptors in motion within the nucleus and to identify the spatial and temporal relationships between type II nuclear receptors and corepressors. 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. Recent collaborative studies with Dr. Prufer revealed the NLSs of the liver X receptor alpha and beta. Another collaborative study with Dr. Fleet demonstrated unique and specific changes in VDR nucleo-cytoplasmic trafficking in the enterocyte-like Caco-2 cell. These changes are associated with changes in responsiveness to the natural hormone and synthetic analogues that helps understanding the low calcemic activities of these new compounds.

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。激素启动受体激活过程，导致受体重新分配并结合到其靶基因启动子区域的特定DNA识别位点。利用显微镜和核受体的荧光蛋白嵌合体，我们率先研究了配体结合调节糖皮质激素（GR）、维生素D （VDR）和类视黄醇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形成多个核灶。突变分析表明，GFP-VDR和GFP-GR的激素依赖性核病灶形成与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。骨化三醇处理后，GFP-VDR和RXR- bfp在核病灶共定位，FRET在这些病灶上形成了VDR/RXR二聚体。二聚化不充分的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，这一发现提示了局灶受体积累与DNA结合之间的相关性。此外，动态显微镜实验，如光漂白后荧光恢复（FRAP）和光漂白中的荧光损失（FLIP），显示VDR和RXR在细胞核内快速移动，激素结合减缓了这种核内运动。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，并确定II型核受体和辅抑制因子之间的空间和时间关系。