Prüfer Kirsten, Barsony Julia
Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Endocrinol. 2002 Aug;16(8):1738-51. doi: 10.1210/me.2001-0345.
Liganded and unliganded vitamin D receptors (VDRs) carry out distinct functions; both types of functions require heterodimerization with retinoid X receptors (RXRs). Our recent studies with fluorescent protein chimeras of VDR and RXR, termed GFP-VDR, YFP-RXR, and RXR-BFP, indicated that RXR regulates VDR functions in part by regulating subcellular localization. Here we explored the mechanisms of this regulation. Photobleaching experiments demonstrated that YFP-RXR and both unliganded and liganded GFP-VDR shuttle constantly between nucleus and cytoplasm. To characterize RXR import, we identified a nuclear localization sequence (NLS) in the DNA-binding domain. Mutations in this NLS caused predominant cytoplasmic localization of nlsYFP-RXR and prevented transcriptional activity. The nlsRXR-BFP retained unliganded GFP-VDR in the cytoplasm and reduced baseline transcriptional activity. After calcitriol exposure, however, both GFP-VDR and nlsRXR-BFP entered the nucleus. We characterized receptor export rates and mechanisms using permeabilization experiments. Mutations in the calreticulin binding region slowed both GFP-VDR and YFP-RXR export. Coexpression of RXR-BFP slowed the export of unliganded GFP-VDR, whereas calcitriol treatment tripled the rate of GFP-VDR export. Treatment with leptomycin B, an inhibitor of CRM-1 receptor-mediated export, inhibited export of unliganded GFP-VDR but did not influence export of liganded GFP-VDR or YFP-RXR. Leptomycin B added before calcitriol similarly decreased hormone-induced luciferase activity but was ineffective when added subsequent to calcitriol. These results indicate that the unliganded and liganded VDR interact differently with the import and export receptors and with RXR. Most likely, the regulation of VDR nuclear import by RXR is essential for ligand-independent functions.
结合配体和未结合配体的维生素D受体（VDR）执行不同的功能；这两种功能都需要与视黄酸X受体（RXR）形成异源二聚体。我们最近使用VDR和RXR的荧光蛋白嵌合体（称为GFP-VDR、YFP-RXR和RXR-BFP）进行的研究表明，RXR部分通过调节亚细胞定位来调节VDR功能。在此，我们探讨了这种调节的机制。光漂白实验表明，YFP-RXR以及未结合配体和结合配体的GFP-VDR在细胞核和细胞质之间持续穿梭。为了表征RXR的核输入，我们在DNA结合域中鉴定了一个核定位序列（NLS）。该NLS中的突变导致nlsYFP-RXR主要定位于细胞质，并阻止转录活性。nlsRXR-BFP将未结合配体的GFP-VDR保留在细胞质中，并降低基线转录活性。然而，在暴露于骨化三醇后，GFP-VDR和nlsRXR-BFP都进入了细胞核。我们使用透化实验表征了受体的输出速率和机制。钙网蛋白结合区域的突变减缓了GFP-VDR和YFP-RXR的输出。RXR-BFP的共表达减缓了未结合配体的GFP-VDR的输出，而骨化三醇处理使GFP-VDR的输出速率增加了两倍。使用CRM-1受体介导的输出抑制剂雷帕霉素B处理可抑制未结合配体的GFP-VDR的输出，但不影响结合配体的GFP-VDR或YFP-RXR的输出。在骨化三醇之前添加雷帕霉素B同样会降低激素诱导的荧光素酶活性，但在骨化三醇之后添加则无效。这些结果表明，未结合配体和结合配体的VDR与核输入和输出受体以及RXR的相互作用不同。很可能，RXR对VDR核输入的调节对于不依赖配体的功能至关重要。
