Importin 4 is responsible for ligand-independent nuclear translocation of vitamin D receptor
Importin 4 is responsible for ligand-independent nuclear translocation of vitamin D receptor
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DOI：
10.1074/jbc.m509347200
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发表时间：
2005-12-09
影响因子：
4.8
通讯作者：
Ozono, K
中科院分区：
文献类型：
--
作者：
Miyauchi, Y;Michigami, T;Ozono, K
维生素D受体（VDR）定位于细胞核中，并作为配体依赖性转录因子发挥作用。为了阐明VDR核转位的分子机制，我们使用毛地黄皂苷透化的半完整细胞进行体外核转运试验。在该测定中，即使在不存在配体的情况下，也将重组完整VDR-（4427）和缺乏VDR羧基末端的截短突变体VDR-（4-232）输入到细胞核。相反，缺乏氨基末端DNA结合结构域的VDR-（91-427）在不存在配体的情况下不输入到细胞核，并且以其配体形式有效输入。这些结果表明，有两种不同的机制的核运输的VDR的配体依赖性和非依赖性的途径，并在不同的区域的VDR负责这些过程。因此，我们以VDR-（4-232）为诱饵进行酵母双杂交筛选，以探索负责VDR的配体非依赖性核转位的分子，并确定importin 4为相互作用蛋白。在将转运因子作为重组蛋白应用的重建实验中，重组importin 4促进VDR的核转位而不管其配体如何，而importin β即使在配体存在下也不能转运VDR。总之，importin 4，而不是importin β，负责VDR的配体非依赖性核转位。
Vitamin D receptor (VDR) is localized in nuclei and acts as a ligand-dependent transcription factor. To clarify the molecular mechanisms underlying the nuclear translocation of VDR, we utilized an in vitro nuclear transport assay using digitonin-permeabilized semi-intact cells. In this assay, recombinant whole VDR-(4427) and a truncated mutant VDR-(4-232) lacking the carboxyl terminus of VDR were imported to nuclei even in the absence of ligand. In contrast, VDR-(91-427) lacking the amino-terminal DNA-binding domain was not imported to nuclei in the absence of ligand, and was efficiently imported in its liganded form. These results suggested that there are two distinct mechanisms underlying the nuclear transport of VDR; ligand-dependent and -independent pathways, and that the different regions of VDR are responsible for these processes. Therefore, we performed the yeast two-hybrid screening using VDR-(4-232) as the bait to explore the molecules responsible for ligand-independent nuclear translocation of VDR, and have identified importin 4 as an interacting protein. In the reconstruction experiments where transport factors were applied as recombinant proteins, recombinant importin 4 facilitated nuclear translocation of VDR regardless of its ligand, whereas importin beta failed in transporting VDR even in the presence of ligand. In conclusion, importin 4, not importin beta, is responsible for the ligand-independent nuclear translocation of VDR.