Petrulis John R, Kusnadi Ann, Ramadoss Preeti, Hollingshead Brett, Perdew Gary H
Center for Molecular Toxicology and Carcinogenesis and the Department of Veterinary Science, Pennsylvania State University, University Park 16802, USA.
J Biol Chem. 2003 Jan 24;278(4):2677-85. doi: 10.1074/jbc.M209331200. Epub 2002 Nov 12.
The mouse aryl hydrocarbon receptor (mAhR) is a ligand-activated transcription factor that exists in a tetrameric, core complex with a dimer of the 90-kDa heat shock protein, and the hepatitis B virus X-associated protein 2 (XAP2). Transiently expressed mAhR-YFP (yellow fluorescent protein fused with the mAhR) localizes throughout cells, with a majority occupying nuclei. Co-expression of XAP2 with mAhR-YFP results in a distinct redistribution to the cytoplasm. We have utilized several approaches to attempt to identify the mechanism by which XAP2 modulates the sub-cellular localization of the mAhR. The nuclear export inhibitor, leptomycin B, was used to demonstrate that XAP2 inhibits ligand-independent nucleocytoplasmic shuttling of the receptor. Results from cytoskeletal disruption and the addition of an alternate nuclear localization sequence (NLS) to mAhR-YFP suggest that XAP2 does not physically tether the complex in the cytoplasm. The use of a rabbit polyclonal antibody raised against a portion of the bipartite NLS of the mAhR revealed that XAP2 does not appear to block access to the NLS. However, XAP2 hinders importin beta binding to the mAhR complex, suggesting that XAP2 alters the conformation of the bipartite NLS of mAhR. XAP2 also represses the transactivation potential of the AhR, in contrast to previously published reports, perhaps by stabilizing the receptor complex and/or blocking nucleocytoplasmic shuttling of the AhR complex.
小鼠芳烃受体（mAhR）是一种配体激活的转录因子，它以四聚体核心复合物的形式存在，该复合物包含一个90 kDa热休克蛋白二聚体和乙肝病毒X相关蛋白2（XAP2）。瞬时表达的mAhR-YFP（与mAhR融合的黄色荧光蛋白）定位于整个细胞，大部分位于细胞核中。XAP2与mAhR-YFP共表达会导致其明显重新分布到细胞质中。我们采用了多种方法来试图确定XAP2调节mAhR亚细胞定位的机制。核输出抑制剂放线菌素B被用于证明XAP2抑制受体的非配体依赖性核质穿梭。细胞骨架破坏实验结果以及向mAhR-YFP添加另一种核定位序列（NLS）的实验结果表明，XAP2并非在物理上把复合物拴在细胞质中。使用针对mAhR二分NLS一部分产生的兔多克隆抗体表明，XAP2似乎不会阻断对NLS的 access。然而，XAP2会阻碍输入蛋白β与mAhR复合物的结合，这表明XAP2改变了mAhR二分NLS的构象。与之前发表的报告相反，XAP2还会抑制AhR的反式激活潜能，这可能是通过稳定受体复合物和/或阻断AhR复合物的核质穿梭来实现的。
