Nuclear Localization and Export Signals of the Human Aryl Hydrocarbon Receptor

Abstract

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the Ahr nuclear translocator (hypoxia-inducible factor 1␤). We found in this study that Ahr contains both nuclear localization and export signals in the NH 2 -terminal region. A fusion protein composed of ␤-galactosidase and full-length Ahr translocates from the cytoplasm to the nucleus in a liganddependent manner. However, a fusion protein lacking the PAS (Per-Ahr nuclear translocator-Sim homology) domain of the Ahr showed strong nuclear localization activity irrespective of the presence or absence of ligand. A minimum bipartite Ahr nuclear localization signal (NLS) consisting of amino acid residues 13-39 was identified by microinjection of fused proteins with glutathione S-transferase-green fluorescent protein. A NLS having mutations in bipartite basic amino acids lost nuclear translocation activity completely, which may explain the reduced binding activity to the NLS receptor, PTAC58. A 21-amino acid peptide (residues 55-75) containing the Ahr nuclear export signal is sufficient to direct nuclear export of a microinjected complex of glutathione S-transferase-Ahr-green fluorescent protein. These findings strongly suggest that Ahr act as a ligandand signal-dependent nucleocytoplasmic shuttling protein.The aryl hydrocarbon receptor (Ahr) 1 binds a variety of environmentally important carcinogens, including polycyclic aromatic hydrocarbons and certain halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Before binding ligands, Ahr is located in the cytoplasm as one component of a complex that has a molecular mass of about 280 kDa (1). This complex is composed of Ahr, two molecules of the 90-kDa heat shock protein, and possibly a 43-kDa protein (2). After ligand binding, Ahr dissociates from the complex and translocates to the nucleus (3). The heterodimer of Ahr and Ahr nuclear translocator (ARNT) constitutes a transcription factor and binds specific DNA sequences called XREs (xenobioticresponsive elements) in the enhancer regions of the CYP1A1 and several other proteins involved in xenobiotic metabolism (4). Because these enzymes are involved in the metabolism of polycyclic aromatic hydrocarbons to active genotoxic metabolites, Ahr plays an important role in carcinogenesis caused by these compounds (5-7).Because ARNT was first cloned as a factor required for ligand-dependent nuclear translocation of Ahr from the cytoplasm to the nucleus (8), the subcellular localization of ARNT was believed to be cytoplasmic. In fact, most ARNT was recovered in the cytosolic fraction by cell fractionation. However, immunohistochemical analysis has shown that ARNT is localized predominantly in the nucleus, regardless of the presence or absence of ligands (9, 10). This controversial subject was clarified by our recent study in which a nuclear localization signal (NLS) of the amino acid residues between 39 and 61 of human ARNT was found to be a novel bipartite type recognized by th...

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal

“…To con®rm the nuclear translocation activity of Ku80(558 ± 574) directly, we examined the fate of puri®ed recombinant protein of GST-Ku80(558 ± 574)-GFP microinjected into the cytoplasm of HeLa-S3 cells as described previously (Ikuta et al, 1998). The GSTKu80(558 ± 574)-GFP fusion protein showed e cient nuclear localization within 30 min incubation at 378C ( Figure 7A), con®rming that the fragment serves as the NLS.…”

“…To construct GST-Ku80-GFP fusion genes, the GST-GFP2 cassette vector was prepared as described previously (Ikuta et al, 1998). After being annealed, the fragments were ligated to EcoRI and BamHI sites of GST-GFP2 vector.…”

“…After being annealed, the fragments were ligated to EcoRI and BamHI sites of GST-GFP2 vector. The GST-NLSc-GFP construct, which contained the core sequence of the NLS of SV40 T-antigen (Kalderon et al, 1984), was prepared as described previously (Ikuta et al, 1998). The junctions of all the constructs were veri®ed by sequencing.…”

“…The puri®cation of expressed GST-Ku80-GFP fusion proteins was carried out as described previously (Ikuta et al, 1998). The puri®ed proteins were dialysed against 20 mM HEPES bu er, pH 7.3, containing 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, and 1 mM EGTA.…”

“…The microinjection experiments were performed essentially as described previously (Ikuta et al, 1998). Various GST-Ku80-GFP fusion proteins were injected into the cytoplasm of HeLa-S3 cells.…”