Aryl hydrocarbon (Ah) receptor DNA-binding activity. Sequence specificity and Zn2+ requirement (original) (raw)
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Archives of Biochemistry and Biophysics, 1996
stitutively express a distinct nuclear DNA-binding form of the AhR complex that may result from the pres-2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) supence of an additional protein or a structural variant presses B lymphocyte proliferation and immunoglobuof the AhR. ᭧ 1996 Academic Press, Inc. lin production. We previously reported that the aryl Key Words: dioxin; TCDD; Ah receptor; B lymphohydrocarbon receptor (AhR) complex, composed of the cytes; DNA binding. AhR ligand binding subunit and the Ah receptor nuclear translocator (ARNT), was constitutively present in nuclear extracts from two human B lymphocyte cell lines (Biochem. Biophys. Res. Commun. 212, 27-34, 1995). The present study compared the AhR complex in The prototype halogenated aromatic hydrocarbon the IM-9 and PJS-91 human B lymphocyte and HepG2 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 4 is a perhuman hepatoma cell lines. AhR mRNA levels in the sistent environmental contaminant which produces a two lymphocyte cell lines were substantially lower wide spectrum of adverse effects in laboratory animals, than those in HepG2 cells, as was immunoreactive AhR including tumor promotion, reproductive and developprotein. In contrast, ARNT mRNA and protein were mental toxicity, and immunotoxicity (1). The immunoexpressed at a high level in all three cell lines. TCDD toxicity of TCDD is well documented and includes efinduction of cytochrome P450 1A1 mRNA and protein fects on both cell-mediated and humoral immunity (2, was detected in only the PJS-91 lymphocyte cell line, 3). Decreased antibody responses to T lymphocyte-indeand at a markedly lower level than that in HepG2 cells.
European Journal of Pharmacology: Environmental Toxicology and Pharmacology, 1995
The aryl hydrocarbon (Ah) responsiveness of the T-47D, Hep G2, LS180, MCF-7, A431, C-4II and MDA-MB-231 human cancer cell lines was determined by the induction of CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity. With the exception of the MDA-MB-231 breast cancer cell line, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly induced CYP1A1 mRNA levels and ethoxyresorufin O-deethylase activity in the remaining six cell lines and, based on their ECso values, for ethoxyresorufin O-deethylase induction, their Ah responsiveness followed the order T-47D > C-4II > MCF-7 > LS180 > Hep G2 > A431. In contrast, all the cell lines expressed the nuclear Ah receptor complex (167.1-24.5 fmol/mg protein) which bound to a 32p-labeled consensus dioxin responsive element (DRE) in a gel mobility shift assay. The results of gel permeation chromatography and sucrose density gradient centrifugation studies showed that the calculated M r values for the nuclear Ah receptor complex varied from 175 kDa (MDA-MB-231 cells) to 221 kDa (MCF-7 cells). In contrast, the photoaffinity labeled nuclear Ah receptor from all the cell lines gave a specifically labeled 110 kDa band and the apparent molecular weight of the nuclear Ah receptor complex cross-linked to a bromodeoxyuridine-substituted DRE was 200 kDa. The data show that the molecular properties and levels of the nuclear Ah receptor complex from seven different human cancer cell lines do not predict Ah responsiveness.
Subunit composition of the heteromeric cytosolic aryl hydrocarbon receptor complex
Journal of Biological Chemistry, 1994
In a previous cross-linking study we have shown that the cytosolic aryl hydrocarbon receptor (AhR) complex has a heterotetrameric structure (Perdew, G. H. (1992) Biochem. Biophys. Res. Commun. 182, 56-62). In this report, both cross-linked and [3SSlmethionine-labeled Hepa lclc7 cytosol were used to characterize the subunit composition of the AhR complex by immunoprecipitation with an AhR polyclonal antibody followed by immunochemical analysis using antibodies against the AhR and 90-kDa heat shock protein (hsp90). Results indicated that the four subunits found in cross-linking experiments were composed of three species: the AhR ligand binding subunit, hsp90, and an unknown 43-kDa protein. The stoichiometry of hsp90 present in each AhR complex was determined in two separate experiments: 1) from cross-linking experiments, stoichiometry was determined by quantitative immunoblotting with anti-AhR and anti-hsp90 antibodies followed by quantitation with i2SI-counterantibody on protein blots; 2) using "Slabeled Hepa 1 cytosol, the hsp9O/AhR stoichiometry was determined by immunopurifying receptor complexes, and the amount of 3sS-labeledAhR and hsp90 was assessed. The stoichiometry values obtained were 2.4 and 1.72 mol of hsp9O/mol of AhR using each experimental approach, respectively. ~~~~~ ~ 2,3,7,8-Tetrachlorodibenzo-p-dioxin and similar compounds (e.g. polychlorinated biphenyl) are environmental contaminants. Their toxic and biological effects occur via the aryl hydrocarbon receptor (AhR)' mediated signal transduction pathway (1). After binding to the agonist the ligand-activated AhR is believed to transform' from a 9 S form to a DNA binding form (6 S form). The 6 S AhR complex is apparently composed of the AhR and Arnt; this heterodimer is capable of binding t o dioxin response elements, resulting in increased transcription of several genes involved in xenobiotic metabolism, such as cyplal mental Health Science Grant ES-04869. This is technical paper 14,311, * This work was supported in part by National Institute of Environ-Indiana Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked
Molecular Pharmacology, 2000
Similar to certain unliganded steroid hormone receptor complexes, the unliganded aryl hydrocarbon receptor has been shown to consist of a multimeric core complex that includes the 90-kDa heat shock protein (hsp90) and the immunophilin-like hepatitis B X-associated protein 2 (XAP2). Immunophilins and XAP2 associated with these complexes bind to the carboxylterminal end of hsp90 through an interaction with their tetratricopeptide repeat (TPR) domains. The consensus TPR binding motif contains two domains, A and B. Recently, the carboxyl terminus of XAP2 has been shown to contain a highly conserved TPR domain that is required for the assembly of XAP2 with both hsp90 and AhR. A search of the murine AhR sequence identified domain B (A-F-A-P) of the consensus TPR sequence directly adjacent to the carboxyl-terminal side of the helix-loop-helix region of the murine and human AhR. We hypothesized that this conserved domain B region may be involved with mediating interactions between either AhR-hsp90, AhR-XAP2, and/or AhR-AhR nuclear translocator protein. Sitedirected mutagenesis of the amino-terminal alanine residue of this region to an aspartic acid (A78D) completely inhibited 2,3,7,8-tetrachloro-p-dioxin (TCDD)-dependent activation of a xenobiotic response element (XRE) driven gene expression construct in transfected COS-1 and BP8 cells. The A82F mutation caused a 40 to 50% decrease in TCDD-dependent activation. The inability of A78D and the reduction of A82F to trans-activate XRE-driven reporter activity did not result from impaired AhR-XAP2-hsp90 interactions, TCDD-dependent AhR translocation to the nucleus, or AhR-AhR nuclear translocator protein interactions. In vitro DNA binding analysis demonstrated that loss of trans-activation potential by the A78D mutation resulted from impaired XRE binding. This study underscores the potential importance of AhR mutations that occur naturally outside of known functional domains. Many of the biochemical and toxic effects of HAHs and PAHs seem to be mediated through cytoplasmic binding to the aryl hydrocarbon receptor (Whitlock, 1987). In its inactive form, the aryl hydrocarbon receptor (AhR) resides in the cytoplasm in a tetrameric 9S core complex consisting of the AhR, a dimer of hsp90 (
Archives of Biochemistry and Biophysics, 1996
matic hydrocarbons which are widespread environ-The Aryl hydrocarbon receptor (AhR) is known to mental contaminants. The AhR is known to bind to a mediate 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-wide range of planar polycyclic aromatic hydrocarbons. induced toxic effects. Immunocytochemical studies re-Exposure to TCDD results in numerous species-and vealed that AhR in HeLa cells is localized throughout tissue-specific toxic and biological effects, including tuthe cell. Upon TCDD treatment most of the cytoplasmic mor promotion, immunotoxicity, hepatotoxicity, teratoreceptor is translocated into the nucleus in a timegenesis, and enzyme induction (1-4). Induction of sevdependent manner. A significant amount of AhR was eral genes have been reported due to TCDD exposure found to be tightly associated with the nuclear fraction including the most widely studied, cytochrome of untreated HeLa cells. The level of receptor in the P4501A1 and its associated monooxygenase activity nuclear fraction was approximately 16% of the total (5). The AhR, like steroid hormone receptors, is known cellular receptor pool. Further characterization of to be a ligand-dependent transcriptional factor that AhR heterocomplex from the HeLa nuclear fraction modulates gene expression through a high-affinity inby sucrose density gradient analysis revealed that the teraction with responsive elements upstream of ligand AhR was present in the 6 S form, and that the nuclear responsive genes. Unlike steroid hormone receptors, AhR could be coimmunoprecipitated using anti-Arnt both AhR and Arnt represent a novel class of basic mAb. The ability of the AhR to specifically interact region/helix-loop-helix transcriptional factors and with dioxin-responsive elements (DRE) was demonthe activated AhR heterodimerizes with Arnt prior to strated utilizing wild-type and two mutant DREs in gel binding to DNA. Activation or transformation 4 of the shift assays. These results would suggest that, in HeLa AhR is known to occur after cells are exposed to ligand. cells, the AhR-Arnt heterodimer is associated with the Upon ligand binding, the AhR dissociates from the 9 nuclear fraction under normal culture conditions. S complex, followed by heterodimerization with Arnt Therefore, HeLa cells can be used as a model system which probably occurs in the nuclear compartment (6, to study the biochemical and molecular function of the Ah receptor and the process that leads to activation 7). Reversible binding studies with [ 125 I]-2-iodo-7,8-diof the AhR in the absence of exogenous ligand. ᭧ 1996 bromodibenzo-p-dioxin have revealed that Arnt does Academic Press, Inc.
2008
The aryl hydrocarbon receptor (AHR) is a ligand-activated member of the bHLH-PAS family of transcription factors. Members of this family include HIF1a, EPAS, and SIM, which are involved in hypoxia and nervous system development. Another member of this family, aryl hydrocarbon nuclear translocator (ARNT), is the dimerization partner for the AHR. The AHR is often classified as a sensor of a wide range of xenobiotics, leading to induction of xenobiotic metabolism through enhanced expression of phase I/II enzymes. The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a prototypic ligand for the AHR and is often used to study the effects of prolonged AHR activation. Rodent exposure to TCDD results in a plethora of toxic effects, including wasting syndrome, tumor promotion, developmental defects, and liver toxicity (reviewed in Vanden Heuvel and
Molecular Pharmacology, 2004
The aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that is responsible for the regulation of several response genes, of which the best characterized is the CYP1A1 gene. The present study was undertaken to elucidate the mechanism of activation of the AhR by omeprazole (OME), 2-mercapto-5-methoxybenzimidazole (MMB), and primaquine (PRQ), compounds that have previously been reported to induce CYP1A1 expression but that are not typical AhR ligands. All compounds caused a significant increase in luciferase activity in rat H4IIE and human HepG2 hepatoma cells transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. Furthermore, MMB and PRQ, but not OME, were capable of transforming cytosolic AhR to a DNA-binding form and displacing AhR-bound [ 3 H]TCDD in rat hepatic cytosol in vitro. By performing site-directed mutagenesis of residues in the ligand-binding domain of the Gal4-AhR, a construct containing a Y320F substitution was found to be resistant to activation by OME, MMB, and PRQ, but not by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Comparable affinities of [ 3 H]TCDD-binding to the wild-type and the Y320F mutant Gal4-proteins, expressed in human embryonic kidney 293 cells, were obtained in the ligand-binding assay. In contrast, the competition of receptor-bound [ 3 H]TCDD by PRQ was absent from Gal4-Y320F but not from Gal4-AhR cell extracts. The results of this study confirm that MMB and PRQ are low-affinity ligands for the AhR and suggest that high-and low-affinity ligands interact with different residues of the AhR ligand-binding pocket. In addition, the data presented here indicate that Tyr 320 plays an important role in AhR activation. The aryl hydrocarbon receptor (AhR) is a member of the growing family of basic helix-loop-helix Per-Arnt-Sim (PAS) transcription factors whose members play key roles in development, adaptation to hypoxia, control of circadian rhythmicity, and metabolism of xenobiotic compounds (Gu et al., 2000). Mechanistically, the AhR functions as a ligand-activated transcription factor that is responsible for the transcriptional activation of several AhR-responsive genes (see reviews by Hankinson, 1995; Whitlock, 1999). A variety of environmental pollutants (e.g., polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons) are high-affinity ligands for the AhR. These ligands, including the prototype AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are characterized by being planar, aromatic, and hydrophobic molecules, fitting into a ligand-binding pocket with a maximal dimension of 14 ϫ 12 ϫ 5 Å. In addition, electronic and thermodynamic properties of the ligand have been shown to be critical for favorable interactions between the ligand and the receptor (Gillner et al., 1993; Kafafi et al., 1993; Waller and McKinney, 1995). Recent identification and characterization of novel AhR ligands or AhR activators (reviewed by Denison and Nagy, 2003), which have physicochemical and structural properties that deviate significantly from these typical AhR ligands, challenge the currently defined ligand-binding model. The structural diversity of these atypical AhR ligands/activators is clearly evident by comparison of the molecular structures of,