Hydrocarbon Receptor Nuclear Translocator Protein Research Articles

Redox properties and PAS domain structure of the Escherichia coli energy sensor Aer indicate a multistate sensing mechanism

The Per-Arnt-Sim (PAS; named for the representative proteins: Period, Aryl hydrocarbon receptor nuclear translocator protein and Single-minded) domain of the dimeric Escherichia coli aerotaxis receptor Aer monitors cellular respiration through a redox-sensitive flavin adenine dinucleotide (FAD) cofactor. Conformational shifts in the PAS domain instigated by the oxidized FAD (FADOX)/FAD anionic semiquinone (FADASQ) redox couple traverse the HAMP (histidine kinases, adenylate cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and kinase control domains of the Aer dimer to regulate CheA kinase activity. The PAS domain of Aer is unstable and has not been previously purified. Here, residue substitutions that rescue FAD binding in an FAD binding-deficient full-length Aer variant were used in combination to stabilize the Aer PAS domain. We solved the 2.4Å resolution crystal structure of this variant, Aer-PAS-GVV, and revealed a PAS fold that contains distinct features associated with FAD-based redox sensing, such as a close contact between the Arg115 side chain and N5 of the isoalloxazine ring and interactions of the flavin with the side chains of His53 and Asn85 that are poised to convey conformational signals from the cofactor to the protein surface. In addition, we determined the FADox/FADASQ formal potentials of Aer-PAS-GVV and full-length Aer reconstituted into nanodiscs. The Aer redox couple is remarkably low at -289.6± 0.4mV. In conclusion, we propose a model for Aer energy sensing based on the low potential of Aer-PAS-FADox/FADASQ couple and the inability of Aer-PAS to bind to the fully reduced FAD hydroquinone.

Transcriptomic and metabolic signatures of diatom plasticity to light fluctuations.

Unlike in terrestrial and freshwater ecosystems, light fields in oceans fluctuate due to both horizontal current and vertical mixing. Diatoms thrive and dominate the phytoplankton community in these fluctuating light fields. However, the molecular mechanisms that regulate diatom acclimation and adaptation to light fluctuations are poorly understood. Here, we performed transcriptome sequencing, metabolome profiling, and 13C-tracer labeling on the model diatom Phaeodactylum tricornutum. The diatom acclimated to constant light conditions was transferred to six different light conditions, including constant light (CL5d), short-term (1 h) high light (sHL1h), and short-term (1 h) and long-term (5 days) mild or severe light fluctuation conditions (mFL1h, sFL1h, mFL5d, and sFL5d) that mimicked land and ocean light levels. We identified 2,673 transcripts (25% of the total expressed genes) expressed differentially under different fluctuating light regimes. We also identified 497 transcription factors, 228 not reported previously, which exhibited higher expression under light fluctuations, including 7 with a light-sensitive PAS domain (Per-period circadian protein, Arnt-aryl hydrocarbon receptor nuclear translocator protein, Sim-single-minded protein) and 10 predicted to regulate genes related to light-harvesting complex proteins. Our data showed that prolonged preconditioning in severe light fluctuation enhanced photosynthesis in P. tricornutum under this condition, as evidenced by increased oxygen evolution accompanied by the upregulation of Rubisco and light-harvesting proteins. Furthermore, severe light fluctuation diverted the metabolic flux of assimilated carbon preferentially toward fatty acid storage over sugar and protein. Our results suggest that P. tricornutum use a series of complex and different responsive schemes in photosynthesis and carbon metabolism to optimize their growth under mild and severe light fluctuations. These insights underscore the importance of using more intense conditions when investigating the resilience of phytoplankton to light fluctuations.

Reconstruction of ARNT PAS-B Unfolding Paths by Steered MD and Artificial Neural Network Reveals New Putative Binding Conformations

Several proteins have been identified that can switch between different folds, expanding their functional role through previously unknown binding interactions. In this context, experimental evidence suggests that the PAS-B domain of the Aryl Hydrocarbon Receptor Nuclear Translocator protein (ARNT) has a fragile β-sheet structure that can undergo a +3 register shift upon a single mutation, changing its dimerization surface. Both unfolding and interconversion mechanisms require the same uncharacterized partially-unfolded state. Single-molecule Atomic Force Microscopy measurements (AFM) confirmed that the ARNT PAS-B domain is mechanically labile and revealed two distinct unfolding pathways, one following a single-stage mechanism, and the other showing an intermediate state. An atomistic characterisation of unfolding paths and relevant intermediate states has been carried out to identify new ARNT conformations possibly involved in partner recruitment. The unfolding pathways for ARNT PAS-B were simulated by steered Molecular Dynamics. Multiple replicas were recorded to explore different paths at low pulling speed. The overall model of unfolding was then reconstructed using a self-organizing map (SOM), a type of artificial neural network with an explicit visual representation. The map was trained on inter-residue distances of native contacts. The SOM model clearly depicts two alternative pathways in a human-understandable two-dimensional map. Retracing of single replicas on the map highlights a two-stage mechanism with initial unfolding of the C-terminal strand in 1/3 of the simulations, in agreement with AFM findings. Shortest path analysis on the map correctly detects replicas with minimal steering work. The partially-folded states extracted from the map could have a role in selectivity for partner recruitment during ARNT transcriptional activity.

A PAS-Containing Histidine Kinase is Required for Conidiation, Appressorium Formation, and Disease Development in the Rice Blast Fungus, Magnaporthe oryzae

Rice blast disease, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most important diseases in rice production. PAS (period circadian protein, aryl hydrocarbon receptor nuclear translocator protein, single-minded protein) domains are known to be involved in signal transduction pathways, but their functional roles have not been well studied in fungi. In this study, targeted gene deletion was carried out to investigate the functional roles of the PAS-containing gene MoPAS1 (MGG_02665) in M. oryzae. The deletion mutant ΔMopas1 exhibited easily wettable mycelia, reduced conidiation, and defects in appressorium formation and disease development compared to the wild type and complemented transformant. Exogenous cAMP restored appressorium formation in ΔMopas1, but the shape of the restored appressorium was irregular, indicating that MoPAS1 is involved in sensing the hydrophobic surface. To examine the expression and localization of MoPAS1 in M. oryzae during appressorium development and plant infection, we constructed a MoPAS1:GFP fusion construct. MoPAS1:GFP was observed in conidia and germ tubes at 0 and 2 h post-infection (hpi) on hydrophobic cover slips. By 8 hpi, most of the GFP signal was observed in the appressoria. During invasive growth in host cells, MoPAS1:GFP was found to be fully expressed in not only the appressoria but also invasive hyphae, suggesting that MoPAS may contribute to disease development in host cells. These results expand our knowledge of the roles of PAS-containing regulatory genes in the plant-pathogenic fungus M. oryzae.

Upregulation of aryl hydrocarbon receptor nuclear translocator 2 in the hippocampi of post-stroke depression rats.

Aryl hydrocarbon receptor nuclear translocator protein 2 (ARNT2), a member of the basic helix-loop-helix superfamily of transcription factors, may serve a vital role in neuronal survival and cell proliferation via formation of heterodimers with hypoxia-inducible factor-1α. Previous studies indicated that ARNT2 levels were elevated in the brains of ischemic rats; however, the involvement of ARNT2 in post-stroke depression (PSD) rats is not well understood. Therefore, the present study aimed to investigate the levels of ARNT2 in the hippocampi of PSD rats, and to clarify the potential association between ARNT2 and behavioral performance. A PSD rat model was established by middle cerebral artery occlusion (MCAO) followed by a 4-week chronic unpredictable mild stress (CUMS) regimen. A sucrose preference test and open field test (OFT) were conducted, and body weight was measured. In addition, reverse transcription-polymerase chain reaction and immunohistochemistry were performed to measure ARNT expression. Results indicated that MCAO+CUMS rats had lower weight gain, consumed less sucrose and moved less compared with controls. Furthermore, the mRNA and protein levels of ARNT in MCAO+CUMS rats were increased compared with in controls. The sucrose preference index and horizontal movement distance in the OFT were positively correlated with ARNT mRNA level. Thus, from these findings it was suggested that ARNT2 may be positively associated with improvement of cognitive impairment, and therefore may be a potential target in PSD treatment.

The Aryl Hydrocarbon Receptor Predisposes Hepatocytes to Fas-Mediated Apoptosis

Liver homeostasis is achieved by the removal of diseased and damaged hepatocytes and their coordinated replacement to maintain a constant liver cell mass. Cirrhosis, viral hepatitis, and toxic drug effects can all trigger apoptosis in the liver as a means of removing the unwanted cells, and the Fas "death receptor" pathway comprises a major physiological mechanism by which this occurs. The susceptibility to Fas-mediated apoptosis is, in part, a function of the hepatocyte's proteome. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to influence apoptosis, conceivably by regulating the expression of genes involved in apoptotic signaling. In this article, we present evidence demonstrating that AhR expression and function promote apoptosis in liver cells in response to Fas stimulation. Reintroduction of the AhR into the AhR-negative BP8 hepatoma cells as well as into primary hepatocytes from AhR knockout mice increases the magnitude of cell death in response to Fas ligand. Enhanced apoptosis correlates with increased caspase activity and mitochondrial cytochrome c release but not with the expression of several Bcl-2 family proteins. In vivo studies showed that in contrast to wild-type mice, AhR knockout mice are protected from the lethal effects of the anti-Fas Jo2 antibody. Moreover, down-regulation of the aryl hydrocarbon receptor nuclear translocator protein in vivo by adenovirus-mediated RNA interference to suppress AhR activity provided wild-type mice partial protection from Jo2-induced lethality.

Identification of a novel dioxin-inducible cytochrome P450.

Representational difference analysis was used to isolate cDNAs corresponding to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible genes from mouse Hepa-1 cells. One cDNA encoded a novel cytochrome P450. The human homolog was also isolated and later proved to be human CYP2S1. The induction of mouse CYP2S1 mRNA by dioxin represents a primary response and required the aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins. The induction of CYP2S1 also occurred in mouse liver and lung, with the highest expression found in lung. CYP2S1 was also inducible in a human lung epithelial cell line. The dioxin-inducibility of CY2S1 is exceptional, because all previously well-characterized cases of the induction of cytochromes P450 by dioxin involve members of the CYP1 family.

Expression of dioxin-related transactivating factors and target genes in human eutopic endometrial and endometriotic tissues

Objective: Although an association between dioxin exposure and endometriosis has been proposed, the effects of this environmental toxin on human endometriosis are not known. To understand the potential underlying molecular mechanisms we studied the expressions of cytochrome P-450 genes ( CYP1A1, CYP1A2, and CYP1B1 ), which are induced by dioxin, and the expressions of cytosolic receptor for dioxin, aryl hydrocarbon receptor, and its nuclear translocator, aryl hydrocarbon receptor nuclear translocator protein, in endometriotic and eutopic endometrial tissues. Study Design: Levels of transcripts of CYP1A1, CYP1A2, CYP1B1, aryl hydrocarbon receptor, and aryl hydrocarbon receptor nuclear translocator protein were determined by a quantitative reverse transcriptase–polymerase chain reaction and Southern blot assay in total ribonucleic acid samples from endometriotic and eutopic endometrial tissues. Eutopic endometrial tissue samples (n = 33) and endometriotic tissue samples (n = 10) were obtained at the time of uterine curettage and laparoscopy from disease-free women and from patients with endometriosis. Portions of these eutopic endometrial and endometriotic tissues were obtained simultaneously from the same patients (n = 8 pairs of samples). Levels of transcripts of CYP1A1, CYP1A2, CYP1B1, aryl hydrocarbon receptor, and aryl hydrocarbon receptor nuclear translocator protein were determined in endometrial and endometriotic tissues during follicular and luteal phases of the cycle and in cultured endometriotic stromal cells treated with forskolin, phorbol diacetate, medroxyprogesterone acetate,and serum. Results: Transcripts of dioxin receptor, its nuclear translocator, and two dioxin-induced target genes (CYP1A2 and CYP1B1) were demonstrated during follicular and luteal phases of the cycle in both eutopic endometrial tissues and tissues affected by pelvic endometriosis, with no readily detectable differences between these tissues. On the other hand, levels of transcripts of another dioxin-induced gene, CYP1A1, were found to be strikingly higher in endometriotic tissues than in the eutopic endometrium. Mean levels in endometriotic tissues were 8.7 times those found in eutopic endometrium. Various hormonal treatments of endometriotic stromal cells did not significantly alter these levels. Conclusion: We demonstrated for the first time the expression of dioxin-related transcription factors aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein and target genes CYP1A1, CYP1A2, and CYP1B1 in endometriotic tissues and stromal cells. Strikingly elevated CYP1A1 transcripts in endometriosis may give rise to significantly increased P-4501A1 enzyme activity and thus promote the development and growth of endometriosis by either activating procarcinogens or inducing the formation of catechol estrogens or both. In fact, the proposed link between dioxin exposure and endometriosis may be explained in part by the up-regulation of the CYP1A1 gene expression in endometriotic tissues. (Am J Obstet Gynecol 2000;182:767-75.)

The aromatic hydrocarbon receptor, transcription, and endocrine aspects of dioxin action

The widespread and persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin elicits adaptive and adverse biological responses by inducing changes in gene transcription. Some of dioxin's effects reflect disruption of endocrine homeostasis. The aromatic hydrocarbon receptor protein, together with its heterodimerization partner, the aromatic hydrocarbon receptor nuclear translocator protein, mediates dioxin action. There are notable similarities between the mechanism of dioxin action and the mechanisms of steroid/retinoid/thyroid hormone action. Studies of dioxin action may provide insights into the regulation of hormone-responsive genes and endocrine physiology.

Aryl Hydrocarbon Receptor Regulation of Ceramide-induced Apoptosis in Murine Hepatoma 1c1c7 Cells: A FUNCTION INDEPENDENT OF ARYL HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR

The relationship between aryl hydrocarbon receptor (AHR) content and susceptibility to apoptosis was examined in the murine hepatoma 1c1c7 cell line and a series of variants having different levels of AHR expression. Exposure of 1c1c7 cultures to N-acetylsphingosine (C2-ceramide) caused a concentration-dependent inhibition of cell proliferation, loss of viability, and induction of apoptosis as monitored by analyses of DNA fragmentation and caspase activation. A variant cell line (Tao) having approximately 10% of the AHR content of 1c1c7 cells also arrested following exposure to C2-ceramide, but did not undergo apoptosis. Modulation of 1c1c7 and Tao AHR contents by transfection of Ahr antisense and sense constructs, respectively, confirmed the relationship between AHR content and susceptibility to C2-ceramide-induced apoptosis. C2-ceramide also induced the apoptosis of an AHR-containing cell line lacking the aryl hydrocarbon receptor nuclear translocator protein. AHR ligands (i.e. 2,3,7,8-tetrachlorodibenzo-p-dioxin and alpha-naphthoflavone) neither induced apoptosis nor modulated the development of apoptosis in C2-ceramide-treated 1c1c7 cultures. AHR content did not affect staurosporine- or doxorubicin-induced apoptosis. These results suggest the AHR modulates aspects of ceramide signaling associated with the induction of apoptosis but not cell cycle arrest, and does so by a mechanism that is independent of its interaction with aryl hydrocarbon receptor nuclear translocator and exogenous AHR ligands.

Expression of the aryl hydrocarbon receptor (AhR) and the aryl hydrocarbon receptor nuclear translocator (ARNT) in fetal, benign hyperplastic, and malignant prostate.

Androgen-dependent tissue has been reported to be affected by chemical ligands of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, which heterodimerizes with the aryl hydrocarbon receptor nuclear translocator protein (ARNT). Fetal (n = 3), benign hyperplastic (BPH) (n = 10), and carcinomatous (CaP) (n = 19) prostate tissues were analyzed using immunohistochemistry. Western blot analysis was used to confirm the identity of the recognized proteins. Immunoblotting of enriched prostatic epithelial cells (EC) and stromal cells revealed constitutive expression of bands at around 110 kDa and 90 kDa, using anti-AhR and anti-ARNT, respectively. Immunohistology of the fetal specimens revealed heterogeneous cytoplasmic and nuclear AhR expression of immature EC and mesenchymal cells. Constitutive expression of AhR (primarily cytoplasmic) and ARNT (nuclear and cytoplasmic) by the majority of adult basal and secretory EC, CaP, and smooth muscle cells was confirmed in situ. The most intense anti-AhR/-ARNT reactivity was found on smooth muscle cells, followed by EC and fibrocytes. Secretory BPH-EC revealed significantly decreased AhR expression when compared to normal tissue segments. By contrast, anti-AhR reactivity was frequently increased in the more dedifferentiated tumor areas. These findings suggest that an undefined physiologic AhR ligand(s) as well as environmental factors may exert effects on EC and smooth muscle cells in the prostate through binding to these receptors.

Signal Transduction-mediated Activation of the Aryl Hydrocarbon Receptor in Rat Hepatoma H4IIE Cells

We have investigated mechanisms of omeprazole (OME)-mediated induction of CYP1A1 and CYP3A, using the rat hepatoma H4IIE cell line, in comparison with mechanisms exerted by traditional aryl hydrocarbon receptor (AhR) ligands such as benso(a)pyrene (B(a)P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). OME did not bind specifically to AhR, and it could not activate the AhR complex in rat cytosol to a xenobiotic-responsive element (XRE)-binding form in vitro. Genistein, a tyrosine kinase inhibitor, and daidzein, an inhibitor of casein kinase II, efficiently inhibited OME-mediated but not B(a)P- or TCDD-mediated induction of CYP1A1, as monitored at the transcriptional, mRNA, and protein levels as well as by analysis of activation of XRE-luciferase reporter constructs transfected into H4IIE cells. The protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and lavendustin A also had similar OME-specific effects. In addition, insulin pretreatment caused an almost complete inhibition of OME-dependent CYP1A1 induction but only partially affected TCDD and B(a)P-mediated induction of CYP1A1. Staurosporine, an inhibitor of protein kinase C, impaired the induction by both B(a)P and OME. OME caused an approximately 2-fold increase in the level of CYP3A expression, but all inhibitors used were ineffective in preventing this induction. Gel shift analysis with radiolabeled XRE and specific peptide antibodies toward AhR and aryl hydrocarbon receptor nuclear translocator protein (Arnt) revealed an OME-mediated translocation of the AhR.Arnt complex into the nuclei. Genistein inhibited the specific nuclear XRE binding caused by OME, but it potentiated the formation of the TCDD-induced XRE.AhR complex. Although daidzein was able to effectively inhibit the OME-stimulated CYP1A1 gene transcription, it did not influence the OME-dependent AhR.XRE complex formation. The data are consistent with a mechanism for OME-mediated induction of CYP1A1 that involves activation of the AhR complex via intracellular signal transduction systems and that is distinct from induction mediated by AhR ligands.

Expression of the Human Aryl Hydrocarbon Receptor Complex in Yeast: ACTIVATION OF TRANSCRIPTION BY INDOLE COMPOUNDS

The human aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator protein (ARNT) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for the study of this heterodimeric transcription factor. Specific transcriptional activation mediated by AHR/ARNT heterodimer, which is a functional indicator of receptor expression, was assessed by beta-galactosidase activity produced from a reporter plasmid. Yeast expressing AHR and ARNT displayed constitutive transcriptional activity that was not augmented by addition of AHR agonists in strains that required exogenous tryptophan for viability. In contrast, strains with an intact pathway for tryptophan biosynthesis responded to AHR agonists and had lower levels of background beta-galactosidase activity. Hexachlorobenzene, benzo(a)pyrene, and beta-naphthoflavone were effective AHR agonists in the yeast system, and had EC50 values of 200, 40, and 20 nM, respectively, for beta-galactosidase activity induction. Tryptophan, indole, indole acetic acid, and tryptamine activated transcription in yeast coexpressing AHR and ARNT (EC50 values approximately 300 microM). Indole-3-carbinol was an exceptionally potent AHR agonist (EC50 approximately 10 microM) in yeast. This yeast system is useful for the study of AHR/ARNT protein complexes, and may be generally applicable to the investigation of other multiprotein complexes.

Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates.

Western blot analysis was used to determine the concentration of the aryl hydrocarbon receptor nuclear translocator (ARNT) protein and aryl hydrocarbon receptor (AHR) in 11 mammalian cell culture lines derived from hepatic and nonhepatic tissues. The strategy was to first use Western blot analysis to determine the expression of ARNT or AHR in each cell line relative to its concentration in murine wild-type Hepa-1c1c7 (Hepa-1) cells. Actual ARNT and AHR concentrations in known amounts of total cell lysates were then determined by generating a standard curve with defined amounts of a highly purified ARNT or AHR protein and performing regression analysis. The results show that the level of ARNT expression in each of the cell lines is similar and represents approximately 0.001-0.002% of total cellular protein. The range of expression was only approximately 3-fold with wild-type Hepa-1 cells expressing the highest level of ARNT (33,000/cell) and canine kidney cells (MDCK line) expressing 14,000 ARNT molecules/cell. In contrast, the concentration of AHR varied by 65-fold over the different cell lines with the wild-type Hepa-1 expressing 323,000 AHR/cell and rat hepatoma cells (H4IIE) expressing 4700. The ratio of AHR to ARNT ranged from 0.3 in H4IIE cells to 10 in the Hepa-1 line with the majority of cells expressing 1-5 times more AHR than ARNT protein. Immunocytochemical staining of each cell line showed that ARNT was exclusively localized to the nuclear compartment and that a conserved nuclear localization signal mapped to the NH-terminal portion of the protein.

Absolute Requirement of Aryl Hydrocarbon Receptor Nuclear Translocator Protein for Gene Activation by Hypoxia

Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding heterodimeric protein complex originally described in the transcriptional activation of the erythropoietin gene by hypoxia. This protein complex is composed of two subunits, HIF-1α and -1β (aryl hydrocarbon receptor nuclear translocator, ARNT). In this study, we used ARNT-deficient cells, derived from the mouse hepatoma cell line Hepa1c1c7, to further characterize HIF-1 complex formation and its relationship with gene activation by hypoxia and desferrioxamine (Df). Gel shift assays revealed that ARNT is absolutely required for the formation of the HIF-1 DNA-binding complex. Results from RNase protection assays and Northern blots showed that the lack of functional HIF-1 complex completely abrogated the response to hypoxia of vascular endothelial growth factor (VEGF) and the glycolytic enzymes aldolase A (ALDA) and phosphoglycerate kinase 1 (PGK-1), genes known to be upregulated by low oxygen tension. Desferrioxamine induction of VEGF and PGK-1 genes was reduced in the ARNT-deficient cells, but at difference with hypoxia, it was not completely suppressed. These results suggest that Df is able to activate gene transcription through HIF-1-independent mechanisms. Exposure to hypoxia or Df did not induce any changes in HIF-1α and -1β mRNA levels, suggesting that posttranscriptional mechanisms are involved in HIF-1 complex activation.

Development of an in Vitro Model for Investigating the Formation of the Nuclear Ah Receptor Complex in Mouse Hepa 1c1c7 Cells

An in vitro assay for investigating factors which modulate formation of the nuclear aryl hydrocarbon (Ah) receptor complex was developed using 9S cytosolic Ah receptor isolated from wild-type Hepa 1c1c7 cells treated with 2,3,7,8-[ 3H]tetrachlorodibenzo- p-dioxin (TCDD) at 4°C and nuclei from Ah-responsive and -nonresponsive wild-type and mutant Hepa 1c1c7 cells. Incubation of the radiolabeled ([ 3H]TCDD) 9S cytosolic Ah receptor with nuclei from untreated wild-type Ah-responsive mouse Hepa 1c1c7 cells resulted in a time- and temperature-dependent formation of the nuclear Ah receptor complex as determined by either velocity sedimentation analysis or gel mobility shift assays using a consensus 32P-labeled dioxin-responsive element. Maximal levels of the nuclear Ah receptor formed within 30 min at 37°C and significantly lower levels were observed after incubation at 4, 15, or 25°C. Complementation studies using nuclei from untreated wild-type and Ah-nonrespensive class II mutant (translocation-deficient) cells and radiolabeled 9S cytosolic receptor (bound with [ 3H]TCDD) from both wild-type and mutant cell lines were also carried out. The results indicated that nuclear translocation was primarily inhibited using cytosol from mutant cells confirming the requirement for the aryl hydrocarbon receptor nuclear translocator protein for formation of the nuclear Ah receptor complex. The effects of a series of Ah receptor antagonists, ATP, vanadate apyrase, phosphatases, and lectin WGA, on formation of the nuclear Ah receptor complex were also investigated in the in vitro model using radiolabeled cytosolic Ah receptor and nuclei from untreated wild-type Hepa 1c1c7 cells. α-Naphthoflavone, ribonuclease, lectin WGA, vanadate, acid and alkaline phosphatase, and apyrase inhibited and ATP enhanced formation of the nuclear Ah re-ceptor complex. The results also show that TCDD-induced transformation of the Ah receptor was inhibited by α-naphthoflavone but not by ATP, lectin WGA, or apyrase. These data suggest that the cytosolic Ah receptor is required for phosphorylation prior to ligand binding and ATP is required for energy-dependent nuclear import of the Ah receptor.

Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT).

The activated aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) bind DNA as a heterodimer. Both proteins represent a novel class of basic helix-loop-helix (bHLH)-containing transcription factors in that (i) activation of AHR requires the binding of ligand (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]), (ii) the xenobiotic responsive element (XRE) recognized by the AHR/ARNT heterodimer differs from the recognition sequence for nearly all other bHLH proteins, and (iii) both proteins contain a PAS homology region, which in the Drosophila PER and SIM proteins functions as a dimerization domain. A cDNA for mouse ARNT has been cloned, and potential functional domains of ARNT were investigated by deletion analysis. A mutant lacking all regions of ARNT other than the bHLH and PAS regions is unimpaired in TCDD-dependent dimerization and subsequent XRE binding and only modestly reduced in ability to complement an ARNT-deficient mutant cell line, c4, in vivo. Both the first and second alpha helices of the bHLH region are required for dimerization. The basic region is required for XRE binding but not for dimerization. Deletion of either the A or B segments of the PAS region slightly affects TCDD-induced heterodimerization, while deletion of the complete PAS region severely affects (but does not eliminate) dimerization. Thus, ARNT possesses multiple domains required for maximal heterodimerization. Mutants deleted for PAS A, PAS B, and the complete PAS region all retain some degree of XRE binding, yet none can rescue the c4 mutant. Therefore, both the PAS A and PAS B segments, besides contributing to dimerization, apparently fulfill additional, unknown functions required for biological activity of ARNT.

Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT).

The activated aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) bind DNA as a heterodimer. Both proteins represent a novel class of basic helix-loop-helix (bHLH)-containing transcription factors in that (i) activation of AHR requires the binding of ligand (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]), (ii) the xenobiotic responsive element (XRE) recognized by the AHR/ARNT heterodimer differs from the recognition sequence for nearly all other bHLH proteins, and (iii) both proteins contain a PAS homology region, which in the Drosophila PER and SIM proteins functions as a dimerization domain. A cDNA for mouse ARNT has been cloned, and potential functional domains of ARNT were investigated by deletion analysis. A mutant lacking all regions of ARNT other than the bHLH and PAS regions is unimpaired in TCDD-dependent dimerization and subsequent XRE binding and only modestly reduced in ability to complement an ARNT-deficient mutant cell line, c4, in vivo. Both the first and second alpha helices of the bHLH region are required for dimerization. The basic region is required for XRE binding but not for dimerization. Deletion of either the A or B segments of the PAS region slightly affects TCDD-induced heterodimerization, while deletion of the complete PAS region severely affects (but does not eliminate) dimerization. Thus, ARNT possesses multiple domains required for maximal heterodimerization. Mutants deleted for PAS A, PAS B, and the complete PAS region all retain some degree of XRE binding, yet none can rescue the c4 mutant. Therefore, both the PAS A and PAS B segments, besides contributing to dimerization, apparently fulfill additional, unknown functions required for biological activity of ARNT.

The role of the aryl hydrocarbon receptor nuclear translocator protein in aryl hydrocarbon receptor action

The aryl hydrocarbon (AH, or dioxin) receptor mediates carcinogenesis by a wide variety of compounds. It acts as a ligand-dependent transcription factor. Many investigators expected that the AH receptor would prove to be a member of the steroid/thyroid/retinoic acid receptor superfamily of proteins. However, recent cloning of the two subunits of the DNA-binding form of the AH receptor has shown that this is not the case. These subunits, the AH receptor nuclear translocator protein (ARNT) and the ligand-binding AH receptor monomer, do not contain zinc finger DNA-binding domains, nor do they have any other sequence similarities with members of the above family. Instead, they both contain basic helix-loop-helix (bHLH) motifs and also another segment of sequence similarity, the “PAS” region. bHLH motifs in other transcription factors are known to function as dimerization and DNA-binding domains. Present experiments are directed toward understanding the mechanisms of action and the roles of the two subunits.