Definition of a Dioxin Receptor Mutant That Is a Constitutive Activator of Transcription
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m105607200
ISSN1083-351X
AutoresJacqueline McGuire, Kensaku Okamoto, Murray L. Whitelaw, Hirotoshi Tanaka, Lorenz Poellinger,
Tópico(s)bioluminescence and chemiluminescence research
ResumoThe intracellular dioxin (aryl hydrocarbon) receptor is a ligand-activated transcription factor that mediates the adaptive and toxic responses to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and structurally related congeners. Whereas the ligand-free receptor is characterized by its association with the molecular chaperone hsp90, exposure to ligand initiates a multistep activation process involving nuclear translocation, dissociation from the hsp90 complex, and dimerization with its partner protein Arnt. In this study, we have characterized a dioxin receptor deletion mutant lacking the minimal ligand-binding domain of the receptor. This mutant did not bind ligand and localized constitutively to the nucleus. However, this protein was functionally inert since it failed to dimerize with Arnt and to bind DNA. In contrast, a dioxin receptor deletion mutant lacking the minimal PAS B motif but maintaining the N-terminal half of the ligand-binding domain showed constitutive dimerization with Arnt, bound DNA, and activated transcription in a ligand-independent manner. Interestingly, this mutant showed a more potent functional activity than the dioxin-activated wild-type receptor in several different cell lines. In conclusion, the constitutively active dioxin receptor may provide an important mechanistic tool to investigate receptor-mediated regulatory pathways in closer detail. The intracellular dioxin (aryl hydrocarbon) receptor is a ligand-activated transcription factor that mediates the adaptive and toxic responses to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and structurally related congeners. Whereas the ligand-free receptor is characterized by its association with the molecular chaperone hsp90, exposure to ligand initiates a multistep activation process involving nuclear translocation, dissociation from the hsp90 complex, and dimerization with its partner protein Arnt. In this study, we have characterized a dioxin receptor deletion mutant lacking the minimal ligand-binding domain of the receptor. This mutant did not bind ligand and localized constitutively to the nucleus. However, this protein was functionally inert since it failed to dimerize with Arnt and to bind DNA. In contrast, a dioxin receptor deletion mutant lacking the minimal PAS B motif but maintaining the N-terminal half of the ligand-binding domain showed constitutive dimerization with Arnt, bound DNA, and activated transcription in a ligand-independent manner. Interestingly, this mutant showed a more potent functional activity than the dioxin-activated wild-type receptor in several different cell lines. In conclusion, the constitutively active dioxin receptor may provide an important mechanistic tool to investigate receptor-mediated regulatory pathways in closer detail. 2,3,7,8-tetrachlorodibenzo-p-dioxin 90-kDa heat shock protein basic helix-loop-helix ligand-binding domain xenobiotic response element dioxin receptor glucocorticoid receptor zinc finger DNA-binding domain polymerase chain reaction green fluorescent protein Chinese hamster ovary phosphate-buffered saline polyacrylamide gel electrophoresis nuclear localization signal The intracellular dioxin receptor also known as the aryl hydrocarbon receptor, is a ligand-dependent transcription factor that mediates the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),1 commonly known as dioxin (1Gu Y.Z. Hogenesch J.B. Bradfield C.A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 519-561Crossref PubMed Scopus (862) Google Scholar). Although disruption of the dioxin receptor gene in mice has not yielded conclusive results, it remains a possible scenario that physiological mechanisms may exist for activation of receptor function,e.g. during critical stages of vertebrate development (2Fernandez-Salguero P. Pineau T. Hilbert D.M. McPhail T. Lee S.S. Kimura S. Nebert D.W. Rudikoff S. Ward J.M. Gonzalez F.J. Science. 1995; 268: 722-726Crossref PubMed Scopus (949) Google Scholar, 3Schmidt J.V. Su G.H. Reddy J.K. Simon M.C. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6731-6736Crossref PubMed Scopus (754) Google Scholar, 4Mimura J. Yamashita K. Nakamura K. Morita M. Takagi T.N. Nakao K. Ema M. Sogawa K. Yasuda M. Katsuki M. Fujii-Kuriyama Y. Genes Cells. 1997; 2: 645-654Crossref PubMed Scopus (546) Google Scholar, 5Lahvis G.P. Lindell S.L. Thomas R.S. McCuskey R.S. Murphy C. Glover E. Bentz M. Southard J. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10442-10447Crossref PubMed Scopus (317) Google Scholar). However, an endogenous ligand for the receptor has not yet been identified, suggesting that alternative pathways for receptor activation may exist. In the absence of ligand, the dioxin receptor is generally found in the cytoplasm associated in high molecular weight complexes comprising a dimer of the molecular chaperone hsp90, an immunophilin homolog known as XAP2 (hepatitis B virus X-associatedprotein-2)/AIP (aryl hydrocarbon receptor-interacting protein)/ARA9 (aryl hydrocarbonreceptor-associated protein-9), and the co-chaperone p23 (6Perdew G.H. Biochem. Biophys. Res. Commun. 1992; 182: 55-62Crossref PubMed Scopus (61) Google Scholar, 7Chen H.S. Perdew G.H. J. Biol. Chem. 1994; 269: 27554-27558Abstract Full Text PDF PubMed Google Scholar, 8Carver L.A. Bradfield C.A. J. Biol. Chem. 1997; 272: 11452-11456Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 9Ma Q. Whitlock J.P. J. Biol. Chem. 1997; 272: 8878-8884Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 10Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Crossref PubMed Scopus (312) Google Scholar, 11Kazlauskas A. Poellinger L. Pongratz I. J. Biol. Chem. 1999; 274: 13519-13524Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In the presence of dioxin, the receptor is converted to a functional DNA-binding species in a multistep process involving nuclear translocation, dissociation from the hsp90 complex, and dimerization with its partner protein Arnt (Ahreceptor nuclear translocator). Formation of the dioxin receptor/Arnt heterodimer is a prerequisite for DNA binding and promotes transcription of target genes including those encoding xenobiotic-metabolizing enzymes such as CYP1A1(1Gu Y.Z. Hogenesch J.B. Bradfield C.A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 519-561Crossref PubMed Scopus (862) Google Scholar). Both the dioxin receptor and Arnt are members of a distinct subclass of the basic helix-loop-helix (bHLH) family of transcriptional regulators known as bHLH/PAS proteins. Members of this potent subfamily mediate diverse biological processes, including response to hypoxia, circadian rhythmicity, and development of the central nervous system (1Gu Y.Z. Hogenesch J.B. Bradfield C.A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 519-561Crossref PubMed Scopus (862) Google Scholar, 12Crews S.T. Genes Dev. 1998; 12: 607-620Crossref PubMed Scopus (301) Google Scholar, 13Taylor B.L. Zhulin I.B. Microbiol. Mol. Biol. Rev. 1999; 63: 479-506Crossref PubMed Google Scholar). Contiguous to the amino-terminal bHLH DNA-binding and dimerization motifs, members of this subfamily are identified on the basis of a second region of homology, the PAS (Per-Arnt-Sim) domain, originally identified in the Drosophila proteins PER and SIM, and Arnt. The PAS domain encompasses a region of ∼250–300 amino acids harboring two degenerate repeat sequences of 44 amino acids termed PAS A and PAS B, respectively, and has been shown to constitute an additional dimerization interface that can function independently of the bHLH motif (14Huang Z.J. Edery I. Rosbash M. Nature. 1993; 364: 259-262Crossref PubMed Scopus (417) Google Scholar, 15Lindebro M.C. Poellinger L. Whitelaw M.L. EMBO J. 1995; 14: 3528-3539Crossref PubMed Scopus (165) Google Scholar). More recently, additional roles in the regulation of heterodimerization and DNA binding specificities have been attributed to the PAS domain (16Pongratz I. Antonsson C. Whitelaw M.L. Poellinger L. Mol. Cell. Biol. 1998; 18: 4079-4088Crossref PubMed Scopus (92) Google Scholar, 17Zelzer E. Wappner P. Shilo B.Z. Genes Dev. 1997; 11: 2079-2089Crossref PubMed Scopus (124) Google Scholar). In the case of the dioxin receptor, the carboxyl-terminal half of the PAS domain, spanning the hydrophobic PAS B repeat motif, has been shown to harbor the core ligand-binding activity of the receptor (18Burbach K.M. Poland A. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8185-8189Crossref PubMed Scopus (723) Google Scholar, 19Whitelaw M.L. Gottlicher M. Gustafsson J.Å. Poellinger L. EMBO J. 1993; 12: 4169-4179Crossref PubMed Scopus (127) Google Scholar). In addition to binding ligand, this region has also been shown to mediate association with the molecular chaperone hsp90 (19Whitelaw M.L. Gottlicher M. Gustafsson J.Å. Poellinger L. EMBO J. 1993; 12: 4169-4179Crossref PubMed Scopus (127) Google Scholar), consistent with a role for hsp90 in regulating signal responsiveness by folding the ligand-binding domain (LBD) into a high affinity ligand binding conformation (20Carver L.A. Jackiw V. Bradfield C.A. J. Biol. Chem. 1994; 269: 30109-30112Abstract Full Text PDF PubMed Google Scholar, 21Coumailleau P. Poellinger L. Gustafsson J.Å. Whitelaw M.L. J. Biol. Chem. 1995; 270: 25291-25300Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 22Whitelaw M.L. McGuire J. Picard D. Gustafsson J.Å. Poellinger L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4437-4441Crossref PubMed Scopus (114) Google Scholar). Taken together, the PAS domain appears to be a complex structure harboring a number of distinct functional activities. In our efforts to understand further the complex interplay between structure and function of the dioxin receptor, we have examined the functional activity of a dioxin receptor deletion mutant lacking the core LBD. Although this protein is constitutively localized to the nucleus, it was functionally inert on a xenobiotic response element (XRE)-driven reporter gene both in the presence and absence of ligand. In contrast, deletion of amino acids 288–421 encompassing the minimal PAS B motif generated an activated form of the receptor that stimulated transcription in the absence of ligand, demonstrating that this mutant functions as a constitutively active regulatory protein. Interestingly, this mutant showed a more potent functional activity than the dioxin-activated wild-type receptor, possibly indicating that exposure to ligand does not induce maximal activation of the receptor. The plasmids used for in vitro translation of the full-length dioxin receptor (mDR/ATG/pSP72) and Arnt (Arnt/pGem) have been described previously (23McGuire J. Whitelaw M.L. Pongratz I. Gustafsson J.Å. Poellinger L. Mol. Cell. Biol. 1994; 14: 2438-2446Crossref PubMed Scopus (116) Google Scholar, 24Mason G.G. Witte A.M. Whitelaw M.L. Antonsson C. McGuire J. Wilhelmsson A. Poellinger L. Gustafsson J.Å. J. Biol. Chem. 1994; 269: 4438-4449Abstract Full Text PDF PubMed Google Scholar). For construction of the dioxin receptor deletion mutants DRΔLBD and DRΔPASB, pSportAhR containing full-length murine dioxin receptor cDNA (18Burbach K.M. Poland A. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8185-8189Crossref PubMed Scopus (723) Google Scholar) was amplified by PCR using primers designed to yield fragments of the dioxin receptor encoding codons 1–229 and 1–287, respectively. Primers were designed to carry restriction sites for ClaI and XhoI, enabling directional insertion into ClaI/XhoI-digested pGem7 to give pDR1–229/Gem and pDR1–287/Gem, respectively. A dioxin receptor fragment encompassing codons 422–805 was obtained by PCR from pSportAhR using specific primers carrying restriction sites for XhoI. The resulting fragment was digested with XhoI and subcloned into XhoI-digested pDR1–229/Gem and pDR1–287/Gem to give pDRΔLBD/Gem and pDRΔPASB/Gem, respectively. Whenever possible, PCR sequence was replaced by natural dioxin receptor sequences from mDR/ATG/pSP72 (23McGuire J. Whitelaw M.L. Pongratz I. Gustafsson J.Å. Poellinger L. Mol. Cell. Biol. 1994; 14: 2438-2446Crossref PubMed Scopus (116) Google Scholar). All constructs were confirmed by DNA sequence analysis.ClaI/XbaI digestion of the corresponding pDR/Gem constructs followed by subcloning into ClaI/XbaI-digested eukaryotic expression plasmid pCMV4 yielded constructs pDRΔLBD/CMV4 and pDRΔPASB/CMV4. The FLAG-tagged expression constructs pDR-FLAG/CMV4 and pDRΔPASB-FLAG/CMV4 were constructed by incorporating a FLAG tag epitope (Sigma), produced by PCR, at the extreme 3′-end of the coding sequence of the respective cDNAs. The pCMX-SAH/Y145F expression vector, encoding a modified and highly chromophoric form of green fluorescent protein (GFP) under the control of the cytomegalovirus promoter, has been described previously (25Kallio P.J. Okamoto K. O'Brien S. Carrero P. Makino Y. Tanaka H. Poellinger L. EMBO J. 1998; 17: 6573-6586Crossref PubMed Google Scholar). To create a GFP fusion construct of the full-length dioxin receptor for expression and visualization in living cells, a PCR fragment was amplified from pSportAhR using primers designed to yield a fragment of the dioxin receptor encoding codons 1–287. Primers were designed to carry restriction sites for BamHI and NheI, enabling in-frame directional insertion into BamHI/NheI-digested pCMX-SAH/Y145F. A CelII/XbaI dioxin receptor fragment encompassing amino acids 72–805 isolated from mDR/ATG/pSP72 was then inserted to provide the final construct pGFP-mDR/CMX. GFP fusion constructs for DRΔLBD and DRΔPASB were produced by digesting pGFP-mDR/CMX with CelII/NotI and replacing with CelII/NotI fragments isolated from pDRΔLBD/Gem and pDRΔPASB/Gem, thereby producing pGFP-DRΔLBD/CMX and pGFP-DRΔPASB/CMX, respectively. To produce the GFP fusion construct containing the minimal LBD of the dioxin receptor, a PCR fragment encoding codons 230–421 was amplified from pSportAhR. The primers were designed to carry restriction sites for XhoI, enabling insertion into SalI-digested pCMX-SAH/Y145F to give pGFP-DRLBD/CMX. pGRDBD/CMV4 and pGRDBDmDR/CMV4 (previously referred to as pGRDBDmDR83–805/CMV4) have been described (26Whitelaw M.L. Gustafsson J.Å. Poellinger L. Mol. Cell. Biol. 1994; 14: 8343-8355Crossref PubMed Scopus (150) Google Scholar). To construct the glucocorticoid receptor/mouse dioxin receptor deletion constructs for expression in mammalian cells, a ClaI/XbaI fragment was isolated from pDRΔLBD/Gem and pDRΔPASB/Gem and subcloned into ClaI/XbaI-digested pGRDBD/CMV4 to give pGRDBD/DRΔLBD/CMV4 and pGRDBD/DRΔPASB/CMV4, respectively. The yeast expression vectors pGRDBD/2HG and pGRDBDmDR/2HG (previously referred to as pGRDBDmDR83–805/2HG) have been described (22Whitelaw M.L. McGuire J. Picard D. Gustafsson J.Å. Poellinger L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4437-4441Crossref PubMed Scopus (114) Google Scholar). To construct the glucocorticoid receptor/mouse dioxin receptor deletion constructs for expression in yeast, an NcoI fragment was isolated from pDRΔLBD/Gem and pDRΔPASB/Gem and subcloned into NcoI-digested pGRDBDmDR/2HG to give pGRDBD/DRΔLBD/2HG and pGRDBD/DRΔPASB/2HG, respectively. The mammalian reporter gene constructs pTX.DIR and p(GRE)2T105LUC and the yeast reporter plasmid pUCΔSS26X have been described previously (27Berghard A. Gradin K. Pongratz I. Whitelaw M. Poellinger L. Mol. Cell. Biol. 1993; 13: 677-689Crossref PubMed Scopus (154) Google Scholar, 28Gradin K. Whitelaw M.L. Toftgård R. Poellinger L. Berghard A. J. Biol. Chem. 1994; 269: 23800-23807Abstract Full Text PDF PubMed Google Scholar, 29Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Crossref PubMed Scopus (650) Google Scholar). CHO cells were grown in Ham's F-12 medium, and HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 2 mml-glutamine, 10% fetal calf serum, 100 IU of penicillin, and 100 mg/ml streptomycin (Life Technologies, Inc.). For reporter gene assays, cells were transiently transfected with 2 μg of reporter plasmid and 1 μg of expression plasmids using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's recommendations. After a 6-h transfection period, cells were induced either with 10 nm TCDD (Cambridge Isotope Laboratories) or with an equivalent volume of vehicle (Me2SO) alone. Following incubation for 48 h, cells were collected and washed with PBS; extracts were prepared; and luciferase activity was measured. Experiments were carried out in duplicate, and extracts were normalized for protein concentration. For immunoblot analysis, transfected CHO cells were collected, washed with PBS, and lysed in 50 μl of whole cell extract buffer (20 mm Hepes, pH 7.9, 1.5 mm MgSO4, 0.2 mm EDTA, 0.42m NaCl, 0.5% (v/v) Nonidet P-40, 25% (v/v) glycerol, 1 mm dithiothreitol, and 1 mmphenylmethylsulfonyl fluoride) on ice for 30 min. Following centrifugation, the resulting supernatants were used as whole cell extracts. Samples containing 30 μg of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto nitrocellulose membranes. Immunodetection was achieved by incubation with mouse monoclonal anti-FLAG antibodies (Sigma), followed by chemiluminescence using the ECL detection system (Amersham Pharmacia Biotech). The yeast strain GRS4 has been described previously (29Picard D. Khursheed B. Garabedian M.J. Fortin M.G. Lindquist S. Yamamoto K.R. Nature. 1990; 348: 166-168Crossref PubMed Scopus (650) Google Scholar). Transformation of GRS4 with expression plasmids for the indicated glucocorticoid receptor/dioxin receptor fusion proteins together with the reporter plasmid pUCΔSS26X was carried out using a modification of the lithium acetate method (30Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar). Quantitation of β-galactosidase reporter gene activity in GRS4 transformants was carried out as described previously (22Whitelaw M.L. McGuire J. Picard D. Gustafsson J.Å. Poellinger L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4437-4441Crossref PubMed Scopus (114) Google Scholar). CHO cells were grown on poly-d-lysine-coated sterile coverslips in 35-mm diameter dishes and transiently transfected with 1 μg of each expression plasmid for GFP fusion proteins using 2 μl of FuGENE 6 transfection reagent (Roche Molecular Biochemicals) per dish. After a 6-h incubation, the medium was replaced with fresh medium, and incubation was continued for a further 24 h. The cells were then treated with 10 nm TCDD or vehicle (Me2SO) for 2 h, washed twice with PBS, and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. After three washes with PBS, the cells were quickly rinsed with deionized water and mounted on glass slides using Gel/Mount (Biomeda Co. Ltd.). Subcellular localization of GFP fusion proteins was observed with a Leica DMRXA fluorescent microscope using the GFP filter set, and images were scanned with a Hamamatsu digital camera, processed with OpenLab Version 3.0 software, and exported as composite PICT files. For each construct and each condition, ∼200 cells expressing GFP fusion proteins were observed, and representative images are presented. At least three independent experiments were carried out for each GFP fusion protein construct. The wild-type dioxin receptor, the dioxin receptor deletion constructs DRΔLBD and DRΔPASB, and Arnt were translated in vitro in the presence or absence of [35S]methionine (Amersham Pharmacia Biotech) in rabbit reticulocyte lysate (Promega) according to the manufacturer's recommendations. For Arnt co-immunoprecipitation experiments, equal concentrations of the indicated in vitro translated, [35S]methionine-labeled proteins were incubated with in vitro translated, unlabeled Arnt (5 μl) in TEG buffer (20 mm Tris-HCl, pH 7.4, 1 mm EDTA, 10% (w/v) glycerol, and 1 mm dithiothreitol) containing CompleteTM mini protease inhibitors (Roche Molecular Biochemicals) in the presence or absence of 10 nm TCDD (dioxin) either for 2 h at 25 °C or overnight at 4 °C in a final volume of 10 μl. Protein mixtures were then precleared by incubation on ice with 10 μl of preimmune serum for 15 min, followed by an additional 15-min incubation with 50 μl of a 50% slurry of protein A-Sepharose in TEG buffer supplemented with 150 mmNaCl, 0.2% Triton X-100, and 2 mg/ml bovine serum albumin. Following rapid centrifugation, the resulting supernatants were incubated with 10 μl of either anti-Arnt antiserum (24Mason G.G. Witte A.M. Whitelaw M.L. Antonsson C. McGuire J. Wilhelmsson A. Poellinger L. Gustafsson J.Å. J. Biol. Chem. 1994; 269: 4438-4449Abstract Full Text PDF PubMed Google Scholar) or preimmune serum for 1 h at room temperature. Protein A-Sepharose was then added (50 μl of a 50% slurry in supplemented TEG buffer), and the samples were incubated on ice for a further 45 min. After brief centrifugation, Sepharose pellets were washed three times with 500 μl of supplemented TEG buffer, followed by a final wash with TEG buffer alone. Co-immunoprecipitated proteins were eluted by boiling in SDS sample buffer and analyzed by SDS-PAGE and chemiluminescence. DNA binding experiments were performed with in vitro translated, unlabeled Arnt together with the wild-type dioxin receptor or the dioxin receptor deletion mutants DRΔLBD and DRΔPASB as described previously (31Antonsson C. Whitelaw M.L. McGuire J. Gustafsson J.Å. Poellinger L. Mol. Cell. Biol. 1995; 15: 756-765Crossref PubMed Google Scholar). Briefly, equivalent concentrations of the indicated in vitrotranslated proteins were incubated with 10 μl of in vitrotranslated Arnt in the presence or absence of 10 nm TCDD for 2 h at 25 °C. DNA binding reactions were carried out by the addition of a 36-base pair 32P-labeled double-stranded oligonucleotide spanning the XRE1 motif of the rat CYP1A1promoter (32Cuthill S. Wilhelmsson A. Poellinger L. Mol. Cell. Biol. 1991; 11: 401-411Crossref PubMed Scopus (49) Google Scholar) in 10 mm Hepes, 5% (v/v) glycerol, 0.05 mm dithiothreitol, 2.5 mm MgCl2, 1 mm EDTA, and 0.08% (w/v) Ficoll in a final volume of 40 μl containing 50 mm NaCl and 1 μg of poly(dI-dC) nonspecific competitor DNA (Amersham Pharmacia Biotech). Following incubation for 20 min at 25 °C, protein-DNA complexes were separated on a 4% low ionic strength native polyacrylamide gel (29:1 acrylamide/bisacrylamide) in Tris/glycine/EDTA buffer at 30 mA and analyzed by autoradiography. Ligand binding assays were carried out essentially as described previously using a modified hydroxylapatite adsorption assay (21Coumailleau P. Poellinger L. Gustafsson J.Å. Whitelaw M.L. J. Biol. Chem. 1995; 270: 25291-25300Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 23McGuire J. Whitelaw M.L. Pongratz I. Gustafsson J.Å. Poellinger L. Mol. Cell. Biol. 1994; 14: 2438-2446Crossref PubMed Scopus (116) Google Scholar). Briefly, equal concentrations of the in vitro translated, unlabeled wild-type dioxin receptor or the indicated dioxin receptor deletion mutants were made up to a final volume of 10 μl with blank reticulocyte lysate translation mixture, followed by dilution with 3 volumes of TEG buffer containing 2 mm dithiothreitol, 5 μg/ml protease inhibitor mixture (aprotinin, leupeptin, and pepstatin A), and 1 mm phenylmethylsulfonyl fluoride. The reaction mixtures were then incubated with 1 nm[3H]TCDD (40 Ci/mmol; Chemsyn, Lenexa, KS) in the presence or absence of a 150-fold molar excess of the specific competitor tetrachlorodibenzofuran at room temperature for 90 min. Following incubation, the reaction mixtures were treated with 50 μl of a 10% slurry (v/v) of dextran-coated charcoal in TEG buffer for 5 min on ice, followed by centrifugation. The resulting supernatants were then incubated with 50 μl of a 50% slurry (v/v) of hydroxylapatite in TEG buffer on ice for 30 min. Following rapid centrifugation, the supernatants were discarded, and the remaining pellets were washed four times with 500 μl of ice-cold TEG buffer containing 0.1% Tween 20. The pellets were eluted by incubating twice with 500 μl of ethanol, and the pooled supernatants were analyzed by scintillation counting. In the absence of ligand, the dioxin receptor exists in the cytoplasm in a latent, non-DNA-binding form characterized by association with the heat shock protein hsp90. Exposure to ligand results in rapid nuclear accumulation of the receptor and conversion to a heterodimeric complex with Arnt, a form that is now competent to bind DNA and to initiate the transcription of target genes (1Gu Y.Z. Hogenesch J.B. Bradfield C.A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 519-561Crossref PubMed Scopus (862) Google Scholar). Thus, the initial step in the activation of dioxin receptor function is binding of ligand. In addition to being a critical determinant of ligand-binding activity, the LBD of the dioxin receptor has also been postulated to be involved in the repression of a number of receptor activities that map outside the LBD itself such as dimerization with Arnt, DNA binding, and transcriptional activity (19Whitelaw M.L. Gottlicher M. Gustafsson J.Å. Poellinger L. EMBO J. 1993; 12: 4169-4179Crossref PubMed Scopus (127) Google Scholar, 26Whitelaw M.L. Gustafsson J.Å. Poellinger L. Mol. Cell. Biol. 1994; 14: 8343-8355Crossref PubMed Scopus (150) Google Scholar, 33Ma Q. Dong L. Whitlock J.P. J. Biol. Chem. 1995; 270: 12697-12703Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). It has not yet been determined how the ligand-binding domain modulates this repressive activity. However, since hsp90 binding has been shown to colocalize within this region, it has been suggested that hsp90 itself may function as the agent of repression either by steric interference or by misfolding of adjacent structures (19Whitelaw M.L. Gottlicher M. Gustafsson J.Å. Poellinger L. EMBO J. 1993; 12: 4169-4179Crossref PubMed Scopus (127) Google Scholar, 22Whitelaw M.L. McGuire J. Picard D. Gustafsson J.Å. Poellinger L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4437-4441Crossref PubMed Scopus (114) Google Scholar). We were interested to determine whether deletion of the minimal region of the dioxin receptor harboring the ligand- and hsp90-binding activities of the receptor would result in a protein that was uncoupled from regulation by dioxin. To this end, we have examined the functional activities, both in vitro and in vivo, of a dioxin receptor deletion mutant (DRΔLBD) lacking the core-delineated LBD that is located between amino acids 230 and 421 (19Whitelaw M.L. Gottlicher M. Gustafsson J.Å. Poellinger L. EMBO J. 1993; 12: 4169-4179Crossref PubMed Scopus (127) Google Scholar, 21Coumailleau P. Poellinger L. Gustafsson J.Å. Whitelaw M.L. J. Biol. Chem. 1995; 270: 25291-25300Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and that spans the C-terminal half of the PAS domain of the mouse dioxin receptor (Fig. 1 A). In control experiments,in vitro translation of DRΔLBD in rabbit reticulocyte lysate resulted in a protein that had lost the ability to bind ligand, consistent with deletion of the minimal domain required for high affinity ligand binding of the dioxin receptor (data not shown). Furthermore, in a specific co-immunoprecipitation assay using monoclonal anti-hsp90 antibodies, this protein showed only low levels of interaction with hsp90 (data not shown) that were attributed to non-LBD interactions via the bHLH motif (31Antonsson C. Whitelaw M.L. McGuire J. Gustafsson J.Å. Poellinger L. Mol. Cell. Biol. 1995; 15: 756-765Crossref PubMed Google Scholar). A bipartite nuclear localization signal (NLS) has been identified in the N terminus of the dioxin receptor, incorporating basic residues within the DNA-binding domain of the bHLH motif. This single N-terminal NLS motif has been shown to be sufficient to mediate ligand-inducible nuclear import of the dioxin receptor in the context of the full-length protein (34Ikuta T. Eguchi H. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1998; 273: 2895-2904Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Using expression vectors carrying GFP fused in frame with either the full-length mouse dioxin receptor or the dioxin receptor deletion mutant DRΔLBD, we examined the effect of deletion of the minimal LBD on intracellular localization of the dioxin receptor in living cells. In control experiments, transient transfection of CHO cells with the parental GFP construct alone revealed a uniform distribution of fluorescence throughout the cell that was unaffected by the presence of ligand (Fig. 1 B). Transient expression of the GFP-dioxin receptor fusion construct resulted in a similar distribution as the parental GFP vector alone in untreated cells. As expected, upon exposure to ligand, fluorescence rapidly accumulated in the nuclear compartment of the cell (Fig. 1 B) (34Ikuta T. Eguchi H. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1998; 273: 2895-2904Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 35Kazlauskas A. Poellinger L. Pongratz I. J. Biol. Chem. 2000; 275: 41317-41324Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 36Kazlauskas A. Sundström S. Poellinger L. Pongratz I. Mol. Cell. Biol. 2001; 21: 2594-2607Crossref PubMed Scopus (170) Google Scholar). In contrast, however, expression of the GFP-DRΔLBD fusion construct showed constitutive nuclear localization in CHO cells even in the absence of ligand (Fig. 1 B). Thus, deletion of the minimal LBD was sufficient to unmask the potent constitutive NLS activity contained within the remaining structure of the receptor. Furthermore, expression of a GFP fusion construct containing the minimal LBD of the dioxin receptor encompassing a
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