title: The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90.
abstract: The aryl hydrocarbon receptor is a member of the nuclear receptor superfamily that associates with the molecular chaperone HSP90 in the cytoplasm. The activation mechanism of the AhR is not yet fully understood. It has been proposed that after binding of ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3methylcholanthrene (3-MC), or β-naphthoflavone (β-NF), the AhR dissociates from HSP90 and translocates to the nucleus. It has also been hypothesized that the AhR translocates to the nucleus and forms a complex with HSP90 and other co-chaperones. There are a few reports about the direct association or dissociation of AhR and HSP90 due to difficulties in purifying AhR. We constructed and purified the PAS domain from AhR. Binding of the AhR-PAS domain to β-NF affinity resin suggested that it possesses ligand-binding affinity. We demonstrated that the AhR-PAS domain binds to HSP90 and the association is not affected by ligand binding. The ligand 17-DMAG inhibited binding of HSP90 to GST-PAS. In an immunoprecipitation assay, HSP90 was co-immunoprecipitated with AhR both in the presence or absence of ligand. Endogenous AhR decreased in the cytoplasm and increased in the nucleus of HeLa cells 15 min after treatment with ligand. These results suggested that the ligand-bound AhR is translocated to nucleus while in complex with HSP90. We used an in situ proximity ligation assay to confirm whether AhR was translocated to the nucleus alone or together with HSP90. HSP90 was co-localized with AhR after the nuclear translocation. It has been suggested that the ligand-bound AhR was translocated to the nucleus with HSP90. Activated AhR acts as a transcription factor, as shown by the transcription induction of the gene CYP1A1 8 h after treatment with β-NF.
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Tsuji
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Fukuda
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Yuhtaroh
authors.initials: Y
authors.lastname: Nagata
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Okada
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Haga
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Shiori
authors.initials: S
authors.lastname: Hatakeyama
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Okamoto
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Hosaka
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Sekine
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Ohtaka
authors.affiliation: Department of Microbiology, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
authors.forename: Soh
authors.initials: S
authors.lastname: Yamamoto
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Bunkyo-Ku, Tokyo 113-8421, Japan.
authors.forename: Michiro
authors.initials: M
authors.lastname: Otaka
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Ewa
authors.initials: E
authors.lastname: Grave
authors.affiliation: Department of Life Science, Graduate School and Faculty of Engineering Science, Akita University, Akita 010-8502, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Itoh
keywords: 17-DMAG, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, dimethylsulfoxide; 3-MC, 3-methylcholanthrene; AhR; AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; Aryl hydrocarbon receptor; CYP1A1, cytochrome P450 1A1; DAPI, 4′,6-diamidino-2-phenylindole; DEPC, dihydrochloride, diethylpyrocarbonated; Dioxin receptor; GST, glutathione, glutathione S-transferase; HSP90; HSP90, 90-kDa of heat shock protein; IPTG, isopropyl-1-thio-β-d-galactopyranoside; Molecular chaperone; NLS, nuclear localization signal; PAS, per-arnt-sim; PLA, proximity ligation assay; RT-PCR, reverse transcription-polymerase chain reaction; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XAP2, hepatitis B virus X-associated protein; XRE, xenobiotic responsible element; bHLH, basic helix-loop-helix; β-NF, β-naphthoflavone