HighlightsWe showed the direct interaction of AhR and HSP90 using purified protein.The ligand 17-DMAG induces a dissociation of HSP90 from AhR.The AhR–HSP90 complex is not affected by the timing of β-naphthoflavone binding to AhR.The AhR–HSP90 complex was translocated to the nucleus after treatment with β-naphthoflavone.

The AhR, so called the dioxin receptor, is a member of the nuclear receptor superfamily. The ligand-free AhR forms a cytosolic protein complex with the molecular chaperone HSP90, co-chaperone p23, and XAP2 in the cytoplasm. Following ligand binding like 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the AhR translocates into the nucleus. Although it has been reported that HSP90 regulates the translocation of the AhR to the nucleus, the precise activation mechanisms of the AhR have not yet been fully understood. AhR consists of the N-terminal bHLH domain containing NLS and NES, the middle PAS domain and the C-terminal transactivation domain. The PAS domain is familiar as a ligand and HSP90 binding domain. In this study, we focused on the bHLH domain that was thought to be a HSP90 binding domain. We investigated the binding properties of bHLH to HSP90. We analyzed the direct interaction of bHLH with HSP90, p23 and XAP2 using purified proteins. We found that not only the PAS domain but also the bHLH domain bound to HSP90. The bHLH domain forms complex with HSP90, p23 and XAP2. We also determined the bHLH binding domain was HSP90 N-domain. The bHLH domain makes a complex with HSP90, p23 and XAP2 via the HSP90 N-domain. Although the NLS is closed in the absence of a ligand, the structure of AhR will be changed in the presence of a ligand, which leads to NLS open, result in the nuclear translocation of AhR.

The AhR binds to contain ligands, such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin, 3-methylcholantrene, or β-naphthoflavone. The activation mechanism of AhRis not yet fully understood, but it is known that AhR associates with the molecular chaperone HSP90 in the cytoplasm. There are a few reports about the association or dissociation of AhR and HSP90, and which domain of HSP90 binds to AhR. We reported the association and activation mechanisms between HSP90 and AhR-PAS or AhR-bHLH. In the current study, we found that cisplatin inhibits the AhR activation. Although ATP and 17-DMAG have no effect on the dissociation of HSP90 from AhR, some contents of HSP90 were dissociated from AhR in the presence of cisplatin. We could detect the increase of CYP1A in the presence of 3-MC. On the contrary, the induction of CYP1A1 was inhibited in the presence of cisplatin. We couldn't detect AhR in the HeLa cell soluble fraction in the presence of 50 μM cisplatin. In the presence of MG-132, we could detect AhR. These results suggested that AhR was dissociated from the HSP90 chaperone complex and processed during the protein proteasome degradation system in the presence of cisplatin.