Aryl hydrocarbon (Ah) receptor levels are selectively modulated by hsp90-associated immunophilin homolog XAP2

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-inducible transcription factor that mediates biological responses to halogenated aromatic hydrocarbons. The unliganded AhR is a cytoplasmic, tetrameric complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2). The role of XAP2 as a member of the AhR core complex is poorly understood. XAP2 shares significant homology with the immunophilins FKBP12 and FKBP52, including a highly conserved, C-terminal, tetratricopeptide repeat (TPR) domain. XAP2 forms a complex with hsp90 and the AhR but can also bind to both independently. This binding is mediated by the conserved TPR domain. Single-point mutations in this region are sufficient to disrupt the association of XAP2 with both the AhR and hsp90 in cells. Cotransfection of the AhR and XAP2 in COS-1 cells results in increased AhR levels compared with cells transfected with the AhR alone. In contrast, coexpression of the AhR with the TPR containing proteins FKBP52, protein phosphatase 5 (PP5), or XAP2 TPR-mutants deficient in binding to the AhR and hsp90 does not affect AhR levels and coexpression of the AhR with the TPR domain of PP5 results in AhR down-regulation. These results demonstrate that XAP2 is apparently unique among hsp90-binding proteins in its ability to enhance AhR levels. A yellow fluorescent protein (YFP)-XAP2-FLAG was constructed and biochemically characterized, and no loss of function was detected. YFP-XAP2-FLAG was transiently transfected into NIH 3T3 and was found to localize in both the nucleus and the cytoplasm when visualized by fluorescence microscopy. Treatment of Hepa-1 cells with the hsp90-binding benzoquinone ansamycin, geldanamycin, and the macrocyclic antifungal compound radicicol resulted in AhR but not XAP2 or FKBP52 turnover. Taken together, these results suggest that XAP2/hsp90 and FKBP52/hsp90 complexes are similar yet exhibit unique functional specificity.

Fig 1.

XAP2 is expressed in various established cell lines. Eight different cell lines were cultured, harvested, and homogenized. Total cytosolic extracts were isolated, and 75 μg of each lysate was resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblot analysis. XAP2 was detected with an XAP2 polyclonal antibody as primary antibody and [¹²⁵I]-DAR as secondary antibody

Fig 2.

XAP2 specifically enhances the level of AhR in COS-1 cells when compared to other hsp90-binding TPR-containing proteins. COS-1 cells were transfected in 6 well dishes with 1 μg of pcDNA3/βmAhR and 0, 0.2, 0.4, 0.6, 0.8, or 1.0 μg of pCI/XAP2, pCI/FKBP52, pCMV6/PP5-FLAG, or pCMV6/PP5-TPR-FLAG (shaded triangle) and brought to a total of 2 μg vector/dish with pCI vector. (A) Total cell lysates were isolated, and 75 μg of each lysate were resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblot analysis. XAP2 and FKBP52 were detected with XAP2 polyclonal and FKBP52 monoclonal antibodies. PP5-TPR and PP5-TPR-FLAG were detected with anti-FLAG M2 antibody. AhR was detected with the anti-AhR monoclonal antibody RPT1. XAP2 was visualized with [¹²⁵I]-DAR, and the AhR, FKBP52, PP5-FLAG, and PP5-TPR-FLAG were visualized with [125I]-SAM. (B) The graph depicts the fold change in AhR levels obtained in the presence of TPR-containing proteins after phosphorimaging of the blots. This experiment has been repeated 3 times with essentially the same results

Fig 3.

Schematic representation of XAP2 TPR mutants and their ability to bind to hsp90 in COS1 cells. (A) Top panel: Black boxes indicate amino acid substitutions in the respective mutant. Bottom panel: Black boxes indicate conserved amino acid residues between XAP2 and FKBP52. The TPR consensus was generated from CDC16, CDC23, CDC27, SSN6, and SK13 (Lamb et al 1995). (B) COS-1 cells were transiently transfected with pCI/XAP2-FLAG, pCI/XAP2-FLAG-TPR mutants, or pCI (control), and cell lysate was isolated, immunoabsorbed with the M2 anti-FLAG affinity resin, eluted with FLAG peptide, resolved by SDS-PAGE, and transferred to PVDF membrane, followed by immunoblot analysis. Hsp90 was visualized with polyclonal antibodies raised against hsp84/86 and [¹²⁵I]-DAR, and XAP2-FLAG was visualized with anti-FLAG M2 antibody and [¹²⁵I]-SAM

Fig 4.

Mutants G272D, G272E, and F288A are unable to interact with the AhR in the absence of hsp90 in a cell-free system, and a conserved glycine in the XAP2 TPR complex is required for assembly of XAP2 in AhR-hsp90 complexes in COS-1 cells. (A) XAP2-FLAG, XAP2-FLAG-TPR mutants, or control (RL) were labeled with [³⁵S] methionine in RL independently, immunoabsorbed with M2 resin, and eluted with FLAG peptide. mAhR was generated in RL (unlabeled) and immunoabsorbed with RPT9/protein G sepharose or with murine IgG/protein G sepharose (control, last 2 lanes). The AhR immunoabsorption was washed in PBS to remove hsp90. RPT9-sepharose-mAhR was mixed with eluted XAP2-FLAG or XAP2-FLAG TPR mutants, and complexes washed, resolved by SDS-PAGE, and transferred to PVDF membrane. Top panel: mAhR visualized with RPT1 and [¹²⁵I]-SAM by autoradiography. Middle panel: XAP2-FLAG visualized by autoradiography. (B) COS-1 cells were transiently cotransfected with pCI/XAP2-FLAG, pCI/XAP2-FLAG-TPR mutants, or pCI (control) and pcDNA3/_mAhR, and cell lysate was isolated, immunoabsorbed with the M2 anti-FLAG affinity resin, eluted with FLAG peptide, resolved by SDS-PAGE, and transferred to PVDF membrane, followed by immunoblot analysis. AhR was visualized with RPT1 and [¹²⁵I]-SAM, hsp90 was visualized with polyclonal antibodies raised against hsp84/86 and [¹²⁵I]-DAR, and XAP2-FLAG was visualized with anti-FLAG M2 antibody and [¹²⁵I]-SAM.><<002>><<002>><<002>>>

Fig 5.

XAP2 mutants G272D and G272E are defective in enhancing the level of AhR in COS-1 cells. COS-1 cells were transfected in 6 well dishes with 1 μg of pcDNA3/βmAhR and 0, 0.2, 0.4, 0.6, 0.8, or 1.0 μg pCI/XAP2-G272D or pCI/XAP2-G272E or with 0, 0.25, 0.5, or 1.0 μg pCI/XAP2. The vector pCI was used to bring each transfection to a total of 2 μg plasmid/dish. Total cell lysates were isolated, and 75 μg of each lysate were resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblot analysis. XAP2 was detected with XAP2 polyclonal antibodies, and AhR was detected with the anti-AhR monoclonal antibody RPT1. XAP2 antibody was visualized with [¹²⁵I]-DAR, and AhR antibody was visualized with [¹²⁵I]-SAM. The graph depicts the fold change in AhR levels obtained in the presence of TPR-containing proteins after phosphorimaging of the blots. This experiment was performed twice with essentially the same results

Fig 6.

Cellular XAP2 is unaffected by geldanamycin or radicicol treatment. Hepa-1 cells were treated with GA or radicicol at 100 nM and 2 μM for 6 h. Following treatment, cytosol was isolated, and 100 μg were resolved by SDS-PAGE and transferred to PVDF membrane, followed by immunoblot analysis with antibodies specific for the AhR, XAP2, and FKBP52. AhR and FKBP52 were visualized with [¹²⁵I]-SAM and XAP2 with [¹²⁵I]-DAR and subsequent autoradiography

Fig 7.

EYFP-XAP2 binds to hsp90 and to AhR/hsp90 complexes in COS-1 cells. Upper panels: pEYFP-XAP2-FLAG or pEYFP alone was transiently transfected in COS-1 cells, cytosol was isolated, and 150 μg of protein were resolved by SDS-PAGE and transferred to PVDF, followed by immunoblot analysis with XAP2 polyclonal antibodies or anti-GFP monoclonal antibodies. Antibodies were visualized with DAR-P and GAM-P, respectively. Lower panels: pEYFP-XAP2-FLAG or pEYFP were transiently transfected in COS-1 cells (left panel); pEYFP-XAP2-FLAG or pEYFP were transiently cotransfected with pcDNA3/βmAhR in COS-1 cells (right panel). In both experiments, cytosol was isolated and immunoabsorbed with the M2 affinity resin, and complexes were eluted with FLAG peptide and resolved by SDS-PAGE, transferred to PVDF membrane, and analyzed by immunoblot analysis. pEYFP-XAP2-FLAG was visualized with the M2 antibody, AhR with the RPT1 antibody, and hsp90 with rabbit polyclonal antibodies against hsp84 and hsp86. M2 antibody was visualized with GAM-P, hsp90 with DAR-P, and AhR with GAM-P by ECL

Fig 8.

YFP-XAP2/Flag and YFP-XAP2/Flag G272D are localized in both the cytoplasmic and nuclear compartments. NIH 3T3 cells were transiently transfected with pEYFP (top panels), pEYFP-XAP2/Flag (middle panels), or pEYFP-XAP2 G272D (bottom panels). After 24 h, the transfected cells were visualized by either epifluorecence (left panels) or scanning confocal microscopy (right panels). The confocal images represent a horizontal midsectional view of the cell. Each image is representative of the population of transfected cells