Prior to ligand activation, the unactivated aryl hydrocarbon receptor (AhR) exists in a heterotetrameric 9S core complex consisting of the AhR ligand-binding subunit, a dimer of hsp90, and an unknown subunit. Here we report the purification of an ϳ38-kDa protein (p38) from COS-1 cell cytosol that is a member of this complex by coprecipitation with a FLAG-tagged AhR. Internal amino acid sequence information was obtained, and p38 was identified as the hepatitis B virus X-associated protein 2 (XAP2). The simian ortholog of XAP2 was cloned from a COS-1 cDNA library; it codes for a 330-amino-acid protein containing regions of homology to the immunophilins FKBP12 and FKBP52. A tetratricopeptide repeat (TPR) domain in the carboxy-terminal region of XAP2 was similar to the third and fourth TPR domains of human FKBP52 and the Saccharomyces cerevisiae transcriptional modulator SSN6, respectively. Polyclonal antibodies raised against XAP2 recognized p38 in the unliganded AhR complex in COS-1 and Hepa 1c1c7 cells. It was ubiquitously expressed in murine tissues at the protein and mRNA levels. It was not required for the assembly of an AhR-hsp90 complex in vitro. Additionally, XAP2 did not directly associate with hsp90 upon in vitro translation, but was present in a 9S form when cotranslated in vitro with murine AhR. XAP2 enhanced the ability of endogenous murine and human AhR complexes to activate a dioxin-responsive element-luciferase reporter twofold, following transient expression of XAP2 in Hepa 1c1c7 and HeLa cells.

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The unliganded aryl hydrocarbon receptor (AhR) exists in the cytoplasm in a tetrameric 9S core complex, consisting of the AhR ligand-binding subunit, a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2), an immunophilin-related protein sharing homologous regions with FKBP12 and FKBP52. Interactions between the recently identified XAP2 subunit and other members of the unliganded AhR complex and its precise role in the AhR signal transduction pathway are presently unknown. Mapping studies indicate that XAP2 requires the PAS, hsp90, and ligand binding domain(s) of the AhR for binding, and that both proteins directly interact in the absence of hsp90. XAP2 is also able to interact with hsp90 complexes in the absence of the AhR, and C-terminal sequences of XAP2 are required for this interaction. XAP2 binds to the C-terminal end of hsp90, which contains a tetratricopeptide repeat domain acceptor site, whereas the AhR binds to a domain in the middle of hsp90. XAP2 was not found to be associated with the AhR-Arnt heterocomplex either in vitro or in nuclear extracts isolated from Hepa 1 cells treated with TCDD. Transient expression of XAP2 in COS-1 cells resulted in enhanced cytosolic AhR levels, suggesting a role for XAP2 in regulating the rate of AhR turnover.

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.