Hepatitis B Virus X-Associated Protein 2 Is a Subunit of the Unliganded Aryl Hydrocarbon Receptor Core Complex and Exhibits Transcriptional Enhancer Activity

ABSTRACT

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 ∼38-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.

ACKNOWLEDGMENTS

We thank Christopher A. Bradfield for pSVSPORT/hAhR, Michael Denison for the DRE-luciferase reporter construct pGudLuc 6.1, Oliver Hankinson for pcDNA3/βmAhR, Webb Miller for helpful assistance in alignments of XAP2 with human FKBP52 and FKBP12, Richard Pollenz for polyclonal AhR antibodies, Steve Safe for TCDD, and Edward Seto for pGST-XAP2 and pGEM-XAP2 constructs. William P. Long and Sheo S. Singh are acknowledged for providing excellent technical assistance. The HHMI W. M. Keck Biopolymer Facility at Yale University performed in-gel tryptic digestions and subsequent microsequencing of p38.

This work was supported by grants ES04869 and ES07799 from NIEHS.