Subcellular Localization of the Aryl Hydrocarbon Receptor Is Modulated by the Immunophilin Homolog Hepatitis B Virus X-associated Protein 2*

The hepatitis B virus X-associated protein 2 (XAP2) is an immunophilin homolog and core component of the aryl hydrocarbon receptor (AhR). Immunophilins are components of many steroid receptor complexes, serv-ing a largely unknown function. Transiently expressed AhR z YFP (yellow fluorescent protein) localized to the nuclei of COS-1 and NIH-3T3 cells. Co-expression of AhR z YFP with XAP2 restored cytoplasmic localization, which was reversed by 2,3,7,8-tetrachlorodibenzo- p -di-oxin treatment (TCDD). The effect of XAP2 on AhR localization was specific involving a nuclear localization signal-mediated pathway. Examination of the ratio of AhR to XAP2 in the AhR complex revealed that ; 25% of transiently expressed AhR was associated with XAP2, in contrast with ; 100% when the AhR and XAP2 were co-expressed. Strikingly, TCDD did not influence these ratios, suggesting that ligand binding initiates nuclear translocation prior to complex dissociation. Analysis of endogenous AhR in Hepa-1 cells revealed that ; 40% of the AhR complex was associated with XAP2, predicting observed AhR localization to cytoplasm and nuclei. This study reveals a novel functional role for the immunophi-lin-like component of a soluble receptor complex and provides new insight into the mechanism of AhR-medi-ated signal transduction, demonstrating the existence of two structurally distinct and possibly functionally unique forms of

Immunophilins are a family of proteins whose biological importance is rapidly becoming apparent. Immunophilins bind to and mediate the effects of immunosuppressant drugs (1), are involved in neural regeneration (2), and are found as components of many steroid receptor complexes (3). The functional role of immunophilins in receptor complexes is largely unknown. The immunophilin homolog hepatitis B virus X-associated protein 2 (XAP2) 1 also ARA9 or AIP, does not appear to bind immunosuppressant drugs (4), and is therefore not strictly an immunophilin. It does, however, share significant homology with immunophilins, particularly FKBP12 and FKBP52 (5,6). XAP2 is a core component of the inactive, cytosolic, aryl hydrocarbon receptor (AhR) complex, together with the AhR (ligandbinding subunit), and a dimer of hsp90 (5,6,7).
The AhR is a ligand-activated member of the bHLH-PAS (basic helix-loop-helix Per-Arnt-Sim) transcription factor family (8). In response to ligand binding, the AhR translocates to the nucleus. Two views exist as to the path taken at this stage. (i) The AhR complex may dissociate in the cytoplasm with free AhR translocating to the nucleus and heterodimerizing with ARNT (AhR nuclear translocator), or (ii) ligand binding may initiate nuclear translocation of the intact complex where hsp90 and XAP2 dissociate prior to, or in concert with, dimerization with ARNT. The AhR/ARNT heterodimer binds to dioxin responsive elements (DRE) in the enhancer regions of genes such as CYP1A1, CYP1A2, CYP1B1, and NADPH quinone oxidoreductase and mediates transcriptional up-regulation (9,10).
In cells, XAP2 appears to be largely bound to hsp90 and thus may act as a chaperone complex for a variety of proteins. XAP2 overexpression in cells has been shown to enhance cytoplasmic AhR levels, suggesting that the amount of XAP2 available to interact with the AhR may limit AhR steady-state levels (11,12). In this report, the role of XAP2 levels in modulating the subcellular localization of the AhR was examined. The presence of a sufficient pool of XAP2 in the cell is necessary to maintain the AhR in the cytoplasm, which appears to require the presence of XAP2 in the AhR core complex. TCDD treatment does not affect AhR⅐XAP2 core complex ratios under conditions that result in near complete nuclear translocation, strongly suggesting that XAP2 does not dissociate from the AhR in the cytoplasm following ligand binding. Examination of endogenous AhR core complex ratios, sedimentation profile, and subcellular localization in Hepa-1 cells strongly supports the hypotheses that XAP2 mediates cytoplasmic localization of the AhR and that the liganded complex undergoes nuclear translocation prior to dissociation and heterodimerization with ARNT.

EXPERIMENTAL PROCEDURES
Construction and Sources of Expression Vectors-pcDNA3-␤mAhR was used for expression of the AhR (13). pCI-FKBP52 was obtained from David Smith. pEYFP-N1 and pEYFP-Nuc were obtained from CLONTECH (Palo Alto, CA). pCI-XAP⅐FLAG and pCI-XAP2-G272D⅐FLAG were previously prepared in our laboratory (14,15). pEYFP⅐AhR was constructed by inserting the mAhR (amplified by polymerase chain reaction from pcDNA3-␤mAhR with XhoI and XbaI sites added to ligate in frame with YFP) into the XhoI and XbaI restriction sites in the multiple cloning site of pEYFP-N1. The NLS mutants (K13A) of the mAhR and AhR⅐YFP were generated using the QuickChange Site-directed Mutagenesis kit (Stratagene, La Jolla, CA) with HPLC purified primers (Operon Technologies Inc., Alameda, CA).
Luciferase Reporter Gene Assay-COS-1 cells grown in 6-well culture dishes were transfected using LipofectAMINE (Life Technologies, Inc., Manassas, VA) according to the manufacturer's instructions. Each transfection included 200 ng of DRE-driven luciferase reporter construct pGudLuc 6.1, 100 ng of AhR construct (or control), and control vector (pcDNA3), for a total of 2 g of DNA per well. The day following transfection, cells were treated with 10 nM TCDD or Me 2 SO (vehicle) for 6 h, and luciferase activity was assayed using a Turner TD-20e luminometer with a luciferase assay system (Promega, Madison, WI).
Fluorescence Microscopy-Cells grown on glass coverslips in 6-well culture dishes were transfected with 2 g of DNA using LipofectAMINE according to the manufacturer's instructions (for NIH-3T3 cells, transfection mixtures were supplemented with 10% fetal bovine serum after 1 h, and cells were incubated in this mixture for an additional 5 h). Before visualization, cells were rinsed twice with phosphate-buffered saline, fixed for 15 min in 4% formaldehyde, phosphate-buffered saline at room temperature, rinsed twice with phosphate-buffered saline, and inverted coverslips mounted onto microscope slides with Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA). Fluorescence micrographs were obtained with a SPOT SP100 cooled CCD camera fitted to a Nikon Optiphot-2 upright microscope with EFD-3 episcopic fluorescence attachment using a Nikon Pan Fluor ϫ 100 oil immersion objective. Indirect immunofluoresence microscopy of Hepa-1 cells was performed using anti-AhR MAb RPT9 as described previously (18). All cells in micrographs are representative of Ͼ80% of the transfected cell population.
Calibration of Relative Sensitivities of AhR and XAP2 Monoclonal Antibodies-AhR⅐FLAG and XAP2 were in vitro translated together with [ 35 S]methionine using the TNT kit (Promega). The translation mixture (5 l) was resolved by TSDS-PAGE and blotted to PVDF membrane as described previously (15). AhR and XAP2 bands were quantitated by phosphorimage analysis, and the signals were corrected for the abundance of methionine molecules in each to give a measure of their relative amounts. The membrane was then analyzed by Western blot with RPT1 (anti-AhR MAb) and anti-ARA9 (anti-XAP2 MAb; Novus Biologicals) primary antibodies, and 125 I-labeled goat anti-mouse IgG (PerkinElmer Life Sciences) secondary Ab. Bands were visualized by autoradiography, excised, and quantitated by ␥-counting. The signal was corrected for the relative amount of each protein giving a measure of the relative sensitivity of each MAb.
Determination of AhR⅐XAP2 Core Complex Ratios-COS-1 cells grown in 100-mm culture dishes were transfected at ϳ70% confluence with LipofectAMINE according to the manufacturer's instructions (Me 2 SO/TCDD treatments were for 1 h prior to harvest). Cell cytosol was prepared as described previously (15). For each immunoprecipitation, 400 g of cytosolic protein was incubated for 1 h on ice with 35 l of packed M2 anti-FLAG affinity resin (RPT9 conjugated to protein G-Sepharose for Hepa-1 AhR) in IP buffer (MENG ϩ 20 mM Na 2 MoO 4 , 100 mM NaCl, 2 mg/ml BSA, 2 mg/ml ovalbumin). Immunoprecipitates were washed 3X with IP buffer and 2X with MENG (ϩ 20 mM NaMoO 4 , 100 mM NaCl). Samples were heated with equal volumes of 2X TSB buffer and resolved by TSDS-PAGE, blotted to PVDF membrane, and analyzed by Western blot as described above. Bands were quantitated by both phosphor image analysis, and ␥-counting (results were comparable), and relative ratios of AhR and XAP2 were determined.
Sucrose Density Gradient Centrifugation-TCDD treated Hepa-1 cytosolic extracts were prepared and analyzed by SDGC as described previously (19). For protein levels, fractions were separated by SDS-PAGE, blotted to PVDF membrane, and bands visualized by Western blot using RPT1/biotin-goat anti-mouse IgG/[ 125 I]-streptavidin system.

RESULTS
Characterization of AhR⅐YFP and AhR-K13A⅐FLAG-To confirm that the AhR⅐YFP fusion and AhR-K13A⅐FLAG point mutation resulted in no loss of function, each was transiently expressed in COS-1 cells. Cytosol was isolated and incubated with anti-AhR MAb RPT9 bound to protein G-Sepharose (AhR⅐YFP), or anti-FLAG M2 MAb affinity gel (AhR-K13A⅐FLAG). Immunoprecipitates were resolved by TSDS-PAGE, electroblotted onto PVDF membrane and visualized by Western blot (Fig. 1, A and B). The AhR, AhR⅐YFP, AhR⅐FLAG, and AhR-K13A⅐FLAG each co-immunoprecipitated hsp90 and XAP2 indicating their competence to assemble into a core complex analogous to that of the AhR. Further confirmation of the functionality of AhR⅐YFP was obtained by examining its ability to activate a DRE-driven luciferase reporter plasmid. COS-1 cells grown in 6-well culture dishes were transfected with pGudLuc 6.1 (DRE-driven luciferase reporter construct) and either pcDNA3 (control), pcDNA3-␤mAhR, or pEYFP⅐AhR. Cells were treated with 10 nM TCDD or Me 2 SO (vehicle) for 6 h, followed by an assay for luciferase activity (Fig. 1C). AhR⅐YFP A, COS-1 cells were co-transfected with pCI-XAP2 and either pcDNA3, pcDNA3-␤mAhR, or pEYFP⅐AhR. AhR was immunoprecipitated with anti-AhR RPT9-protein G-Sepharose and the immunoprecipitates were resolved by TSDS-PAGE, electroblotted to PVDF membrane, and visualized by Western blot analysis, using either peroxidase-conjugated goat anti-mouse or donkey anti-rabbit IgG, followed by visualization with Vector VIP detection kit. B, COS-1 cells were co-transfected with pCI-XAP2 and either pcDNA3, pcDNA3-␤mAhR⅐FLAG, or pcDNA3␤mAhR-K13A⅐FLAG. AhR⅐FLAG was immunoprecipitated with M2 anti-FLAG affinity gel and analyzed as described for A. C, COS-1 cells were co-transfected with 40 ng of pcDNA3-␤mAhR or pEYFP⅐AhR and 80 ng of pGudLuc 6.1 DRE-driven luciferase reporter vector (total DNA 800 ng/well made up with pcDNA3) in 12-well culture plates. Approximately 18 h after transfection, cells were treated with 10 nM TCDD or Me 2 SO (DMSO vehicle) for 6 h, following in which luciferase activity was assayed. Values were corrected for protein content as determined by BCA assay. demonstrated activation of the luciferase reporter construct similar to the AhR.
AhR⅐YFP Localization in COS-1 and NIH-3T3 Cells Is Specifically Modulated by XAP2-NIH-3T3 and COS-1 cells grown on glass coverslips were transfected with pEYFP⅐AhR. AhR⅐YFP alone consistently localized to the nuclei of transfected cells in the presence and absence of TCDD (Fig. 2). Co-expression of AhR⅐YFP and XAP2 resulted in distinct cytoplasmic localization of AhR⅐YFP with near complete nuclear translocation occurring upon a 1-h treatment with 10 nM TCDD. To examine the specificity of the effect of XAP2 on AhR⅐YFP localization, cells were co-transfected with pEYFP⅐AhR and pCI-FKBP52 or pCI-XAP2-G272D⅐FLAG, which contains a point mutation in the C-terminal tetratricopeptide repeat (TPR) domain and has previously been shown to be incapable of assembling into the AhR core complex (15). Neither protein had an effect on the subcellular localization of AhR⅐YFP, suggesting that the altered localization of AhR⅐YFP is specifically because of assembly of exogenous XAP2 into the AhR⅐hsp90 complex.
Localization of YFP and YFP-Nuc Are Unaffected by Coexpression of XAP2 and TCDD Treatment-COS-1 cells were grown on glass coverslips and transfected with either pEYFP or pEYFP-Nuc (containing three tandem repeats of the nuclear localization signal of the simian virus 40 large T-antigen fused to its C terminus) (CLONTECH, Palo Alto, CA). YFP localized throughout cells, whereas YFP-Nuc was visible only in nuclei (Fig. 3A). The localization of YFP and YFP-Nuc was unaffected by treatment with 10 nM TCDD for 1 h. COS-1 cells were co-transfected with either pEYFP or pEYFP-Nuc and pCI-XAP2 or pCI (control) (Fig. 3B). In both cases, XAP2 had no effect on localization, further demonstrating that the effect of XAP2 on AhR localization is specific and is not an artifact of altered nuclear import.
AhR-K13A⅐YFP Localizes to Cytoplasm and Is Unable to Undergo Ligand-dependent Nuclear Translocation-To determine whether the subcellular localization of AhR⅐YFP was mediated by the nuclear localization signal (NLS) of the AhR, a point mutation was made in the NLS, to yield AhR-K13A⅐YFP, which has been previously shown to abolish nuclear translocation (20). In both NIH-3T3 (Fig. 3C) and COS-1 cells (Fig. 3D), AhR-K13A⅐YFP localized to cytoplasm and did not undergo detectable nuclear translocation following treatment with 10 nM TCDD for 1 h, confirming that AhR⅐YFP localization in both COS-1 and NIH-3T3 cells is mediated by the NLS of the AhR.
XAP2 Stoichiometry in the AhR Core Complex Transiently Expressed in COS-1 Cells-The stoichiometry of the interaction of the AhR with XAP2 was examined by first calibrating the relative sensitivities of MAbs to each protein (Fig. 4). The anti-ARA9 MAb was determined to have a sensitivity of 0.95 relative to the anti-AhR MAb RPT1. COS-1 cells were trans-fected with pcDNA3/pCI (control), pCI-XAP2 alone (control), pcDNA3-␤mAhR⅐FLAG alone, and pCI-XAP2 ϩ pcDNA3-␤mAhR⅐FLAG. Cytosol was prepared, immunoprecipitated with M2 anti-FLAG resin, resolved by TSDS-PAGE, and blotted to PVDF membrane. AhR and XAP2 bands were visualized by Western blot with the appropriate primary and 125 I-labeled secondary antibodies and quantitated by ␥-counting (Fig. 4B). Values were corrected for nonspecific association with the affinity resin and the sensitivity correction factor to determine the ratio of XAP2 to AhR (Fig. 4C). Transient expression of AhR alone resulted in an AhR/XAP2 ratio of 4:1, whereas co-expression of AhR with XAP2 resulted in a ratio of 1:1.
Examination of Endogenous AhR in Hepa-1 Cells-Hepa-1 cells, fixed with paraformaldehyde, were incubated with affinity purified anti-AhR MAb RPT9 (500 g/ml) and subsequently with goat anti-mouse IgG conjugated to lissamine-rhodamine sulfonyl chloride (LSRC). The AhR localized throughout cytoplasm and nuclei, and demonstrated near complete nuclear localization following treatment with TCDD (Fig. 5A). Fractionation of cells treated with TCDD revealed that the majority of liganded AhR was present in the cytosolic fraction, an apparent artifact of leaching during fractionation, as immunofluorescence microscopy revealed near complete nuclear localization (Fig. 5B). SDGC analysis revealed the liganded AhR to be exclusively in the 9 S, oligomeric form (Fig. 5C), demonstrating the presence of intact AhR complex in Hepa-1 nuclei following TCDD treatment. Analysis of total cytosolic levels and AhR⅐XAP2 core complex ratios demonstrated a 1.5-fold excess of AhR over XAP2 in Hepa-1 cytosol and the presence of XAP2 in ϳ40% of the immunoprecipitated AhR complexes. DISCUSSION An AhR⅐YFP fusion protein was constructed and biochemically characterized to demonstrate the validity of using it to model the AhR (Fig. 1, A and C). Transient expression of AhR⅐YFP in NIH-3T3 and COS-1 cells (Fig. 2) resulted in nuclear localization of AhR⅐YFP, in the presence and absence of TCDD. One hypothesis to explain our observations is that transiently expressed AhR⅐YFP may overwhelm endogenous levels of a factor required for cytoplasmic localization. Two obvious candidates for this factor were hsp90 and XAP2. Coexpression of AhR⅐YFP with hsp90 had no effect (data not shown), however XAP2 co-expression resulted in a clear redistribution of AhR⅐YFP to the cytoplasm (Fig. 2). The relocalized, cytoplasmic AhR⅐YFP was responsive to TCDD, undergoing near complete nuclear translocation following treatment for 1 h. These data suggest that XAP2 modulates the subcellular localization of AhR⅐YFP and that transiently expressed AhR appears to be functionally distinct from endogenous AhR. Localization of transiently expressed AhR to the nucleus (and

FIG. 2. Localization of AhR⅐YFP in NIH-3T3 and COS-1 cells.
Cells were grown on glass coverslips in 6-well culture dishes. Cells were transfected with either pEYFP⅐AhR (500 ng), pEYFP⅐AhR (500 ng) ϩ pCI-XAP2 (1 g), pEYFP⅐AhR (500 ng) ϩ pCI-XAP2-G272D (1 g), or pEYFP⅐AhR (500 ng) ϩ pCI-FKBP52 (1 g). Cells were treated with either 10 nM TCDD or Me 2 SO (DMSO) for 1 h prior to visualization where indicated. restoration of cytoplasmic localization by co-expression of XAP2) has been previously observed in COS-1 cells by indirect immunofluorescence microscopy (12). Histochemical studies of AhR localization in tissue slices have shown time and tissue specific nuclear, perinuclear, and cytoplasmic staining (16,21), suggesting that AhR localization may be regulated in a tissuespecific manner, possibly through regulation of XAP2 expression. The specificity of the effect of XAP2 on AhR⅐YFP localization was confirmed by co-expression with the immunophilin FKBP52, and XAP2-G272D⅐FLAG. No effect was seen in either case, demonstrating the specific requirement for functional XAP2.
The effects of TCDD treatment and transient XAP2 expression on NLS-mediated nuclear uptake were examined by transiently expressing YFP and YFP-Nuc in COS-1 cells. YFP localized diffusely throughout cytoplasm and nuclei, whereas YFP-Nuc localized exclusively to nuclei (Fig. 3). TCDD treatment did not alter localization of either protein. To examine for a possible modulation of NLS-mediated nuclear import by XAP2, COS-1 cells were co-transfected with either pEYFP or pEYFP-Nuc and pCI-XAP2. XAP2 did not alter localization in the presence or absence of TCDD (Fig. 3B), demonstrating that neither XAP2 nor TCDD appear to modulate nuclear import pathways in our system. The possibility that transiently expressed AhR⅐YFP may bypass NLS-mediated nuclear uptake pathways was examined using pEYFP⅐AhR-K13A. AhR-K13A⅐YFP localized exclusively to the cytoplasm of both NIH-3T3 and COS-1 cells in the presence and absence of TCDD (Fig.  3, C and D), suggesting that acquisition of nuclear AhR⅐YFP through a mechanism other than NLS-mediated import is unlikely, thus localization of AhR⅐YFP is mediated through the NLS of the AhR and is modulated by XAP2.
Modulation of AhR⅐YFP localization by XAP2 was further explored by determination of ratios of AhR/XAP2 in the AhR core complex. AhR and XAP2 MAbs were calibrated to determine their relative sensitivities to quantitatively determine the ratio of XAP2 to AhR in the AhR core complex (Fig. 4). Western blot analysis revealed that only 25% of transiently expressed AhR in COS-1 cells was associated with XAP2, in contrast with ϳ100% association when the AhR was co-expressed with XAP2. Strikingly, TCDD had no detectable effect on AhR/XAP2 ratios, lending strong support to the hypothesis that ligand binding initiates nuclear translocation of the intact AhR core complex prior to complex dissociation and dimerization with ARNT. In terms of the role of XAP2 in the subcellular localization of the AhR, transient expression of the AhR appears to overwhelm the limited pool of XAP2 that is in equilibrium with the AhR. In vitro translated AhR complexes that lack XAP2 are functional (22), however, in cells XAP2 appears to mediate the subcellular localization of the unliganded AhR. To complement these studies, localization and XAP2 stoichiometry were examined for endogenously expressed AhR in Hepa-1 cells. Indirect immu- nofluorescence/laser scanning confocal microscopy revealed the AhR in Hepa-1 cells to be localized throughout cytoplasm and nuclei, with near complete nuclear localization observed following TCDD treatment (Fig. 5A). These data contradict an earlier report showing predominantly cytosolic localization of unliganded AhR in Hepa-1 cells (23). Stoichiometric analysis found ϳ40% of the AhR in Hepa-1 cells complexed with XAP2 (Fig.  5D), supporting localization data and the hypothesis that XAP2 inclusion in the core complex is required for cytoplasmic localization of the AhR. In addition, fractionation of TCDD treated Hepa-1 cells followed by SDGC analysis further supports the hypothesis that the liganded AhR translocates to the nucleus intact. The majority of AhR in TCDD treated Hepa-1 cells is nuclear, but is found in the cytosolic fraction after cell disruption and is still in the 9 S core complex.
Together, the results suggest that an important role of XAP2 in the AhR core complex is to maintain the receptor in the cell cytoplasm. To the best of our knowledge, this is the first report of a distinct function of a receptor-associated immunophilinlike protein. The data also suggest that XAP2 incorporation into the AhR core complex, although resulting in apparently increased levels of the AhR complex, is not necessary for significant expression of the AhR, and the lack of stoichiometric amounts of XAP2 does not result in rapid degradation as is seen with disruption of functional hsp90 (24). The AhR appears to exist in two distinct forms in cells, one containing and one lacking XAP2. It is possible that these two forms are functionally unique, and the presence or absence of XAP2 in the AhR core complex may help explain tissue-specific AhR differences. The finding that TCDD does not alter AhR/XAP2 ratios, combined with cytochemistry data, demonstrates that essentially all of the AhR translocates to the nucleus upon TCDD treatment, strongly suggesting that the AhR core complex translocates to the nucleus intact. Furthermore, these data indicate that, whereas TCDD invokes a rapid accumulation of AhR core complex in the nucleus, TCDD binding appears to be insufficient to induce hsp90/XAP2 dissociation. Ligand-induced trans-location of the AhR core complex prior to dissociation has been previously addressed and evidence utilizing different approaches has consistently supported the data reported here (19,25,26,27). Interestingly, the rate-limiting step in dissociation of hsp90/XAP2 and subsequent heterodimerization with ARNT is not known.
Modulation of the localization of the AhR by XAP2 is not a relationship that is without precedent. The family of small acidic proteins designated as 14-3-3 (see Ref. 28 for review) have been shown to have similar activities. Phosphorylated Cdc25 binds to 14-3-3 proteins, which appear to function to sequester Cdc25 in cytoplasm, resulting in functional inhibition (29). The mechanism by which XAP2 modulates the subcellular localization of the AhR is unknown, but may result from a number of possible scenarios. These include the following. 1) XAP2 may physically mask the NLS of the AhR, thereby inhibiting nuclear translocation pending a ligand-induced conformational change that results in exposure of the NLS and subsequent binding of the appropriate NLS recognition molecules. 2) XAP2 may mediate sequestration of the AhR in the cytoplasm by an unknown mechanism. 3) XAP2 may inhibit nuclear retention of the AhR, and 4) XAP2 may enhance the nuclear export of the AhR, leading to apparent cytoplasmic localization. Several of these possibilities are currently under investigation to fully elucidate the mechanism by which XAP2 modulates the subcellular localization of the AhR.