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INTRODUCTION |
The aryl hydrocarbon receptor
(AhR)1 is a ligand-activated
transcription factor that binds to a diverse group of compounds, with
TCDD being the most studied and highly toxic AhR ligand (1-3). The
TCDD·AhR complex mediates a wide range of biological responses in rodents, such as a wasting syndrome, hepatotoxicity, teratogenesis, and tumor promotion. The unliganded AhR exists in cytosolic extracts as
a core tetrameric complex, composed of a ligand binding subunit (AhR),
a dimer of hsp90, and the immunophilin homolog XAP2 (4-6). Upon
binding ligand, the AhR translocates to the nucleus where it forms a
heterodimer with ARNT, concurrent with the loss of hsp90 from the
complex (7). The AhR·ARNT heterodimer is capable of regulating
transcription of a number of genes, such as CYP1A1, CYP1B1, and NADPH
quinone oxidoreductase, upon binding to DREs in their enhancer regions.
The ability of XAP2 to interact with the AhR was originally established
independently by three laboratories (6, 8, 9). XAP2, also referred to
as AIP (for AhR-interacting protein), or ARA9 (for AhR-associated
protein 9), is a tetratricopeptide repeat motif domain containing
immunophilin homolog that has significant sequence homology with
FKBP52. The immunophilin FKBP52 is found bound to hsp90 and also exists
in larger complexes with certain steroid receptors, including the
glucocorticoid and progesterone receptors (10). Subsequent studies have
established that XAP2 is able to stabilize and enhance cellular levels
of the mAhR (11), which may occur through protection from proteolysis
(12). In contrast, FKBP52 was unable to modulate AhR levels (13).
Interestingly, transient expression of XAP2 also leads to sequestration
of the mAhR in the cytoplasm (12, 14, 15). Regulation of intracellular movement of the AhR both in the presence and absence of ligand is a
potentially important aspect of Ah receptor function that warrants
further investigation.
For many proteins larger than 40 kDa, import or export across the
nuclear envelope is controlled by the presence of nuclear localization
sequences (NLS) and leucine-rich nuclear export signals (NES). An NLS
is usually a short cluster of basic amino acids, such as those found in
SV40 large T antigen, or it can be two clusters of basic amino acids
separated by 10-12 amino acids, known as a bipartite NLS (16). The NLS
is recognized by importin
, and this complex is then recognized by
importin
, which mediates docking of the ternary complex to the
cytoplasmic face of the nuclear pore complex. However, importin
is
capable of binding directly to arginine-rich NLS of a number of
proteins and mediate nuclear import (17, 18). The NES are short,
hydrophobic, leucine-rich amino acid sequences
(XLXXLXXLXLX)
and are found in a variety of proteins (e.g. p53, I
B
,
and glucocorticoid receptor). In the nucleus chromosomal region
maintenance protein 1 (CRM-1) binds to the NES, followed by cooperative
binding of RanGTP, which results in a complex competent for export
(17). Proteins that carry both an NLS and NES often dynamically shuttle
between the cytoplasm and the nucleus by a process termed
nucleocytoplasmic shuttling. In these cases, the relative localization
seen is the result of the relative rate of import and export combined.
Shuttling provides a rapid and reversible means to regulate protein
localization through protein-protein interactions, phosphorylation, or
other post-translational modifications, in the microenvironment of the cytoplasm or nucleus. An example is the regulation of FKHRL1, a member
of the Forkhead family of transcription factors, which is retained in
the cytoplasm after Akt phosphorylation and subsequent 14-3-3 protein
binding (18).
A bipartite NLS between amino acid residues 13 and 39 of the human AhR
(hAhR) has been identified using a GFP fusion protein expression system
(19). In addition, a NES has been identified between residues 55 and 75 in helix 2 of the helix-loop-helix domain of the hAhR and has been
shown to interact with CRM-1 (20). Considering that the AhR has both a
NLS and a NES, it is not surprising that it can undergo
nucleocytoplasmic shuttling; indeed, this has been demonstrated through
the use of the nuclear export inhibitor, leptomycin, and microinjection
techniques (20-22). The primary objective of the studies presented
here was to examine the mechanism of cytoplasmic retention of the mAhR
in the presence of XAP2. The initial observation in this study
indicated that XAP2 effectively blocks ligand-independent
nucleocytoplasmic shuttling. There are a number of hypothetical
mechanisms that could explain the ability of XAP2 to retain the mAhR in
the cytoplasm. The most likely hypotheses are that XAP2 blocks importin
access to the NLS, XAP2 locks the bipartite NLS of AhR into an
unfavorable conformation for importin binding, XAP2 sequesters the mAhR
in the cytoplasm by binding to cytoskeletal matrix proteins, or XAP2
may enhance nuclear export, leading to apparent cytoplasmic
localization. The results outlined in this report demonstrate that
XAP2·mAhR complexes are not sequestered in the cytoplasm through
docking on tubulin or actin filaments. In addition, any other type of
anchoring seems unlikely, as XAP2 did not promote sequestration of
mAhR-YFP-Nuc, which contains a NLS distinct from the one present in the
native receptor. Interestingly, XAP2 prevented active nucleocytoplasmic
shuttling of the mAhR, which would suggest that XAP2 may block the NLS
or alter the conformation of the bipartite NLS. Antibodies directed
against the NLS were capable of binding to mAhR·XAP2 complexes,
indicating that the NLS of mAhR is not blocked by the presence of XAP2.
However, XAP2 does reduce importin
binding to the mAhR·hsp90
complex in vitro. These studies taken together would suggest
that XAP2 alters the ability of importin
to recognize the bipartite
NLS sequence of the mAhR, and this appears to be the primary mechanism
of XAP2-mediated redistribution of the mAhR to the cytoplasm. Another
hypothesis that we wanted to test is whether blocking of
nucleocytoplasmic shuttling could lead to a repression of AhR
transcriptional activity. Transient transfection experiments indicated
that XAP2 is capable of repressing AhR activity. In summary, these
studies would suggest that XAP2 cause a functionally significant
alteration in the conformation of the mAhR.
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EXPERIMENTAL PROCEDURES |
Construction and Sources of Expression Vectors--
The vector
pcDNA3-
mAhR provided by Oliver Hankinson (University of
California, Los Angeles, CA) was used for mammalian expression of the
mouse AhR (23). As a control for importin binding experiments, amino
acid residues 13-15 of the mAhR were mutated to alanine residues using
pcDNA3-
mAhR and a QuikChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA). The vector was designed as
pcDNA3-
mAhR/M-NLS. The nucleotide sequence corresponding to
the amino acids 31-44 of the mAhR/FLAG was deleted using a
site-directed mutagenesis approach. Two alanine residues were inserted
between amino acids 30 and 45, and this construct was designated as
pcDNA3-
mAhR/FLAG/
NLS. The plasmids pEYFP-N1, pEYFP-actin,
pEYFP-tubulin, pEYFP-Nuc, and pECFP-Nuc were obtained from
Clontech (Palo Alto, CA). The pCI-XAP2,
pCI-XAP2-FLAG, and pCI-XAP2-G272D-FLAG vectors were previously prepared
in our laboratory (11, 13). The pEYFP-mAhR and pEYFP-mAhR-K13A were
constructed as previously described (14). The pEYFP-mAhR-YFP-Nuc and
pEYFP-mAhR-K13A-YFP-Nuc were constructed by excising mAhR-YFP and
mAhR-K13A-YFP from the vectors described above with NheI and
BsrGI and inserted directly into the
NheI/BsrGI sites of pEYFP-Nuc. The DRE-driven
reporter vector pGudLuc 6.1 was obtained from Mike Denison (University
of California, Davis, CA). Human importin
-myc-pET30a and
GST-importin
and
constructs were obtained from Stephen Adam
(Northwestern University Medical School, Chicago, IL).
Cell Culture--
Cells were grown in
-minimal essential
medium supplemented with 10% fetal bovine serum (HyClone Laboratories,
Logan, UT), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin
(Sigma) at 37 °C in 95% air, 5% CO2.
Fluorescence Microscopy--
Fluorescence micrographs were
obtained directly from cells grown in six-well microplates that were
transfected with 1.5 µg of DNA using LipofectAMINE with PLUS reagent
(Invitrogen) according to the manufacturers instructions.
Approximately 18 h following transfection, fluorescence was
visualized with a Nikon TE300 inverted microscope with TE-FM
epifluorescence attachment using a Nikon Pan Fluor 60X objective and a
SPOT RT Color model 2.2.0 cooled CCD camera. For examination of
cytoskeletal disruption, cells grown on glass cover slips in six-well
culture dishes were transfected with 2 µg of DNA using LipofectAMINE
(Invitrogen) according to the instructions from the manufacturer.
Approximately 18 h after transfection, cells were treated with
either 5 µM colchicine or 10 µM
cytochalasin B (Sigma) for 1 h. Before visualization, cells were
rinsed twice with PBS, fixed for 15 min in 4% formaldehyde/PBS at room
temperature, and rinsed twice with PBS, and inverted coverslips were
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 100× oil immersion objective. All
cells in micrographs are representative of >80% of the transfected
cell population.
Production of Affinity-purified Rabbit Polyclonal Anti-AhR-NLS
Antibodies--
A 15-residue peptide corresponding to the
C-terminal portion of the bipartite NLS of the AhR (amino
acids 31-44) (19) was synthesized with an N-terminal cysteine
residue (H2N-CKSNPSKRHRDRLNT-COOH) by New England Peptide
(Fitchburg, MA). The peptide was conjugated to keyhole limpet
hemocyanin and injected into rabbits using standard techniques at New
England Peptide. The AhR-NLS peptide (1 mg) was conjugated to 3 ml of
Sulfolink resin (Pierce) following the instructions from the
manufacturer. A 3-ml column was washed and stored at 4 °C in M/N
buffer (20 mM MOPS, 0.02% sodium azide) until use.
Affinity purification was carried out entirely at 4 °C. Before use,
the column was washed with three volumes of ice-cold M/N buffer. Rabbit
serum (6 ml) was applied to the column by gravity flow, collected, and
reapplied. The column was then washed as follows: 10 volumes of M/N, 5 volumes of M/N + 500 mM NaCl, and 2 volumes of M/N. The
antibody was eluted with two volumes of 0.1 M glycine-HCl,
pH 2.5, and 1-ml fractions were collected into 1.5-ml microcentrifuge
tubes containing 100 µl of 1 M Tris, pH 8.0. Protein
content of fractions was determined using the BCA assay (Pierce). The
specificity of this peptide antibody was assessed by in
vitro translating pcDNA3-
mAhR/FLAG and
pcDNA3-
mAhR/FLAG/
NLS in the presence of
[35S]methionine, which were subjected to SDS-PAGE,
transferred to PVDF membrane. The presence of the AhR was visualized
using either mAb RPT 1 or anti-AhR-NLS peptide antibody and goat
anti-mouse peroxidase conjugate. The ability of the anti-AhR-NLS
polyclonal antibody bound to protein G-Sepharose to immunoprecipitate
in vitro translated mAhR/FLAG or mAhR/FLAG/
NLS was
assessed using standard techniques.
Cytosol Preparation and Immunoprecipitations--
Transfected
COS-1 cells grown in 100-mm plates were harvested by trypsinization,
rinsed with PBS, and mechanically homogenized with 30 strokes of a
stainless steel Dounce homogenizer. Immunoprecipitations were carried
out for 1 h at 4 °C in IP buffer (MENG (25 mM MOPS, 2 mM EDTA, 0.02% NaN3, 10% glycerol, pH 7.4)
with 20 mM sodium molybdate, 50 mM NaCl, 2 mg/ml bovine serum albumin, 2 mg/ml ovalbumin), using rabbit polyclonal
anti-AhR-NLS antibodies bound to protein G-Sepharose (Pierce).
Immunoprecipitates were rinsed three times with IP buffer, twice with
wash buffer (MENG with 20 mM sodium molybdate, 50 mM NaCl), resolved by Tricine SDS-PAGE, and electroblotted to PVDF membrane (Millipore, Bedford, MA) as previously described (13).
The AhR and XAP2 were visualized by Western blot analysis using RPT1
mAb (24), and anti-ARA9 mAb (Novus Biologicals, Littleton, CO),
respectively. Primary antibodies were detected with either 125I-labeled goat anti-mouse IgG or
125I-labeled donkey anti-rabbit polyclonal IgG (Amersham
Biosciences), visualized by autoradiography, and quantitated with a phosphorimager.
Importin/AhR Interaction Assay 1--
COS-1 cells in
three 100-mm dishes were transfected with pcDNA3-
mAhR/FLAG, with
pcDNA3-
mAhR/FLAG and pCI-XAP2, or with pcDNA3-
mAhR/FLAG-M-NLS (as a importin specificity control) using LipofectAMINE PLUSTM transfection method as described by
the manufacturer (Invitrogen). After 24 h cells were trypsinized
and washed three times with PBS. Cells from each set of three plates
were lysed in 1 ml of MENG containing 20 mM
NaMoO4, 1% Nonidet P-40, protease inhibitor mixture
(Sigma), and 1 mM DTT for 15 min at 4 °C, and
centrifuged at 100,000 × g for 30 min at 4 °C. The
lysate (~350 µl) was treated with TCDD to a final concentration of
10 nM or Me2SO for 30 min at ambient
temperature followed by 10 min on ice. To the TCDD- or
Me2SO-treated lysate 350 µl of immunoprecipitation buffer
(MENG containing 20 mM NaMoO4, 300 mM NaCl, 10 mg/ml bovine serum albumin, 5 mg/ml ovalbumin,
1 mM DTT) was added and then transferred to 25 µl of
pre-washed anti-FLAG M2-agarose (Sigma). The immunoprecipitations were
incubated with agitation for 1 h at 4 °C and washed three times
with MENG containing 100 mM NaCl and 20 mM
NaMoO4. The FLAG-tagged proteins were displaced by
incubating with 200 µg of FLAG peptide (Sigma) in 125 µl of 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl and
0.1% Nonidet P-40 for 15 min at ambient temperature. The displacement
was repeated once more, and supernatants were pooled. Each of the
displaced FLAG-tagged proteins (200 µl) was transferred into 200 µl
of importin-binding buffer (PBS containing 10 mg/ml bovine serum
albumin, 5 mg/ml ovalbumin, 0.2% Nonidet P-40, 10% glycerol, and 1 mM DTT), and 22 µg of GST-importin
was added. The
mixture was incubated for 30 min on ice, transferred to 25 µl of
pre-washed glutathione-Sepharose (Amersham Biosciences), incubated with
agitation for 1 h at 4 °C, and washed three times with 1 ml of
PBS. GST-importin
was displaced from the resin by incubating with
80 µl of 20 mM glutathione in 50 mM Tris-HCl, pH 7.5, plus 1 mM DTT for 5 min at ambient temperature. The
displacement was repeated once more, and the pooled supernatant was
subjected to Tricine SDS-PAGE. Protein was transferred onto PVDF
membrane (Millipore, Bedford, MA) as previously described. The mAhR,
XAP2, and GST-importin
were visualized by protein blot analysis
using mAb RPT1, mAb anti-ARA9 (Novus Biologicals), and mAb B-14
anti-GST (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
respectively. Primary antibodies were detected with
125I-labeled goat anti-mouse IgG (Amersham Biosciences),
visualized by autoradiography, and quantitated with a phosphorimager
and/or a
counter.
Importin/AhR Interaction Assay 2--
COS-1 cells
(100-mm dishes) were transfected with pcDNA3,
pcDNA3-
mAhR/FLAG, or pcDNA-
mAhR/FLAG and pCI-XAP2 using
LipofectAMINE PLUSTM transfection method as described by
the manufacturer (Invitrogen). After 24 h cells were harvested and
lysed in MENG + 20 mM sodium molybdate + 1% Nonidet P-40
and centrifuged at 100,000 × g for 30 min. The
supernatants were transferred to three tubes, each containing 50 µl
of M2-agarose (Sigma), and incubated with agitation for 90 min. The
immunoprecipitations were washed twice with MENG + 20 mM
sodium molybdate, followed by two additional washes with 20 mM MOPS, 50 mM potassium acetate, 50 mM NaCl, 2 mM magnesium acetate, 0.02%
NaN3, pH 7.4 (binding buffer). The human importin
-myc-pET30a construct was transcribed and translated in a
TNT coupled translation system (Promega, Madison, WI) in
the presence of [35S]methionine. To the
immunoprecipitates 5 µl of in vitro translated importin
and 45 µl of binding buffer was added. Each sample incubated for
20 min at 30 °C and gently mixed every 5 min, followed by incubation
at 4 °C for 45 min. The immunoprecipitates were washed quickly three
times and incubated with 50 µl of FLAG peptide (5 mg/ml), and these
mixtures were incubated for 30 min at room temperature. After
centrifugation the FLAG peptide-displaced AhR-FLAG present in the
supernatant was collected. This displacement was repeated once more and
the pooled supernatants subjected to Tricine SDS-PAGE. Proteins were
transferred to PVDF membrane, and the presence of
[35S]methionine-labeled importin
was visualized and
quantitated by phosphorimaging. The presence of XAP2 and AhR was
detected using the monoclonal antibodies described above and peroxidase conjugated to goat anti-mouse IgG as the secondary antibody. The presence of peroxidase was visualized with a Vector VIP peroxidase kit
(Vector Laboratories, Burlingame, CA).
Luciferase Reporter Gene Assay--
COS-1 cells in 100-mm plates
were co-transfected using LipofectAMINE PLUSTM (Invitrogen)
with 0.5 µg of pcDNA3-
mAhR, 100 ng of luciferase reporter
construct pGudLuc 6.1, 30 ng of pCMV-
Galactosidase, either 2 or 4 µg of pCI-XAP2, and pCI was added for a total of 8 µg of DNA.
Transfected cells after 9 h were transferred to 12-well plates;
13 h later cells were treated with iodoflavone. Cells were lysed
after an 8-h exposure to ligand, and reporter activity was assayed
using a Turner TD-20e luminometer and a luciferase assay system
(Promega).
 |
RESULTS |
XAP2 Inhibits Ligand-independent Nucleocytoplasmic Shuttling of the
mAhR--
COS-1 cells were utilized in this study because of their
ability to be transfected with greater than 50% transfection
efficiency; additionally, they have low levels of endogenous AhR, and
our previous studies examining regulation of mAhR localization were performed in this cell line (14). COS-1 cells were transiently transfected with pEYFP-mAhR, pEYFP-mAhR + XAP2, or pEYFP-mAhR-K13A (containing a single point mutation that renders the NLS nonfunctional) and treated with either vehicle (MeOH) or 10 nM leptomycin
B (Fig. 1). As previously reported (14),
control-treated cells expressing mAhR-YFP showed localization of the
receptor throughout cells, with a higher level in cell nuclei than in
the cytoplasm. Co-expression of XAP2 resulted in redistribution of the
wild-type receptor to the cytoplasm, whereas mAhR-K13A-YFP is
cytoplasmic with or with out co-expression of XAP2 (19). Treatment with
leptomycin B, a specific inhibitor of CRM-1-mediated nuclear export,
resulted in enhanced nuclear accumulation of mAhR-YFP, suggesting that the unliganded receptor is capable of undergoing nucleocytoplasmic shuttling as has previously been suggested using N-terminal fragments of the human AhR fused to glutathione S-transferase and GFP
(20). Nucleocytoplasmic shuttling was eliminated by mutation of the NLS
of the AhR, but quite unexpectedly, co-expression of XAP2 with mAhR-YFP
also resulted in inhibition of nucleocytoplasmic shuttling. When XAP2
was co-expressed with mAhR-YFP and cells treated with leptomycin B,
nuclear accumulation was not observed after 1 h (Fig. 1), and
little or no increase in nuclear accumulation was seen at the longest
observed time point of 6 h (data not shown). Inhibition of
nucleocytoplasmic shuttling of the mAhR by XAP2 suggests that the
observed effect of XAP2 on mediating cytoplasmic retention of mAhR-YFP
is not caused by either an enhanced rate of nuclear export or an
inhibition of nuclear retention. This information has allowed us to
narrow down the possible hypotheses to explain XAP2-mediated
sequestration of the mAhR; these hypotheses are schematically
represented in Fig. 2A.

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Fig. 1.
XAP2 blocks ligand-independent
nucleocytoplasmic shuttling of the mAhR-YFP. COS-1 cells grown in
six-well microplates were transiently transfected with either
pEYFP-mAhR, pEYFP-mAhR + pCI-XAP2, or pEYFP-mAhR-K13A. Control (carrier
solvent) and 10 nM leptomycin B treated cells were
visualized after 1 h of treatment by fluorescence microscopy. The
mAhR-YFP alone localized throughout cells and showed nuclear retention
following 1 h treatment with the export inhibitor leptomycin B. Co-expression of XAP2 resulted in cytoplasmic localization, and
leptomycin B-mediated nuclear accumulation was inhibited. A mutation in
the NLS abolished nuclear accumulation of the AhR-YFP in the absence of
XAP2 and was unaffected by treatment with leptomycin B.
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Fig. 2.
Possible mechanisms by which XAP2 may mediate
cytoplasmic localization of the mAhR. A, (i) XAP2 may
physically anchor the mAhR to actin/tubulin-based components of the
cytoskeleton; (ii) XAP2 may physically anchor the mAhR to some unknown
cytoplasmic component; (iii) XAP2 may block access to the NLS of the
mAhR; (iv) XAP2 may stabilize a conformation of the receptor in which
the bipartite NLS is incapable of recognition by importin molecules.
B, schematic representation of mAhR constructs used to
examine the role of XAP2 in mediating cytoplasmic localization of the
AhR complex.
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XAP2 Does Not Promote Anchoring of the mAhR to the Cytoskeletal
Components Actin or Tubulin--
We hypothesized that XAP2 may
sequester the mAhR in the cytoplasm through interaction with components
of the cytoskeleton. To examine this possibility, COS-1 cells were
co-transfected with either pEYFP-actin and pECFP-Nuc or pEYFP-tubulin
and pECFP-Nuc, followed by treatment with either 10 µM
cytochalasin B or 5 µM colchicine (Fig.
3A). Initial experiments were
conducted to (a) demonstrate specific disruption of the
cytoskeleton by each agent, and (b) demonstrate that the
integrity of the nuclear membrane was maintained following each
treatment. The actin cytoskeleton was disrupted by 10 µM
cytochalasin B, but was unaffected by 5 µM colchicine,
whereas the tubulin cytoskeleton was disrupted by 5 µM
colchicine, but was unaffected by 10 µM cytochalasin B. In each case, nuclear retention of CFP-Nuc (nuclear localized cyan
fluorescent protein) was preserved. COS-1 cells were next co-transfected with pEYFP-mAhR and pCI-XAP2 and subjected to treatment with cytoskeletal disruption agents in the presence and absence of TCDD
(Fig. 3B). Control cells showed mAhR-YFP localization to
cell cytoplasm, whereas 1-h treatment with 10 nM TCDD
resulted in nuclear uptake of the receptor. Treatment of cells with
cytochalasin B or colchicine did not result in altered subcellular
localization of the unliganded receptor and did not disturb
ligand-dependent nuclear accumulation, which occurred at a
similar rate in control-treated cells. These results suggest that actin
or tubulin cytoskeletal systems are not involved in mediating the
translocation of the AhR complex.

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Fig. 3.
Role of actin and tubulin structures in the
localization and movement of the mAhR in COS-1 cells.
A, COS-1 cells expressing YFP-actin and CFP-Nuc or
YFP-tubulin and CFP-Nuc were treated for 1 h with either 5 µM colchicine to disrupt tubulin or 10 µM
cytochalasin B to disrupt actin. In each case, the agent was specific
and did not appear to adversely influence the integrity of the nuclear
envelope. B, COS-1 cells expressing mAhR-YFP and XAP2 were
treated with either colchicine or cytochalasin B in the presence or
absence of 10 nM TCDD for 1 h. Treatment with
cytoskeletal disrupting agents did not affect localization of
mAhR-YFP, and TCDD treatment resulted in nuclear accumulation in a
timeframe similar to that for control-treated cells. DMSO,
Me2SO.
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XAP2 Does Not Alter Nuclear Accumulation of the mAhR Directed by an
Alternate NLS--
To examine whether XAP2 was capable of modulating
the localization of the mAhR directed by an alternate NLS, two
constructs were generated. They consisted of the mAhR-YFP and
mAhR-K13A-YFP with three repeats of the NLS from SV40 large T-antigen
added at the C terminus of YFP (Fig. 2B). The resulting
localization of these constructs transiently expressed in COS-1 cells
is summarized in Fig. 4. As has been
previously demonstrated, the mAhR-YFP localized mostly in nuclei, and
co-expression of XAP2 resulted in redistribution of the mAhR-YFP to the
cytoplasm, whereas mAhR-K13A-YFP remained in cytoplasm. Addition of an
NLS to the C terminus of mAhR-YFP resulted in strong nuclear
localization; in addition, co-expression of XAP2 did not alter nuclear
accumulation of the AhR in either case, suggesting that XAP2 is only
able to modulate localization of the mAhR directed by its endogenous
NLS. Thus, XAP2 does not have an "anchoring" effect on the mAhR
complex. YFP-Nuc, lacking the mAhR, shows essentially complete
localization to nuclei (data not shown).

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Fig. 4.
XAP2 does not influence mAhR-YFP nuclear
accumulation directed by an alternate NLS. COS-1 cells were
transiently transfected with various constructs expressing the noted
proteins. The mAhR-YFP localized predominantly in the nuclei and was
retained in the cytoplasm upon co-expression of XAP2. The NLS mutant of
mAhR-YFP (K13A) also localized exclusively to the cytoplasm. Addition
of an NLS at the C terminus of the mAhR fusion protein produced a
species that localized almost exclusively to cell nuclei with and
without the NLS of mAhR (compare mAhR-YFP-Nuc with mAhR-K13A-YFP-Nuc).
In both cases, co-expression of XAP2 had no visible effect on
subcellular localization of the mAhR as directed by an alternate
NLS.
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XAP2 Does Not Block the Ability of an Anti-mAhR NLS Antibody to
Bind to the mAhR--
To determine whether XAP2 plays a role in
altering/masking the mAhR NLS, we used a rabbit polyclonal antibodies
directed against the C-terminal portion of the bipartite NLS, amino
acid residues 31-44, of the mAhR. The critical residues of the NLS of
AhR necessary for function are between amino acid residues 13 and 39 (19). The specificity of this polyclonal antibody was examined on a
protein blot and was found to be highly specific for the AhR
transiently expressed in COS 1 cells (Fig.
5A). In addition,
this antibody fails to recognize the mAhR after the amino acids 31-44
are deleted (Fig. 5, B and C). Thus, this
antibody is highly specific and only binds to the NLS sequence of the
mAhR. These antibodies were bound to protein G-Sepharose and used to
immunoprecipitate the mAhR from transiently transfected COS-1 cells.
The COS-1 cells were transfected with either pcDNA3-
mAhR (3 µg) and pCI-XAP2 (6 µg) or pcDNA3-
mAhR (3 µg) alone.
Cytosolic extracts from control cells and cells treated with TCDD (10 nM, 1 h) were incubated for 1 h on ice with
protein G-Sepharose-bound anti-AhR-NLS antibodies or protein
G-Sepharose-bound mouse IgG as control. Immunoprecipitates were
resolved by Tricine SDS-PAGE, electroblotted to PVDF membrane, and
visualized by Western blot with mAbs to the AhR and XAP2 (Fig. 5D). Co-expression of XAP2 with the mAhR resulted in a clear
increase in expression of the AhR; however, neither ligand treatment
nor the presence of excess XAP2 resulted in significant differences in
the ability of anti-AhR-NLS polyclonal antibodies to immunoprecipitate the receptor. The key observation to note is that these antibodies efficiently immunoprecipitate the AhR complexed with XAP2.

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Fig. 5.
XAP2 does not block access to the NLS of the
mAhR. A rabbit polyclonal antibody directed against the C-terminal
half of the bipartite NLS of the mAhR was produced and purified by
affinity chromatography and was designated anti-AhR/NLS rabbit
polyclonal antibody. A, cytosolic extracts from COS 1 cells
transfected with pcDNA3/mAhR/FLAG (lane 1) or
pcDNA3 (lane 2) were resolved by SDS-PAGE and
transferred to membrane. The presence of the AhR was detected with the
anti-AhR/NLS or control polyclonal antibodies. B, in
vitro translated mAhR-FLAG or mAhR-FLAG/ NLS were subjected
to immunoprecipitation analysis using anti-AhR/NLS polyclonal
antibody. A 1/3 input of in vitro translated
mAhR-FLAG/ NLS and mAhRFLAG are shown in lanes
1 and 2. Immunoprecipitations were subjected to
SDS-PAGE, transferred to membrane, and
[35S]methionine-labeled receptor visualized by
autoradiography. C, detection of mAhRFLAG but not
mAhR-FLAG/ NLS by anti-AhR/NLS rabbit polyclonal antibody on
membranes containing SDS-PAGE resolved in vitro translated
[35S]methionine-labeled proteins. Panel 1,
immunochemical visualization of the AhR with anti-AhR/NLS rabbit
polyclonal antibody or monoclonal antibody RPT 1. Panel 2,
autoradiography of the in vitro translated
[35S]methionine-labeled AhR/FLAG and mAhR-FLAG/ NLS.
D, the anti-AhR antibody (or mouse IgG control) bound to
protein G-Sepharose was used to immunoprecipitate the mAhR from cytosol
prepared from COS-1 cells transiently transfected with either the mAhR
or the mAhR and XAP2, as well as in the presence or absence of TCDD.
The left lanes show 20% of the input for each
immunoprecipitation.
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Importin
Binding to the mAhR Is Inhibited by XAP2--
Two
in vitro assays were developed to test whether or not the
presence of XAP2 in the AhR-FLAG complex results in reduced ability to
bind importin
. In the first approach, the ability of immunopurified
mAhR·FLAG complexes isolated for transfected COS-1 cells to bind to
GST-importin
immobilized on glutathione-agarose was assessed. The
presence of XAP2 in the mAhR complex resulted in a 40% decrease in
receptor binding to importin
(Fig.
6). The very low level of mAhR/M-NLS
binding to GST-importin
indicates that the receptor interacts with
importin
through its NLS. The second assay used a different
approach and examined whether or not in vitro translated
importin
could bind to immobilized AhR-FLAG. AhR-FLAG expressed
alone or co-expressed with XAP2 in COS-1 cells was immunoprecipitated
from cytosolic extracts with M2-agarose, and, after washing the
agarose, in vitro translated importin
was added for 1h.
After washing the immunoprecipitates, the isolated receptor complexes
were eluted from the agarose with FLAG peptide. The eluted protein was
subjected to Tricine SDS-PAGE, protein was transferred to membrane, and
the presence of importin
, AhR, and XAP2 was assessed
(Fig. 7). In preliminary experiments a
higher level of importin
binding to the mAhR was obtained compared with importin
1 (data not shown). A 2.5-fold increase in binding of
importin
to the AhR complex was detected relative to background binding. In contrast, AhR complexed with a relatively large amount of
XAP2 exhibited 3-fold less specific binding of importin
to the AhR.
Whether importin
is binding directly to the NLS of AhR or
indirectly, by binding to importin
complexed with the receptor in
cells was not determined. Nevertheless, this result, considered along
with the inability of XAP2 to block access to the NLS of the receptor,
suggests that XAP2 inhibits importin
binding by altering the
conformation of the bipartite NLS of the mAhR, yielding an unfavorable
conformation for importin binding.

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Fig. 6.
XAP2 inhibits importin
binding to the AhR. COS-1 cells were
transfected with pcDNA3- mAhR-FLAG in the presence or absence of
pCI-XAP2 and AhR-FLAG complexes were immunoprecipitated from cytosolic
extracts and displaced from the M2 resin with FLAG peptide. The
isolated AhR complexes were incubated with GST-importin bound to
glutathione-agarose. AhR·FLAG complexes were displaced from the
glutathione-agarose with glutathione and subjected to SDS-PAGE followed
by transfer to membrane. The presence of the AhR, GST-importin , and
XAP2 were visualized as described under "Experimental Procedures".
A, upper panel, the presence of mAhR
bound to GST-importin ; lower panel, the
amount of GST-importin in each sample. The percentage of mAhR bound
values given has been corrected for the amount of GST-importin and
mAhR present or placed into each assay. B, upper
panel, the relative amount of mAhR incubated with
GST-importin -glutathione-agarose in each assay, respectively. The
lower panel shows the relative amount of XAP2
incubated with GST-importin -glutathione-agarose in each assay.
Assays were performed three times, and essentially the same results
were obtained.
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Fig. 7.
Importin binding to
the AhR is inhibited by XAP2. COS-1 cells were transfected with
pcDNA3- mAhR-FLAG in the presence or absence of pCI-XAP2 and
AhR-FLAG complexes were immunoprecipitated from cytosolic extracts. The
isolated AhR complexes were incubated with in vitro
translated [35S]methionine-labeled importin . The
AhR-FLAG complexes were displaced from the agarose with an excess of
FLAG peptide, resolved by Tricine SDS-PAGE, and transferred to PVDF
membrane. The presence of [35S]methionine-labeled
-importin was assessed by phosphorimaging, whereas the AhR and XAP2
were visualized using standard immunochemical techniques as described
under "Experimental Procedures."
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XAP2 Represses mAhR Transactivation Potential at Low Agonist
Levels--
Previous published reports from several laboratories have
shown that transient expression of XAP2 in established cell lines increases AhR transcriptional activity upon treatment with saturating levels of TCDD (6, 8). Considering that XAP2 is able to stabilize the
AhR and block nucleocytoplasmic shuttling, we wanted to explore whether
under the appropriate conditions XAP2 can repress AhR transcriptional
activity. In addition, it is reasonable to hypothesize that inhibition
of ligand-mediated transformation of the AhR by XAP2 would be greater
in the presence of a relatively weak ligand. To test this hypothesis,
COS-1 cells were transfected with either pcDNA3/
mAhR or
pcDNA3/
mAhR plus pCI/XAP2 at two different levels, followed by
treatment with various concentrations of iodoflavone. Iodoflavone was
chosen because it has intermediate affinity for the AhR (25). AhR
transcriptional activity was assessed using a DRE-driven luciferase
reporter. A significant level of inhibition of reporter activity is
observed in the presence or absence of ligand upon co-transfection with
XAP2 (Fig. 8, A and
C). The graph in panel A
(Fig. 8) reveals an inhibition of reporter activity except at
relatively high ligand concentrations (e.g. 5 µM). This result is even more striking when you consider that, at high levels of co-transfected XAP2, receptor levels actually increase (Fig. 8B). In addition, expression of an
intermediate amount of XAP2 leads to repression of iodoflavone-mediated
mAhR activity even at a saturating dose of ligand (Fig. 8C).
It is important to note that both constitutive and inducible mAhR
activity are repressed by XAP2 expression. These results would indicate that XAP2 does repress transcriptional activity of the mAhR.

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Fig. 8.
XAP2 represses AhR transcriptional activity
at low agonist levels. COS-1 cells in 100-mm plates were
co-transfected with 0.5 µg of pcDNA3- mAhR, 100 ng of pGudLuc
6.1, 30 ng of pCMV- Gal, either 2 or 4 µg of pCI/XAP2, and pCI was
added, for a total of 8 µg of DNA. After 9 h transfected cells
were transferred to 12-well plates; 13 h later cells were treated
with iodoflavone. After 8 h in the presence of ligand, cells were
lysed, and reporter activity was assessed. A, COS-1 cells
were co-transfected with 4 µg of pCI-XAP2. B, the level of
AhR and XAP2 in cells transfected with the same vector composition as
in A was assessed by protein blot analysis. C,
COS-1 cells were co-transfected with 2 µg of pCI-XAP2. An
asterisk above the bar corresponds to a
statistical difference (p < 0.03) using a paired
Student's t test. The error bars
represent the standard deviation obtained from triplicate
samples.
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DISCUSSION |
Upon transient expression in cells, the mAhR erroneously localizes
to the nuclear compartment of cells. This apparent artifact is
corrected upon co-expression of the XAP2, resulting in a dramatic re-localization of the mAhR exclusively to the cytoplasm (12, 14, 15).
The mechanism by which XAP2 modulates the subcellular localization of
the mAhR is unknown, but may result from a number of possible
scenarios. Prior to undertaking these studies, some of the
possibilities included, but were not limited to, the following. 1) XAP2
may enhance the nuclear export of the mAhR, leading to apparent
cytoplasmic localization; 2) XAP2 may inhibit nuclear retention of the
mAhR; 3) XAP2 may mediate sequestration or tethering of the mAhR in the
cytoplasm by an unknown mechanism; 4) XAP2 may physically mask the NLS
of the mAhR, 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; 5) XAP2 may stabilize the mAhR in a conformation in which
the NLS is exposed, but not recognized by import machinery. A variety
of experiments based on the above hypotheses were performed and are
presented in this report.
Leptomycin B is a specific inhibitor of CRM-1-mediated nuclear export;
CRM-1 has been demonstrated to be responsible for nuclear export of the
AhR (20). The experiments documented in Fig. 1 examined the possibility
that XAP2 might enhance nuclear export of the mAhR, thus resulting in
cytoplasmic localization of the mAhR. As a possible mechanism
underlying the re-localization of the mAhR by XAP2, this theory was
quickly eliminated. As can be seen in Fig. 1, co-expression of XAP2
with mAhR-YFP resulted in a strong inhibition of ligand-independent
nucleocytoplasmic shuttling. The mAhR-YFP localized throughout cells
with higher concentrations in the nucleus, a further increase in
nuclear accumulation followed after 1 h of treatment with 10 nM leptomycin B. Co-expression of XAP2 resulted in
cytoplasmic localization of mAhR-YFP, and receptor localization remains
largely unaffected after 1 h of leptomycin B treatment. The cells
were monitored for 6 h following treatment, and no significant
nuclear accumulation was observed (data not shown). This result has
been recently observed in similar experiments performed in HeLa cells
(22). As a control, mAhR-K13A-YFP (mutation in NLS) was also examined.
Lack of a functional NLS resulted in strong cytoplasmic localization,
and no nuclear accumulation was observed following leptomycin B
treatment, suggesting that the NLS of the mAhR is required for
nucleocytoplasmic shuttling. The observation that the mAhR undergoes
rapid ligand-independent nucleocytoplasmic shuttling strongly suggests
that the NLS of the mAhR is exposed and can be recognized by components
of the nuclear import pathway in the absence of ligand. Both GST-GFP fusions of an N-terminal fragment of the hAhR as well as the endogenous mAhR have been reported to undergo ligand-independent nucleocytoplasmic shuttling (19, 22).
We have reported that transiently expressed mAhR, in the absence of
co-expressed XAP2, is capable of existing in a core complex that lacks
the XAP2 component, which we hypothesized may somehow lack the ability
to be retained in the cytoplasm (14). This observation, together with
the inhibitory effect of XAP2 on nucleocytoplasmic shuttling, suggests
that shuttling may only be seen in complexes lacking XAP2, and further
raises the possibility that the mAhR may exist in multiple forms. In
addition, XAP2 probably exists in a dynamic equilibrium with mAhR
complexes, with XAP2 dissociation enhancing receptor translocation to
the nucleus, whereas hsp90 probably remains bound to the mAhR upon
ligand-dependent and -independent translocation into the
nucleus (26). The occurrence of ligand-independent nucleocytoplasmic
shuttling itself suggests that we need to modify how we look at the
mechanism of action of the mAhR. The rapid nucleocytoplasmic shuttling
of the mAhR would indicate that the observed localization of the
receptor at any given point in time is actually a snapshot of a dynamic
process. Thus, the relative rates of import and export will determine
the apparent subcellular distribution observed. The traditional view
that ligand binding initiates nuclear translocation seems to be
represented more realistically by a shift in the equilibrium to favor
accumulation in the nuclear compartment, as translocation appears to be
continually occurring in the presence and absence of ligand. The
nuclear accumulation that is observed in response to ligand binding is
beyond the scope of this report, but may reflect either enhanced import
of the receptor, diminished export, or a combination of both scenarios. Ligand binding may confer these changes in receptor movement by directly inducing a conformational change and/or by promoting an event
such as phosphorylation/dephosphorylation that may mediate the altered localization.
The clear inhibition of nucleocytoplasmic shuttling of the mAhR by XAP2
revealed the possibility that XAP2 might promote or mediate association
of the mAhR with cytoskeletal components. XAP2-mediated association of
the receptor with cytoskeletal components is a possible mechanism for
the altered subcellular localization that is induced by XAP2, as well
as the inhibition of nucleocytoplasmic shuttling. This type of a role
has been ascribed to FKBP52, the immunophilin component of the
glucocorticoid receptor (GR) complex, which has been shown to associate
with microtubules and is required for nucleocytoplasmic trafficking of
the GR along the cytoskeleton (27, 28). We chose to examine both
tubulin and actin components of the cytoskeleton. Treatment of cells
with the cytoskeletal disrupting agents did not result in altered
localization of mAhR-YFP, nor was ligand-dependent nuclear
translocation affected when cells were treated with TCDD. These results
suggest that XAP2 is not modulating the subcellular localization of the
mAhR via interaction with actin- or tubulin-based cytoskeletal
components, although association with some other cytoplasmic cellular
component remains a possibility at this point. In contrast, Berg and
Pongratz (29) have recently reported that actin is necessary for
XAP2-mediated retention of the AhR. The reason for this difference in
the results they obtained compared with this report is unknown. Their
experiments were performed in HeLa cells, whereas COS-1 cells were used
in this study. Nevertheless, our results are also consistent with the
inability of cytoskeletal disrupting agents to alter the induction of
CYP1a1 in murine Hepa 1c1c7 cells by AhR ligands (27). The lack of an
effect of cytoskeletal disruption on ligand-induced nuclear
accumulation suggests that the mAhR does not rely on targeted movement
along microtubules as has been reported for the glucocorticoid receptor
(28). Disruption of microtubule, microfilament, and intermediate
filament networks does not cause a significant reduction in the rate of
nuclear accumulation of the GR. However, geldanamycin is able to
markedly reduce the rate of translocation of steroid-bound GR, whereas
disruption of cytoskeletal structures leads to no observed inhibition
of the rate of GR translocation upon geldanamycin treatment (29). This
would suggest that hsp90·GR complexes utilize the cytoskeleton during
transport to the nucleus, yet transport to the nucleus does not require
an intact cytoskeleton. Although geldanamycin induces translocation of
the AhR to the nucleus (data not shown), geldanamycin does not induce
translocation of the unliganded GR complex. However, geldanamycin does
inhibit liganded GR movement into the nucleus (30). Thus, despite the
fact that both the GR and AhR are chaperoned by hsp90, regulation of
nucleocytoplasmic shuttling appears to be distinct.
To further explore the possibility that XAP2 may facilitate a
physical tethering of the mAhR in the cytoplasm, and to test whether or
not the effect of XAP2 on mAhR localization was mediated via the NLS of
the mAhR, we created mAhR-YFP-Nuc and mAhR-K13A-YFP-Nuc. These fusion
proteins are identical to mAhR-YFP and mAhR-K13A-YFP, except for the
addition of three tandem repeats of the NLS of SV40 large T antigen to
the C terminus of YFP of each fusion protein. The results of transient
expression of these constructs and the appropriate controls in COS-1
cells are shown in Fig. 4. Again, mAhR-YFP is found localized
predominantly in the nucleus and to a lesser extent in the cytoplasm,
whereas co-expression of XAP2 results in mostly cytoplasmic
localization. The mAhR-K13A-YFP mutant localizes exclusively to the
cytoplasm. Both mAhR-YFP-Nuc and mAhR-K13A-YFP-Nuc were found to
localize to nuclei, demonstrating that the added NLS was capable of
directing nuclear import of the receptor, even when the NLS of the mAhR
is nonfunctional. Significantly, co-expression of XAP2 did not result
in altered localization, as both forms of mAhR-YFP-NLS can be seen to
clearly localize to nuclei. The lack of an effect of XAP2 on the
cellular distribution of mAhR-K13A-YFP-Nuc argues against a tethering
mechanism underlying cytoplasmic sequestration. Taking into
consideration these results, some type of structural modulation of the
receptor such as altering the conformation of the bipartite NLS, or a
physical masking of the NLS by XAP2 inclusion in the complex, becomes
more attractive as a theory to explain XAP2-mediated cytoplasmic
sequestration of the mAhR.
To explore the possibility that the presence of XAP2 in the core
complex may result in physical masking of the NLS of the mAhR, a rabbit
polyclonal antibody against the C-terminal portion of the bipartite NLS
(amino acid residues 31-44) of the AhR was produced. The anti-NLS
antibodies were then utilized to immunoprecipitate the AhR from
cytosolic extracts of COS-1 cells, transiently expressing either the
mAhR or mAhR together with XAP2. We have previously reported that,
under the transfection conditions used, ~25% of transiently
expressed mAhR complexes contain XAP2 when the mAhR is expressed alone,
whereas co-expression of the mAhR with XAP2 results in XAP2 inclusion
in nearly 100% of the mAhR complexes (14). The results summarized in
Fig. 5D revealed that neither TCDD nor co-expression of XAP2
altered the ability of these antibodies to recognize the NLS-based
epitope. Perhaps most important, the results clearly indicated that
XAP2 is co-immunoprecipitated with the AhR. In addition,
immunoprecipitation of the mAhR with the anti-AhR
monoclonal antibody Rpt 9 (24), directed against the N-terminal portion
of the NLS of the AhR (amino acid residues 12-31), resulted in
co-immunoprecipitation of XAP2 (14), further suggesting that access to
the N-terminal portion of the NLS is not blocked by XAP2. The lack of
effect of XAP2 on the ability of AhR antibodies to bind the NLS of the
mAhR clearly suggests that XAP2 does not physically mask the NLS when
present in the complex. The mechanism of XAP2-mediated sequestration of
the mAhR appears to be quite different from the model that has emerged explaining the mechanism of cytoplasmic sequestration of NF-
B subunit p65 by I
B. Using antibodies to the NLS of p65, several laboratories have clearly shown that I
B physically masked the NLS of
p65 (31-33).
The results in Fig. 6 would indicate that the ability of importin
to bind to the NLS of the AhR is reduced when XAP2 is found complexed
with the mAhR. This result suggests that XAP2 alters the conformation
of the bipartite NLS of the AhR and leads to reduced recognition by
importins. Considering that the site on the AhR required for XAP2
binding is near the ligand binding domain, it is tempting to speculate
that the ligand binding pocket could also be altered by the presence of
XAP2. Indeed this possibility is supported by the results in Fig. 8,
which demonstrate that XAP2 represses the ligand-mediated activity of
the mAhR, although this result may be because of an overall
stabilization of the hsp90·mAhR complex or the blocking of
nucleocytoplasmic shuttling in the presence of XAP2. Considering that
XAP2 levels vary considerably from tissue to tissue, it is plausible
that XAP2 could mediate tissue-specific differences in mAhR activity.
In particular, the very low level of XAP2 in liver is consistent with
the concept that the mAhR should be capable of being rapidly activated
in response to dietary exposure to AhR ligands (9, 34). In
summary, the data presented here support the assertion that XAP2 is
capable of altering importin
recognition of the AhR, and this
appears to be the primary mechanism of XAP2-mediated retention of the AhR in the cytoplasm. However, it is still possible that other mechanisms may also play a contributing role in the ability of XAP2 to
block nucleocytoplasmic shuttling of the AhR. Finally, the work
presented here is one of the first demonstrations of a clear functional
role for an hsp90 co-chaperone protein in influencing the activity of a
chaperoned soluble receptor.