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INTRODUCTION |
The bHLH-PAS dioxin (aryl
hydrocarbon)1 receptor (1, 2)
is a member of a growing family of transcription factors that includes,
among others, a number of circadian rhythmicity regulatory proteins (3,
4) and proteins such as the hypoxia-inducible factor HIF-1
(5), its
endothelial cell-specific homologue E-PAS/HLF (6, 7), and Arnt (8),
which is a partner factor both of the dioxin receptor and of
hypoxia-inducible factors. These factors, with the possible exception
of Arnt, appear to be conditionally regulated and respond to different
stimuli. The dioxin receptor is activated by dioxin (9) and related
environmental pollutants, whereas HIF 1
is activated under
conditions of oxygen deprivation to induce the expression of genes
encoding, e.g. erythropoietin and vascular endothelial
growth factor (10).
In the absence of ligand the dioxin receptor is present in a latent
conformation in the cytoplasmic compartment of the cell (11) associated
with the molecular chaperone hsp90 (12). Hsp90 is required both for
maintaining the dioxin receptor in a latent non-DNA binding state and a
ligand binding conformation (13). Expression of the dioxin receptor in
mutant yeast cells containing reduced levels of hsp90 abolishes ligand
responsiveness demonstrating the critical importance of hsp90 for
dioxin receptor function (14, 15). The nuclear form of the dioxin
receptor interacts with Arnt (16-18) and does no longer posses the
ability to bind ligand and does not interact with the molecular
chaperone hsp90 (12, 13). This form of the receptor specifically binds
to enhancer elements known as XREs (xenobiotic response elements) of a
number of genes encoding drug-metabolizing enzymes (17, 18). Release of
hsp90 from the latent form of the dioxin receptor is therefore a
critical step in the activation process of the dioxin receptor.
Recently the hepatitis virus X protein-associated protein (XAP-2) also
known as ARA 9 or AIP of 38 kDa has been shown to interact with the
latent form of the dioxin receptor (19-21). This protein has been
reported to increase the transcriptional activity of the dioxin
receptor, although the mechanism of action has not been elucidated
(22). The co-chaperone protein p23 (23) has been shown to be associated
with the N-terminal ATP binding domain of hsp90 (24). p23 has also been
found to be associated with non-activated form of selected members of
the steroid receptor superfamily such as the glucocorticoid and
progesterone receptors (25). Moreover, p23 has been reported recently
to be associated with the dioxin receptor (26). The role of p23 in
modulating target protein function is, however, not clear. Given the
background that interaction with p23 correlates with high affinity
ligand binding activity of certain steroid receptors (27), it seems plausible that p23 is involved regulation and stabilization of the
ligand binding conformation of these receptors. In addition to hsp90
and p23, other protein factors are involved in the formation of the
high affinity ligand binding steroid receptor form. In fact, formation
of such a complex appears to involve p60 (28), also known as HOP
(Hsp organizing Protein), several
immunophilins, and possibly factors that are found in association with
proteins such as hsp70 (25).
We have observed previously that ligand-dependent release
of hsp90 in vitro requires the interaction of the dioxin
receptor with additional cellular factors, including Arnt (29).
Interestingly, in the present study, fractionation of cellular extracts
through sucrose density gradients yielded an hsp90-associated form of the dioxin receptor, which did not require ligand to generate the DNA
binding complex with Arnt. This loss of ligand dependence correlated
with dissociation of p23 from the dioxin receptor-hsp90 complex. It was
possible to reconstitute ligand dependence in receptor activation by
addition of molybdate, an agent that has been shown stabilize the
interaction between hsp90 and p23. Thus, these results indicate a role
of p23 in modulating ligand responsiveness in receptor activation.
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MATERIALS AND METHODS |
Recombinant Plasmids and Protein Expression--
The
construction of pGemArnt, pSP72 mDR, and pGEX 4T3 hArnt has been
described previously (17, 30). In vitro translation of the
dioxin receptor and Arnt was performed according to the manufacturer's
recommendations using coupled transcription/translation reactions in
rabbit reticulocyte lysate (Promega Biotech). Bacterial expression of
glutathione S-transferase-tagged Arnt has been described in
detail elsewhere (30).
Cells and Preparation of Cellular Extracts--
Hepa 1c1c7 mouse
hepatoma cells and the mutant derivative C4 were grown in minimum
essential medium as described previously (12). Cells were grown to near
confluence in an atmosphere of 5% CO2 until harvested.
Cytosolic extracts were prepared by scraping untreated cells and
washing them twice in phosphate-buffered saline and once in TEG buffer
(20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10% (w/v)
glycerol, and 1 mM dithiothreitol). After washing the
pellet, the cells were homogenized in 1 volume of TEG buffer and
centrifuged at 120,000 × g for 45 min. The resulting
supernatant was taken as the cytosolic fraction and used either
immediately or frozen in small aliquots at
70 °C.
Fractionation of Cytosol by Sucrose Density Gradient
Centrifugation--
Cytosolic extracts (400 µl; about 2-10 mg of
protein/ml of cytosol) or in vitro translated dioxin
receptor was layered on 10-40% (w/v) linear sucrose gradients
prepared in TEG buffer containing 25 mM NaCl and 10 mM sodium molybdate as indicated. The gradients were
centrifuged at 300,000 × g to a cumulative centrifugal
effect of 1.7 × 1012 radian2/s in a
Beckman L8-80 ultracentrifuge. Fractions were collected by gravity flow
starting from the bottom of the gradients. IgG (6.6 S) and bovine serum
albumin (4.4 S) were used as external sedimentation marker proteins.
In Vitro DNA Binding Assay--
The dioxin receptor was
noncovalently labeled by incubation of crude or fractionated cytosol
for 2.5 h at 30 °C with 10 nM [3H]TCDD (Chemsyn, Lenexa, KS) or 10 nM
nonradioactive dioxin. Dioxin receptor-dependent DNA
binding activities were analyzed by a gel mobility shift assay (EMSA)
performed essentially as described (31). Briefly, DNA binding reactions
were assembled with the indicated protein fractions in 10 mM Hepes, pH 7.9, 5% (v/v) glycerol, 0.5 mM
dithiothreitol, 2.5 mM MgCl2, 1 mM
EDTA, 0.08 (w/v) Ficoll, and NaCl to a final concentration of 60 mM. The total volume of the DNA binding reactions ranged
between 20 and 50 µl. A 32P-3'-end-labeled,
double-stranded 36-base pair oligonucleotide XRE (32) spanning a
dioxin-responsive XRE element of the rat cytochrome P-4501A1
upstream/promoter region was added to the reactions as specific probe
in the presence of 1 µg of nonspecific poly(dI-dC) competitor DNA.
The reactions were incubated for 15 min at 25 °C, and protein-DNA
complexes were resolved on 4% (acrylamide/bisacrylamide ratio of 29:1)
low ionic strength native polyacrylamide gels at 30 mA and 0-4 °C
using a Tris-glycine EDTA buffer (31). In indicated DNA binding
experiments, polyclonal antibodies against the dioxin receptor (17, 33)
or Arnt (34), or preimmune serum, were added to the binding reaction
mixtures together with protein fractions and the radiolabeled XRE probe
to assess the specificity of protein-DNA complexes.
Co-immunoprecipitration Experiments--
In vitro
[35S]methionine-labeled dioxin receptor or Arnt were
mixed with 3G3 anti-hsp90 antibodies (Santa Cruz Technologies), anti-p23 (JJ3) antibodies (23), or anti-dioxin receptor polyclonal antibodies. Subsequently 40 µl of 50% protein A-Sepharose in
phosphate-buffered saline in JJ3 or anti-dioxin receptor antibody
experiments or Sepharose-coupled IgM antibodies in 3G3 antibody
experiments was added and incubated under gentle rotation for 1 h
at 0-4 °C. Bound material was washed five times in
phosphate-buffered saline, and proteins were eluted by addition of
SDS-polyacrylamide gel electrophoresis sample buffer.
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RESULTS |
Ligand-independent Activation of the Dioxin Receptor to a DNA
Binding Form--
Fractionation of the dioxin receptor on sucrose
density gradients provides a convenient tool to separate different
functional forms of the receptor. Typically the dioxin receptor can be
recovered both in the 9 S region of the gradient corresponding to an
approximate molecular mass of 300 kDa or in the 5-6 S region with an
approximate mass of 200 kDa (12, 13, 35). These different complexes represent distinct functional forms of the dioxin receptor; the 9 S
form represents the ligand binding, hsp90-containing receptor complex,
whereas the 5-6 S form represents the DNA binding heterodimeric complex between the receptor and Arnt (12, 17). In the present experiments we have used cytosolic extracts from Hepa-1 C4 (36) cells
expressing a mutant form of Arnt (37) that does not enable the dioxin
receptor to bind DNA (8). Addition of wild-type Arnt to this extract
restores the DNA binding activity of the receptor in vitro
(16, 17). The Hepa-1 C4 cytosolic extract was treated with 10 nM TCDD for 2 h at 25 °C and subsequently fractionated on a 10-40% linear sucrose density gradient. The individual fractions were incubated with Arnt for 30 min, and XRE
binding activity was assayed by EMSA. The only region of the gradient
that displayed XRE binding activity were fractions in the 9S region of
the gradient (Fig. 1A),
corresponding to the sedimentation position of the receptor-hsp90
complex (12). Thus, consistent with the model that ligand alone is not
sufficient to induce release of hsp90 but requires functional Arnt
(29), ligand treatment did not induce any change in the sedimentation profile of the dioxin receptor from 9 S to the 5-6 S region of the
gradient, corresponding to the sedimentation position of hsp90-free receptor forms (13).

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Fig. 1.
Ligand-independent activation of the dioxin
receptor to an XRE binding form. A, Hepa-1 C4 cell
cytosol was treated with 10 nM TCDD prior to fractionation
on a linear 10-40% sucrose density gradient. Following fractionation
the individual fractions were incubated with in vitro
translated Arnt and analyzed by EMSA. The sedimentation positions of
external sedimentation markers (bovine serum albumin and IgG) and the
top of the gradient are indicated. B, upon sucrose gradient
fractionation dioxin receptor-containing fractions were pooled and
incubated in the presence or absence of 10 nM TCDD, Arnt,
or a combination of both treatments. Subsequently, the DNA binding
activity of the dioxin receptor-Arnt complex was assessed by EMSA. The
arrow indicates the specific dioxin receptor-Arnt complex,
whereas asterisks indicate nonspecific protein-DNA
complexes.
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To assess the role of ligand in activating the receptor to an XRE
binding form, we next fractionated untreated Hepa-1 C4 cytosolic extracts on sucrose gradients and pooled the receptor-containing fractions in the 9 S region of the gradient. This material was incubated with 10 nM dioxin or vehicle alone for 2 h
at 25 °C in the presence of bacterially expressed glutathione
S-transferase-Arnt, and XRE binding activity was monitored
by EMSA. Interestingly, an XRE binding complex was generated both in
the presence or absence of ligand (Fig. 1B, compare
lanes 3 and 5). This complex harbored both the dioxin
receptor and Arnt, as assessed by specific antibodies (data not shown).
In contrast, the dioxin receptor in non-fractionated extracts requires
ligand to interact with in vitro translated (38) or
bacterially expressed (data not shown) Arnt to generate the XRE binding
complex. Thus, these data suggest that the ligand-dependent mechanism of receptor activation had been lost upon fractionation of
the receptor.
Given the loss of ligand dependence to induce DNA binding activity by
the dioxin receptor, we examined the effect of Arnt on ligand binding
activity by sucrose gradient-fractionated receptor preparations. As
schematically outlined in Fig.
2A, we fractionated in
vitro translated dioxin receptor on a sucrose gradient and pooled
the dioxin receptor-containing fractions in the 9 S region of the
gradient. The fractions were then dialyzed to remove sucrose and
incubated in the presence or absence of Arnt for 1 h at 30 °C.
This material was subsequently incubated with 10 nM
[3H]TCDD for 2 h at 25 °C and loaded on a second
sucrose density gradient. Following centrifugation the individual
fractions were assayed for radioactivity. As shown in Fig.
2A the fractionated dioxin receptor was able to bind
[3H]TCDD. However, upon addition of Arnt, ligand binding
activity was significantly decreased. In view of these results we also examined the effect of Arnt to induce release of hsp90 from in vitro translated 35S-labeled dioxin receptor upon
fractionation on sucrose gradients. Consistent with the results using
nonfractionated receptor material (29), we observed no release of hsp90
from the 9 S dioxin receptor upon incubation of the 9 S receptor with
ligand alone in the absence of Arnt (Fig. 2B, compare
lanes 2 and 3). Interestingly, however, addition
of Arnt to the isolated 9 S dioxin receptor form induced dissociation
of hsp90 from the receptor both in the presence or absence of dioxin
(Fig. 2C, compare lanes 2-5), consistent with the observed reduction in ligand binding activity (Fig.
2A).

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Fig. 2.
Arnt impairs ligand binding activity of the 9 S dioxin receptor form and induces ligand-independent release of
hsp90. A, upper panel: schematic outline of
the experimental protocol. In vitro translated dioxin
receptor was fractionated on a sucrose density gradient, and the 9 S
fraction was collected and treated with Arnt or vehicle alone for
1 h at 30 °C prior to incubation with 10 nM
[3H]TCDD for 2 h at 25 °C. Excess TCDD was
removed by incubation with dextran-coated charcoal, and the fraction
was loaded on top of a second linear 10-40% sucrose density gradient
and centrifuged as before. Lower panel, following
fractionation bound TCDD was counted in a liquid scintillation counter.
B and C, unfractionated lysate (B) or
the 9 S dioxin receptor-fraction (C) containing in
vitro translated 35S-labeled dioxin receptor was
incubated as described above in the absence or presence of bacterially
expressed Arnt and/or 10 nM TCDD for 2 h at 30 °C.
Co-immunoprecipitaion experiments (hsp90 ippt) were
performed with 3G3 hsp90 monoclonal antibodies as described under
"Materials and Methods." Precipitated proteins were analyzed on a
7.5% SDS-polyacrylamide gel electrophoresis gel, which was analyzed by
fluorography. The position of the dioxin receptor (DR) is
indicated.
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The Dioxin Receptor Interacts with the Co-chaperone p23--
The
experiments presented above suggest that the latent form of the dioxin
receptor becomes destabilized upon fractionation on sucrose gradients,
resulting in ligand-independent release of hsp90 and ensuing receptor
activation by Arnt. It has been extensively documented that hsp90 binds
additional co-chaperone proteins, which are important for functional
activities of certain hsp90 associated proteins, such as a number of
steroid receptors (25). We therefore investigated the potential
involvement of hsp90-associated factors in stabilization of the dioxin
receptor-hsp90 complex. To this end we in vitro translated
the dioxin receptor in the presence of [35S]methionine
and performed co-immunoprecipitation experiments with p23 antibodies.
The dioxin receptor was efficiently co-precipitated by p23 antibodies,
whereas in control experiments Arnt, which does not interact with
hsp90, failed to interact with p23 (Fig. 3A, compare lanes 3 and
6). We next studied the role of ligand and Arnt on release of p23
from the dioxin receptor complex. In vitro translated
[35S]methionine-labeled receptor was incubated in the
absence or presence of 10 nM dioxin, Arnt, or both
treatments. Interestingly, addition of Arnt or ligand alone did not
induce release of p23 from the dioxin receptor complex (Fig.
3B, compare lanes 3-5). Only in the presence of
a combination of Arnt and ligand we observed partial dissociation of
p23 from the dioxin receptor (Fig. 3B, compare lanes 3 and 6).

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Fig. 3.
The dioxin receptor but not Arnt interacts
with p23. A, Arnt and the dioxin receptor were in
vitro translated in the presence of [35S]methionine
and immunoprecipitated using monoclonal antibodies directed against p23
and protein A-Sepharose. B, in vitro translated
dioxin receptor was incubated in the absence or presence of Arnt, 10 nM TCDD, or a combination of both treatments, for 1 h
at 30 °C prior to immunoprecipitation with p23 monoclonal antibodies
( -p23) and analysis as in Fig. 2. The position of the
dioxin receptor (DR) is indicated. Lanes 1 and 4 show 50% of the input (I) material.
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Loss of Ligand-inducible XRE Binding Activity Correlates with
Dissociation of p23 during Gradient Centrifugation--
In view of the
background that p23 has been shown to stabilize binding between hsp90
and other target proteins (27), we investigated whether sucrose
gradient centrifugation affected the interaction of p23 with the 9 S
dioxin receptor form. In vitro translated
[35S]methionine-labeled dioxin receptor was fractionated
on a sucrose density gradient, and receptor-containing fractions in the
9 S region were pooled and used in p23 immunoprecipitation experiments. As expected, experiments performed with nonfractionated in
vitro translated dioxin receptor showed highly efficient
co-immunoprecipitation of the labeled receptor using p23 antibodies
(Fig. 4A, compare lanes
1-3). However, following gradient centrifugation a significant decrease in recovery of dioxin receptor by precipitation with p23
antibodies was detected (Fig. 4A, compare lanes 3 and
6). In control reactions, dioxin receptor antibodies precipitated similar amounts of dioxin receptor from either the unfractionated material or the 9 S gradient fraction (Fig. 4B, compare
lanes 3 and 6). In conclusion, these experiments
indicate that the association of the dioxin receptor-hsp90 complex with
p23 becomes destabilized upon fractionation, correlating with the loss
of requirement of ligand to induce XRE binding activity.

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Fig. 4.
Fractionation of the dioxin receptor on a
sucrose gradient induces release of p23. In vitro
translated, [35S]methionine-labeled dioxin receptor was
fractionated on a sucrose gradient, and receptor-containing material in
the 9 S region of the gradient was collected. This fraction (9S
DR) or nonfractionated input (Non-fract.) material was
used for immunoprecipitation experiments using p23 ( -p23)
(A), dioxin receptor ( -DR) (B), or control (C)
antibodies and analyzed as in Fig. 2. Lanes 1 and 4 show
50% of the input (I) material.
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Reconstitution of Ligand-dependent Activation of the 9 S Dioxin Receptor Form--
Molybdate ions and other transition metal
oxides have been shown to stabilize the interaction between hsp90 and a
number of hsp90 interacting proteins, most notably different members of the steroid hormone receptor family (27). We therefore supplemented cytosolic extracts from Hepa-1C4 cells with 10 mM
MoO3 prior to fractionation through sucrose density
gradients containing 10 mM MoO3. As expected
the dioxin receptor was recovered as a 9 S sedimenting complex. This
material was subsequently incubated with Arnt both in the presence or
absence of 10 nM dioxin and induction of the XRE binding
activity of the dioxin receptor-Arnt complex was monitored by EMSA.
Upon exposure to Arnt the dioxin receptor-Arnt complex did not require
ligand to bind the XRE target sequence following fractionation of the
receptor in a sucrose density gradient lacking MoO3 (Fig.
1B; Fig. 5, compare
lanes 2 and 6). Strikingly, however, addition of 10 mM MoO3 to the gradient rendered formation of
the DNA binding complex strictly ligand-dependent (Fig. 5,
compare lanes 4 and 8).

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Fig. 5.
Reconstitution of
ligand-dependent activation of the XRE binding activity by
the 9 S dioxin receptor form. Hepa-1 C4 cell cytosol was
fractionated through a sucrose gradient in the presence or absence of
10 mM Na2MoO4. The 9 S dioxin
receptor containing fractions were subsequently incubated with 10 nM TCDD or with vehicle alone prior to addition of Arnt for
30 min at room temperature and anlysis by EMSA.
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DISCUSSION |
The process of activation of the dioxin receptor from a
cytoplasmic latent form to a nuclear transcriptionally active form includes release of hsp90 (12, 13). We have shown previously that, in
addition to ligand, Arnt facilitates disruption of the dioxin
receptor-hsp90 complex (29). In contrast to our results, Fukunaga
et al. (39) have observed partial release of hsp90 from the
dioxin receptor in the absence of Arnt upon addition of dioxin in
vitro. The reason behind these discrepancies in results is
presently unclear but may reflect different experimental conditions, most notably very significant dilution of the samples in the
immunoprecipitation protocol used by Fukunaga et al. (39).
Dilution is known to destabilize steroid hormone receptor-hsp90
complexes (25). In the present study treatment of cytosolic extracts
from Hepa-1C4 cells expressing a mutant form of Arnt with 10 nM dioxin prior to fractionation through a sucrose density
gradient yielded only one fraction of dioxin receptor sedimenting at
the position (9 S) of the receptor-hsp90 complex, indicative of a
failure to induce a significant release of hsp90 under these
conditions. Although it remains unclear whether hsp90 is released from
the receptor prior or subsequent to nuclear translocation, the receptor
has been observed to be associated with hsp90 upon extraction from purified nuclei (12). Moreover, the receptor shows
ligand-dependent nuclear translocation in Hepa-1C4 cells
(40) where we failed to produce ligand-induced release of hsp90
in vitro. Taken together, these data indicate that the
receptor may be imported into the nucleus in association with hsp90.
Obviously, it will be important to further investigate this issue.
Fractionation of the dioxin receptor through a sucrose density gradient
resulted in a loss of the requirement of ligand to generate an XRE
binding receptor form, whereas the interaction between hsp90 and the
dioxin receptor was not disrupted by this procedure. Moreover,
consistent with the ligand-independence in activation of DNA binding
activity, addition of Arnt to the sucrose gradient fraction containing
the receptor-hsp90 complex resulted in release of hsp90 in the absence
of ligand. We interpret these results to indicate that factor(s)
involved in stabilization of the latent form of the receptor may have
been dissociated following sucrose gradient centrifugation. In an
effort to identify such a putative factor we have examined the role of
p23 in dioxin receptor activation. p23 is a protein that has been found
to be associated with hsp90 (24, 41, 42). In the case of the
progesterone receptor and the glucocorticoid receptors, p23 has been
found to be associated with the high affinity ligand binding
conformations of these receptors (23). It has been shown that formation
of the ultimate ligand binding forms of these receptors represents a
well ordered process where a number of different protein are involved,
e.g. p23, hsp90, hsp70, and Hop (p60) (25, 43, 44).
Here we demonstrate that p23 is associated with the ligand binding form
of the dioxin receptor. Moreover, as outlined in the model in Fig.
6, p23 was not displaced upon occupation
of the ligand binding domain of the receptor by ligand but remains
together with hsp90 associated with the receptor in an intermediary
complex prior to dimerization of the receptor with Arnt and release of hsp90. Whereas certain steroid receptors show low ligand binding affinity in the absence of p23 (27), dissociation of p23 from the
dioxin receptor by fractionation of the receptor on sucrose gradients
did not appear to affect its ligand binding activity. Remarkably,
however, in the absence of p23, release of hsp90 by Arnt and the
formation of an XRE binding form did not require ligand. It is
therefore possible that the role of ligand may be to counteract the
function of p23 by binding to the dioxin receptor and possibly inducing
a conformational change that will enable the receptor to dimerize with
Arnt and subsequently release hsp90. This interpretation is
strengthened by the reconstitution of ligand-dependent XRE
binding activity of the dioxin receptor by addition of molybdate. Molybdate is a widely used compound that stabilizes the interaction between for example the glucocorticoid receptor and hsp90 (41). In our
system molybdate may supplement the functions of unknown factors that
in nonfractionated cytosolic extracts stabilize the interaction between
the dioxin receptor and hsp90. Alternatively, molybdate may mimic the
function of other, hsp90-p23 stabilizing factors. The nature of these
putative factor(s) stabilizing the interaction between p23 and hsp90 is
presently unknown, but it may be a protein, possibly a chaperone, or a
different type of agent such as the glucocorticoid receptor stabilizing
factor modulator (45-47). Clearly, the precise role of ligand in
dioxin receptor activation and the possible involvement of novel
factors in this process needs to be further elucidated. As
schematically represented in the model in Fig. 6, a candidate factor is
p23, the role of which may be to stabilize an intermediary complex that
contains the ligand-occupied hsp90-receptor complex. In summary, we
provide evidence suggesting that the role of ligand in dioxin receptor activation may be to overcome the inhibitory effects of hsp90 associated factors such as the co-chaperone p23 and thereby
facilitating release of hsp90 by Arnt, resulting in generation of the
DNA binding form.

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Fig. 6.
Model of distinct steps in the dioxin
receptor activation pathway. p23 is associated with the ligand
binding hsp90-dioxin receptor complex. Upon binding of ligand a stable
intermediary complex exists consisting of ligand-occupied dioxin
receptor, hsp90 and p23. This form of receptor requires Arnt for
release of hsp90 (and possible co-release of p23) and formation of the
DNA binding receptor-Arnt heterodimeric complex. See text for
details.
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