(Received for publication, March 3, 1995; and in revised form, July 12, 1995)
From the
The dioxin receptor is a cytoplasmic basic helix-loop-helix/Per-Arnt-Sim homology (bHLH/PAS) protein known to bind planar polycyclic ligands including polycyclic aromatic hydrocarbons, benzoflavones, heterocyclic amines, and halogenated aromatic hydrocarbons, e.g. dioxins. Ligand-induced activation of the dioxin receptor initiates a process whereby the receptor is transformed into a nuclear transcription factor complex with a specific bHLH/PAS partner protein, Arnt. In analogy to the glucocorticoid receptor, the latent dioxin receptor is found associated with the molecular chaperone hsp90. We have defined and isolated a minimal ligand binding domain of the dioxin receptor from the central PAS region, comprising of amino acids 230 to 421, and found this domain to interact with hsp90 in vitro. Expression of the minimal ligand binding domain in wheat germ lysates or bacteria, systems which harbor hsp90 homologs unable to interact with the glucocorticoid or dioxin receptors, resulted in non-ligand binding forms of this minimal 230 to 421 fragment. Importantly, affinity of the minimal ligand binding domain for dioxin was similar to the affinity inherent in the full-length dioxin receptor, and a profile of ligand structures which specifically bound the minimal ligand binding domain was found to be conserved between this domain and the native receptor. These experiments show that the minimal ligand binding domain maintains the quantitative and qualitative aspects of ligand binding exhibited by the full-length receptor, implying that the central ligand binding pocket may exist to accommodate all classes of specific dioxin receptor ligands, and that this pocket is critically dependent upon hsp90 for its ligand binding conformation.
Signal transduction by dioxins, related halogenated
hydrocarbons, and polycyclic aromatic hydrocarbons is mediated by the
dioxin receptor (also known as the aryl hydrocarbon receptor), an
intracellular bHLH/PAS ()protein which, in response to
exogenous ligands, forms a transcription factor complex with a specific
bHLH/PAS co-factor, Arnt (for recent reviews, see Swanson and
Bradfield(1993), Hankinson(1994), and Whitlock(1994)). The dioxin
receptor and Arnt are distinguished from other members of the bHLH
family of transcription factors by virtue of PAS (Per-Arnt-Sim)
homology regions, segments of 250 to 300 amino acids juxtaposed to the
bHLH motifs which share similarity with the bHLH/PAS Drosophila transcription factor Sim (Nambu et al., 1991), and Drosophila PAS circadian oscillator Per (Huang et
al., 1993). The ligand-activated dioxin receptor/Arnt heterodimer
recognizes a specific core DNA sequence, the XRE (xenobiotic response
element, TNGCGTG, Lusska et al. (1993)), which bears some
resemblance to the E-box core (CA(C/G)(C/G)TG) recognized by the
majority of bHLH proteins (Kadesh, 1993). XRE sequences function as
dioxin responsive enhancers and are present in upstream regions of
several dioxin-regulated genes (e.g. cytochrome P450IA1,
glutathione S-transferase, and various other genes encoding
xenobiotic metabolizing enzymes; for a review, see Landers and
Bunce(1991)). Recent studies designed to investigate the transcription
activating capabilities of the dioxin receptor and Arnt subunits have
revealed these proteins to harbor potent transactivation domains in
their C termini (Whitelaw et al., 1994; Jain et al.,
1994; Li et al., 1994). Furthermore, phosphorylation states of
the dioxin receptor and Arnt may be a critical factor in dioxin
signaling, as modulation of protein kinase activities has been
demonstrated to inhibit dioxin induced XRE binding activities and
responsiveness of XRE driven reporter genes (Okino et al.,
1992; Berghard et al., 1993; Carrier et al., 1992;
Gradin et al., 1994).
In untreated cells, the dioxin receptor is held in a latent cytoplasmic complex with the molecular chaperone hsp90 (Perdew, 1988; Denis et al., 1988) and a 43-kDa protein (Chen and Perdew, 1994). Ligand signaling initiates a transformation process whereby the receptor translocates to the nucleus (Pollenz et al., 1994), hsp90 is released, and dimerization with Arnt is achieved. As Arnt has been reported to be a nuclear protein (Pollenz et al., 1994; Hord and Perdew, 1994), and Arnt may play an active role in releasing hsp90 from the dioxin bound receptor (McGuire et al., 1994), disruption of the hsp90-dioxin receptor complex may be a nuclear rather than cytoplasmic event. Consistent with this hypothesis, dioxin receptor-hsp90 complexes have been isolated from nuclear extracts of ligand-treated cells (Wilhelmsson et al., 1990; Perdew, 1991). Hsp90 is an intrinsic component of the dioxin receptor ligand signaling mechanism, as ligand responsiveness of chimeric dioxin receptor constructs in a genetically manipulated strain of Saccharomyces cerevisiae has been abrogated in depleted hsp90 environments (Carver et al., 1994; Whitelaw et al., 1995). Multiple roles for hsp90 in maintaining a functional dioxin receptor form are suggested by in vitro studies, which have shown hsp90 to (i) impose repression on the ligand free dioxin receptor, preventing premature interaction with Arnt (Matsushita et al., 1993; Probst et al., 1993; Whitelaw et al., 1993b), (ii) be a critical ``chaperoning'' factor in formation of a receptor capable of binding ligand (Antonsson et al., 1995; Pongratz et al., 1992), and (iii) possibly also chaperone a DNA binding conformation of the ligand-activated receptor (Antonsson et al., 1995).
In a previous strategy to identify regions of the mouse dioxin receptor which could convey dioxin responsiveness onto heterologous transcription factors, a core ligand binding domain between amino acids 230 and 421 was found to bind dioxin in the context of a glucocorticoid receptor/dioxin receptor chimera (Whitelaw et al., 1993a). We have now isolated and studied this core ligand binding domain in detail and find that upon in vitro translation in reticulocyte lysate binding of dioxin and other specific ligands is quantitatively and qualitatively similar to that observed with the full-length native dioxin receptor. Importantly, this 192-amino acid region also binds hsp90 in vitro, strengthening initial implications that hsp90 binding localizes to the ligand binding domain to convey a repression upon the ligand free dioxin receptor (Whitelaw et al., 1994). In vitro translation of this ligand binding domain in wheat germ lysates, which contain an hsp90 homolog unable to interact with the dioxin receptor (Antonsson et al., 1995), failed to produce a protein with ligand binding activity, implying a role for hsp90 in chaperoning a functional ligand binding conformation of this domain. Consistent with this hypothesis, bacterial expression and purification of the ligand binding domain to homogeneity also produced a non-functional protein. These studies confirm the ligand binding domain of the dioxin receptor to reside between amino acids 230 and 421, and establish the interaction of this domain with hsp90 to be essential in formation of a functional ligand binding entity.
Figure 1:
Schematic representation of the dioxin
receptor, glucocorticoid receptor/dioxin receptor chimeras, and
truncated dioxin receptor fragments. A, molecular architecture
of the murine dioxin receptor showing basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) homology, and
glutamine-rich (Q-rich) domains. B, chimeric receptor
constructs containing the N-terminal 500 amino acids of the human
glucocorticoid receptor harboring a zinc finger DNA binding domain and
a transactivation domain (termed DBD) fused to varying
C-terminal segments of the murine dioxin receptor. C,
schematic of the minimal ligand binding domain (LBD), defined
as a region of the dioxin receptor encompassing amino acids 230 to 421,
modified to include a histidine (His) tag and the
hemagglutinin (Ha) epitope (HisHaLBD) at the N
terminus, and a modified dioxin receptor containing a C-terminal
histidine tag (HisDR).
To ascertain whether a smaller region
within this 230 to 421 amino acid domain was capable of binding dioxin,
we have now made further chimeras where the N-terminal and C-terminal
boundaries of the 230 to 421 region were truncated, providing fusion
proteins DBD/DR300-400 and
DBD/DR280-421 (Fig. 1). As hydroxylapatite ligand binding assays with
[
H]dioxin have previously shown that in vitro translated
DBD does not bind ligand (Whitelaw et
al., 1993a), we therefore performed this in vitro assay
with
DBD/DR300-400,
DBD/DR280-421,
DBD/DR230-421, and
DBD/DR83-805 fusion proteins
translated in reticulocyte lysates. Translation of chimeras in the
presence of [
S]methionine gave radiolabeled
fusion proteins which ran as single bands on SDS-PAGE analysis.
Importantly, translation efficiencies were similar for the different
chimeras (Fig. 2A), allowing direct comparison of
[
H]dioxin retention to provide a crude
approximation of binding affinities.
Figure 2:
In vitro ligand binding assays of
DBD-dioxin receptor fusion proteins and the DR230-421
minimal LBD. A, chimeric
DBD/dioxin receptor proteins
were in vitro translated in reticulocyte lysates in the
presence of [
S]methionine and aliquots (2
µl) analyzed by 7.5% SDS-PAGE and fluorography. Positions of the
molecular mass standard proteins are indicated. B,
hydroxylapatite ligand binding assays performed with unlabeled
reticulocyte lysate(-) or translation mixtures containing the
indicated chimeric protein or LBD using 2 nM [
H]TCDD as specific ligand and 250-fold M excesses of TCDF as specific
competitor.
Binding of
[H]dioxin by
DBD/DR83-805, a chimera
lacking only the bHLH domain of the dioxin receptor, is similar to that
of
DBD/DR230-421 (Whitelaw et al., 1993a, Fig. 2B), verifying that the dioxin binding region is
contained within residues 230 to 421. As expected, competition with an
excess of TCDF, another dioxin receptor-specific ligand, displaced
binding of [
H]dioxin by these chimeras to the
background levels shown by unprogrammed lysates (Fig. 2B). Truncation of dioxin receptor residues from
both the N-terminal and C-terminal ends of
DBD/DR230-421
provided chimera
DBD/DR300-400 (Fig. 1), a fusion
protein devoid of dioxin binding activity (Fig. 2B). A
second chimera,
DBD/DR280-421 (Fig. 1), consisting of
a 50 amino acid truncation from the N-terminal end of the core
230-421 dioxin receptor domain, produced a fusion protein with
greatly reduced dioxin binding capability (Fig. 2B), as
did a C terminally truncated chimera
DBD/DR230-400 (data not
shown). These results indicate that the region of the dioxin receptor
between amino acids 230 and 421 provides an accurate demarcation of the
core ligand binding domain and that this domain, exhibiting a ligand
binding capability similar to that of
DBD/DR83-805 (which
contains 90% of dioxin receptor residues), very likely maintains most
if not all of the ligand binding activity shown by the native dioxin
receptor. The accuracy of these chimeras in estimating ligand binding
capabilities of native dioxin receptor residues is illustrated by an in vitro translation of the isolated ligand binding domain,
DR230-421 (Fig. 1), showing similar
[
H]dioxin binding capacity to the chimera
DBD/DR230-421 (Fig. 2B).
Figure 3:
Association of the dioxin receptor LBD
with hsp90 is necessary for ligand binding activity. A, aliquots of the full-length dioxin receptor (lane 1) and
the minimal ligand binding domain (lane 2) translated in
reticulocyte lysates in the presence of
[S]methionine were analyzed by 10% SDS-PAGE and
fluorography. B, aliquots (10 µl) of the
[
S]methionine-labeled dioxin receptor (lanes
1 and 2) or LBD (lanes 3 and 4)
translation mixtures were incubated with specific (lanes S)
anti-hsp90 antibody 3G3 (5 µl) or control (lanes C) IgM
which had been preadsorbed to a resin of goat anti-mouse IgM Sepharose.
After extensive washing, immunoprecipitated proteins were analyzed by
10% SDS-PAGE and fluorography. The positions of the molecular mass
standard proteins are indicated. C, aliquots (10 µl) of
either unprogrammed(-) or LBD containing reticulocyte or wheat
germ lysates were subjected to hydroxylapatite ligand binding assays
with 2 nM [
H]TCDD in the presence or
absence of a 100-fold M excess of TCDF, as
indicated.
Figure 4:
Saturation binding analysis of HisDR and
HisHaLBD. A, HisHaLBD (lane 1) and HisDR (lane
2) were translated in reticulocyte lysates in the presence of
[S]methionine and aliquots (2 µl) were
analyzed by 10% SDS-PAGE and fluorography. B, aliquots (10
µl) of the [
S]methionine-labeled dioxin
receptor (lanes 1 and 2) or HisHaLBD (lanes 3 and 4) translation mixtures were incubated with specific (lanes S) anti-hsp90 antibody 3G3 (5 µl) or control (lanes C) IgM which had been preadsorbed to a resin of goat
anti-mouse IgM-Sepharose. After extensive washing, immunoprecipitated
proteins were analyzed by 10% SDS-PAGE and fluorography. C,
hyroxyapatite ligand binding experiments were performed with the
indicated in vitro translation mixtures using 2 nM
[
H]TCDD in the presence (+) or absence
(-) of 250-fold M excesses of TCDF. Panels D and E show specific saturation curves for HisDR and
HisHaLBD, respectively, using in vitro translated proteins and
increasing concentrations (0.2 to 2.5 nM) of
[
H]TCDD. Nonspecific binding was determined by
using a 250 M excess of TCDF. Scatchard plots and K
(dissociation constants) or r (linear regression coefficient) values are shown for each
protein.
After establishing that the exogenous amino acid tags were
not detrimental to ligand binding, we performed a quantitative and
comparative analysis of affinities the in vitro expressed
minimal ligand binding domain and the full-length dioxin receptor
exhibited for dioxin. Hydroxylapatite ligand binding assays were
therefore carried out with in vitro translated HisDR and
HisHaLBD proteins, using increasing concentrations of
[H]dioxin, providing reproducible saturation
binding curves representatively shown in Fig. 4, D and E. Scatchard analysis of these binding curves revealed
dissociation constant (K
) values of 0.41 nM for the full-length dioxin receptor and 0.47 nM for the
ligand binding domain, respectively. These values are in excellent
agreement with K
values previously calculated for
the native dioxin receptor in crude cell extracts (Ema et al.,
1994), verifying fidelity of the in vitro ligand binding assay
and establishing that the core domain between amino acids 230 and 421
of the dioxin receptor mediates full ligand binding activity including
wild type affinity for dioxin.
Figure 5:
Qualitative ligand binding specificity of
the dioxin receptor is maintained by the minimal LBD. Reticulocyte
lysates containing in vitro translated LBD were subjected to
hydroxylapatite ligand binding experiments using 1 nM [H] TCDD and 100-fold M excesses of
competitors TCDF, 5,6-benzoflavone (BNF),
indolo[3,2-b]carbazole (ICZ), and
dexamethasone (DEX) as indicated.
Figure 6:
Bacterially expressed and purified
HisHaLBD is unable to bind dioxin. A, bacterial expression of
HisHaLBD from the pET19 vector is induced by IPTG. Protein extracts
from bacteria transformed with HisHaLBD/pET19b (lanes 1 and 3) or empty expression vector (lanes 2 and 4), grown in the absence(-) or presence (+) of
IPTG, were subjected to SDS-PAGE and Western blotting. Immunodetection
of HisHaLBD was performed with anti-Ha antibody 12CA5. Positions of the
molecular mass standard proteins are indicated. B,
purification of HisHaLBD was performed by elution from a Ni-Sepharose
column with increasing concentrations of imidazole. Input,
flow through (F.T.) and elution aliquots were subjected to
analysis by silver staining of an SDS-PAGE gel. Purified HisHaLBD
eluted at imidazole concentrations of 100 mM (lane 7)
and 150 mM (lane 8) is indicated by the arrow. C, purified bacterial HisHaLBD (1 µg) was
subjected to hydroxylapatite ligand binding experiments with
[H]TCDD either before or after attempted
renaturation with hsp90 or crude reticulocyte lysate.
[
H]TCDD binding of in vitro translated
HisHaLBD in the absence or presence of 250 nM of the specific
competitor TCDF was included as a positive ligand binding
control.
Ligand binding assays on the bacterially
expressed, purified LBD showed a lack of any specific
[H]dioxin binding, with levels of total
[
H]dioxin binding activity in the hydroxylapatite
assay being identical in the absence or presence of specific competitor
TCDF (Fig. 6C). Since we have demonstrated HisHaLBD to
bind hsp90 upon in vitro expression in reticulocyte lysate (Fig. 4B), and this interaction is possibly essential
for chaperoning a ligand binding conformation of the LBD (Fig. 3, B and C), the lack of ligand binding
shown by the purified bacterially expressed LBD is likely to result
from malfolding of the LBD in the absence of hsp90. Consistent with
this notion, expression of the glucocorticoid receptor in bacteria
produces a receptor which does not interact with the bacterial hsp90
homolog, and shows significantly attenuated affinity for ligand
(Ohara-Nemoto et al., 1990). In attempts to renature the
purified LBD in the presence of hsp90, we preincubated the purified LBD
with purified hsp90, as described by Wiech et al.(1992),
before performing the [
H]dioxin binding assay.
Total ligand binding activity following this procedure was not
increased above that observed for the purified, untreated LBD (Fig. 6C). One possibility for this apparent lack of
renaturation of the LBD is that other heat shock proteins or molecular
chaperones may play critical roles in the postulated renaturation
process. Consistent with this idea, reconstitution of the semipurified
glucocorticoid receptor into a ligand binding oligomeric complex has
been successful by incubation in reticulocyte lysates, with roles for
several heat shock proteins being proposed as essential (for a review,
see Pratt, 1993). Moreover, this reticulocyte lysate procedure has been
successful in renaturing high affinity ligand binding conformations of
bacterially expressed mineralocorticoid receptor (Caamano et
al., 1993). However, incubation of the purified LBD with
reticulocyte lysate according to the steroid receptor protocols
(Scherrer et al., 1990) failed to provide a receptor species
exhibiting either ligand binding (Fig. 6C) or hsp90
binding (data not shown) activities, in direct contrast to the ligand
and hsp90 binding seen when the LBD is translated in reticulocyte
lysate (Fig. 4, B and C). These results are
consistent with a need for the LBD to be chaperoned into a native
conformation by hsp90 and perhaps other heat shock proteins, and
indicate that once the LBD has become extensively denatured or
malfolded, renaturation, if possible at all, may require several
factors and/or reagents in a multi-faceted process.
We have found the ligand binding domain of the mouse dioxin receptor to reside between amino acids 230 and 421, a segment of 192 amino acids which includes within its borders a region which was cross-linked with a radiolabeled photoaffinity dioxin ligand (amino acids 230 to 337; Burbach et al.(1992)) and a polymorphic residue (amino acid 375) which influences mouse strains to harbor low (DBA/2J) or high (C57/BL) affinity receptor phenotypes (Ema et al., 1994). Attempts to define a shorter ligand binding domain, by deletion to the boundaries of amino acids 300 to 400, or to between amino acids 280 and 421, failed to provide fragments with significant dioxin binding activity. Our delineation between amino acids 230 and 421 therefore represents a close approximation to the concise limits which would define the minimal ligand binding domain. Definition of a minimal ligand binding domain has also been attempted for the structurally unrelated but functionally similar glucocorticoid receptor. A 16-kDa tryptic fragment, consisting of amino acids 537 to 673 of the rat glucocorticoid receptor, was found to bind a profile of glucocorticoid receptor ligands with a similar qualitative specificity as the native receptor (Chakroborti and Simons, 1991). The affinity of this 16-kDa fragment for the prototypic dexamethasone agonist was, however, approximately 20-fold lower than that of the native receptor (Simons et al., 1989). In contrast, the ligand binding domain of the dioxin receptor not only maintains the qualitative ligand binding specificity shown by the native receptor, but also binds dioxin with an affinity similar to the full-length receptor. The 192-amino acid ligand binding domain of the dioxin receptor is therefore a more precisely defined entity than the broad 300 amino acid C terminus of the glucocorticoid receptor that has been found necessary to provide full ligand binding activity (Ohara-Nemoto et al.(1990), and references therein).
In analogy to the dioxin receptor, the glucocorticoid receptor is a ligand activated transcription factor which exists in a latent cytoplasmic complex with hsp90. Expression of the glucocorticoid receptor in yeast strains containing low hsp90 levels (Picard et al., 1990) or mutants of hsp90 (Bohen et al., 1993) produces a compromise in sensitivity of the receptor for ligand activation, while expression in bacteria, an organism where an hsp90 homolog (C62.5, Bardwell and Craig, 1987) shows only 42% identity to mammalian hsp90 and does not interact with the receptor, provides a form of the glucocorticoid receptor with a severely lowered affinity for ligand (Ohara-Nemoto et al., 1990). It therefore seems that interaction of hsp90 with the dioxin and glucocorticoid receptors may perform similar functions in the chaperoning of ligand binding domains. In support of this idea, the 16-kDa core ligand binding domain of the glucocorticoid receptor was also found to interact with hsp90 (Chakroborti and Simons, 1991). Attempts to further delineate the ligand and hsp90 binding domains within the glucocorticoid receptor have revealed a minimal hsp90 binding region to reside within the core steroid binding sequences (Howard et al., 1990; Dalman et al., 1991). Despite this striking mechanistic similarity between the bHLH/PAS dioxin receptor and zinc finger glucocorticoid receptor, no structural homology is evident when aligning their ligand/hsp90 binding domains. The need for hsp90 interaction to impart high affinity ligand binding activity, while observed for both the glucocorticoid (Ohara-Nemoto et al., 1990) and mineralocorticoid receptors (Caamano et al., 1993), does not seem to be general for the zinc finger superfamily of nuclear receptors. Bacterial expression of the large C-terminal progesterone (Eul et al., 1989) or retinoid X receptor (Cheng et al., 1994) ligand binding domains produce species which display wild type progesterone and 9-cis-retinoic acid binding activities, respectively, indicating no evident requirement for hsp90 to chaperone ligand binding conformations for these receptors. Recent studies of steroid hormone receptor activation in yeast model systems have found a homolog of the DnaJ chaperone, Ydj1, to be an essential component of hsp90-mediated signal transduction mechanisms (Kimura et al., 1995; Caplan et al., 1995). As these studies imply Ydj1 also exerts its affects via the ligand binding domains of steroid hormone receptors, it will now be important to investigate the potential influence of the Ydj1 chaperone on signaling through the dioxin receptor LBD.
While the ligand binding domains of the dioxin and glucocorticoid receptors are mechanistically similar in requiring interaction with hsp90 to enable ligand binding, these distinct sequences do not seem to be equally amenable to renaturation from a denatured state. When the glucocorticoid receptor was stripped of hsp90 and associated proteins, reassociation with hsp90 was achieved by incubation with rabbit reticulocyte lysate, a medium rich in heat shock protein complexes (Scherrer et al., 1990), or by incubation with a semipurified heat shock protein complex isolated from reticulocyte lysate (Hutchison et al., 1994). Reassociation with the hsp90-heat shock protein complex was accompanied by reconstitution of ligand binding, a phenomenon also observed with the bacterially expressed mineralocorticoid receptor, which was found to bind aldosterone with wild type affinity only after incubation with reticulocyte lysate (Caamano et al., 1993). Using these protocols, we have failed to renature the dioxin binding state of the bacterially expressed and purified dioxin receptor LBD. When expressed in bacteria, the dioxin receptor LBD is recovered from extracts in a particulate fraction, requiring high urea concentrations for solubilization. This process may be severely denaturing, resulting in a totally malformed protein fragment which resists renaturation by the mild reticulocyte lysate conditions used when renaturing glucocorticoid and mineralocorticoid receptors, which are recovered from the soluble fractions of bacterial extracts. We are currently developing expression systems which will enable the dioxin receptor ligand binding domain to be recovered in the soluble fraction of bacterial extracts, and may therefore prove more amenable to reconstitution with hsp90 and show subsequent recovery of ligand binding activity.
Somewhat paradoxically, the interaction of hsp90 with the ligand binding domain allows the dioxin receptor to be activated, as translation in systems where the receptor does not interact with hsp90, either in vitro (wheat germ lysate) or in vivo (engineered S. cerevisiae), produces inert, dioxin nonresponsive proteins. The LBD-hsp90 interaction may therefore be inextricably entwined with both positive and negative regulatory functions of the dioxin receptor. Interestingly, there exists a very heterogeneous population of ligands for the dioxin receptor, seemingly all binding to the same central LBD core of the receptor. The nature of these ligand-receptor interactions is poorly understood, with planarity and size exclusion limits being the only common denominators employed in deriving a conceptual model to explain binding for the different ligand classes (Gillner et al.(1993), and references therein). Identification of the minimal LBD will facilitate exploration of the ligand binding pocket by detailed point mutagenesis, allowing a determination of critical residues, or perhaps subsets of residues, which may confer differential specific binding to the dioxin, indole, flavone, and polycyclic aromatic hydrocarbon ligand classes.