Agonist and Antagonists Induce Homodimerization and Mixed Ligand Heterodimerization of Human Progesterone Receptors in Vivo by a Mammalian Two-Hybrid Assay
Susan A. Leonhardt,
Magda Altmann and
Dean P. Edwards
Department of Pathology and Program in Molecular
Biology University of Colorado Health Sciences Center Denver,
Colorado 80262
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ABSTRACT
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This study utilizes the mammalian two-hybrid
system to examine the role of ligand in the dimerization of human
progesterone receptor (hPR). The GAL4 DNA-binding domain and the herpes
simplex virus VP16 transactivation domain were fused to the amino
terminus of full-length hPR (both the A and B isoforms) to produce
chimeric proteins. PR dimerization was detected by the ability of
cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce
expression of a reporter gene under the control of GAL4-binding sites
(pG5CAT). Hormone agonist-dependent interactions were observed between
the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B
(A-B), indicating that hormone can stimulate the formation of the three
possible dimeric forms of PR within cells. In contrast, neither type I
(ZK98299) nor type II (RU486, ZK112993) progestin antagonists
stimulated interaction between these same hybrid PR proteins. However,
activation of the VP16/PR chimera by antagonists on a progesterone
response element-controlled reporter gene (DHRE-E1b-CAT) was only a
fraction (413%) of that stimulated by agonist R5020. One
possibility for the failure to detect an induction in the two-hybrid
assay is antagonist-induced repression of the activity of the VP16/PR
fusion protein rather than a failure of antagonists to stimulate
interaction between the hybrid proteins. To test this idea, an UP-1
carboxyl-terminal truncation mutant of PR was used to construct the
two-hybrid proteins. PR-UP-1 selectively binds antagonists, but not
agonists, and is fully activated in response to antagonists. Both types
of progestin antagonists stimulated interactions between GAL4/PR(UP-1)
and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are
capable of stimulating PR dimerization in cells and do not function by
disrupting or preventing dimerization. To determine whether PR bound to
an antagonist can dimerize in whole cells with PR bound to agonist,
GAL4/PR(UP-1) was paired in the two- hybrid assay with a VP16/PR
fusion protein harboring a point mutation in PR at amino acid 722
(Gly-Cys) that specifically binds progestin agonist but not antagonist.
Neither R5020 nor RU486 alone stimulated interaction between these
ligand-specific PR hybrid proteins. However, strong interaction was
detected by addition of both agonist and antagonists, indicating the
formation of mixed ligand heterodimers and that both PR partners
require ligand for dimerization to occur. Based on electrophoretic gel
mobility shift assays (EMSAs), these heterodimers appear to have
substantially reduced DNA binding activity. Progestin antagonists
inhibit agonist activation of PR at concentrations that are too low to
be accounted for by a simple competition mechanism for binding to PR.
We propose that antiprogestin inactivation of PR in trans
by heterodimerization contributes to the biological potency of these
compounds.
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INTRODUCTION
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Human progesterone receptor (hPR) is a ligand-activated
transcription factor that mediates the actions of the sex steroid
progesterone in target cells. A member of the steroid receptor
superfamily, hPR is expressed as two distinct isoforms of 94 kDa, PR-A,
and 120 kDa, PR-B (1). The two isoforms have identical sequence in
their carboxyl-terminal ligand-binding domain (LBD) and the centrally
located DNA-binding domain (DBD). PR-A differs from PR-B only by
lacking 164 amino acids (aa) of the extreme N terminus (1). In the
absence of hormone, PR is found in oligomeric complexes with several
other proteins, including heat shock protein 90 (hsp90), hsp70, p59,
and immunophilins (2). Ligand binding dissociates receptors from this
inactive complex to allow receptor dimerization and binding to
hormone-response elements (HREs) that are typically located in the
5'-regulatory region of steroid-responsive genes (3). After binding
DNA, PR activates or represses transcription of target genes by yet
incompletely understood mechanisms. This process appears to involve
direct interaction with the general transcription machinery (4) and or
association with coactivators/corepressors that link the receptors with
the general transcriptional machinery (5, 6, 7).
For the sex steroid receptors and glucocorticoid receptor (GR),
dimerization appears to be a critical factor controlling receptor
binding to palindromic HREs (8, 9). Steroid receptors have been shown
to be able to form dimers in solution, and as full-length proteins they
bind preferentially to HREs as preformed dimers. In general, steroid
receptors interact with target palindromic HREs as a single type of
homodimer. Exceptions to this are hPR, which binds to target DNA as
three dimeric species composed of AA, AB, and BB subunits (10, 11, 12, 13) and
the more recent demonstration that GR can form a heterodimer complex on
DNA with mineralocorticoid receptor (MR) (14, 15). In addition, the
newly discovered estrogen receptor-ß (ERß) can heterodimerize with
ER
(16). In contrast, receptors for nonsteroid hormones, such as
vitamin D, thyroid hormone, and retinoids, preferentially bind DNA as
heterodimers with retinoic acid X receptor (17).
Two dimerization domains have been described within steroid receptors.
A DNA-dependent dimerization interface located within the DBD is
important for restricting steroid receptor recognition of palindromic
HREs separated by three intervening nucleotides and for stabilization
of receptor dimers on DNA (18). A second dimerization function
resides in a region(s) outside the DBD that is involved in solution
dimerization in the absence of DNA (18, 19, 20). The LBD of ER contains a
strong hormone-dependent dimerization function that is required for
solution dimerization of ER (8, 21, 22). Other studies with GR,
androgen receptor, and PR have suggested that the LBD alone is
not sufficient for stable solution dimerization and that amino-terminal
sequences are also involved (23, 24, 25, 26, 27). Recently, we have analyzed the
sequence regions involved in solution dimerization and have found that
both the hinge and amino-terminal sequences contribute to PR
dimerization (28).
Several synthetic ligands have been developed that compete effectively
for binding of progestins to PR and are capable of inhibiting receptor
activation. The mechanism by which antiprogestins inhibit activation
remains incompletely understood. Original studies based on in
vitro electrophoretic gel mobility shift assays (EMSA) classified
ZK98299 (Onapristone) as a type I compound that failed to promote PR
binding to DNA. Other antiprogestins such as RU486 (Mifepristone) were
classified as type II compounds that efficiently enhance PR binding to
DNA (13) and thus impair receptor transactivation at step(s) downstream
of DNA binding. However, assays based on detection of PR-DNA binding in
whole cells (29, 30) and our more recent results with altered EMSA
conditions (30) indicate that ZK98299 does enhance PR binding to DNA.
It has been well documented that antiprogestins induce conformational
changes within the LBD of PR that are distinct from that induced by
agonists (11, 12, 31, 32, 33, 34). Similar results have been shown with ER and
estrogen antagonists (35). One consequence of the different
conformation is to prevent the interaction of specific steroid receptor
coactivators with the hormone agonist-dependent transcription
activation function 2 (AF-2) (5, 6, 7). ZK98299 and RU486 have been shown
to have different effects on PR conformation, suggesting that they may
represent different classes of progestin antagonists based on inducing
distinct conformational changes within PR (30, 36, 37).
The biological activity of antiprogestins as assessed by transfection
experiments indicates that these compounds inhibit transcriptional
activation of PR at substoichiometric concentrations with hormone. This
suggests that antiprogestins do not act by simple competition with
hormone for binding to PR (38). This unusually high potency of
antiprogestins could, in part, be explained by the PR-antiprogestin
complex functioning to repress the PR-agonist complex in
trans. Since PR requires dimerization for function, this
could be achieved by heterodimerization between a PR bound to agonist
with a PR bound to antagonist.
Numerous studies have recently taken advantage of the two-hybrid
protein-protein interaction assay to examine dimerization of eukaryotic
transcription factors (39). Originally developed in yeast (40) and
later adapted to mammalian cells (41), the assay involves coexpression
of two proteins of interest fused to an autonomous DBD (from the GAL4
or LEXA transcription factors) and a strong transcriptional activation
domain (from GAL4 or VP16). Stable protein-protein interactions can
functionally reconstitute the separate DBD and activation domains of
the transcription factor, resulting in activation of a reporter gene
bearing DNA-binding sites for the DBD fusion protein. Previously, a
yeast two-hybrid assay was used to show ligand-inducible dimerization
of ER in vivo (42) and, more recently, a mammalian based
two-hybrid assay was used to demonstrate estrogen-dependent
heterodimerization between ERß and ER
in whole cells (16). In the
present study, we used a mammalian two-hybrid assay to investigate
whether the three detected PR dimer forms complexed to DNA
in vitro can be formed in whole cells and whether or not
this is ligand dependent. In addition, we have used this system to
examine the effect of progesterone antagonists on receptor dimerization
and whether PR bound to agonist can heterodimerize in the cell with PR
bound to antagonist.
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RESULTS
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Expression of GAL4/PR and VP16/PR Fusion Proteins
To examine PR dimerization in whole cells, we have used a
mammalian based two-hybrid protein-protein interaction assay. Either
the GAL4 DBD or the VP16 heterologous transcriptional activation domain
were fused to the amino terminus of full-length hPR (both the A and B
isoforms) (see Fig. 1A
). The chimeric
constructs were transiently transfected into PR- negative COS-1 cells
along with a reporter plasmid, pG5CAT, that contains five GAL4
consensus binding sites and the minimal E1b promoter driving the
chloramphenicol acetyl transferase (CAT) gene. Interaction between two
PR molecules was assessed by the ability of cells transfected with
GAL4/PR and VP16/PR constructs to activate the pG5CAT reporter gene as
compared with cells transfected with either PR chimera alone.

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Figure 1. Schematic and Expression of GAL4 DBD and VP16 PR
Chimeras
A, PR chimeric constructs used in the mammalian two-hybrid assay. PR-B
(aa 1933) and PR-A (aa 165933) were fused at their amino terminus
to the GAL4 DBD (aa 1147) and the VP16 activation domain (aa
411455). DNA binding (cross-hatched boxes) and ligand
binding (dark solid boxes) domains of the receptors are
also shown. B, The expression level of the GAL4/PR and VP16/PR fusion
proteins was analyzed by transient transfection into COS-1 cells,
followed by immunoblot analysis with the PR-specific MAb, 1294/H9. For
comparison, equal amounts of proteins from T47D breast cancer cells and
COS-1 cells that had been transfected with the plasmid pSVhPR-B, to
express wild-type PR-B, were also analyzed. The sizes of the molecular
mass markers in kilodaltons are indicated by line
markings, and the sizes of native PR-A and PR-B in T47D cells
are indicated by arrows.
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We first analyzed transfected COS-1 cells for expression levels and the
ability of the receptor fusion proteins to bind hormone. As shown by
immunoblot assay of COS-1 cell lysates with a PR-specific monoclonal
antibody (MAb), the chimeric PR constructs produced immunoreactive
proteins of larger apparent mol wt than native PR-A and PR-B from T47D
breast cancer cells and full-length PR-B (transfected with pSVhPR-B)
expressed in COS-1 cells as a nonfusion protein (Fig. 1B
). In addition,
each of the PR fusion proteins was expressed at approximately the same
level as native PR in T47D cells (Fig. 1B
). It should be noted that
even though the VP16 domain is of smaller mol wt than the GAL4 DBD, the
VP16 fusion proteins exhibit slower mobility than the GAL4 fusion
proteins. A similar phenomenon was reported for androgen receptor
two-hybrid proteins suggesting that the highly acidic nature of VP16
causes anomalous electrophoretic mobility (43). The immunoreactive
doublets observed in the PR-A and PR-B GAL4 lysates may be a result of
proteolytic cleavage of the fusion proteins; however, these lower mol
wt species do not seem to interfere with our ability to detect
interactions with the VP16 fusion proteins (see below). The expressed
PR fusion proteins were also characterized for steroid binding activity
by a whole cell-binding assay. The results summarized in Table 1
show that all PR fusion proteins
exhibit similar specific binding to the synthetic progestin
[3H]R5020 with equal capacity, again approaching the
level of native PR in T47D cells. Thus, fusion of PR to either the GAL4
DBD or VP16 did not adversely affect the expression or the steroid
binding activity of PR.
Hormone-Dependent Dimerization of the A and B Isoforms of hPR
The A and B forms of PR have been shown to bind in
vitro, in a hormone-dependent manner, to target progesterone
response elements (PREs) in three dimeric forms: AA, AB, and BB dimers
(11, 12, 44, 45). PR dimerization in solution has also been
demonstrated in vitro by coimmunoprecipitation (45, 46, 47),
nondenaturing gel electrophoresis (48), and pull-down assay with his
tagged PR (28). However, results are variable as to whether solution
dimerization in vitro is hormone dependent, and these
in vitro assays have not been able to distinguish between
the different possible isoform compositions of the dimers. Therefore,
we have used a mammalian two-hybrid assay to ask whether the three
possible dimers of PR (AA, AB, and BB) are able to form in whole cells
and to what extent these dimerization interactions are hormone
dependent.
When COS-1 cells were cotransfected with either, or both, parental
vectors that express GAL4 DBD or VP16 without PR sequences, activation
of the GAL4- controlled pG5CAT reporter gene was negligible (Fig. 2
). Also, little or no activation of
pG5CAT was observed when cells were cotransfected with a single
VP16/PR-B chimeric vector cotransfected with a GAL4 DBD empty vector
control (Fig. 2
). The low CAT activity observed under these conditions
was considered as the basal activity of the pG5CAT promoter in COS-1
cells. However, the GAL4/PR-B chimeric construct as a single hybrid
(cotransfected with VP16 empty control vector) did activate pG5CAT 2-
to 4-fold above the basal level in response to R5020 (Fig. 2
).
Activation of the pG5CAT reporter by the single GAL4/PR-B chimeric
receptor is not entirely unexpected. Full-length PR-B harbors multiple
transactivation domains (49, 50) and has the potential, through direct
utilization of these domains, to mediate transcription when
tethered to the pG5CAT through its GAL4 DBD fusion sequences. In
contrast, PR-A, which is generally a weaker transcriptional activator
than PR-B (51, 52), mediates no induction of the pG5CAT reporter as a
single hybrid in response to hormone (not shown and Fig. 3
). The data in Fig. 2
illustrate the
importance of factoring CAT expression mediated by the single GAL4/PR
hybrid to reliably detect induction of PR dimerization as reflected by
interaction of the two PR fusion proteins. In later two-hybrid
experiments, the GAL4/PR-B chimeric construct as a single hybrid
mediated a higher activation of the pG5CAT reporter in response to
R5020 (see below) than shown in Fig. 2
, further emphasizing the need to
factor in the contribution of this single hybrid when calculating CAT
expression that represents PR dimerization.

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Figure 2. Background Activity of PR-B Single Hybrids in
Mammalian Cells
COS-1 cells were transiently transfected with the indicated constructs
and the pG5CAT reporter. Twenty four hours posttransfection, cells were
treated for another 24 h with either vehicle (ethanol) or 100
nM R5020. CAT activity was measured as described in
Materials and Methods and was calculated as counts per
min per µg of protein from duplicate treatment wells. The results are
from a single representative experiment of three independent
determinations.
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Figure 3. Hormone-Dependent Dimerization of Different PR
Isoforms In Vivo
COS-1 cells were cotransfected with GAL4/PR chimeric vectors and VP16
(empty vector) construct or with GAL4/PR chimeric vectors and VP16/PR
chimeric vectors and the pG5CAT reporter plasmid. Twenty four hours
posttransfection, cells were treated with either vehicle or 100
nM R5020 for 24 h. CAT activity was determined and
calculated as counts per min per µg of protein from duplicate
treatment wells. Within each group of PR hybrid interactions (A-A, A-B,
or B-B), relative CAT activity was determined as described in
Materials and Methods. Values are averages from multiple
independent experiments (± SEM; n = 7). The fold
hormone stimulation of dimerization was calculated as the difference in
relative CAT activity mediated by cells expressing two hybrid proteins
plus ligand and the relative CAT activity mediated by the GAL4 single
hybrid plus ligand (see Materials and Methods). The fold
stimulations were: 3.44 for PR-A-PR-A, 7.16 for PR-A-PR-B, and 3.20 for
PR-B-PR-B interactions. Statistical analysis using a t
test revealed a significant difference between the relative CAT
activity mediated by two hybrid proteins plus R5020 and the activity
mediated by one hybrid protein plus R5020 for all three types of PR-PR
interactions (P < 0.05). No significant difference
was measured between two hybrid and one hybrid mediated CAT activity in
the absence of ligand. The schematic underneath the
graph illustrates CAT activity mediated by the one hybrid
protein in response to R5020 and CAT activity mediated by two hybrid
proteins in response to R5020 treatment.
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We next compared the ability of the different isoforms of hPR to
interact with each other in the two-hybrid assay in the presence and
absence of R5020. Reporter gene induction in the two-hybrid interaction
experiments was determined as relative CAT activity by arbitrarily
setting the CAT enzyme activity obtained with the single GAL4/PR hybrid
in the absence of ligand (ethanol) to one (see Materials and
Methods). GAL4/PR single hybrids were cotransfected with empty
VP16 vector controls (see Fig. 3
). As shown in Fig. 3
, hormone (R5020)
treatment of cells transfected with the GAL4/PR-A single hybrid did not
stimulate CAT activity above the ethanol control, whereas R5020
stimulated a significant (6-fold) increase of CAT expression in cells
transfected with the GAL4/PR-B single hybrid. When cells were
cotransfected with both GAL4/PR and VP16/PR fusion proteins, no
significant stimulation of CAT activity above the single hybrid
controls was obtained in the absence of ligand. Hormone (R5020)
addition to cells cotransfected with the different two hybrid
combinations in each case stimulated a significant increase of CAT
activity as compared with ethanol controls and, more importantly,
hormone also stimulated a significant increase of CAT activity over the
single hybrid controls treated with R5020 (Fig. 3
). The two-hybrid
combinations included GAL4/PR-A plus VP16/PR-A (A-A interaction),
GAL4/PR-A plus VP16/PR-B (A-B interaction), and GAL4/PR-B plus
VP16/PR-B (B-B interaction). To determine the fold hormone stimulation
due to two-hybrid protein interaction (i.e. dimerization),
CAT activity contributed by the single GAL4/PR hybrid in the presence
of R5020 was factored out by calculating the relative CAT activity
obtained from the two-hybrid plus hormone divided by the relative CAT
activity obtained by the single hybrid plus hormone (see
Materials and Methods). By this analysis, hormone stimulated
a 3.44-fold increase in CAT activity mediated by PR-A/PR-A interaction,
a 7.16-fold increase mediated by PR-A/PR-B interaction, and 3.2-fold
increase mediated by PR-B-/PR-B interactions. Hormone-dependent
interaction between the A and B form of PR was also observed when PR-B
was fused to GAL4 DBD and PR-A was fused to VP16. However, in this
orientation the fold hormone (3-fold) stimulation was less than that
obtained with the fusion proteins in the opposite orientation in Fig. 3
(data not shown). This result, taken together with the fact that PR-B
has a higher intrinsic activity than PR-A, makes it difficult to
interpret whether the higher 7-fold stimulation mediated by GAL4/PR-A
and VP16/PR-B in Fig. 3
actually reflects a stronger dimerization
between PR-A and PR-B as compared with A-A and B-B interactions.
Nonetheless, the results in Fig. 3
strongly support the conclusion that
formation of the three possible forms of PR dimers (AA, AB, and BB)
occurs in the cell in a hormone-dependent manner.
Antagonist-Stimulated PR Dimerization in Whole Cells
Using the same PR chimeric constructs, we next asked how different
progesterone antagonists would effect PR-PR interactions in the intact
cell. The antagonists included RU486 and closely related ZK112993 (type
II), which behave as strong antagonists but display partial agonist
activity under certain conditions, and the more complete antagonist
ZK98299 (type I). None of these compounds stimulated pG5CAT activity
above basal levels in cells transfected with both GAL4/PR-B and
VP16/PR-B chimeras (data not shown). This was surprising since these
same antagonists have been shown to promote efficient binding of PR to
target DNA in whole cells (29, 30). We therefore considered the
possibility that antagonists may reduce the transcriptional activity of
the VP16 fusion proteins in such a manner that interaction between
VP16/PR and GAL4/PR chimeras is undetected, due to the failure of the
VP16 fusion protein recruited to the promoter to mediate a strong
transcriptional response. To test this idea, we analyzed the ability of
the VP16/PR-B chimera to function as a positive transactivator of a
PRE-controlled reporter gene (DHRE-E1b-CAT) in response to agonists and
antagonists. As shown in Fig. 4
, R5020
stimulated a substantial 125-fold induction of DHRE-E1b-CAT reporter
activity in cells transfected with VP16/PR-B. VP16/PR-B transactivation
of the DHRE-E1b-CAT gene was also stimulated by all antagonists
examined, including ZK98299. However, the fold induction was only
413% (range is from three independent experiments) of that mediated
by hormone agonist (Fig. 4
). The reduced ability of antagonists to
stimulate CAT activity does not appear to be due to promoting a less
efficient binding of the VP16/PR fusion protein to DNA than hormone
agonist. We have performed a control experiment by cotransfecting
VP16/PR-B with a carboxyl-terminal truncation UP-1 mutant of PR that
binds and responds to RU486 as an agonist (Ref. 33 and see below).
Cotransfected VP16/PR-B inhibited the transcriptional activity of
VP16/PR-B(UP-1) in a dose-dependent manner in cells treated with RU486
(not shown). This inhibitory effect suggests that the VP16/PR-B
chimeric receptor dimerizes and efficiently binds to DNA in the cell in
response to RU486. In further support of antagonists reducing the
transcriptional activity of the VP16/PR-B fusion protein as opposed to
reducing its binding efficiency for DNA, we recently showed by promoter
interference assays that all progestin antagonists used in this study
stimulate binding of PR to PREs in vivo (whole mammalian
cells) with the same efficiency as the agonist R5020 (30). How
antagonists reduce the transcriptional activity of VP16/PR-B fusion
protein is not known. A possible mechanism is suggested by recent
studies showing that PR and ER bound to antagonists strongly associates
with corepressors that can functionally silence partial agonist effects
of antagonists (53, 54, 55).

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Figure 4. Effects of PR Antagonists on Transactivation
Mediated by VP16/PR-B and VP16/PR-B(UP-1) Chimeric Receptors
COS-1 cells were transiently cotransfected with either VP16/PR-B (10
ng) or VP16/PR-B(UP-1) (10 ng), the progestin responsive DHRE-E1b-CAT
reporter (500 ng), and the CMV-ß-gal expression vector used as an
internal control for transfection efficiency. At 24 h
posttransfection, the cells were treated with vehicle (ETOH), R5020
(100 nM), RU486 (100 mM), ZK112993 (100
nM) or ZK98299 (500 nM), incubated for another
24 h, and then harvested. CAT activity was normalized to the
ß-gal activity, and the data are represented as fold hormone
induction over the response of cells containing VP16/PR-B treated with
vehicle only. The data are from a representative experiment of n =
3 independent experiments.
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Because of the substantially weakened transcriptional activity of the
VP16/PR chimera bound to antagonists, we turned to the use of a
carboxyl-terminal deletion UP-1 mutant of hPR for assessing the effects
of antagonists on PR dimerization in the two-hybrid assay. Deletion of
42 aa from the carboxyl terminus of human PR results in a receptor that
is unable to bind hormone agonists but is capable of binding and being
activated by antagonist to a level that approaches agonist activation
of wild-type PR (33). These properties of PR-UP-1 suggest that agonist
and antagonists contact different aa residues within the LBD of hPR and
that the carboxyl-terminal tail contains a repressive function that
antagonists are unable to overcome. For purposes of our study, the
functional behavior of PR-UP-1 also predicts, although not formally
demonstrated by in vitro assays, that antagonists stimulate
dimerization and DNA binding of PR-UP-1. Therefore, we reasoned that
use of PR-UP-1 mutants may be one way to detect the effects of
antagonists on PR dimerization in two-hybrid assays. The UP-1 mutant of
PR-B was inserted into the GAL4 DBD fusion vector (GAL4/PR-B(UP-1)) and
into the VP16 fusion vector (VP16/PR-B(UP-1)). We first compared
VP16/PR-B(UP-1) with VP16/PR-B as transactivators to determine whether
VP16/PR-B(UP-1) exhibits better activation potential than VP16/PR-B in
response to antagonists. As expected, VP16/PR-B(UP-1) is not capable of
being activated by hormone agonist (Fig. 4
). In contrast, when COS-1
cells were transfected with VP16/PR-B(UP-1), all antagonists tested
stimulated a substantial induction of DHRE-E1b-CAT. The fold induction
stimulated by RU486 and ZK112933 was equal to that of R5020 induction
mediated by the VP16/PR-B construct (Fig. 4
). VP16/PR-B(UP-1)
transactivation induced by ZK98299 was approximately 50% lower than
other antagonists but was still substantially greater than the level of
transactivation mediated by wild-type PR-B fused to VP16 in response to
ZK98299 (Fig. 4
).
We next analyzed the effect of antagonists on interaction between the
two PR-UP-1 chimeras. As expected R5020, which does not bind the UP-1
PR mutant, did not stimulate CAT activity above the ethanol controls in
cells transfected with either the single hybrid GAL4/PR-B(UP-1) control
or with both GAL4 and VP16 fusions of PR-B(UP-1) (Fig. 5
). In contrast, the antagonists RU486
and ZK98299 each stimulated a significant increase (3.2-fold and
2.8-fold, respectively) of CAT activity in cells cotransfected with
both fusion proteins over the single hybrid GAL4/PR-B(UP-1) control
treated with the same antagonist (Fig. 5
). Although ZK112993 also
stimulated CAT activity (2.6-fold) in cells transfected with both
hybrid PR-B(UP-1) proteins over the single-hybrid control (Fig. 5
), the
increase was statistically significant only at a P value of
0.08 suggesting a trend toward stimulating PR-PR interaction.

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Figure 5. Antagonists Induce PR Homodimerization in Whole
Cells
COS-1 cells were transiently cotransfected with GAL4/PR-B(UP-1) and
VP16 (empty vector control) or with GAL4/PR-B(UP-1) and
VP16/PR-B(UP-1), the pG5CAT reporter, and the CMV-ß-gal expression
internal control for correcting differences in transfection efficiency.
At 24 h posttransfection, the cells were treated with vehicle
(ETOH), R5020 (100 nM), RU486 (100 nM),
ZK112993 (100 nM), or ZK98299 (500 nM),
incubated for another 24 h, and then harvested. Relative CAT
activity was determined, and the data were represented as described in
Materials and Methods. The values for relative CAT
activity are averages (±SEM) from n = 6 independent
experiments. The fold ligand induction of relative CAT activity due to
dimerization was calculated as described in Materials and
Methods and was as follows: 3.2-fold for RU486, 2.62-fold for
ZK112993, and 2.82-fold for ZK98299. Statistical analysis using a
t test revealed that there were no significant
differences in CAT activity between the one hybrid control and two
hybrid proteins in the absence of ligand and with R5020
treatment. RU486 and ZK98299 produced a significant increase in CAT
activity mediated by two-hybrid interaction compared with the
one-hybrid control (P < 0.05), while the stimulation
observed with ZK112993 was significant only at a P value
of 0.08.
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Because ZK98299 has a 10-fold lower affinity for PR than RU486 (29),
whole cell binding experiments were performed with
[3H]ZK98299 to determine whether it can stably bind and
fully occupy PR-B(UP-1) in cells. Both PR-B(UP-1) fusion proteins
expressed in COS-1 cells bound to ZK98299 with the same capacity as
[3H]RU486 under conditions of the two-hybrid experiments
(ZK98299 added at 500 nM and other ligands at 100
nM). Whole-cell binding experiments also confirmed that
PR(UP-1) fusion proteins, as expected, failed to bind
[3H]R5020 but did bind to [3H]RU486 and
[3H]ZK98299 (data not shown). These results strongly
suggest that progestin antagonists are capable of stimulating PR
homodimerization within cells.
Heterodimerization between PR Bound to Agonist and PR Bound to
Antagonist in Whole Cells
An interesting and unexplained property of these progestin
antagonists is their ability to inhibit activation of PR at
concentrations well below that of the hormone, indicating that
antagonists do not act by simple competition with hormone for PR
binding sites (11, 38). Despite the fact that RU486, ZK112993, and
ZK98734 all have the same binding affinity for PR as the agonist R5020
(Ref. 29 and unpublished observations), these compounds inhibit
activation of PR at substoichiometric concentrations with R5020. This
is illustrated in Fig. 6
with ZK112993
and ZK98299 in a breast cancer cell line containing a stably integrated
mouse mammary tumor virus (MMTV)-CAT, a progestin-inducible reporter
gene (11). Cells were treated with a saturating amount of R5020 (5
nM) or were treated with 5 nM R5020 and
increasing amounts of ZK112993 (Fig. 6A
) or ZK98299 (Fig. 6B
). Reporter
gene activity stimulated by 5 nM R5020 was inhibited by
50% at a 10-fold lower concentration of ZK112993 (average
IC50 = 0.4 nM, n = 5), while maximal
inhibition was obtained at approximately 3 nM ZK112993
(Fig. 6A
). The IC50 for the antagonist ZK98299 was between
310 nM (average 7 nM, n = 5), and
maximal inhibition was obtained between 100 and 500 nM of
ZK98299 (Fig. 6B
). When the 10-fold lower affinity for PR as compared
with R5020 is corrected for, ZK98299 also antagonizes at
substoichiometric concentrations with R5020. Thus, both antagonists
have an observed IC50 that is approximately 10-fold lower
than the expected.

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Figure 6. Inactivation of PR by Antiprogestins Occurs at
Substoichiometric Concentrations with Progestin Agonists
T47D cells containing a stably integrated MMTV-CAT reporter gene (11 )
were treated for 24 h with 5 nM R5020 alone or with 5
nM R5020 and increasing amounts as indicated of ZK112993
(A) or ZK98299 (B). CAT enzyme activity was measured as described in
Materials and Methods. CAT activity obtained with R5020
treatment alone was set as 100%, and activity in the presence of
antagonists was calculated as relative to the 100% control. For each
antagonist, the data are from a single representative experiment of
multiple (n = 5) independent determinations.
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One possible mechanism contributing to the biological potency of these
progestin antagonists is repression of PR bound to agonist in
trans by heterodimerization with PR bound to antagonist. To
test whether such mixed ligand agonist/antagonist heterodimers can form
in whole cells, we have adapted the mammalian two-hybrid assay to
direct the binding of agonist and antagonist to one or the other
receptor chimera. To do this, we have taken advantage of the PR-UP-1
mutant that binds antagonists selectively and a previously described
mutation at aa position 722 (Gly
Cys) of hPR, which abolishes binding
of RU486 but retains normal progestin agonist binding affinity and
functional response to progestins (56). In the context of
full-length PR-B, we inserted the 722 (Gly
Cys) mutant into the GAL4
or VP16 fusion vectors and used these fusion proteins partnered with
PR-B(UP-1) fusion proteins to create a mammalian cell two-hybrid system
that expresses receptor hybrids with the desired agonist/antagonist
ligand specificities (see Fig. 7A
). To
confirm that these mutant PRs as fusion proteins possess the ligand
specificities expected, a steroid binding assay in whole cells was
performed with [3H]R5020 and [3H]RU486 in
COS-1 cells transfected with each receptor GAL4 and VP16 chimeric
construct. The VP16/PR-B(722m)-fusion protein bound
[3H]R5020 specifically and exhibited no specific binding
to [3H]RU486. Conversely, the GAL4/PR-B(UP-1) fusion
protein bound [3H]RU486 in whole cells but not
[3H]R5020. As expected, native PR in T47D cells as a
control bound R5020 and RU486 equally (Fig. 7A
). It should be noted
that whole-cell binding assays used a 50 nM concentration
of R5020 and RU486, which is approximately equal to the
concentrations used in two-hybrid assays (see Fig. 7B
). Even at
these high concentrations, the mutant PRs show complete agonist
(PR722m) and antagonist (PR(UP-1)) specificity. The whole-cell steroid
binding assays also show (Fig. 7A
) that PR(UP-1) and PR722m fusion
proteins are expressed at approximately equal levels in transfected
COS-1 cells, which was also confirmed by immunoblot analysis of the
cell lysates (not shown).

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Figure 7. Heterodimerization between PR Bound to Agonist and
PR Bound to Antagonists
A, Schematic of the mutant chimeric receptors and their ligand
specificities. The GAL4 DBD was fused to the amino terminus of PR-B
containing deletion of 42 aa from the carboxyl terminus to yield
GAL4/PR-B(UP-1). VP16 was fused to the amino terminus of PR-B with a
change of aa 722 from Gly to Cys to yield VP16/PR-B(722 m). Whole-cell
steroid binding assays were performed by incubating transfected COS-1
or T47D cells with 50 nM [3H]R5020 or 50
nM [3H]RU486 as described in Materials
and Methods. The specific binding was calculated as picomoles
of steroid binding/mg protein. Data from a representative experiment
are shown. B, Mammalian two-hybrid assay using PR ligand specificity
mutants. COS-1 cells were transiently transfected with single hybrid
GAL4/PR-B(UP-1) plus empty VP16 vector control (one hybrid) or
GAL4/PR-B(UP-1) and VP16/PR-B(722m) (two hybrid), along with the pG5CAT
reporter and the CMV-ß-gal plasmid for determining transfection
efficiency. At 24 h posttransfection, the cells were treated with
either vehicle (ETOH), 100 nM R5020, 100 nM
RU486, or cotreated with 100 nM R5020 and 50 nM
RU486. Cells were incubated for another 24 h and then harvested.
Relative CAT activity was calculated by normalizing the single hybrid
in the absence of ligand to 1, and the data are represented as in Figs. 3 and 5 and as described in Materials and Methods. The
values are averages (±SEM) from four independent
experiments. Statistical analysis using a t test
revealed that were no significant differences in CAT activity between
the one hybrid control and two hybrids for ethanol, R5020, and
RU486 treatment groups; a significant increase in CAT activity was
observed with the two hybrids cotreated with R5020 and RU486
compared with the single hybrid treated with both ligands. The increase
in relative CAT activity due to dimerization was determined to be
4.2-fold. The schematic underneath the graph illustrates
mixed ligand heterodimer formation between antagonist-bound PR-B and
agonist-bound PR-B.
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We next assessed the ability of the GAL4/PR-B(UP-1) fusion
protein to interact with VP16/PR-B(722m) in a two-hybrid setting in
response to treatment with agonist, antagonist, or both compounds. When
cells were transfected with GAL4/PR-B(UP-1) alone as a single hybrid,
RU486 stimulated a 6-fold increase of the pG5CAT reporter gene
expression over the no-ligand (ethanol) control. As expected, no
induction of CAT expression was mediated by GAL4/PR-B(UP-1) as a single
hybrid in response to R5020, and the stimulation obtained by
cotreatment with R5020 and RU486 was no different from RU486 alone
(Fig. 7B
). In COS-1 cells cotransfected with both GAL4/PR-B(UP-1) and
VP16/PR-B(722m) hybrid constructs, no further increase in pG5CAT
activity over the single hybrid control (GAL4/PR-B(UP-1) + VP16) was
observed by addition of RU486 alone. Addition of R5020 alone also did
not stimulate a significant increase in CAT activity over the single
hybrid also treated with R5020. A significant 4.2-fold stimulation of
CAT activity over the single hybrid control was observed only when
cells were cotreated with R5020 and RU486 (Fig. 7B
). Thus, both agonist
and antagonist were required for interaction between GAL4/PR-B(UP-1)
and VP16/PR-B (722m). These results provide strong evidence that PR
bound to an antagonist is capable of heterodimerization in the cell
with PR bound to antagonist. These results also underscore that
dimerization in whole cells requires ligand binding to both PR
partners.
Mixed Agonist/Antagonist PR Heterodimers in Vitro
Exhibit Reduced Ability to Bind to DNA
The two-hybrid assay results indicate that PR bound to an
antagonist can heterodimerize with PR bound to an agonist but does not
determine whether these mixed ligand heterodimers are functional. As
one means to examine this question, we have performed EMSAs of
separately expressed A and B receptors bound to either agonist or
antagonist. Because of the molecular size difference of the A (90 kDa)
and B receptors (120 kDa), mixing of the two PR isoforms results in the
formation of an intermediate mobility PR-A/PR-B heterodimer complex
that we and others have shown previously to be easily discernible by
EMSA from the faster mobility AA and slower mobility BB dimers (11, 12, 13, 38, 44, 45, 46). The A and B receptors were expressed in the baculovirus
insect cell system as previously described, and ligands were added to
Sf9 insect cell cultures for the last 68 h of expression to allow
binding to PR in the cell before extraction of receptor from nuclei
with 0.4 M NaCl. Free ligand was removed by treatment of
Sf9 cell extracts with dextran-coated charcoal as previously
described, and assays were performed at 04 C under conditions that
keep ligand dissociation to a minimum (45). When a constant amount of
PR-B bound to R5020 was mixed with increasing amounts of PR-A bound to
R5020 in a DNA binding reaction with a 32P-labeled PRE
oligonucleotide probe, a single PR-B-PRE complex was detected at the
lowest amount of PR-A, while two additional mobility complexes were
detected at higher amounts of PR-A (Fig. 8A
, lanes 16). As shown previously by
supershifts with appropriate PR antibodies (11, 12, 44, 45), the
intermediate mobility complex is composed of PR-A-PR-B heterodimers,
while the fastest mobility complex contains PR-A homodimers. In
contrast, little or no intermediate mobility complexes were detected
when PR-B bound to R5020 was mixed with increasing amounts of PR-A
bound to the antagonist ZK112993 (Fig. 8A
, lanes 712) or with
increasing amounts of PR-A bound to ZK98734 (Fig. 8A
, lanes 1318). We
and others previously observed similar results when one PR isoform
bound to RU486 was mixed with another bound to R5020. The amount of
intermediate mobility heterodimer complex was minimal compared with the
intermediate mobility heterocomplexes obtained when PR-A and PR-B were
bound to the same ligand (either R5020 or RU486) (12, 45). It should be
noted that the amount of PR-B (R5020) DNA complex decreased as
increasing amounts of PR-A antagonist were added to the EMSA assays,
albeit the decrease is more predominant with PR-A-ZK98734 than with
PR-A-ZK112993 (Fig. 8A
). This decrease of the PR-B complex at the
expense of the PR-A complex, without the appearance of a third
intermediate mobility complex [which does occur when mixing PR isoform
bound to the same ligand (R5020)], is consistent with the formation of
mixed ligand heterodimers in solution that exhibit reduced DNA binding
affinity.

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Figure 8. Agonist/Antagonist PR Heterodimers Formed In
Vitro have Impaired DNA Binding Activity
A, EMSA. PR-A and PR-B were separately expressed in Sf9 insect cells by
the baculovirus system as previously described (44 ). PR-B was bound to
R5020 (100 nM) and a separately expressed preparation of
PR-A was bound to either R5020, ZK112993, or ZK98734. PR in whole cell
extracts was incubated with a [32P]PRE oligonucleotide in
a DNA binding reaction previously described (11 ), and binding was
detected by EMSA. Lanes 16 shows PR-B bound to R5020 mixed with
increasing amounts of PR-A bound to R5020; lanes 712 show PR-B bound
to R5020 with increasing amounts of PR-A bound to ZK112993; lanes
1318 show PR-B bound to R5020 with increasing amounts of PR-A bound
to ZK98734. AA, AB, and BB indicate the position of DNA complexes
containing PR-A homodimers, PR-A-PR-B heterodimers, and PR-B-PR-B
homodimers, respectively. B, Heterodimerization detected in
vitro by coimmunoprecipitation assay. PR-A expressed in Sf9
insect cells was bound to the different ligands indicated while
expressed PR-B was bound to R5020. Whole-cell extracts were prepared
and PR levels were estimated by immunoblot blot. Equal amounts of PR-B
and PR-A were mixed and immunoprecipitated with the PR-B specific MAb,
B-30, under conditions at 04 C that minimize exchange of ligands
(45 ). The immunoprecipitates were analyzed by immunoblot with the PR-A
and PR-B reactive MAb, AB-52. Coimmunoprecipitation of PR-A in the
presence of PR-B (+PR-BR5020, lanes 15) indicates the formation of
PR-A-PR-B dimers in solution. As controls for nonspecific binding of
PR-A to the B-30 MAb protein A-Sepharose resins, each PR-A preparation
was subjected to immunoprecipitation with B-30 in the absence of PR-B
(lanes 610).
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We next considered the possibility that failure to detect
agonist/antagonist PR heterodimers bound to DNA may be due to an
inability to form solution dimers in vitro despite the fact
that heterodimerization was detected in whole cells by the two-hybrid
assay. To determine whether agonist/antagonist PR heterodimers can form
in solution in vitro, coimmunoprecepitation assays were
performed with the baculovirus-expressed PR-B bound to R5020 and PR-A
bound to either R5020 or different antagonists including RU486,
ZK98299, ZK98734, and ZK112993. A and B receptors were mixed together
for 30 min at 04 C and immunoprecipitated with a MAb (B-30) to the
unique amino terminus of PR-B. The immunoprecipitates were analyzed by
immunoblot with a MAb (AB-52) that reacts with both PR-A and PR-B. As
shown in Fig. 8B
, PR-A was efficiently coimmunoprecipitated with the
PR-B-R5020 complex whether it was bound to R5020 or any of the
antagonists (lanes 15). Specificity of PR-A interaction with PR-B is
shown by the lack of PR-A coimmunoprecipitation with the B-30 MAb in
the absence of added PR-B (Fig. 8B
, lanes 610). These results suggest
that PR bound to hormone agonist is capable of forming a solution
heterodimer with PR bound to antagonist in the absence of DNA. These
in vitro results, taken together with the results of the
two-hybrid assay with the ligand specificity mutant PRs, strongly
support the conclusion that PR bound to antagonist is capable of
forming a heterodimer with PR bound to agonist and that these
heterodimers have impaired ability to bind to target DNA sequences.
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DISCUSSION
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The A and B isoforms of PR have been shown to bind in
vitro, in a hormone-dependent manner, to target PREs as three
dimeric forms: AA, AB, and BB dimers (11, 12, 44, 45, 46). The present
study using the mammalian two-hybrid system demonstrates that the three
possible forms of PR dimers can also form in whole cells in a
hormone-dependent manner.
The two-hybrid system has been used previously to examine dimerization
of steroid receptors. Using a yeast two-hybrid assay, Wang et
al. (42) showed that dimerization of human ER
in
vivo is estrogen dependent. A mammalian-based two-hybrid system
was used to show ligand-dependent interactions between the
- and
ß-subtypes of mouse ER (16). Similar to our results with PR in a
mammalian-based system, a estrogen-dependent rise in reporter gene
activity was observed with a single GAL4/mER
hybrid (and VP16 empty
vector). This activity was attributed to the ligand-inducible
transactivation function of ER
itself, when directed to the promoter
of the GAL4 reporter construct by binding via the GAL4 DBD (16).
Reporter gene activity induced by the single GAL4/ER
hybrid was
approximately 5-fold, which is similar to the induction level observed
to be mediated by the GAL4/PR-B single hybrid treated with hormone
(6-fold) (Fig. 3
). Interestingly, we did not observe an R5020 induction
of reporter gene activity (pG5CAT) with the A isoform as a single GAL4
fusion protein. Both the A isoform of PR and ER
contain two
transactivation functions, whereas the B isoform of PR contains an
additional third activation domain (49, 50). It would, therefore,
appear that the two activation functions (AF-1 and AF-2) of ER
are
stronger than those of PR, at least in the context of the two-hybrid
assay. In our initial two-hybrid experiments, we attempted to reduce
the assay background activity of the single GAL4/PR-B hybrid protein by
taking advantage of known mutations in AF-2 of PR. Replacement of
conserved glutamate residues at positions 907 and 911 with alanine
results in a mutant receptor that is able to bind progesterone but has
greatly reduced AF-2 hormone-dependent activity (51). By using
PR-B(907, 911m) to construct the GAL4 DBD fusion, we observed a
reduction in hormone-dependent activity of the single hybrid protein.
However, the fold hormone induction of pG5CAT dependent on coexpression
of the second VP16/PR-B hybrid protein was no greater than that
obtained with wild-type PR-B as the GAL4 DBD fusion construct (not
shown). In the study of ER dimerization by the yeast two-hybrid assay,
no assay background activity was detected with the single GAL4/hER
hybrid (42). This clearly contrasts with our data with PR and with the
reported assay background detected with the GAL4 DBD/mER
as a single
hybrid protein in mammalian-based two-hybrid assays (16). This apparent
discrepancy between yeast- and mammalian-based two-hybrid assays is
likely due to receptors in yeast possessing lower intrinsic
transcriptional activity due to the absence of steroid receptor
coactivators that are normally present in mammalian cells (6, 7, 57).
Our initial two-hybrid experiments using wild-type PR-B to analyze the
effects of antagonists on PR dimerization suggested that these
compounds do not promote dimerization in vivo. Recent
results showing that PR and ER bound to antagonists inappropriately
forms a strong association with nuclear receptor corepressors (NCOR or
SMRT) provides a likely explanation for the lack of antagonist-induced
two-hybrid dependent activation of the pG5CAT reporter (53, 54, 55). In the
context of the two-hybrid assay, we would argue that recruitment of
corepressors suppresses the transcriptional activity of the VP16/PR
fusion protein even though interactions may occur efficiently between
GAL4/PR and VP16/PR hybrids in the presence of antagonists. In support
of this idea we observed that the ability of the VP16/PR fusion protein
to transactivate a PRE-controlled reporter gene in response to
antagonists was reduced as compared with agonist (Fig. 4
). To resolve
this issue we turned to the PR-UP-1 mutants in the two-hybrid assay.
The VP16/PR-B(UP-1) fusion protein, in response to antagonist, mediated
transactivation of the PRE-controlled reporter gene to a similar extent
as agonist-induced transactivation mediated by the VP16 fusion protein
constructed with wild-type PR-B (VP16/PR-B). This suggested to us that
the PR-UP-1 mutant would be useful to detect antagonist-dependent PR
dimerization in whole cells by two-hybrid assay. This indeed was the
case. The progestin antagonists RU486 and ZK98299 both stimulated a
significant interaction between GAL4/PR-B(UP-1) and VP16/PR-B(UP-1) as
detected by the induction of CAT reporter gene activity over that
obtained by the single GAL4/PR-B(UP-1) hybrid in response to
antagonists. Although statistical analysis did not show a significant
stimulation, the antagonist ZK112993 also exhibited a trend of
stimulating interaction between the GAL4/PR-B(UP-1) and VP16/PR-B(UP-1)
fusion proteins (Fig. 5
). These two-hybrid results support the
conclusion that antagonists induce PR dimerization in whole cells and
do not appear to act by impairing receptor dimerization.
Antiestrogens were reported to induce dimerization of human ER
when
measured in a yeast two-hybrid assay (42). Both tamoxifen and ICI
182,780 were observed to induce ER-ER interactions. However, the level
of reporter ß-galactosidase (ß-gal) activity induced by tamoxifen
and ICI 182,780 was only 15% and 20%, respectively, of the activity
induced by estradiol-17ß (42). Additionally, it was shown that
dimerization is perturbed when yeast cells were cotreated with estrogen
and antiestrogens. Our data with PR antagonists would suggest that the
lower induction of ß-gal observed by antiestrogens is due to
suppression of the heterologous transcriptional activation domain of
the GAL4 AAD/ER construct as opposed to a reduction of ER-ER
interaction per se. It is interesting that
antagonist-induced ER dimerization was detected using wild-type ER as
the fusion protein constructs, whereas we observed no dimerization
using wild-type PR to construct hybrid proteins. Antiprogestin-induced
PR-PR interactions were detected only with PR-UP-1 mutants. This
apparent difference could be explained as a difference between yeast-
and mammalian-based two-hybrid assays since it appears that yeast does
not express an NCOR- or SMRT-like molecule (53, 57).
It is now well established that binding of PR to antagonists induces a
conformational change in the LBD of the receptor that is different from
that induced by agonist (31, 32, 33, 34, 35, 36, 37). These conformational differences were
initially suggested to prevent the formation of heterodimers between
receptors bound to agonist and receptors bound to antagonist. This was
based on EMSA experiments in which mixing of one hPR isoform liganded
with RU486 and another liganded with R5020 failed to produce an
intermediate heterodimer complex bound to a PRE (12). However, the
ability of RU486- and R5020-liganded receptors to form heterodimers in
solution in the absence of DNA was not examined (12). In a subsequent
study, we examined whether mixed agonist/antagonist heterodimers could
form in solution by a coimmunoprecipitation assay. We observed that
PR-A bound to RU486 can heterodimerize as efficiently with PR-B bound
to R5020 as the two PR isoforms bound to the same ligand: either R5020
or RU486 (45). Similar to results of Meyer et al. (12), we
also observed a dramatically reduced ability of mixed R5020/RU486
heterodimers to bind to PREs by EMSA (45). By use of ligand specificity
PR mutants, we show here that mixed R5020/RU486 PR heterodimers can
form in whole cells as detected by the mammalian two-hybrid assay (Fig. 7B
). This suggests that antagonists do not alter the dimer interface
sufficiently to prevent dimerization with PR bound to agonist. In
addition, the fact that both R5020 and RU486 were needed to form these
heterodimers provides new information that both PR partners must be
bound to ligand in order for dimerization to occur; ligand binding to
one partner is not sufficient. In further support of the compatibility
of dimer interfaces is the crystal structure of the ER LBD bound to
estradiol and the antiestrogen raloxifene (58). The dimerization
interfaces appear to be similar for the LBD bound to estradiol and
raloxifene; the major conformational difference induced by agonist and
antagonist is the position of helix 12, which contains the conserved
sequences in AF-2 (58). The recently published crystal structure of PR
LBD bound to progesterone, and modeling with the antagonist RU486, also
suggest that dimerization interfaces are similar for the LBD bound to
progesterone and RU486 (59). Similar to ER, it was predicted that the
major difference between the PR LBD bound to progesterone and RU486 is
the displacement of helix 12 and the C-terminal tail.
The fact that PRs bound to RU486 and R5020 are capable of forming
heterodimers in vitro (in solution) and in whole cells may
aid in the interpretation of the mechanistic basis for the biological
potency of RU486 and other structurally related progestin antagonists.
All antagonists that we have tested, including ZK98299 after correction
for its 10-fold lower affinity than R5020 for PR, inhibit activation of
PR at substoichiometric concentrations with R5020 (Refs. 11, 38 and
Fig. 6
). This suggests that these compounds do not work solely by a
simple mechanism of competing with agonist for binding to PR. One
possible mechanism to explain these biological results is that
receptors bound to antagonists can inactivate receptors bound to
agonist in trans by heterodimerization. The implication is
that all receptors do not need to be occupied by antagonist for
complete antagonism to occur. This is diagrammed schematically in Fig. 9
, where the agonist/antagonist
heterodimer forms in solution and then is unable to bind efficiently to
a PRE.

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Figure 9. Progestin Antagonist Transrepression by
Heterodimerization
Treatment of cells with progestin agonist or antagonist
stimulates PR homodimerization and binding to progesterone response
elements (PREs). Antagonists fail to stimulate transcription because
they induce an altered conformational change in the LBD (black
filled symbols) that does not permit PR interaction with
coactivators required to transmit the receptor signal into a
transcriptional response. When cells are treated with both agonist and
antagonists, heterodimers can be formed between PR bound to antagonist
and PR bound to agonist. Based on EMSA results, these heterodimers do
not efficiently bind to PREs and thus appear to be inactive. The basis
for the inability of these heterodimers to bind to DNA is not known.
Although differences in conformation in the LBD does not prevent
heterodimerization, the mixed ligand heterodimers may be asymmetric
such that the DBDs (hatched symbols) are not oriented
correctly over the inverted repeat palindromic PRE. The amino-terminal
domain of PR is represented by open symbols. Thus we
propose that progestin antagonists can act in trans to
inhibit hormone agonists by heterodimerization and that this accounts
in part for the ability of these compounds to inhibit PR activation at
concentrations that are substoichiometric with progestin agonists.
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MATERIALS AND METHODS
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Materials
[3H]R5020 (promegestone:
[17
-methyl-3H]17
,
21-dimethyl-19-norpregna-4,9-diene-3, 20-one; 87 Ci/mmol) and unlabeled
R5020 were obtained from Dupont/NEN Research Products (Boston, MA).
[3H]RU486 (Mifapristone: [17
-
hydroxy-11[4-dimethyl-aminophenyl]17-propenyl-estra-4,5-
diene-3-one) and unlabeled RU486 were gifts from R. Daraedt
(Roussel UCLAF, Romanville, France). ZK98299 (Onapristone), ZK112993,
ZK98734, and [3H]ZK98299 were provided by David Henderson
(Schering, Berlin, Germany). 1294/H9 is an unpublished MAb that
recognizes the A and B forms of hPR. MAbs B-30 and AB-52 to PR have
been described previously (60).
Construction of Two-Hybrid Fusion Plasmids for PR
The Mammalian MATCHMAKER Two-Hybrid Assay Kit was obtained from
CLONTECH (Palo Alto, CA). Included within the kit is the pM cloning
vector for expressing GAL4 DBD (aa 1147) fusion proteins and pVP16
(aa 411455) for expressing the VP16 transcriptional activation
domain. Also included is pG5CAT, a reporter vector that contains the
CAT gene downstream of five consensus GAL4 binding sites and the
minimal promoter of the adenovirus E1b gene. Human PR-B was cloned into
the pM and pVP16 vectors in the following manner. A 2.93-kb
AflII-Asp718 fragment of YEphPR-B (33) was filled
in with Klenow and inserted into the unique BamHI
restriction site of pGAD424 (CLONTECH) that had been blunt ended with
Klenow. This plasmid, pGAD424.PR-B, was used as the source of hPR-B
cDNA for fusion with the GAL4 DBD and the VP16 protein of the herpes
simplex virus. A 2.9-kb EcoRI-PstI fragment from
pGAD424.hPR-B was ligated into the EcoRI-PstI
restriction sites in the multiple cloning cassette of both the pM and
pVP16 vectors to yield, respectively, pM.PR-B (referred to subsequently
as GAL4/PR-B) and pVP16.PR-B (referred to subsequently as VP16/PR-B).
This places the GAL4 DBD and the activation domain of VP16 immediately
amino-terminal to hPR cDNA. Cloning junctures were sequenced (Sequenase
2.0, US Biochemical, Cleveland, OH) and PR-B was determined to be
correctly oriented and in-frame with GAL4 DBD or VP16. For PR-A
constructs, a 2.4 kb BspHI-Asp718 fragment of
YEphPR-B (33) was filled-in with Klenow and inserted into the unique
SmaI restriction site of pGBT9 (CLONTECH). This plasmid was
used as the source of hPR-A cDNA for fusion with the GAL4 DBD and the
VP16. A 2.4 kb EcoRI-PstI fragment from
pGBT9.hPR-A was cloned into the EcoRI-PstI of pM
and pVP16 to yield respectively pM.PR-A (referred to subsequently as
GAL4/PR-A) and pVP16.PR-A (referred to subsequently as VP16/PR-A).
Cloning junctures were sequenced (Sequenase 2.0, US Biochemical) and
hPR-A was determined to be correctly oriented and in-frame with the
GAL4 DBD or VP16.
A C-terminal truncation mutant of hPR-B (pSVhPR-B 891) was provided by
Donald McDonnell (Duke Medical Center, Durham, NC). To construct
fusions with hPR-B 891, a 2.8-kb BamHI fragment from
pSVhPR-B 891 was inserted into GAL4/PR-B and VP16/PR-B vectors that had
been previously digested with BamHI to drop out the 2.8-kb
PR-B fragment. This hPR cDNA contains a reading-frame mutation that
generates a stop codon at aa 892, resulting in a carboxyl-terminal
truncated receptor by 42 aa (33). The resulting plasmids,
GAL4/PR-B(UP-1) and VP16/PR-B(UP-1), were sequenced to confirm the
presence of the nucleotide deletion at base 2849.
A two-hybrid vector expressing PR-B containing a point mutation at aa
722 was constructed by digesting VP16/PR-B with the restriction enzymes
StuI and EcoN I and replacing the resulting
1.3-kb fragment with a 1.3-kb StuI-EcoN I
fragment from pBK·CMVhPR-BRU (provided by Dawn Wen, Ligand
Pharmaceutical, San Diego, CA). pBK·CMVhPR-BRU contains
full-length hPR-B cDNA with a G to T substitution at nucleotide
position 2339. This mutation results in a Gly
Cys change at aa 722 in
the human PR-B amino acid sequence. This generates a receptor that has
lost detectable RU486 binding, but retains wild-type binding affinity
for progestin agonists and is fully activated by progestin agonists
(56). The resulting plasmid, VP16/PR-B(722m), was sequenced to confirm
the presence of the G to T substitution.
For PR transactivation experiments, the progesterone responsive
reporter, DHRE-E1b-CAT, was used. This contains two synthetic PREs
linked to the TATA box of the adenovirus E1b (61) and CAT (30).
Transient Transfections
Monkey kidney COS-1 cells were plated in DMEM (GIBCO-BRL) with
10% FBS at a density of 1.75 x 105 to 2.0 x
105 cells per well in six-well dishes (Falcon, Franklin
Lakes, NJ) at 37 C. After 24 h the cells were transfected
by an nonrecombinant adenovirus-mediated DNA transfer technique as
previously described (62). This method involves the use of a
replication-defective adenovirus that is coupled with
poly-L-lysine to bind the plasmid DNA noncovalently. This
process facilitates cellular uptake of the plasmid DNA by
receptor-mediated endocytosis. For mammalian two-hybrid experiments, 50
ng/well of plasmids encoding either GAL4 DBD or VP16 fusion proteins
were used and 500 ng/well of the pG5CAT reporter plasmid. In early
two-hybrid experiments, we used charcoal-treated serum and found that
it made no difference compared with whole FBS, so we used whole FBS for
our studies. For experiments to measure PR as a transactivator, the
DHRE-E1b-CAT was added at 500 ng/well, and the PR expression plasmid
was added at 10 ng/well. To treat cells with various PR-ligands,
24 h after transfection, the medium was replaced with DMEM + 10%
FBS containing vehicle (0.01% ethanol) or ligand at the concentration
indicated in the figure legends, followed by incubation at 37 C. After
an additional 24 h, the cell monolayers were rinsed with CAT wash
buffer (40 mM Tris-Cl, pH 7.4, 150 mM NaCl, and
1 mM EDTA) and lysed in the well by the addition of 300
µl of cell lysis buffer (20 mM potassium phosphate, pH
7.4, 5 mM MgCl2, and 0.5% Triton X-100). To
control for variation in transfection efficiency, cells were
cotransfected with a CMV-ß-gal reporter plasmid, and ß-gal was
measured in a luminometer (Monolight 2001) with the Galacto-Light Plus
kit (Tropix, Bedford, MA) according to manufacturers
instructions.
Whole-Cell Steroid-Binding Assay
COS-1 cells plated in six-well dishes at a density of 1.75
x 105 cells per well were grown overnight at 37 C in DMEM
+ 10% FBS. Cells were then transfected with a single concentration (25
ng) of the indicated expression plasmids by the adenovirus-mediated DNA
transfer technique as described above. PR-rich T47D human breast cancer
cells (60) were also plated in six-well dishes at a density of
1.75 x 105 cells per well in MEM + 5% FBS. After
44 h the cells were incubated for an additional 4 h at 37 C
with 12 nM of [3H]R5020,
[3H]RU486, or [3H]ZK98299 ± unlabeled
100 nM homologous ligand. Radioactive steroid was extracted
from cells with ethanol and quantitated by liquid scintillation
counting. Parallel transfected cells were lysed and assayed for protein
concentration. Receptor expression levels were normalized to protein
and calculated as picomoles of steroid binding per mg of protein.
Nonspecific binding was determined by parallel incubations of
mock-transfected COS-1 cells. Data represents specific binding taken as
total binding minus nonspecific binding.
Immunoblot Analysis
COS-1 cells were plated in six-well dishes at a density of
2 x 105 cells per well in DMEM + 10% FBS for 24
h. Wells were transiently transfected by the adenovirus method (62)
with 25 ng of the appropriate plasmid (indicated in the figure
legends). After 44 h, the medium was removed and replaced with
medium containing either 100 nM of R5020 or 100
nM of RU486. After an additional 4 h at 37 C, the cell
monolayers were washed with CAT wash buffer, and cells from triplicate
wells were harvested and combined in 0.4 ml of 0.5% Triton X-100 lysis
buffer. The lysates were removed from the wells and centrifuged at
12,000 rpm for 10 min at 4 C. The supernatants were assayed for protein
concentration by Bradford assay (63). Total protein in 1% SDS sample
buffer (250 µg) was boiled and electrophoresed on 7.5%
polyacrylamide SDS gels. Separated proteins were transferred to
nitrocellulose and incubated with the receptor-specific (1 µg/ml)
MAb 1294/H9. Immune complexes were decorated with rabbit
antimouse immunoglobins (N. L. Cappel Laboratories, Cochranville,
PA) and [35S] protein A (Amersham) and detected by
autoradiography.
T47D human breast cancer cells were plated in six-well dishes at a
density of 2 x 105 cells per well in MEM (GIBCO-BRL)
with 5% FBS. At 44 h after plating, the cells were treated with
100 nM R5020. Cells from triplicate wells were
harvested in a total of 0.4 ml of 0.5% Triton X-100 lysis buffer and
prepared as the COS-1 lysates.
CAT Assays
CAT enzyme activity was assayed by a radiometric/organic phase
extraction method as previously described (64). Enzyme activity was
calculated as counts per min of [3H]acetylcoenzyme A
converted per µg of protein in the cell lysate. CAT assays were done
in duplicate for each lysate. Cell treatment groups were also performed
in duplicate. Protein concentration was measured by the method of
Bradford (63), and equal amounts of protein (1030 µg) were added to
the assay. Specific CAT activity was determined by subtraction of assay
background obtained from lysates of nontransfected cells. To compensate
for variation in transfection efficiency, the cells were also
transfected with a CMV-ß-gal reporter plasmid. CAT results were
calculated as the ratio of CAT activity per unit of ß-gal
activity.
Baculovirus Expression of Human PR-A and PR-B
Human PR-A and PR-B were expressed as nonfusion full-length
proteins from baculovirus vectors in Sf9 insect cells as previously
described (44). To bind ligands to baculovirus-expressed receptors,
they were added at 200 nM to Sf9 cell cultures during the
last 68 h of expression. To prepare whole-cell extracts, Sf9 cells
were lysed in TEDG buffer [10 mM Tris-base, pH 7.4, 1
mM EDTA, 1 mM dithiothreitol, and 10%
glycerol], containing 0.5 M NaCl and a mixture of protease
inhibitors (60). Cell lysates were centrifuged at 100,000 xg for 30 min to yield a soluble supernatant and then dialyzed
against lysis buffer containing no NaCl.
Coimmunoprecipitation Assay
Protein A-Sepharose was prebound noncovalently with
receptor-specific MAbs and used as an immunoabsorbent as described
previously (28, 45). Resins were prebound to rabbit anti-mouse IgG
(Cappel) and used as a bridging antibody. Receptor-specific MAbs were
then bound to the immobilized rabbit antimouse IgG. Sf9 cell extracts
containing PR-A or PR-B were mixed at 10 µg/100 µl of resin in
siliconized microcentrifuge tubes and incubated on ice for 30 min.
Equal amounts of receptors were added to each assay as determined by
ligand-binding assay and immunoblot analysis. MAb-coated protein
A-Sepharose beads (100 µl) were added to each tube and incubated at 4
C for 1 h on an end-over-end rotator. Resins were then washed four
times by centrifugation in TEG (TEDG minus dithiothreitol) containing
100 mM NaCl, transferred to a new microcentrifuge tube, and
washed twice more. Immobilized proteins were eluted with 2% SDS
loading buffer and then analyzed by immunoblot, using
[35S]protein A and autoradiography as the detection
method.
EMSAs
For EMSA, we used a 28-bp oligonucleotide containing a
PRE/glucocorticoid response element derived from the MMTV-long terminal
repeat (11, 65). Sf9 insect cell extracts containing PR-A or PR-B were
incubated for 1 h at 4 C with 32P-labeled DNA (0.3 ng)
in a total reaction volume of 25 µl. Also included was 1 µg of poly
(dA-dT) as nonspecific competitor DNA. The DNA binding buffer contained
10 mM Tris-base, pH 7.4, 50 mM NaCl, 5
mM dithiothreitol, 2 mM MgCl2, 10%
glycerol, and 50 ng/ml of a carrier protein. Samples (25 µl) were
electrophoresed on 5% polyacrylamide gels prepared at 40:1 (wt/wt)
acrylamide-bis-acrylamide ratio using 20 mM Tris-acetate,
0.5 mM EDTA as the electrode buffer. To maintain constant
temperature during electrophoresis, 4 C water was recirculated through
the gel apparatus. Gels were dried and subjected to autoradiography
(65).
T47D Breast Cancer Cells Stably Transfected with MMTV-CAT to
Measure Biological Activity of Progestin Antagonists
T47D human breast cancer cells were cultured as described
(11) in MEM supplemented with 5% FBS. A cloned derivative (B-11) of
T47D, stably transfected with a construct containing the CAT gene
linked to the MMTV promoter/enhancer, has been previously described
(11). Cells plated in six-well dishes at a density of 0.5 x
106 cells per well were grown for 3 days at 37 C and then
incubated for another 24 h with medium stripped of steroid
hormones by treatment with dextran-coated charcoal (11). This was
followed by an additional 24 h incubation with hormone and other
compounds in the same medium. Harvested cells were washed twice in 40
mM Tris (pH 7.6), 150 mM NaCl, and 1
mM EDTA and lysed in buffer containing 20 mM
potassium phosphate (pH 7.8), 5 mM MgCl2, and
0.5% (vol/vol) Triton X-100. Cell lysates were analyzed for CAT enzyme
activity as described above.
Data Analysis
For two-hybrid experiments presented in Figs. 3
, 5
, and 7
, CAT
activity mediated by the GAL4/PR construct as a single hybrid in the
absence of ligand was normalized to 1.0, and the CAT activity mediated
by other constructs and construct pairs was calculated relative to the
single-hybrid control. These data are expressed as relative CAT
activity. The two-hybrid CAT data represent mean values
(±SEM) from four to seven independent experiments. To
calculate the fold CAT induction mediated by PR dimerization in
response to ligand, we used the following calculation:
The values for fold induction were analyzed by the Students
t test using Excel 5.0 (Microsoft) to determine whether
there was a statistically significant difference between the treatment
groups. Results were considered statistically significant at
P < 0.05.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Lori Sherman for expert technical assistance
and the University of Colorado Cancer Center Tissue Culture Core
(P30CA46934) for assistance with baculovirus-expressed PR and cell
culture media.
This work was supported in part by Public Health Services Grant
R01DK-49030 (to D.P.E.) and National Research Service Award
fellowship F32DK-09662 (to S.A.L.).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dean P. Edwards, Department of Pathology and Program in Molecular Biology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail: edwardsd{at}defiance.uchsc.edu
Received for publication December 22, 1997.
Revision received August 3, 1998.
Accepted for publication September 8, 1998.
 |
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