Download Estrogen Receptor Mutants Which Do Not Bind 17P

Estrogen Receptor Mutants Which
Do Not Bind 17P-Estradiol Dimerize
and Bind to the Estrogen Response
Element in Viva
Yao Zhuang,
Benita
S. Katzenellenbogen,
and David
J. Shapiro
Department
of Biochemistry
(Y.Z and D.J.S) and
Department
of Physiology
and Biophysics
(B.S.K.)
University of Illinois
Urbana, Illinois 61801
To investigate the stage in estrogen receptor (ER)
action at which hormone functions,
we prepared
human ER mutants unable to bind 176-estradiol.
In
transfected
Chinese Hamster Ovary (CHO) cells,
two of the ER mutants exhibited less than 5% of
the ability to activate transcription
shown by wild
type ER. lmmunoprecipitation
followed by Western
blotting with monoclonal
antibodies was used to
examine the ability of the ER mutants to form heterodimers with a truncated form of wild type ER.
The non-hormone-binding
mutants formed heterodimers with the truncated
ER as efficiently as
wild type ER. We used a promoter
interference
assay to measure the interaction of the ER with the
estrogen response element (ERE) in viva. Expression plasmids encoding the ER mutants and wild
type ER were transfected
into CHO cells across a
range of concentrations,
resulting in both high and
low levels of promoter interference.
The ER mutants and wild type ER elicited similar levels of
promoter
interference,
indicating
that although
they were unable to bind ligand, the ER mutants
bound to the ERE in viva as effectively as wild type
ER. Additional
evidence that the non-hormonebinding ER mutants are not in a functionally
inactive complex comes from their ability to suppress
the activity of wild type ER, when they were coexpressed in the same cells.
These data support a model for ER action in
which the unliganded
ER is free to dimerize and
bind to the ERE. In this model, the primary role of
176-estradiol
in ER action is to induce a conformational change which activates the ligand-dependent transactivation
domain. (Molecular
Endocrinology 9: 457466, 1995)
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INTRODUCTION
The estrogen receptor (ER) is a member of the steroid/
nuclear receptor superfamily of ligand-regulated transcription factors. Members of this superfamily possess common
structural
features, with discrete
regions of the receptor responsible for ligand binding,
nuclear localization, dimerization, DNA binding, and
transcription activation (reviewed in Refs. l-4). The
hormone binding domain of ER and other steroid hormone receptors interacts specifically with ligand and
contains a dimerization function and a ligand-dependent transactivation domain (5-12).
Transcription activation by steroid hormone receptors is largely dependent on the presence of bound
hormone ligand (reviewed in Refs. l-3 and 13). Studies of the glucocorticoid and progesterone receptors
(GR and PR) have led to a multistep model for conversion of steroid receptors from an inactive form to
one in which they are effective transcription activators.
In this model, hormone binding induces a conformational change, which both releases the receptor from
an inactive complex with several heat shock proteins,
and exposes the dimerization function in the hormone
binding domain. By facilitating receptor dimerization,
the bound hormone greatly increases the ability of the
receptor to bind to its response element on DNA. Once
bound to its response element, the ligand-induced
conformational change enables the C-terminal liganddependent transactivation
domain to function and
converts the receptor to a form in which it is an effective transcription activator (reviewed in Ref. 3). While
there is substantial evidence to support this model for
the role of ligand in GR and PR action (14-25), the
question of whether hormone functions similarly in ER
action has been more controversial. The ER has been
reported to possess substantial ligand-independent
ability to activate transcription both in viva (26) and in
some cell-free systems (27). In most in vitro studies,
binding of the ER to the estrogen response element
(ERE) was not dependent on the presence of added
ligand (7, 12, 28-30). However, in vitro binding of the
457
MOL ENDO.
Vol 9 No. 4
1995
458
ER to the ERE may be influenced by the salt used to
extract the receptor, and by the ionic and temperature
conditions of the binding reaction (11, 31). In vivo
studies using promoter interference assays in yeast
and amphibian ceils reported that binding of ER to the
ERE was ligand dependent (32, 33). However, substantial in vivo binding to the ERE in the absence of
ligand was observed in several mammalian cell lines
(34).
Analysis of the role of 17/3-estradiol (EJ binding in
ER action is complicated by the presence of traces of
estrogens in the charcoal-treated
serum used to cuiture cells. To evaluate the ability of the unliganded ER
to dimerize and to bind to the ERE under conditions
that excluded a contribution from traces of bound
ligand, we prepared ER mutants that were unable to
bind E,. Using immunoprecipitation
and Western blotting to analyze their ability to form heterodimers in
vivo, we found that the non-hormone-binding
ER mutants effectively formed heterodimers with ER. In vivo
promoter interference assays showed that these ER
mutants bound to the ERE as well as wild type ER.
Although the ER mutants were able to dimerize and
bind to the ERE in the absence of bound E,, they were
unable to activate transcription.
Another way to determine whether the unliganded
ER is located in an inactive complex with other proteins is to determine whether the inactive non-hormone-binding ER mutants can suppress the activity of
wild type ER, when they are coexpressed in the same
cells. Several dominant negative mutants of nuclear
receptors have been described (35-39). The v-erb A
oncogene is a potent dominant negative mutant of
thyroid receptor, which is unable to bind ligand, but
retains the ability to bind to the thyroid response element (40, 41). The ER mutants we constructed, which
were unable to bind estrogen, showed moderate ability to suppress the activity of wild type ER, demonstrating that they were not sequestered in an inactive
complex with other proteins.
These data suggest that, at least in the case of the
ER in Chinese hamster ovary (CHO) cells, the major
function of the hormone may be to induce a conformational change that exposes or activates
the
ligand-dependent
AF2 transactivation
domain in the
hormone-binding
domain of the ER.
tions and deletions in the ER mutants are shown at the
top of Fig. 1. The ability of the mutants to activate
transcription
was determined by cotransfection
in
CHO cells. Two of the mutants, NHB3 and NHBS,
retained substantial ability to activate transcription of
the (ERE),-TATA-chloramphenicol
acetyltransferase
(CAT) reporter plasmid (42) and were not further studied. The other two mutants, NHBl and NHB7, exhibited less than 5% of the ability of wild type ER to
activate transcription (Fig. 1). Since these experiments
were carried out at a high 1O-’ M concentration of E,,
and the wild type ER reaches maximal activity in these
cells between 0.1 and 1 nM E, (35), it seemed probable
that the NHBl and NHB7 ER mutants had little or no
ability to bind estrogen.
The Failure of the NHBl and NHB7 Mutants to
Activate Transcription
Is Not Due to Extremely
Rapid Degradation
One potential explanation for the inability of the NHBl
and NHB7 ER mutants to activate transcription is that
they are unstable in cells and are rapidly degraded. We
analyzed the levels of expression of wild type and
mutant ERs by Western blot analysis using an ERspecific monoclonal antibody. When equal amounts of
ER MUTANT
hER
(W-l.)
Transactivation
Mutants
by ER Ligand-Binding
Domain
Amino acids in the region 515-525 of the ER have
been shown to play an important role in ligand binding
(7). To construct ER mutants likely to have lost the
ability to bind E,, amino acid replacements and deletions were generated in this region of the human ER
(hER). These hER mutants were designated non-hormone-binding (NHB) mutants. The amino acid substa-
‘I5 RHMSNKGMEHL=
NHBl
@S516,G521 R)
RHMZNKRMEHL
NHB3
(H516A,M517A)
FiIiA_SNKGMEHL
NHB5
NHB7
(K520A)
(K520D,G521V
E523R,H524L)
RHMSNAGMEHL
RHMSNDVMRLL
---
7
0
RESULTS
AMINO ACID SEQUENCE
,L
WT
NHBI
NHB3
NH85
NH87
ESTROGEN RECEPTOR
Fig. 1. Structure and Activity of the hER Mutants
The amino acid sequence and designation of each of the
NHB mutants is shown at the top. For wild type hER and each
of the NHB ER mutants, 5 ng CMV expression plasmid, 0.8
pg (ERE),-TATA-CAT
reporter plasmid (42), and 1 fig thymidine kinase-luciferase internal standard (pT109) were cotransfected into CHO cells. The cells were treated with 10 ’
M E, for 48 h before harvesting. Cell extracts were prepared
and CAT assays were performed as described in Materials
and Methods. The data represent the mean 5 SEM for three
separate transfections.
Unliganded ER Dimerizes and Binds to the ERE
459
DNA were used in the transfections, the level of NHB7
was comparable to, or slightly higher than, the level of
wild type ER. However, the level of NHBl was only one
fifth the level of wild type ER (Fig. 2). When we used 5
times more of the expression plasmid encoding NHBl
in the transfections, the levels of NHBl and wild type
ER were comparable (Fig. 2). To ensure that equal
amounts of the receptor proteins were present, in
subsequent experiments, we used 5 times more of the
NHBI expression plasmid than of the expression plasmids encoding wild type ER or NHB7. Even at the
5-fold higher level of expression plasmid, NHBI had
less than 5% the activity of wild type ER (data not
shown).
The NHBl
Bind E,
and NHB7 ER Mutants
Are Unable to
Although NHBl and NHB7 are related to ER mutants
that do not bind estrogen (7) and are unable to activate
transcription at high concentrations of E,, it was necessary to directly evaluate their ability to bind E,. Wild
type ER, NHBl, and NHB7 were expressed in COS-1
cells, and specific binding of E, to extracts containing
equal amounts of each of the receptors was determined. The wild type ER exhibited a binding curve (Fig.
3) and Scatchard plot (data not shown) typical of ER,
with a dissociation constant (Kd) of 0.55 nM. Using
extracts that were shown by Western blotting to contain levels of NHBI and NHB7 equal to those of wild
type ER, neither NHBl nor NHB7 exhibited significant
ability to bind E, (Fig. 3). Since the ER mutants have
not even begun to show specific binding at the highest
concentration of E, tested (10 nM), it seems clear that
they exhibit at least a 1OO-fold reduction in their ability
to bind estrogen.
NHBI and NHB7 Bind to the ERE in Gel Mobility
Shift Assays
We used gel mobility shift assays to investigate the
ability of NHBI and NHB7 to interact with the ERE in
DNA
2ug
2ug
2ug
2ug
1oug
Fig. 2. Determination of the Levels of Wild Type and Mutant
ERs by lmmunoblot Analysis of Transfected Cells
Whole cell extracts were prepared from COS-1 cells transfected with the indicated amounts of expression plasmids
encoding wild type ER or the NHBl or NHB7 ER mutants.
Proteins were resolved by SDS polyactylamide gel electrophoresis, transferred to nitrocellulose, and probed with the
ER- specific monoclonal antibody D547. When 5 times more
of the expression vector encoding NHBl (10 wg) was used in
the transfection, the expression level of NHBl was equivalent
to that of wild type ER.
0.264 -
0
2
6
10
ES~RADIOLYnM)
Fig. 3. Hormone Binding Profile of Wild Type ER, NHBl , and
NHB7
Cell extracts were prepared from COS-1 cells transfected
with expression plasmids encoding wild type ER, or either the
NHBl or NHB7 mutant. The levels of ER in each extract were
determined by Western blotting with monoclonal antibody
D547 (see Fig. 2 legend). Extracts containing the same
amount of receptor were incubated for 2 h at 22 C with
varying concentrations of rH]E, in the presence or absence
of a 200-fold excess of radioinert E,. Unbound pH]E, was
removed by charcoal-dextran treatment, and rH]E, bound to
the ERs was quantitated by scintillation counting.
vitro. Extracts from COS-1 cells transfected
with expression plasmids encoding either wild type ER,
NHBl, or NHB7 all bound efficiently to a 51 -base pair
fragment containing a consensus ERE (42) (Fig. 4.).
Since unliganded and liganded ER exhibit similar binding to the ERE in most in vitro studies (7, 12, 28-30),
the ability of the NHBI and NHB7 mutants to bind to
the ERE in gel mobility shift assays did not clearly
establish that they bind to the ERE in cells. We therefore examined the ability of the ER mutants to interact
with the ERE in intact cells.
The NHB ER Mutants
In Vivo
Efficiently
Bind to the ERE
To investigate the ability of the NHBl and NHB7 mutants to bind to the ERE in vivo, we used a promoter
interference assay (32-34,43). In this assay, ER bound
to two EREs near the transcription initiation site of the
strong cytomegalovirus (CMV) promoter competes for
binding with basal transcription factors. The extent of
interference with transcription provides a measure of
the interaction of the ER with the ERE (34). Transfections were carried out in the presence or absence of
E,, with levels of the expression plasmids encoding ER
and the ER mutants that produce similar levels of
protein (Fig. 2). In agreement with recent data (34, 35),
the wild type ER exhibited a slight increase in intetference in the presence of hormone (Fig. 5). The NHBl
and NHB7 ER mutants exhibited similar levels of promoter interference in the presence and absence of
hormone. Promoter interference by the NHBl and
NHB7 mutants was similar to interference by unliganded wild type ER and very slightly lower than the level
MOL END0 . 1995
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460
I
0
hER
Fig. 4. Analysis of in Vitro Binding to the ERE by the ER and
the NHBl and NHB7 Mutants
For gel mobility shift assays, cell extracts were prepared
from COS-1 cells transfected with expression plasmids
encoding wild type ER (WT), or either the NHBl or NHB7
mutant. Aliquots of extracts containing the same amount of
either wild type ER, or NHBl, or NHB7 were preincubated
with poly deoxyinosine/deoxycytosine on ice followed by
incubation with 32P-labeled ERE fragment at room temperature as detailed in Materials and Methods. DNA-protein
complexes were resolved on a 5% polyacrylamide gel in a
low ionic strength buffer. The figure contains the autoradiogram of a 12 h exposure at -70 C with an intensifying
screen.
NHB7
Fig. 5. In Viva Binding to the ERE by Wild Type ER and by
the ER Mutants
CHO cells were cotransfected with 2 ng of the expression
plasmid encoding wild type ER or NHB7, or 10 ng of the
expression plasmid encoding NHBl and 400 ng of the CMV(ERE),-CAT promoter interference reporter plasmid in the
presence (+E2) or absence (-E2) of 1O-’ M E,. The cells were
harvested after 24 h, and extracts were prepared and assayed for CAT activity. The level of CAT activity in cells
transfected with the CMV-(ERE),-CAT reporter plasmid alone
was set at 0% inhibition. The data represent the mean ir SEM
for three separate transfections.
and clearly establish that, in CHO cells, at a variety of
concentrations
of intracellular ER, the NHB mutants
and liganded wild type hER exhibit similar abilities to
bind to the ERE in vivo.
The ER Mutants
Type ER
of interference exhibited by liganded wild type ER (Fig.
5). If the promoter interference assays were performed
at levels of transfected DNA that produce levels of ER
substantially higher than are required to saturate the
promoter, then even if NHBl and NHB7 actually exhibited considerably reduced in vivo binding to the
ERE, they could produce results equivalent to those
for wild type ER.
To eliminate this possibility, we carried out promoter
interference assays using a broad range of levels of
the ER expression plasmids. Since 5 times more of the
NHBl expression plasmid is required to produce levels of ER equivalent to those of wild type ER or the
NHB7 mutant, we compared results for levels of transfected DNA that produced equivalent intracellular levels of NHBl, NHB7, and wild type ER. Across the
entire range of levels of interference with the promoter,
which extend from IO%-55% inhibition of promoter
activity, the NHBl and NHB7 mutants and liganded
wild type ER exhibited similar promoter interference
profiles (Fig. 6). These data extend earlier studies (34)
NHBI
Form Heterodimers
with Wild
Previous studies on wild type ER and on NHB mutants
did not directly address the question of whether hormone is required for ER dimerization. In a previous
study in which E, increased the binding of the ER to
the ERE in vivo, one proposal was that the ligand might
stimulate or stabilize ER dimerization (34). To test the
ability of the NHB ER mutants to form dimers, we
needed to be able to distinguish between the NHBl or
NHB7 mutants and wild type ER. We therefore employed a truncated ER (ERl-554) in which the F domain is deleted (44). ERl-554 contains the hormonebinding and dimerization domains of the full-length
ER, but lacks the epitope for recognition by the ERspecific monoclonal antibody D75 (Fig. 7 , panel A).
Only homodimers of wild type ER, NHBl, or NHB7,
and heterodimers between ERl-554 and a full-length
ER will be immunoprecipitated
by this antibody. We
cotransfected COS-1 cells with expression plasmids
encoding a full-length ER (either wild type ER, NHBl,
or NHB7) and the truncated receptor, ERl-554. Ex-
461
Unliganded El3 Dimerizes and Binds to the ERE
WT hER : NHB7
NHBl
0
0
0.1
0.5
0.2
1.0
EXPRESSION
0.3
1.5
VECTOR
0.4
2.0
0.5
2.5
(ng)
Fig. 6. Subsaturating Levels of Plasmids Encoding the NHB
Mutants and Wild Type El3 Exhibit Similar Levels of Promoter
Interference
CHO cells were cotransfected with O-O.5 ng of the expression plasmid encoding wild type ER or NHB7, or O-2.5 ng of
the expression plasmid encoding NHBl (which produces 5
times less receptor protein for a given amount of transfected
DNA; see Fig. 2) and 400 ng of the CMV-(ERE),-CAT promoter interference reporter plasmid in the presence (+E2)
(wild type ER), or absence (NHBl and NHB7) of 10m9 M E,.
The cells were harvested after 24 h and extracts were prepared and assayed for CAT activity. The level of CAT activity
in cells transfected with the CMV-(ERE),-CAT reporter plasmid alone was set at 0% inhibition. The data represent the
mean + sEM for five separate transfections.
tracts were prepared and immunoprecipitated
with
D75 monoclonal antibody. Precipitated proteins were
fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and blotted on to nitrocellulose, and the ERs were visualized by reaction
with the ER-specific monoclonal antibody D547. The
D547 antibody recognizes an epitope in the hinge
region of ER and can therefore be used to visualize
both the full-length ER and ERi-554. The data presented in Fig. 7 demonstrate that ERl-554 is efficiently immunoprecipitated
from extracts of cells containing ERl-554
and either unliganded
NHBl or
NHB7, or liganded wild type ER. Heterodimer formation between the unliganded NHBl or NHB7 mutants
and unliganded ERl-554 is about as efficient as heterodimer formation between liganded wild type ER
and liganded ERl-554.
The level of ERl-554 in the immunoprecipitates
is
lower than the level of full-length ER, since homodimers of ERl-554 will not be immunoprecipitated
by the D75 antibody. It is also possible that binding of
bivalent antibody to full-length ER homodimer stabilizes it and blocks exchange of monomers. This is
consistent with the observation that antibody to ER
can increase the binding of wild type ER to the ERE in
gel shift assays (28).
The ER Mutants Antagonize
ER in Intact Cells
Transactivation
by
Another way to determine whether the NHB ER mutants are sequestered in an inactive complex is to
determine whether they exhibit biological activity in
vivo. Since the NHB mutants are unable to activate
transcription, but retain their capacity for DNA binding
and heterodimer formation, we examined the question
of whether they could act as dominant negative mutants in CHO cells. The ability of the NHB mutants to
antagonize estrogen-dependent
transcription by wild
type ER was evaluated in a series of transfections in
which different ratios of the expression plasmids encoding wild type ER or one of the NHB mutants was
used. With increasing ratios of mutant protein to wild
type ER protein, there was a progressive decline in the
activity of wild type ER. At 1 :l, 5:1, and 1O:l ratios of
the two proteins, NHBl suppressed ER activity by
37%, 64%, and 82%, while NHB7 suppressed activity
by 33%, 46%, and 55% (Fig. 8). The most straightforward explanations for how an ER mutant might act as
a dominant negative mutant are based on its ability to
compete with wild type ER for binding to the ERE and
to form inactive heterodimers with wild type ER (see
below). If the NHBl and NHB7 ER mutants were sequestered in an inactive complex, it is difficult to see
how they could suppress the activity of wild type ER.
These data provide additional support for the view that
while ligand binding is required for transactivation, it is
not required for DNA binding or dimerization of the
receptor.
DISCUSSION
There are two basic models to account for the role of
ligand in steroid/nuclear receptor action. Members of
the thyroid and retinoic acid receptor subfamily do not
appear to exist in inactive complexes with heat shock
proteins and are, therefore, able to form homodimers
and heterodimers in the absence of ligand and to bind
to their response elements on DNA (reviewed in Ref.
3). The unliganded forms of these nuclear receptors
often act as repressors of transcription (40,41, 45-48).
In apparent contrast, there is substantial evidence that
the unliganded forms of GR and PR exist in a complex
with heat shock proteins (14, 22, 24, 25, 49) and are
usually unable to bind to their response elements on
DNA (12, 16, 19-21, 50). A plausible model has been
proposed, in which binding of the ligand causes the
dissociation of this complex and exposes the major
dimerization region of the receptor (3). This enables
the liganded receptor to dimerize and bind with high
affinity to the glucocorticoid
response element/progesterone
response
element.
These
structural
changes and related covalent modifications enable the
bound receptor to function as a ligand-dependent
transcription activator (3). There has been considerable disagreement on the applicability of these models
to the role of ligand in ER action (7, 12, 26-31). Since
some of the apparent disagreement between different
studies may have been due to the presence of traces
of estrogen in the medium used in the studies, we
generated ER mutants unable to bind E,. Binding
MOL END0 . 1995
Vol 9 No. 4
462
D75 HZ22
ERl-554 -
WT hER\,
EM-554 /-*
.,,
+./NH67
“*-‘. ERl-59
B
A
Fig. 7. NHBl and NHB7 Efficiently Heterodimerize with a Truncated ER in Viva
Extracts were prepared from COS-1 cells transfected with expression plasmids encoding wild type ER or the NHBl or NHB7
mutants. The extracts were immunoprecipitated
as described in Materials and Methods with monoclonal antibody D75. The
immunoprecipitates
were dissolved in SDS sample buffer, fractionated by SDS polyacrylamide gel electrophoresis, and then
subjected to Western blot analysis using antibody D547, which recognizes both the full length ERs and the truncated receptor
ERl-554. In segment A, in a control experiment, extracts from COS cells were transfected with expression vector for the
truncated ER (ERI-554) and immunoprecipitated with either antibody D75 or H222 (which binds to the ERI-554 mutant), and the
immunoprecipitates were fractionated by SDS gel electrophoresis and analyzed by Western blotting with monoclonal antibody
D547. These data show that antibody D75 does not react with the ERl-554 mutant. In segment B, COS-1 cells were cotransfected
with expression vectors encoding ERl-554 and wild type ER. Cells were treated with lo-’ M E, after transfection. Cell extracts
were immunoprecipitated
with antibody D75, proteins were resolved by SDS polyacrylamide gel electrophoresis, and the ERs
were visualized by Western blotting with antibody D547. C, COS-1 cells were cotransfected with expression vectors for ERl-554,
and for either NHBl (lane 1) or NHB7 (lane 2) in the absence of E,. Cell extracts were immunoprecipitated
with monoclonal
antibody D75, and the immunoprecipitates
analyzed by Western blotting with monoclonal antibody D547.
1 T
1
km
1:1
5:l
1O:l
1:1
5:l
10:1
MlJTANT:WILDTYPE
Fig. 8. Examination of the Abilities of ER Mutants to Block
Wild Type ER Activity in CHO Cells
CHO cells were transfected with the (ERE),-TATA-CAT
reporter plasmid, 5 ng of expression vector encoding wildtype ER and increasing amounts of expression vector encoding either NHBl or NHB7. The ratios shown in the panel are
the ratios of wild type and mutant receptor.protein.
After
transfection, cells were treated with lo-’ M E, for 48 h and
then harvested. Cell extracts were prepared and analyzed for
CAT activity. Values represent the mean t SEM for at least
three separate transfections.
studies showed that two of these mutants, NHBI and
NHB7, exhibited at least a lOO-fold decrease in their
ability to bind estrogen (Fig. 3). These ER mutants,
therefore, have negligible ability to bind estrogens at
physiologically relevant concentrations. These ER mutants also lacked significant ability to activate transcription. We were therefore able to carry out studies
aimed at determining the site at which their inability to
bind estrogen renders them inactive. Since receptor
extracted from cells might have been inadvertently
freed from a complex with other proteins, and ER
made in a cell-free translation system might not be
efficiently assembled into such a complex, we carried
out most of our studies using assays of ER function in
intact mammalian cells.
We used an in vivo promoter interference assay to
monitor the ability of the NHBl and NHB7 ER mutants
to interact with the ERE. To accurately compare the
abilities of the NHBl and NHB7 mutants and wild type
ER to interact with the ERE, we carried out our experiments across a range of levels of transfected expression vector DNA. The unliganded NH61 and NHB7
mutants and liganded wild type ER exhibited similar
abilities to inhibit the activity of the promoter across
the entire range of DNA concentrations
(Fig. 6). It
therefore seems clear that the unliganded NHBl and
NHB7 ER mutants are as effective in binding to the
ERE in vivo as liganded wild type ER. There have been
a few other studies in which promoter interference
assays were used to measure the interaction of hER
with the ERE. McDonnell et al. (32) reported that unliganded ER produced in yeast from low copy number
plasmids did not bind to the ERE, while ER produced
from high copy number plasmids bound to the ERE in
the absence of ligand, and a recent report (51) indicates that it dimerizes in vitro. In apparent contrast,
substantial binding to the ERE was observed in mammalian cells containing both high and low levels of
endogenous ER (34). Reese and Katzenellenbogen
(34) attempted to exclude the possibility that low concentrations of estrogen in the culture medium were
responsible for binding by showing that the ER exhibited little ability to activate transcription in the absence
of added estrogen. However, it was not possible to
completely exclude the possibility that there are different E, dose-response curves for promoter interference and transcription activation. Our finding that ER
mutants which do not bind estrogen show efficient
binding to the ERE in the promoter interference assay
extends those studies by excluding any potential contribution of residual estrogen to ERE binding. It is not
Unliganded ER Dimerizes and Binds to the ERE
presently clear why the ER in yeast and Xenopus cells
exhibits ligand-dependent
binding to the ERE, while
ER in mammalian cells is able to bind to the ERE in the
absence of ligand. It is possible that this reflects actual
differences in ER action in Xenopus, whose ER must
function at 18 C, rather than at 37 C. Alternatively, the
differences may be related to differences in the way
DNA is packaged into chromatin after transient transfections in these systems. What is clear from these
differences is that binding of the ER to the ERE does
not represent the critical determining step that converts the liganded form of hER into an effective
transcription activator.
There are, in principle, at least two ways in which the
efficient binding of the NHB ER mutants to the ERE
could represent an experimental artifact. If the ER was
proteolyzed to a form that does not bind to heat shock
proteins, the ER might exhibit constitutive binding. Our
observations that the wild type ER and the ER mutants
exhibit a single band on gel mobility shift assays and
that immunoprecipitation
of both the ER mutants and
wild type ER from COS-1 cells followed by Western
blotting reveals only the full-length receptor argue
against this possibility. Also, deletion of significant
segments of the ligand-binding
domain that would
prevent association with heat shock proteins would
also prevent ER dimerization. It seems unlikely, therefore, that our data are due to partial proteolysis of the
ER.
The second possibility is that we have carried out
the promoter interference assays at concentrations of
transfected DNA far beyond those required for liganded ER to saturate the ERE. To eliminate this possibility we carried out promoter interference at subsaturating levels of transfected DNA. Since subsaturating
levels of the NHBl and mutants and wild type ER
exhibited similar abilities to interfere with the promoter
at levels of inhibition ranging from IO%-55%, it seems
clear that we are not carrying out our studies at concentrations of the expressed ERs far beyond those
required to saturate the EREs.
The related possibility that the level of ER in these
transfected cells is sufficient to exhaust the supply of
heat shock proteins also seems improbable. HSPSO
and HSP70 are among the most abundant proteins in
eukaryotic cells. Unliganded glucocorticoid
receptor
overexpressed in CHO cells is efficiently bound to heat
shock proteins (52).
The observation that E, increased the ability of ER to
bind to the ERE in promoter interference assays led to
the proposal that it either stabilized the receptor
against degradation or facilitated or stabilized receptor
dimerization (34). ER mutants unable to dimerize show
a dramatic reduction in their ability to bind to DNA in
vitro (7). Since we found that the NHB mutants effectively bound to the ERE, it seemed possible that they
were able to dimerize in the absence of ligand. We
cotransfected DNA encoding either of the NHB mutants and a truncated ER into cells, prepared extracts,
and carried out immunoprecipitation
and Western
463
blotting. Under conditions in which the NHB mutants
and the truncated ER did not contain bound ligand,
they formed heterodimers that were immunoprecipitated as efficiently as heterodimers between the liganded forms of wild type ER and the truncated ER. These
data indicated that the unliganded ER retained an
unimpaired ability to dimerize. Since these experiments were carried out in COS-I cells, our observation
that the unliganded ER is able to interact with other cell
components are not restricted to CHO cells. The immunoprecipitation
and Western blotting assays are
extremely difficult to use to quantitate the percentages
of the various heterodimers that are immunoprecipitated. What is critical is the data demonstrating that
the unliganded and liganded heterodimers are immunoprecipitated to a similar extent (Fig. 7).
The third line of evidence supporting the view that
the NHB mutants are able to interact with other cell
components stems from their activity as dominant
negative mutants. Three potential sites of action have
been proposed to account for the ability of inactive
nuclear receptor mutants to suppress the activity of
the wild type receptor when they are coexpressed in
the same cells: 1) Competition for binding to the hormone response element; 2) the formation of inactive
heterodimers; 3) binding to a limiting component of the
transcription apparatus. The ability of the NHB mutants to act as dominant negative mutants therefore
provides additional evidence that the unliganded ER is
not in an inactive complex with other proteins.
Although the NHBl and NHB7 ER mutants were
able to suppress the activity of wild type ER, both
NHBl and NHB7 were considerably less potent than
three recently described dominant negative mutants
that contained deletions or mutations in the AF2 transactivation domain (35). At equal levels of NHBl or
NHB7 and wild type ER, the activity of wild type ER
was suppressed by approximately 35%. In contrast,
the mutations in the ER transactivation domain suppressed activity by 60-80%. Similarly, in a recent
study of the ability of dominant negative mutants to
suppress the activity of the endogenous ER in MCF-7
cells, NHB7 was effective only at much higher levels of
transfected DNA than the dominant negative mutants
containing mutations or deletions in the AF2 transactivation domain (B. A. Ince, D. Schodin, D. Shapiro,
and B. S. Katzenellenbogen,
submitted for publication). The structural basis for the large differences in
the ability of different ER mutants to act as dominant
negative mutants is presently unknown.
While our studies clearly demonstrate that ligand
binding is not a prerequisite for receptor dimerization
and binding to the ERE, they do not resolve the question of the role of heat shock proteins in ER action.
Several studies have shown that ER binds to heat
shock proteins (53, 54). An attractive possibility has
been proposed by Schlatter et al. (54) to explain their
data comparing the binding of heat shock proteins to
ER and GR. Although heat shock proteins bound to in
vitro synthesized ER, binding required additional se-
MOL END0
464
1995
Vol 9 No. 4
quences not important in the interaction of GR with
heat shock proteins. Based on their observation that
the interaction of the heat shock proteins with the ER
seemed weaker than with the GF?, and the observation
by several laboratories that unliganded ER, but not
GR, is translocated into the nucleus (5.5, 56), they
proposed that the association of heat shock proteins
with ER is weak and transitory. They theorized that this
association might be related to the ability of heat
shock proteins to facilitate correct protein folding. In
this model, interaction of heat shock proteins with ER
plays a role in preventing ER from forming inactive
complexes with other proteins while it is folding, and in
assuming a correct protein conformation. Once the
protein is correctly folded the heat shock proteins
would either dissociate from the receptor or remain
bound but not affect its ability to dimerize or bind to
the ERE.
In this work we clearly demonstrate that ER mutants
unable to bind estrogen effectively dimerize and bind
to the ERE. The failure of these mutants to activate
transcription is therefore not due to their inability to
bind to the ERE and may be a consequence of their
inability
to induce
ligand-dependent
structural
changes required for the ER to function as a transcription activator.
MATERIALS
AND METHODS
Materials
rH]Acetyl CoA (0.5 mCi/ml) and [3H]E2 were from DuPont/
New England Nuclear (Boston, MA). Radioinert E, was obtained from Sigma Chemical Co (St. Louis, MO). Monoclonal
antibodies to ER were a generous gift of Dr. Geoffrey Greene
(University of Chicago, Chicago, IL). Restriction enzymes and
other enzymes used in cloninq and sequencinq were from
GIBCO/BRL, (Gaithersburg, MD) and from U.S. Biochemicals
(Cleveland, OH).
Preparation
Plasmids
of ER Mutants
and Reporter
charcoal dextran-treated fetal calf serum. CHO cells were
plated at 2.10s cells/60 mm plate (Falcon), maintained at 37
C in a 5% CO, incubator for 48 h and transfected by CaCI,
coprecipitation
with glycerol shock (34, 61). Each 60-mm
plate was treated with 0.4 ml coprecipitation solution containing 1 pg pATC2 reporter plasmid, 1 pg PT109 luciferase
internal standard, 5-50 ng expression plasmids in pCMV5
encoding wild type ER (58), or the ER mutants, and 6 pg
PTZ18U carrier DNA. Transfections and glycerol shock were
essentially as described (35). CAT assays were carried out by
our mixed phase assay (62). Luciferase and protein assays
were as described (42).
For promoter interference assays CHO cells were transfected with 0.4 wg CMV-(ERE),-CAT plasmid, 1 pg SV40luciferase DNA as internal control, 2 ng of the expression
plasmids encoding wild type ER or NHB7, or 10 ng of the
plasmid encoding NHBl, and 6.6 Fg pTZ18U carrier. Cells
were then harvested 24 h after transfection and assayed for
CAT activity as described above.
COS-1 cells were used to overexpress the ER using a
modification of the method of Reese and Katzenellenbogen
(58). Cells were maintained at 37 C in 5% CO,, in DME/F12
medium supplemented with 10% charcoal-dextran treated
fetal calf serum. COS-1 cells were plated at 3.1 O5 cells per
loo-mm dish and maintained for 40 h, and the medium was
replaced. After 5 h in fresh medium, the plates were treated
with 1 ml DNA precipitate containing 2-10 pg of the CMV5
expression vector encoding ER or one of the ER mutants, and
PTZ18U carrier DNA to a total of 20 pg DNA. Chloroquine
was then added to each plate to a final concentration of 50
FM. After 5 h, the cells were shocked with 10% dimethylsulfoxide in serum-free media for 3 min, followed by two washes
with Hanks balanced salt solution. Cells were then incubated
in 10 ml complete medium for 40 h, washed twice with cold
PBS, and then harvested in cold PBS. The cells were pelleted
(3,000 rpm for 5 min), dissolved in 75 ~1 of extraction buffer
containing 20 mM Tris, pH 7.5, 2 mM dithiothreitol (Dll), 0.5
mM NaCI, 10% glycerol, 0.1 mM EDTA, 50 pg/ml leupeptin, 5
@g/ml phenylmethylsulfonylfluoride
, 1 Kg/ml pepstatin, 5
wg/ml aprotinin. Cell extracts were prepared either by two
freeze-thaw cycles, or by homogenization,
followed by
ultracentrifugation at 45,000 rpm at 4 C for 20 min.
Hormone Binding
the El3 Mutants
Assays for Wild Type ER and
Whole cell extracts were prepared from COS-1 celis transfected with either wild type ER or one of the ER mutants. The
aliquots were normalized by Western blot analysis to contain
the same amount of receptor. Samples were incubated at 22
C for 2 h with various concentrations of [3H]E2 in the presence
or absence of a 200-fold excess of radioinert E,. Free [3H]E2
was removed by adsorption on to 0.5% charcoal-0.05%
dextran, and the [3H]E2 bound to ER or ER mutants was
quantitated by scintillation counting.
Oligonucleotide-primed
site-directed
mutagenesis of the
hER cDNA was performed using the dut ung- method (57)
using M13K07 as helper phage. Single-stranded DNA templates were prepared from the pCMV.5 phagemid vector (58,
59). The identity of the mutants was confirmed by DNA sequencing.
We have previously described the (ERE),-TATA-CAT reporter plasmid [which we previously referred to as pATC2
(42)]. The luciferase reporter plasmid pT109 (60) was used as
the internal control. pTZ18U was used as carrier DNA in the
transfections. The CMV-(ERE),-CAT promoter interference
plasmid has been described (34). The plasmid encoding
ERl-554, which lacks the F domain, was recently reported
(44).
Samples were fractionated on either a 12% or a 7.5% SDS
polyacrylamide gel (63) followed by overnight transfer of proteins to a nitrocellulose sheet (Schleicher & Schuell, Keene,
NH), which was subjected to Western blotting using anti-ER
monoclonal antibody D547 as described (64), except that the
chemiluminescence-based
ECL system (Amersham Corp.,
Arlington Heights, IL) was used.
Cell Cultures
Gel Mobility
and Transfections
Chinese Hamster Ovary (CHO) cells were maintained in phenol-red free Dulbecco’s modified Eagle’s (DME)/F12 tissue
culture medium (Sigma, No. 2906) supplemented with 5%
lmmunoblots
Shift Assays
A 51 -base pair oligonucleotide probe containing a consensus
ERE was cut from the plasmid PM-2 (42) and was labeled
with [3’P]ATP by filling in the sticky ends with Klenow frag-
Unliganded ER Dimerizes and Binds to the ERE
ment. Cell extracts containing 20 Kg protein were incubated
with 1-2 pg of poly(deoxyinosine-deoxycytosine)
on ice in a
20 ~1 reaction containing: 15 mM Tris, pH 7.9, 80 mM KCI, 4
mM DTT, 0.2 mM EDTA, and 10% glycerol. Radiolabeled
probe (10,000 cpm) was added to the mixture, and the reaction was incubated for 20 min at room temperature. Free
probe and ER-DNA complex were separated on a 5% low
ionic strength ployacrylmide gel as described (65).
lmmunocoprecipitation
Containing Truncated
of Heterodimers
ER and Mutant ER
COS-1 cells were cotransfected with expression plasmids
encoding a truncated ER (ERl-554) and one of the NHB ER
mutants. Western blots were used to confirm that the proteins were expressed at equal levels in the cells. Cell extracts
containing 100-200 pg total protein were incubated with
anti-ER monoclonal antibody D75 on ice for 1 h, in buffer
containing 20 mM HEPES, pH 7.8, 50 mM KCI, 10% glycerol,
1 mM DTT, as well as protease inhibitors (50 pg/ml leupeptin,
5 Kg/ml phenylmethylsuifonyl
fluoride, 1 Fg/ml pepstatin, 5
pg/ml aprotinin), followed by incubation with rabbit-anti-rat
immunoglobulin G on ice for 0.5 h; the reaction mixture was
then incubated with a 10% slurry of zysorbin, which contains
protein A on the membrane surface and thus has high affinity
for rabbit immunoglobulin G. The immunoprecipitates
were
pelleted by centrifugation at 12,000 x g at 4 C for 5 min,
released into SDS sample buffer by boiling for 5 min, and
analyzed on Western blots.
Acknowledgments
We are grateful to Dr. S. Nordeen for the gift of the luciferase
plasmid, to Dr. G. Greene who generously provided the
monoclonal antibodies, and to Dr. B. W. O’Malley for supplying a preprint of a review of nuclear receptor action.
Received August 2, 1994. Revision received December 16,
1994. Accepted December 28, 1994.
Address requests for reprints to: Professor David Shapiro,
Department of Biochemistry, B-4 RAL, University of Illinois,
600 South Mathews Avenue, Urbana, Illinois 61801,
This research was supported by NIH Grants HD-16720 (to
D.J.S.) and CA-60524 (to B.S.K. and D.J.S.).
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