Mouse Estrogen Receptor ß Forms Estrogen Response Element-Binding Heterodimers with Estrogen Receptor
Katarina Pettersson,
Kaj Grandien,
George G. J. M. Kuiper and
Jan-Åke Gustafsson
Department of Medical Nutrition and Center for Biotechnology,
Karolinska Institute, S-14186 Huddinge, Sweden
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ABSTRACT
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The recent discovery that an additional estrogen
receptor subtype is present in various rat tissues has advanced our
understanding of the mechanisms underlying estrogen signaling. Here we
report on the cloning of the cDNA encoding the mouse homolog of
estrogen receptor-ß (ERß) and the functional characterization of
mouse ERß protein. ERß is shown to have overlapping DNA-binding
specificity with that of the estrogen receptor-
(ER
) and
activates transcription of reporter gene constructs containing
estrogen-response elements in transient transfections in response to
estradiol. Using a mammalian two-hybrid system, the formation of
heterodimers of the ERß and ER
subtypes was demonstrated.
Furthermore, ERß and ER
form heterodimeric complexes with retained
DNA-binding ability and specificity in vitro. In addition,
DNA binding by the ERß/ER
heterodimer appears to be dependent on
both subtype proteins. Taken together these results suggest the
existence of two previously unrecognized pathways of estrogen
signaling; I, via ERß in cells exclusively expressing this subtype,
and II, via the formation of heterodimers in cells expressing both
receptor subtypes.
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INTRODUCTION
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Nuclear receptors represent a large family of transcription
factors that regulate the activity of target genes by direct binding to
specific DNA recognition elements located in the vicinity of the
transcription start site of genes. Members of this gene family have an
evolutionary and functionally conserved structure with a hypervariable
N-terminal region that contributes to the transactivation function, a
centrally located, well conserved DNA-binding domain (DBD), and a
C-terminal domain involved in ligand binding, dimerization and
transactivation functions (1).
Steroid hormone receptors constitute a distinct subgroup within the
nuclear receptor family (2), which includes receptors for
glucocorticoids, mineralocorticoids, androgens, progestins, and
estrogens [glucocorticoid receptor, mineralocorticoid receptor,
androgen receptor, progesterone receptor, and estrogen receptor (ER),
respectively]. In addition two orphan nuclear receptors, the ERR-1 and
2 (ER-related receptors) (3) have been referred to this group (4). The
steroid hormone receptors bind as homodimers to palindromic DNA
response elements (2). Another important feature of steroid hormone
receptors is the interaction with the molecular chaperone hsp 90
(5).
Estrogens influence growth, differentiation, and function of many
target tissues, including tissues of the female and male reproductive
tract (6). Estrogens also play an important role in the maintenance of
bone mass and in the cardiovascular system where estrogens have certain
protective effects (7, 8). The ER-encoding cDNAs have been cloned from
several species (9, 10, 11, 12). Important examples of genes regulated by
estrogens are the PR, epidermal growth factor receptor, certain growth
factors (insulin-like growth factor-I, transforming growth factor-
and -ß) and several protooncogenes (c-fos,
c-myc, c-jun) (13). Loss of ER function has long
been postulated to be incompatible with life, and therefore the
successful generation of ER-deficient mice came as a surprise (14).
These mice are viable but display severe dysfunction of the
reproductive organs, and both sexes are sterile. The females have
hypoplastic uteri and hyperemic ovaries with no detectable corpora
lutea. The fact that disruption of the ER gene did not completely
eliminate the ability of small follicles to grow, as was evident from
the presence of secondary and antral follicles in the knock-out mouse
ovary, pointed to the possible existence of alternative ER mediating
the intraovarian effects of estradiol. In some tissues from the ER
knock-out mice residual binding of estradiol with an affinity and
specificity reminiscent of an ER protein could be measured (14, 15). We
have recently cloned a novel ER cDNA from rat prostate (16), which was
suggested to be named rat ERß subtype to distinguish it from the
previously cloned ER cDNA (consequently ER
subtype). The rat ERß
protein was found to be highly homologous to the rat ER
protein,
particularly in the DBD (>95% amino acid identity) and in the
C-terminal ligand-binding domain (55% amino acid identity). In
ligand-binding assays rat ERß binds estrogens with an affinity and
specificity resembling that of ER
, and ERß is able to activate
transcription of an estrogen-response element containing reporter gene
construct (16, 17). In subsequent studies it was shown that ERß is
the primary ER subtype expressed in rat ovary and that ERß message is
down-regulated by gonadotropins in granulosa cells, suggesting that the
functional significance of estrogen action in the rat ovary may be
mediated primarily by ERß (18).
The detailed biological significance of the existence of two ERs is
presently unclear. Perhaps the existence of two ER subtypes may
provide, at least in part, an explanation for the selective actions of
estrogens and certain antiestrogens in different target tissues (19, 20).
In this paper we describe the cloning of the mouse ovary ERß-cDNA and
the characterization of the mouse ERß protein with respect to DNA
binding, homo- and heterodimerization, and transactivational
functions. Finally, cotransfection of both ER subtypes with an estrogen
response element (ERE) containing reporter gene construct showed that
the formation of heterodimeric ER
/ERß complexes may indeed
constitute a novel estrogenic gene-regulatory pathway.
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RESULTS AND DISCUSSION
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A Homolog of Rat ERß Is Expressed in Mouse Ovary
The recent discovery of a novel ER protein present in rat
prostate and ovary (16) has given a new perspective to studies of
estrogen action. The mouse represents an important model system for
studies of gene function in mammals. We therefore investigated whether
a homolog to the previously cloned rat ERß (rERß) is present in the
mouse. Oligonucleotides, constructed to encompass the coding sequence
of the rERß cDNA, were used in an RT-PCR amplification of total RNA
isolated from mouse ovaries. Amplification products with the expected
size (
1.5 kbp) were subcloned and sequenced. The open reading frame
of these clones displayed a high degree of amino acid identity with the
rERß protein and were therefore recognized as the mouse homolog of
rERß and will hereafter be referred to as the mouse ERß (mERß).
As shown in Fig. 1
, the mERß amino acid
sequence also manifests considerable similarities to mouse and rat
ER
in the DNA- and ligand-binding domains (Fig. 1
). Several amino
acid residues that have been demonstrated to be required for
high-affinity binding of estradiol by ER
(21) were found to be
conserved in rat ERß. The rat ERß binds estradiol (E2)
with an affinity very comparable to that of ER
(17). Since the same
amino acid residues are also conserved in the ligand-binding domain of
mERß, we concluded that mERß should bind E2 in a
similar manner as the rat ERß.
mERß Protein Binds to an ERE
Members of the nuclear receptor superfamily bind to specific
DNA motifs, generally palindromic or direct repeats of the sequence
AG(A/G)(A/T)CA (22) located in the vicinity of the promoter of target
genes. The specificity of DNA recognition by nuclear receptors is
mediated essentially through the so called P-box, a short stretch of
amino acids located in the N-terminal zinc finger in the DNA binding
domain (DBD) (Ref. 1 and references therein). Amino acids in the P-box
have been demonstrated to make direct contacts with bases in the DNA
response elements, thus participating in dictating the DNA binding
specificity (23). The P-box of ER
(EGCKA) differs from the P-box of
other members of the steroid receptor subgroup, such as glucocorticoid
receptor and progesterone receptor, with the result that ER
recognizes DNA elements contrasting from the sequences recognized by GR
and PR. The consensus ERE consists of a palindromic repeat of the core
sequence AGGTCA spaced by three nucleotides (24). The high degree of
conservation in the DBD of the
and ß ER subtypes (
96%, Fig. 1
) and the absolute identity of the P-box sequences strongly suggest a
shared DNA recognition specificity between the two ER subtypes. We
consequently performed DNA binding studies with mERß using
radiolabeled consensus ERE oligonucleotides.
Mouse ERß and human ER
protein were synthesized in
vitro in a rabbit reticulocyte lysate (RRL) system before
incubation with E2 and a radiolabeled double-stranded ERE
oligonucleotide. The resulting DNA-protein complexes were analyzed by
electrophoretic gel mobility shift assay (Fig. 2
). ERß binds to the wild type
consensus ERE both in the absence or presence of E2 (lanes
2 and 3), but not to an ERE mutated in both half-sites (Fig. 2
, lanes 7
and 8). This coincides with the binding specificity of ER
(Fig. 2
, lanes 4 and 5 and lanes 9 and 10, respectively). In contrast to results
obtained in a recent study by Tremblay and co-workers (25), we did not
observe a reduced affinity for the ERE by ERß when equal amounts of
ER
and ERß protein were used (as quantitated by
[35S]methionine labeling). The reason for this
discrepancy is unclear.

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Figure 2. In Vitro Synthesized Mouse ERß
Binds to an ERE
32P-labeled DNA fragments corresponding to a consensus ERE
(lanes 15) or a derivative ERE mutated in both half-sites (lanes
610) were incubated with unprogrammed lysate (lanes 1 and 6) or ERß
(lanes 23 and 78) or ER (lanes 45 and 910) synthesized in
the RRL system. DNA-protein complexes were fractionated on a
nondenaturing acrylamide gel. The gel was dried and autoradiographed at
-70 C.
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ERß Binds to the ERE as a Homodimer
Efficient DNA binding and transactivation function of nuclear
receptors are often dependent on the formation of dimers. Members of
the steroid receptor subgroup have been shown to interact with DNA
predominantly as homodimers, whereas the receptors for retinoic acid,
thyroid hormone, and vitamin D, as well as several orphan nuclear
receptors, form heterodimeric complexes with the ubiquitous partner RXR
(retinoid X receptor) (26). Importantly, some orphan nuclear receptors
have been demonstrated to bind to DNA as monomers (27). To establish
whether mERß binds to DNA as a homodimer or as a monomer, we
generated a deletion mutant construct in which the 91 most N-terminal
amino acids of the A/B domain of ERß were removed (Fig. 3A
). This region of steroid receptors has
not been shown to participate in DNA recognition, and we therefore
anticipated that the truncated mERß would be able to bind to DNA as
efficiently as the wild type receptor. The mERß construct was also
tagged with a nine-amino acid HA1-epitope, which is recognized by the
monoclonal 12CA5 antibody (28). The truncated epitope-tagged mERß
(ERß-TAG) in electrophoretic mobility shift assays gave rise to a
DNA-protein complex clearly distinguishable from the complex formed by
the wild type ERß (ERß-wt), (Fig. 3B
). After mixed synthesis of
ERß-TAG and ERß-wt in the RRL system, a third complex of
intermediate mobility was visible (Fig. 3B
, lanes 24) representing
the dimer between wtERß and ERß-TAG. This experiment clearly shows
that ERß binds to DNA as a homodimer, similar to the binding of ER
(Refs. 1 and 2 and references therein).

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Figure 3. ERß Binds to the ERE as a Dimer
A, Schematic presentation of the wild type ERß (ERß-wt) and the
truncated receptor (ERß-TAG). The DBD and the LBD are
overlined in the wild type receptor. The inserted
hemaglutenine (HA) epitope of the ERß-TAG is indicated
(dotted) and overlined. B,
32P-labeled consensus ERE was incubated with wild type
ERß alone (ERß-wt, lane 1), or ERß-TAG alone (lane 5), or both
proteins together in 3:1, 1:1, and 1:3 ratios (lanes 2, 3, and 4,
respectively) after synthesis in the RRL system. DNA-protein complexes
were analyzed as described in the legend to Fig. 2 .
Arrows point to complexes formed by ERß-wt and
ERß-TAG as indicated. The arrow without label
indicates the band with intermediate migration, which represents the
heterodimer of wild type and truncated receptors (lanes 24).
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Coexpression of ERß and ER
Does Not Inhibit ER
-Stimulated
Activity of an ERE-Reporter Gene Construct
Having established the ability of mERß to bind to an ERE, we
proceeded to characterize the capacity of mERß to activate
transcription of an ERE-containing reporter gene in transient
cotransfections of mammalian cells. We also wanted to study whether
cotransfection of both ER subtypes would alter the activity of the ERE
reporter construct. Human fetal kidney 293 cells were cotransfected
with a luciferase enzyme reporter gene construct containing two copies
of a consensus ERE in front of a thymidine kinase promoter, together
with human ER
- or mERß-expressing plasmids, or both. As shown in
Fig. 4
, the E2-stimulated
activity of the reporter gene construct by mERß was lower when
compared with the activity obtained with ER
when studied under the
same transfection conditions. The 2-fold lower
E2-stimulated activity was not due to squelching of mERß,
since the reporter activity was dose-dependent with regard to the
amount of cotransfected mERß-expression plasmid (data not shown).
Although ERß and ER
are highly homologous in the DBD and in parts
of the ligand-binding domain (LBD), there remain substantial
differences, particularly in the N-terminal A/B domain (Fig. 1
). This
domain contains the AF-1 transcriptional activity function of ER
(29) and may have a similar function in ERß. The diverging A/B
domains and/or dissimilarities in the LBD of ERß and ER
may result
in differences in maximal transactivational activity of both ER
subtypes. The slightly lower maximal transactivational activity of
ERß compared with ER
has also been observed by other investigators
(25, 30). When ER
and ERß were cotransfected, however, the
reporter activity did not change significantly, when compared with the
activity observed with ER
alone (Fig. 4
). Based on these
observations we conclude that ERß does not repress the activity of
ER
. Furthermore, if the two receptors were competing as homodimers
for the ERE-binding sites, higher concentrations of ERß would be
expected to result in a decrease in reporter activity, toward the
activity pattern of ERß alone. Since no sign of such a competition
was observed, we speculated that an interaction was taking place
between the two ER subtypes (although we cannot rule out that ER
alone is responsible for the transcriptional activity of the reporter
gene). To be able to monitor a possible interaction between ERß and
ER
, we used a mammalian cell two-hybrid system.

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Figure 4. ERß and ER Activate Transcription from an
ERE-Containing Reporter Construct
Human fetal kidney 293 cells were transfected with a luciferase
reporter construct containing a tandem ERE and ERß or ER
expression plasmids as indicated (described in Materials and
Methods). Cells were treated with vehicle (-, gray
bars) or 10 nM 17ß-estradiol (10 nM
E2, black bars). The results are presented
as fold induction over values obtained from cells transfected with only
the luciferase reporter and treated with vehicle, which were
arbitrarily set to 1. Values represent the mean ± SD
of three independent experiments.
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ERß Interacts with ER
in Vivo and in
Vitro
The two-hybrid system provides a powerful technique for studying
potential interactions between two proteins within a cell. The
principle is, in short, to fuse protein A to an autonomous DBD and
protein B to a strong transactivation domain. The hybrid protein
constructs are cotransfected into cells together with a reporter gene
construct containing the cognate response element. The activity of the
reporter gene will depend on an interaction between the fusion
proteins, which will direct the transactivation domain to the promoter.
The system has been widely used in yeast, but is also applicable in
mammalian cells. For our studies we chose to use the DBD of the yeast
protein Gal4 and the transactivation domain of the viral factor VP16.
The Gal4-DBD was fused to the full-length mER
, and the VP16
transactivation domain was coupled to full-length mER
and mERß,
respectively (Fig. 5A
). The chimera
constructs were then cotransfected together with a Gal4-luciferase
reporter construct into COS 7 cells. Comparisons were made between
cells with or without E2 incubation. Reporter activity
remained low when cells were transfected with the vectors expressing
only the Gal4-DBD or the VP16 transactivation domain, or when either of
these constructs was transfected together with any of the hybrid
constructs (Fig. 5B
). In contrast, when the Gal4-mER
construct was
transfected together with the VP16-mER
construct or the VP16-mERß
construct, the activity of the reporter was induced approximately 3- to
4-fold, respectively (Fig. 5B
, two most right-hand panels,
white bars), compared with reporter transfected alone,
indicating an interaction between the chimeric proteins. In the
presence of E2 the induction levels rose to about 18-fold
(Fig. 5B
, two most right-hand panels, black
bars). These results clearly demonstrate an interaction between
the mERß and mER
proteins in vivo, suggesting that the
transcriptional activity observed during coexpression of ER
and
ERß (Fig. 4
) is, at least in part, due to the formation of
ER
/ERß heterodimers. The rise in reporter activity observed,
especially in the presence of E2 (
5-fold), with the
Gal4-mER
hybrid and the original VP-16 construct (Fig. 5B
, second panel) is in all likelihood due to the
ligand-inducible transactivation function of ER
itself, when
directed to the promoter of the Gal4 reporter gene construct by binding
via the Gal4 DBD.

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Figure 5. ERß Interacts with ER in a Mammalian
Two-Hybrid Protein System
A, Schematic description of the fusion proteins used in the two-hybrid
assay. The Gal4-DBD was fused to the full-length ER , creating
Gal4-ER , and the transactivating domain of VP16 (VP16-TA) was linked
to full-length ER and ERß, creating VP16-ER and VP16-ERß,
respectively. B, COS-7 cells were transfected with a luciferase
reporter plasmid
containing Gal4-binding sites and expression plasmids for
Gal4 (Gal4-), VP16 (VP16-), Gal4-ER , VP16-ER , and VP16-ERß as
indicated. The cells were treated with vehicle (-, white
bars) or 1 µM 17ß-estradiol (1 µM
E2, black bars). Data are presented as fold
induction and represent the mean ± SD of three
separate experiments performed in duplicate. The values obtained from
cells transfected with reporter alone and treated with vehicle were
arbitrarily set to 1. C, ER was labeled with
[35S]methionine by translation in RRL and incubated with
GST-mERß or GST-protein. Samples were subsequently incubated with
GST-Sepharose, washed, eluted in SDS buffer, and separated on 10%
SDS-PAGE gels.
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We also performed glutathione S-transferase (GST) pulldown
experiments with [35S]methionine-labeled ER
and a
GST-mERß fusion protein, in order to detect a direct interaction
between the two proteins in vitro. As shown in Fig. 5C
, ER
could be successfully coprecipitated with the GST-mERß fusion
protein but not with the GST alone (compare lane 2 to lane 3, lane
1 = input of ER
, 20%), demonstrating a direct interaction
between both ER subtypes.
ERß and ER
Form DNA-Binding Heterodimers
The results from the two-hybrid assay and from the pulldown
experiment suggested that ER
and ERß are able to form
heterodimers. In combination with the results from the cotransfection
experiments (Fig. 4
), it appeared likely that the putative ERß/ER
heterodimer would be able to bind to an ERE.
We performed electrophoretic mobility shift assay with in
vitro synthesized ER
and ERß to examine this possibility.
Because the wild type ERß migrates closely with ER
on native gels
(see Fig. 2
), we decided to use the truncated ERß-TAG (described
above and in Materials and Methods) in order to identify
DNA-protein complexes. ERß-TAG and ER
were synthesized in
vitro and then mixed at increasing and decreasing amounts,
respectively, incubated on ice, followed by incubation with the
32P-labeled ERE. An ERE-protein complex of intermediate
mobility was formed in samples in which the two ER subtypes were
coincubated (Fig. 6A
, lanes 25),
probably representing a heterodimeric complex between ERß-TAG and
ER
. This putative heterodimerization was also evident when
full-length wt ERß was used instead of ERß-TAG, but the complexes
were not as easily distinguished (not shown).

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Figure 6. ERß Forms DNA-Binding Heterodimers with ER
A, ER or ERß-TAG alone (lanes 1 and 7, respectively) or together
as indicated (lanes 26) were incubated with a 32P-labeled
consensus ERE, and DNA-protein complexes were separated on a
nondenaturing acrylamide gel. The arrow indicates the
intermediate complex formed in the presence of both ER and ERß. B,
ER or ERß-TAG alone or together were incubated with a mouse
monoclonal ER -specific antibody (ER ab, lanes 3, 58, and 10) or
with no antibody (lanes 2 and 9) together with 32P-labeled
ERE. Complexes were analyzed as above. C, Essentially the same
experiment as in B except that the 12CA5-TAG antibody recognizing the
epitope-tagged ERß (TAG ab, lanes 2, 47 and 9), or no antibody was
used (lanes 1, 3, and 8). D, 32P-labeled DNA fragments
corresponding to either a consensus ERE (lanes 14) or a derivative
ERE mutated in one of the half-sites (lanes 58) were incubated with
unprogrammed lysate (lanes 1 and 5) or ER (lanes 2 and 6) or ERß
(lanes 3 and 7) or a mixture of both ER and ERß (lanes 4 and 8).
DNA-protein complexes were analyzed as described above.
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To confirm the presence of both ER subtypes in the heterodimeric
complex, the DNA-binding assay was also performed in the presence of an
ER
antibody and the 12CA5 antibody. Figure 6B
shows the result of
incubation with the monoclonal ER
antibody 1D5. The ER
homodimer
is efficiently supershifted with this antibody as expected (Fig. 6B
, lane 3 compared with lane 2), whereas the ERß homodimer is not
affected (lanes 9 and 10). The intermediate complex formed in the
presence of both ER
and ERß-TAG is also supershifted with the
ER
antibody (Fig. 6B
, lanes 58 compared with lane 4), thus
demonstrating the presence of ER
within the heterodimeric complex.
In Fig. 6C
essentially the same experiment has been repeated using the
12CA5 monoclonal antibody directed against the HA-epitope of
mERß-TAG. The 12CA5 antibody successfully interacts with both the
ERß homodimer and the intermediate complex previously shown to
contain also ER
(Fig. 6C
, lanes 10 and 47). The 12CA5 antibody
does not cross-react with the ER
homodimer (lane 2). These results
clearly demonstrate that the intermediate complex that is formed when
ER
and ERß are coincubated contains both receptors and is a true
heterodimeric complex.
To determine whether both partners of the heterodimer participate in
DNA binding, experiments were performed with an ERE mutated in one of
the half-sites at a position previously demonstrated to be crucial for
efficient binding by the ER
protein (31). In crystallographic
studies of the DBD of ER
bound to the ERE (31), it was established
that a mutation in one of the half-sites resulted in reduced
cooperativity in binding to the second half-site, due to lack of proper
interaction between the dimer interfaces present in the DBD. Binding by
ER
to such a mutated ERE was therefore less efficient. We found that
ERß did not bind to this mutated ERE in analogy to the ER
(Fig. 6D
, lanes 7 and 6, respectively). In addition, no protein-DNA complex
was formed with the heterodimer (lane 8), indicating that cooperativity
in DNA binding is also required for efficient DNA binding by
ERß/ER
heterodimers.
Furthermore, the ERß as well as ER
were unable to bind to
oligonucleotides containing direct repeats of the core sequence AGGTCA
spaced by one or four nucleotides (DR1 or DR4), irrespective of the
presence or absence of retinoid X receptor (data not shown).
Our findings on ER
/ERß heterodimerization and the recent
demonstration of GR/MR heterodimerization (32, 33) challenge the
commonly held view that steroid receptors form only homodimers.
Previous biochemical and structural evidence indicated that steroid
receptors form homodimers through a dimerization interface within their
zinc finger DNA binding domain, and a generally much stronger
dimerization interface within the ligand binding domain (Refs. 1 and 22
and references therein). Further studies will be required to localize
the dimerization interfaces involved in the formation of ER
/ERß
heterodimers.
The rat tissue distribution and/or relative level of ER
and ERß
mRNA seems to be quite different; that is, moderate to high expression
in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal
for ER
and prostate, ovary, uterus, lung, bladder, brain, and testis
for ERß (17). This may imply that in testis and ovary both subtypes
are expressed to some extent. In the mouse, both ER mRNAs can be found
in ovary and uterus (not shown). In the rat, hypothalamus ER
and
ERß are coexpressed in certain regions, most likely in the same
neurons (34). The coexpression of ER
and ERß in the same tissue
and/or cells suggests the interesting possibility that ER
and ERß
proteins may interact with each other. In this study we have indeed
shown that the two ER subtypes have the ability to form heterodimers.
The discovery of an ERß protein and the ability of ER
and ERß to
form heterodimers strongly suggest the existence of two previously
unrecognized pathways of estrogen signaling: via ERß homodimers in
cells exclusively expressing this subtype and via ER
/ERß
heterodimers in cells expressing both subtypes (Fig. 7
). The ERß homodimers and the
ER
/ERß heterodimers may possibly interact with novel response
elements, different from the known EREs. By such a mechanism the
physiological regulatory potential of estrogenic hormones may be
greatly expanded. Different target tissues may respond differently to
the same hormonal stimulus due to alternative composition of receptors.
Varying ratios of ER
and ERß in different cells, resulting in
different populations of homo- and heterodimers, could constitute a
hitherto unrecognized mechanism involved in the tissue- and cell
type-specific effects of estrogens and certain antiestrogens (19, 20).
Future studies will be required to determine the physiological
significance of the existence of more than one ER protein.
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MATERIALS AND METHODS
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Cloning of Mouse ERß cDNA
Total RNA from ovaries dissected from 5-week-old mice was
prepared as described (35). Complementary DNA was synthesized using
Super-Script reverse transcriptase (GIBCO BRL, Paisley, Scotland) as
described previously (36) using 1 µg of total RNA. PCR amplification
of the cDNA was carried out with 35 cycles of repeated denaturation for
15 sec at 95 C, 15 sec of annealing at 57 C, and 60 sec of extension at
72 C with Taq-polymerase (Pharmacia, Uppsala, Sweden) under
the conditions described by the manufacturer with 1:20 of synthesized
cDNA and oligonucleotides Erbkg3 5'-ATGAGTATTCAGCCATGGCATTCTACAG and
Erbkg4 5'-CAGGCCTGGCCATCACTGAGACTG, which were constructed to encompass
the entire coding region of rat ERß cDNA. To facilitate subsequent
subcloning, an NcoI restriction enzyme recognition site was
introduced over the start codon (bases -2 to +4). The PCR product was
visualised on a 1% agarose gel, revealing a single band of
approximately 1450 bp, corresponding in size to the rat ERß open
reading frame. The DNA band was excised and the DNA was purified using
the QIAEX gel purification kit (QIAGEN, Hilden, Germany). The resulting
DNA was phosphorylated with T4 polynucleotide kinase (Amersham, Solna,
Sweden) and cloned into the T-overhang vector pTKS (37). The insert of
pTKS-mERß was sequenced by the dideoxy method (38) with T7
DNA-polymerase (Pharmacia).
Plasmid Constructs
For in vitro transcription/translation in RRL,
pTKS-mERß was digested with NcoI and EcoRI,
which yielded a fragment encompassing the entire coding sequence (cds),
which was inserted Sp6-sense into
NcoI/EcoRI-digested pSP72 (Promega, Madison, WI),
thus generating pSP72-mERß. pSP72-mERß-TAG was made by replacement
of nucleotides 1273 of the mERß cds with oligonucleotides
5'-CATGGGCTACCCCTACGACGTGCCCGACTACGCCGTGAACA and
5'-CTAGTGTTCACGGCGTAGTCGGGCACGTCGTAGGGGTAGCC, which encode the HA1
epitope recognized by the 12CA5 monoclonal antibody. The plasmid
pSP72-hER
has been described elsewhere (36). For transfections of
mammalian cells the XhoI/BglII-fragment from
pSP72-mERß was inserted into the pSG5 expression vector (Stratagene,
La Jolla, CA) digested with EcoRI/BglII; the
XhoI-site of the mERß-fragment and the
EcoRI-site of pSG5 were filled in with Klenow fragment to
allow blunt-ended ligation. The vector pSG5-hER
was made by excising
hER
cDNA from pSP72-hER
with EcoRI and SacI
and inserting it into pSG5 digested with EcoRI and
BglII. To enable this ligation, the SacI site was
filled in with T4 DNA-polymerase and BglII with Klenow
fragment. The reporter construct 2xERE-TK-Luc was constructed by
subcloning of a tandem ERE (39) with XhoI overhangs into the
SalI site of the p19-TK-Luc reporter plasmid (40). The
Gal4-mER
and VP16-mER
two-hybrid constructs were made through PCR
amplification of the plasmid MOR101 (containing the mouse ER
cDNA)
(41) to introduce a KpnI site upstream of the start codon of
the mER
, with the use of oligonucleotides mER-ATG
(5'-GCCAGGTACCATGGCCATGACC) and mER-EagI
(5'-CCCAGGCTGTTGGCACTGAAGGC). The 275-bp long PCR product was cut with
KpnI and EagI, MOR101 was digested with
EagI (nucleotide 460 in the mouse ER
cDNA) and
BamHI 3' of the mER
cDNA), and both fragments were
subcloned into the KpnI and BamHI sites of
pCMX-Gal4 or pCMX-VP16 (42). VP16-ERß was made by in-frame insertion
of the NcoI/EcoRI fragment of pSP72-mERß into
the EcoRV/EcoRI sites of pCMX-VP16, and the
NcoI site of mERß was filled in with Klenow fragment to
enable the ligation. The Gal4-luciferase reporter construct used in the
two-hybrid assay has been described elsewhere (43). The pGST-mERß was
generated by in-frame ligation of the NcoI/EcoRI
fragment from pSP72-mERß into the corresponding sites of pGST (I.
Pongratz and F. Delauney, unpublished data) creating a GST-mERß
fusion that could be translated in the RRL system.
Cell Culture and Transient Transfections
Cells from the human fetal kidney cell line 293 were routinely
cultured in a 1:1 mixture of Hams Nutrient mixture F12 (F12, GIBCO
BRL) and DMEM (GIBCO-BRL) supplemented with 7.5% FBS, 0.5%
nonessential amino acids (NEA, GIBCO BRL) and 1% PEST (100 U
penicillin/ml and 100 µg streptomycin/ml). Cells were seeded in
six-well plates 24 h before transfection. Transfections using the
Lipofectin (GIBCO BRL) reagent were performed as described by the
manufacturer in a serum- and antibiotic-free mixture of 1:1 of F12 and
phenol-red free DMEM with 0.75 µg of the 2xERE-TK-Luc reporter and
0.10.4 µg of pSG5-ER
or pSG5-ERß as indicated. The pSG5 vector
was used to equalize plasmid concentrations, and 0.1 µg of a
placental alkaline phosphatase (AP) expression plasmid (44) was
included to control for differences in transfection efficiences. Medium
was changed to a phenol red-free mixture of F12 and DMEM containing
7.5% dextran-coated charcoal-treated FBS, 0.5% NEA, and 1% PEST
after 24 h. Hormone or vehicle (0.1% ethanol) was added
simultaneously. Cells were allowed to stand for 48 h with a
renewed change of media and hormone after 24 h. Media were
collected for assaying of AP activity. The cells were harvested in 10
mM Tris-HCl/10 mM EDTA/150 mM NaCl
and centrifuged for 4 min at 4000 rpm, supernatant was removed, and
cell pellets were lysed in Lysis Buffer 2 (Bio-Orbit, Turku, Finland).
Luciferase activity was measured using the GenGlow system (Bio Orbit).
The results are presented as the mean ± SD of fold
induction of three separate transfections performed in duplicate.
COS 7 cells were routinely maintained in DMEM (GIBCO BRL) supplemented
with 5% FBS and 1% PEST. For transient transfections, cells were
seeded in six-well plates 24 h prior to transfection.
Transfections were carried out with Lipofectin reagent in phenol red-
free DMEM without serum and antibiotics, using 0.5 µg of the
GAL4-luciferase reporter construct and 0.1 µg of each of the
two-hybrid expression plasmids as indicated in the legend to Fig. 5B
.
Expression vector concentrations were kept constant in all
transfections by addition of the original pCMX-Gal4 or pCMX-VP16
plasmids, and 0.2 µg of the AP expression vector was included in all
transfections as an internal control for transfection efficiency. Cells
were left in the Lipofectin-DNA mixture for 24 h after which the
medium was changed to phenol red-free DMEM supplemented with 5%
dextran-coated charcoal-treated FBS and 1% PEST. Hormone (1
µM E2) or vehicle (0.1% ethanol) was added.
After 24 h cells were harvested as described previously, and
luciferase activity was measured. All samples were normalized against
the activity of the AP internal standard. Transfections were carried
out in duplicate, and the results are presented as fold induction and
represent the mean value ± SD of three separate
experiments.
In Vitro Translation, GST Pulldown, and DNA-Binding
Assays
For the ERE-binding studies, 1 µg pSP72-mERß or 1 µg
pSP72-hER
was transcribed/translated in the TNT-coupled RRL system
(Promega) with Sp6 RNA polymerase, according to the manufacturers
instructions, in the presence of 100 nM 17ß-estradiol or
vehicle (0.01% ethanol). Five microliters of the lysate were used in
each DNA-binding reaction with a 32P end-labeled wild type
or double-mutated ERE as indicated in the legend to Fig. 2
. Protein-DNA
complexes were separated on 5% polyacrylamide/0.25x Tris-borate-EDTA
gels at
10 V/cm, followed by drying and autoradiography at -70
C.
In the homodimerization experiments, increasing amounts of
pSP72-mERß-TAG (0, 0.1, 0.2, 0.3, and 0.4 µg) were translated in
the RRL together with decreasing amounts of pSP72-mERß (0.4, 0.3,
0.2, 0.1, and 0 µg). Five microliters of programmed lysate were used
in each DNA-binding reaction with radiolabeled ERE.
For the heterodimerization studies, 1 µg pSP72-hER
or pSP72-mERß
was translated in RRL. In the experiment of Fig. 6A
, 5
, 4
, 3
, 2
, 1
or 0
µl of ER
-containing lysate were mixed with 0, 1, 2, 3, 4, or 5
µl of ERß lysate and incubated 15 min on ice before the DNA-binding
reaction with radiolabeled ERE.
Translations were carried out in the same manner for the
antibody-upshift experiments, and 4 µl ER
or ERß lysate,
respectively, or a mixture of 2 µl of each was incubated for 15 min
on ice. Thereafter, 1.5 µl monoclonal ER
antibody 1D5 (Dako,
Carpinteria, CA) or 12CA5 TAG antibody (BAbCOf) were added to the
respective homodimers and 0.5, 1, 2, or 3 µl of each antibody were
added to the heterodimer reactions. The DNA-binding reaction was
started immediately. The single-mutated ERE used in the band shift
assay in Fig. 6D
has been described previously (36). Four microliters
of ER
or ERß protein containing RRL or a mix of 2 µl of each
were used in the DNA-binding assay.
For the GST pulldown experiments, pSP72-hER
was translated in the
presence of [35S]methionine in RRL and pGST-mERß or the
original pGST was translated in RRL in the absence of radiolabeled
amino acids. Five microliters of ER
-containing lysate were mixed
with 5 µl lysate containing GST-mERß or GST-protein. Samples were
incubated for 15 min on ice before 50 µl GST-Sepharose diluted in PBS
were added to each sample followed by 30 min of incubation on ice. The
Sepharose beads were washed four times in PBS/0.1% Triton X-100, and
bound proteins were eluted by incubation in 2x SDS-buffer for 5 min at
100 C. ER
lysate (=20%) was loaded as input together with the
eluted samples on a 10% SDS-PAGE and run at 150 V. The gel was
immersed in 1 M salicylic acid for 20 min, dried, and
autoradiographed at -70 C.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Drs. Ingemar Pongratz and Franck
Delauney for the generous gift of the pGST plasmid, Göran
Bertilsson for technical assistance, and Jane Thomsen for critical
reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Katarina Pettersson, Department of Medical Nutrition, Karolinska Institute, NOVUM, S-14186 Huddinge, Sweden.
This work was supported by a grant from the Swedish Cancer Society to
J-ÅG and GGJMK was supported by a visiting scientist fellowship from
the Karolinska Institute.
The sequence reported in this paper has been deposited in the GenBank
database (AJ000220).
Received for publication December 18, 1996.
Revision received May 7, 1997.
Accepted for publication June 2, 1997.
 |
REFERENCES
|
---|
-
Gronemeyer H, Laudet V 1995 Transcription factors 3:
nuclear receptors. Protein Profile 2:11731308[Medline]
-
Beato M, Truss M, Chavez S 1996 Control of transcription by
steroid hormones. Ann NY Acad Sci 784:93123[Medline]
-
Giguere V, Yang N, Segui P, Evans RM 1988 Identification of a
new class of steroid hormone receptors. Nature 331:9194[CrossRef][Medline]
-
Laudet V, Hanni C, Coll J, Catzeflis F, Stehelin D 1992 Evolution of the nuclear receptor gene superfamily. EMBO J 11:10031013[Abstract]
-
Pratt WB, Welsh MJ 1994 Chaperone functions of the heat shock
proteins associated with steroid receptors. Semin Cell Biol 5:8393[CrossRef][Medline]
-
Clark JH, Schrader WT, OMalley BW 1992 Mechanisms of action
of steroid hormones. In: Wilson JD, Foster DW (eds) Textbook of
Endocrinology. WB Saunders, New York, pp 3590
-
Farhat MY, Lavigne MC, Ramwell PW 1996 The vascular
protective effects of estrogen. FASEB J 10:615624[Abstract/Free Full Text]
-
Turner RT, Riggs BL, Spelsberg TC 1994 Skeletal effects of
estrogen. Endocr Rev 15:275300[Medline]
-
Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P,
Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and
homology to v-erb-A. Nature 320:134139[Medline]
-
Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J 1986 Sequence and expression of human estrogen receptor complementary
DNA. Science 231:11501154[Medline]
-
Koike S, Sakai M, Muramatsu M 1987 Molecular cloning and
characterization of rat estrogen receptor cDNA. Nucleic Acids Res 15:24992513[Abstract]
-
White R, Lees JA, Needham M, Ham J, Parker M 1987 Structural
organization and expression of the mouse estrogen receptor. Mol
Endocrinol 1:735744[Abstract]
-
Ciocca DR, Roig LM 1995 Estrogen receptors in human nontarget
tissues: biological and clinical implications. Endocr Rev 16:3562[Medline]
-
Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies
O 1993 Alteration of reproductive function but not prenatal sexual
development after insertional disruption of the mouse estrogen receptor
gene. Proc Natl Acad Sci USA 90:1116211166[Abstract]
-
Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS,
Lubahn DB, Smithies O, Korach KS 1995 Analysis of transcription and
estrogen insensitivity in the female mouse after targeted disruption of
the estrogen receptor gene. Mol Endocrinol 9:14411454[Abstract]
-
Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson
J-Å 1996 Cloning of a novel estrogen receptor expressed in rat
prostate and ovary. Proc Natl Acad Sci USA 93:59255930[Abstract/Free Full Text]
-
Kuiper GGJM, Carlsson B, Grandien K, Enmark E,
Häggblad J, Nilsson S, Gustafsson J-Å 1997 Comparison of
the ligand binding specificity and transcript distribution of estrogen
receptors
and ß. Endocrinology 138:863870[Abstract/Free Full Text]
-
Byers M, Kuiper GGJM, Gustafsson J-Å, Park-Sarge OK 1997 Estrogen receptor-ß mRNA expression in rat ovary: downregulation by
gonadotropins. Mol Endocrinol 11:172182[Abstract/Free Full Text]
-
Katzenellenbogen JA, OMalley BW, Katzenellenbogen BS 1996 Tripartite steroid hormone receptor pharmacology-interaction with
multiple effector sites as a basis for the cell- and promoter-specific
action of these hormones. Mol Endocrinol 10:119131[Medline]
-
Kuiper GGJM, Gustafsson J-Å The novel estrogen receptor ß
subtype: potential role in the cell, promoter specific actions of
estrogens, antiestrogens (mini-review). FEBS Lett, in press
-
Ekena K, Weis KE, Katzenellenbogen JA, Katzenellenbogen BS 1996 Identification of amino acids in the hormone binding domain of the
human estrogen receptor important in estrogen binding. J Biol Chem 271:2005320059[Abstract/Free Full Text]
-
Glass CK 1994 Differential recognition of target genes by
nuclear receptor monomers, dimers, and heterodimers. Endocr Rev 15:391407[Medline]
-
Zilliacus J, Carlstedt-Duke J, Gustafsson J-Å, Wright AP 1994 Evolution of distinct DNA-binding specificities within the nuclear
receptor family of transcription factors. Proc Natl Acad Sci USA 91:41754179[Abstract]
-
Klein-Hitpass L, Schorpp M, Wagner U, Ryffel GU 1986 An
estrogen-responsive element derived from the 5' flanking region of the
Xenopus vitellogenin A2 gene functions in transfected human cells. Cell 46:10531061[Medline]
-
Tremblay GBA, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA,
Labrie F, Giguere V 1997 Cloning, chromosomal localization and
functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11:353365[Abstract/Free Full Text]
-
Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan
receptors. Cell 83:841850[Medline]
-
Laudet V, Adelmant G 1995 Nuclear receptors. Lonesome orphans.
Curr Biol 5:124127[Medline]
-
Field J, Nikawa J, Broek D, MacDonald B, Rodgers L, Wilson IA,
Lerner RA, Wigler M 1988 Purification of a RAS-responsive adenylyl
cyclase complex from Saccharomyces cerevisiae by use of an
epitope addition method. Mol Cell Biol 8:21592165[Medline]
-
Tora L, White J, Brou C, Tasset D, Webster N, Scheer E,
Chambon P 1989 The human estrogen receptor has two independent
nonacidic transcriptional activation functions. Cell 59:477487[Medline]
-
Mosselman S, Polman J, Dijkema R 1996 ERß-identification and
characterisation of a novel human estrogen receptor. FEBS Lett 392:4953[CrossRef][Medline]
-
Schwabe JW, Chapman L, Finch JT, Rhodes D 1993 The crystal
structure of the estrogen receptor DNA-binding domain bound to DNA: how
receptors discriminate between their response elements. Cell 75:567578[Medline]
-
Liu W, Wang J, Sauter NK, Pearce D 1995 Steroid receptor
heterodimerization demonstrated in vitro and in
vivo. Proc Natl Acad Sci USA 92:1248012484[Abstract]
-
Trapp T, Rupprecht R, Castren M, Reul JM, Holsboer F 1994 Heterodimerization between mineralocorticoid and glucocorticoid
receptor: a new principle of glucocorticoid action in the CNS. Neuron 13:14571462[Medline]
-
Shughrue PJ, Komm B, Merchenthaler I 1996 The distribution of
estrogen receptor-ß mRNA in the rat hypothalamus. Steroids 61:678681[CrossRef][Medline]
-
Chomczynski P, Sacchi N 1987 Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156159[CrossRef][Medline]
-
Pettersson K, Svensson K, Mattsson R, Carlsson B, Ohlsson R,
Berkenstam A 1996 Expression of a novel member of estrogen response
element-binding nuclear receptors is restricted to the early stages of
chorion formation during mouse embryogenesis. Mech Dev 54:211223[CrossRef][Medline]
-
Ichihara Y, Kurosawa Y 1993 Construction of new T vectors for
direct cloning of PCR products. Gene 130:153154[CrossRef][Medline]
-
Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with
chain-terminating inhibitors. Proc Natl Acad Sci USA 74:54635467[Abstract]
-
Dana SL, Hoener PA, Wheeler DA, Lawrence CB, McDonnell DP 1994 Novel estrogen response elements identified by genetic selection
in yeast are differentially responsive to estrogens and antiestrogens
in mammalian cells. Mol Endocrinol 8:11931207[Abstract]
-
Schule R, Muller M, Kaltschmidt C, Renkawitz R 1988 Many
transcription factors interact synergistically with steroid receptors.
Science 242:14181420[Medline]
-
Hillier SG, Saunders PT, White R, Parker MG 1989 Oestrogen
receptor mRNA and a related transcript in mouse ovaries. J Mol
Endocrinol 2:3945[Abstract]
-
Perlmann T, Umesono K, Rangarajan PN, Forman B, Evans RM 1996 Two distinct dimerization interfaces differentially modulate target
gene specificity of nuclear hormone receptors. Mol Endocrinol 10:958966[Abstract]
-
Forman BM, Umesono K, Chen J, Evans RM 1995 Unique response
pathways are established by allosteric interactions among nuclear
hormone receptors. Cell 81:541550[Medline]
-
Alksnis M, Barkhem T, Strömstedt PE, Ahola H, Kutoh E,
Gustafsson J-Å, Poellinger L, Nilsson S 1991 High level expression of
functional full length and truncated glucocorticoid receptor in Chinese
hamster ovary cells. Demonstration of ligand-induced down-regulation of
expressed receptor mRNA and protein. J Biol Chem 266:1007810085[Abstract/Free Full Text]