(Received for publication, May 9, 1997, and in revised form, June 6, 1997)
From the Molecular Endocrinology Laboratory, Imperial Cancer
Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United
Kingdom and Department of Biotechnology and Biochemistry,
N. V. Organon, P. O. Box 20, 5340 BH Oss, The Netherlands
The estrogen receptor (ER) is expressed in two
forms, ER and ER
. Here we show that ER
and ER
, expressed
both in vitro and in vivo, form heterodimers
which bind to DNA with an affinity (Kd of
approximately 2 nM) similar to that of ER
and greater
than that of ER
homodimers. Mutation analysis of the hormone binding
domain of ER
suggests that the dimerization interface required to
form heterodimers with ER
is very similar but not identical to that
required for homodimer formation. The heterodimer, like the homodimers,
are capable of binding the steroid receptor coactivator-1 when bound to
DNA and stimulating transcription of a reporter gene in transfected
cells. Given the relative expression of ER
and ER
in tissues and
the difference in DNA binding activity between ER
/ER
heterodimers
and ER
it seems likely that the heterodimer is functionally active
in a subset of target cells.
Estrogen receptors (ER)1
were recently shown to be encoded by two distinct genes,
ER and ER
(1, 2). Reverse
transcription-polymerase chain reaction (PCR) analysis indicates that
ER
is highly expressed in prostate and ovary (1, 2), but moderate
expression was detected in many other tissues including testis and
uterus, some of which also seem to express ER
(3). The two receptors
which share about 95% homology in the DNA binding domain and 55%
homology in the ligand binding domain, both bind to a consensus
estrogen response element (ERE) (4) and exhibit similar ligand binding properties (3). They are poorly conserved in the N-terminal domain but
ER
, like ER
, appears to contain a similar activation domain,
activation function 1 (AF-1) sensitive to a mitogen-activated protein
kinase pathway (4-6). In addition, both receptors contain a second
activation domain, activation function 2 (AF-2) (7, 8), whose activity
is enhanced by the coactivator SRC-1 (4, 9, 10). Thus, although the
relative expression of ER
and ER
varies in cells, their ligand
binding, DNA binding, and transactivation properties are rather similar
to one another.
Steroid hormone receptors usually bind to inverted DNA repeats as
homodimers, although the glucocorticoid and mineralocorticoid receptors
have been reported to form heterodimers, at least in vitro
(11, 12). In the classically accepted model of steroid hormone action,
the estrogen receptor is sequestered in an inactive state in a
multiprotein complex in the absence of hormone (13). Upon estrogen
binding, the receptor forms homodimers which then interact with
response elements in the vicinity of target genes and modulate rates of
gene transcription. In view of the similarity of the ligand binding
domain of ER and ER
we investigated the possibility that the two
receptors may form functional heterodimers in target cells. ER
and
ER
were capable of forming heterodimers on DNA that could bind the
coactivator, SRC-1, and appeared to stimulate transcription of a
reporter gene. Moreover, we demonstrate that while the region of ER
required for homodimerization overlaps with that required for
heterodimerization the two regions are not coincident.
The isolation and construction of cDNA clones
that encode the mouse ER and a series of point mutants for analyzing
receptor dimerization have been described previously (14, 15). To
express human ER
the 1.5-kilobase ER
cDNA (1) was subcloned
into the BamHI site of pSP65 for in vitro
transcription and translation and pSG5 for mammalian cell expression.
The human ER
cDNA from pSG5HEGO, kindly provided by Pierre
Chambon, was subcloned into the EcoRI site of pSP65.
Glutathione S-transferase (GST)-SRC-(570-780) was generated
by subcloning a PCR fragment of SRC-1 into pGEX2TK.
ER and ER
protein was synthesized in vitro using the
TNT-coupled reticulocyte lysate system (Promega) in the
presence of 0.1 mM methionine as described previously (16).
To quantitate the relative amount of receptors produced, 1 µCi/µl
[35S]methionine (Amersham) was included in the reaction
mixture.
For biochemical analysis,
wild-type and mutant receptors were overexpressed in COS-1 cells by
electroporation using a Bio-Rad gene pulser at 450 V and 250 millifarads as described previously (17). Cells were transfected with
20 µg of expression plasmid, either pSG5HEGO or pSG5ER as
indicated. After 2 days, cells were harvested, and whole cell extracts
were prepared using a high salt extraction buffer (400 mM
KCl, 20 mM HEPES, pH 7.4, 1 mM dithiothreitol,
20% glycerol, plus protease inhibitors) as described (17). Their
protein concentration was determined using the Bio-Rad protein assay
kit (Bio-Rad). For transient transfection assays, HeLa cells were
plated into 24-well microtiter plates in phenol red-free medium
containing 5% charcoal/dextran-stripped fetal calf serum. Cells were
transfected using a modified calcium phosphate coprecipitation method
(18) with 1 µg of pEREBLCAT reporter plasmid, a total of 10 ng of
ER
and ER
expression plasmids as described under "Results,"
150 ng of pJ7lacZ plasmid as an internal control, and pSG5 plasmid to a
total of 1.5 µg of DNA per well. After 24 h, the cells were
washed with Dulbecco's modified Eagle's medium and then maintained in
medium with or without 1 × 10
8 M
17
-estradiol for 24 h. The cells were then washed with
phosphate-buffered saline and harvested in lysis buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, and 0.65% Nonidet P-40). Extracts were assayed
for CAT activity (19) or
-galactosidase activity using a
Galacto-Light chemiluminescent assay (Tropix).
DNA binding was assayed using a gel
shift assay. Aliquots of receptors, either translated in
vitro or expressed in COS-1 cells, were incubated with a
32P-labeled double-stranded oligonucleotide probe
containing a consensus ERE sequence
(5-CTAGAAAGTCAGGTCACAGTGACCTGATCAAT-3
) as described previously (17).
The human ER
monoclonal antibody H226 or ligands were added as
indicated. In some experiments GST-SRC-(570-780), expressed and
purified as described previously (20), was added. The samples were
applied directly onto prerun nondenaturing 7% polyacrylamide gels
(17), and complexes were detected by autoradiography or scanning
with a PhosphorImager (Molecular Dynamics).
The DNA binding activity of ER and ER
was tested using
in vitro translated receptors and a consensus estrogen
response element in a gel shift assay. Both ER
and ER
bound to
the element, and the mobility of ER
, but not that of ER
, was
retarded in the presence of the hER
antibody, H226 (Fig.
1). When the two receptors were
cotranslated we were able to detect a complex with an intermediate mobility corresponding to ER
/ER
heterodimers, in addition to ER
homodimers (Fig. 1, tracks 7-12). Their mobility was
retarded by H226 consistent with the presence of ER
in both
complexes. Their relative amounts varied depending on the input ratio
of the two receptors but it is noteworthy that ER
homodimers were barely detected even when ER
was expressed in 2-fold excess over ER
(Fig. 1, track 11). Their affinity for DNA was then
determined by carrying out gel shift experiments over a wide range of
probe concentrations (Fig. 2). We found
that ER
homodimers and ER
/ER
heterodimers bound to DNA with a
similar Kd of approximately 2 nM whereas
that of ER
homodimers was about 4-fold greater.
We next analyzed the DNA binding activity of ER and ER
when they
were expressed in COS-1 cells. When ER
alone was expressed, we
observed two complexes, a major upper complex, corresponding to the
ER
homodimer, and an additional complex that is probably generated
by proteolysis. It seems to lack N-terminal sequences since it is
recognized by a monoclonal antibody specific for the C-terminal F
region (21) (data not shown). Similar results have been reported
previously (22). As expected, the mobility of ER
but not ER
was
retarded in the presence of the specific hER
antibody, H226. When
equivalent amounts of ER
and ER
expression vector were
coexpressed, heterodimers were the predominant form observed, and ER
homodimers were not detected (Fig.
3A). We then used these
extracts to compare the effect of 17
-estradiol and 4-hydroxytamoxifen on the DNA binding activity of the three dimeric forms (Fig. 3B). As previously demonstrated for ER
(7),
the DNA binding activity of both ER
and ER
/ER
heterodimers was unaffected by ligand binding, but their mobilities were slightly increased in the presence of 17
-estradiol (Fig. 3B,
tracks 2, 5, and 8). Thus, we conclude
that ER
/ER
heterodimers, expressed in intact cells, are capable
of forming on DNA and that ER
homodimers and ER
/ER
heterodimers are preferentially formed.
Previous work with a series of ER mutants has identified a region
within the ligand binding domain of the estrogen receptor which is
required for both receptor dimerization and high affinity DNA binding
(15). We have used these mutants to determine whether the region
required to form homodimers with a truncated version of ER
(mouse
estrogen receptor (MOR)-182-599) is similar to that required to form
heterodimers with ER
on DNA. We find that the ability of R507A to
form either ER
homodimers or ER
/ER
heterodimers is markedly
reduced (Fig. 4, compare tracks
4 and 11) while L511R and I518R, which show negligible
homodimer formation, retain some ability to form ER
/ER
heterodimers (Fig. 4, compare tracks 6 with 13 and 7 with 14). In contrast, mutation of Q510A
had no affect on the dimerization of either receptor. A series of other mutations in this region of the receptor (A509R, L512V, L513G, I514R,
L515G, L516A, H517A, R519G) was then screened to attempt to identify
additional residues which could discriminate between homo- and
heterodimerization, but all the mutants retained their DNA binding
activity both as ER
homodimers and ER
/ER
heterodimers (data
not shown). We conclude that the region of ER
required for
homodimerization overlaps that required for heterodimerization, but the
two regions are not coincident.
We next assessed the transcriptional activity of ER/ER
heterodimers in transiently transfected COS-1 cells using the pEREBLCAT reporter gene. ER
and ER
expression vectors were tested
individually or in combination at a ratio of 1:1 or 1:2 to minimize the
relative amount of ER homodimers formed (see Fig. 3). The ability of
ER
to stimulate transcription was slightly greater than that of
ER
(Fig. 5A), as previously
reported for this reporter (4). Coexpression of ER
and ER
resulted in an intermediate level of transcription that was blocked by
the addition of the antiestrogens, 4-hydroxytamoxifen and ICI 182780. Similar results were obtained in HeLa cells (Fig. 5B).
Therefore, since the heterodimer is the major dimeric form of the
receptor under these conditions, it appears to retain its ability to
stimulate transcription.
To obtain additional evidence to support our suggestion that
ER/ER
heterodimers are capable of stimulating transcription we
tested whether they were able to bind the coactivator, SRC-1, as
previously demonstrated for ER
(10) and ER
homodimers (4). This
was achieved by analyzing the ability of the receptors, bound to DNA,
to interact with a fragment of SRC-1, residues 570-780, that binds
nuclear receptors in a ligand-dependent
manner.2 As shown in Fig.
6, when ER
, ER
, or both were
incubated with increasing amounts of GST-SRC-(570-780) in the presence
and absence of ligand we could detect additional complexes in the gel
shift assay. The interaction of SRC-(570-780) with ER
was dependent on the presence of ligand (Fig. 6, compare tracks 7 and
8) whereas there was an appreciable interaction with ER
in the absence of ligand (Fig. 6, compare tracks 13 and
14). The interaction of SRC-1 with the heterodimer was
enhanced in the presence of ligand (Fig. 6, compare tracks
19 and 20). As a control, we showed that these retarded
complexes were not due to the binding of SRC-1 directly to an ERE (Fig.
6, tracks 1 and 2) but required the presence of
receptor. Thus ER
/ER
heterodimers, bound to DNA, are capable of
recruiting SRC-1.
The main conclusion from our study is that human ER and ER
are capable of forming functional heterodimers on DNA. The relative distribution of ER homodimers and heterodimers will, at least in part,
be dependent on the relative expression of the two receptors which
varies widely in different cell types. Both ER
and ER
have been
detected in many tissues by reverse transcription-PCR or in
situ hybridization (3, 23) but the relative amounts of receptor
protein in specific cell types have yet to be determined. Nevertheless
this preliminary analysis suggests that the expression of ER
may be
greater than that of ER
in epididymis, testis, pituitary ovary,
uterus, adrenals, and heart. Given that ER
homodimers and
ER
/ER
heterodimers are preferentially formed over ER
homodimers it seems that heterodimers are more likely to be formed than
ER
homodimers in these tissues. On the other hand, ER
is
expressed at higher levels in prostate, bladder, lung, thymus, and
certain hypothalamic cells (3, 23), and so ER
homodimers may be formed in these tissues.
The molecular basis for the reduced DNA binding activity of ER
compared with that of ER
and ER
/ER
heterodimers is unclear, but recent work indicates that the mouse ER
also binds to an ERE
less strongly than ER
(4). Differences in the DNA binding domains of
the two receptors are unlikely to account for the variation since they
differ by only two residues (1, 2), neither of which seems to be in a
position that is likely to affect its DNA binding properties (24). An
alternative possibility is that the receptors differ in their ability
to dimerize. The major dimer interface in ER
has been mapped to a
region of the hormone binding domain (15, 25) which is conserved and
likely to correspond to helix 10 in nuclear receptors (26). The crystal
structure of the ligand binding domain of RXR also indicates that the
dimer interface comprises this helix and, to a lesser extent, helix 9 and the loop between helix 7 and 8 (27). Comparison of the sequence of
the corresponding region in ER
and ER
indicates that helix 10 is
similar (13/18 residues are identical) but the loop region and helix 9 are poorly conserved in the two receptors. Thus, it seems likely that
the residues required to form the dimer interface in ER
and ER
homodimers are distinct. Although the precise dimerization interfaces
in these receptors have yet to be identified, functional analysis of a
series of mutations in helix 10 of ER
indicates that the residues
required for the formation of ER
homodimers are similar but not
identical to those required for ER
/ER
heterodimerization.
Both ER and ER
are capable of stimulating the transcription of
reporter genes in transfected cells and the activation functions, AF-1
and AF-2 characterized in ER
(5, 6, 8, 28), appear to be conserved
in ER
(4). When ER
and ER
are expressed, under conditions when
heterodimers are the predominant dimeric species, transcription of an
ERE reporter gene is stimulated to an intermediate level compared with
that of either homodimer suggesting that ER
/ER
heterodimers are
transcriptionally active. This is supported by our observation that
ER
/ER
heterodimers are capable of binding the coactivator SRC-1
(9, 29). We found that SRC-1 interacts with all three dimeric states of
ER bound to DNA although its interaction with heterodimers was less
dependent on ligand than that observed with ER
homodimers. The
interaction of ER
homodimers with SRC-1 was dependent on ligand in
solution (4)3 but not on DNA
and, consistent with this, SRC-1 was found to augment the
transcriptional activity of ER
in the absence of ligand (4).
Finally, the ability of the heterodimer to stimulate transcription was
blocked by the antiestrogens, 4-hydroxytamoxifen and ICI 182780, as
previously demonstrated for ER homodimers (4, 7, 8, 30).
The discovery of a second estrogen receptor raises many questions, most
notably relating to their respective functions. The ability of ER
and ER
to form heterodimers suggests that estrogen receptor may
function in different dimeric states, and it is possible that they
could be activated by selective ligands. In view of the similarity of
their DNA binding domains it is doubtful whether different forms bind
to distinct response elements, but they could activate different genes
in different target cells given their distinct expression patterns.
We thank I. Goldsmith for oligonucleotides, A. Wakeling (Zeneca Pharmaceuticals) for 4-hydroxytamoxifen, and C. Nolan (Abbott Laboratories) for the monoclonal antibody H226. We are also extremely grateful to C. Dickson, H. Hurst, E. Kalkhoven, R. White, and members of the Molecular Endocrinology Laboratory for discussions and comments on the manuscript.