Functional Analysis of a Novel Estrogen Receptor-ß Isoform
Bettina Hanstein1,
Hong Liu2,
Molly C. Yancisin and
Myles Brown
Department of Adult Oncology Dana-Farber Cancer Institute
Boston, Massachusetts 02115
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ABSTRACT
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A new level of complexity has recently been added
to estrogen signaling with the identification of a second estrogen
receptor, ERß. By screening a rat prostate cDNA library, we detected
ERß as well as a novel isoform that we termed ERß2. ERß2 contains
an in-frame inserted exon of 54 nucleotides that results in the
predicted insertion of 18 amino acids within the ERß hormone-binding
domain. We also have evidence for the expression of both ERß1 and
ERß2 in human cell lines. Competition ligand binding analysis of
bacterially expressed fusion proteins revealed an 8-fold lower affinity
of ERß2 for 17ß-estradiol (E2)
[dissociation constant (Kd
8
nM)] as compared with ERß1
(Kd
1 nM). In
vitro transcribed and translated ERß1 and ERß2 bind
specifically to a consensus estrogen responsive element in a gel
mobility shift assay. Furthermore, we show heterodimerization of ERß1
and ERß2 with each other as well as with ER
. In affinity
interaction assays for proteins that associate specifically with the
hormone-binding domain of these receptors, we demonstrate that the
steroid receptor coactivator SRC-1 interacts in an estrogen-dependent
manner with ER
and ERß1, but not with ERß2. In cotransfection
experiments with expression plasmids for ER
, ERß1, and ERß2 and
an estrogen-responsive element-containing luciferase reporter, the dose
response of ERß1 to E2 was similar to that of
ER
although the maximal stimulation was approximately 50%. In
contrast, ERß2 required 100- to 1000-fold greater
E2 concentrations for maximal activation. Thus,
ERß2 adds yet another facet to the possible cellular responses to
estrogen.
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INTRODUCTION
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The estrogen receptor (ER) is a member of the nuclear receptor
superfamily of ligand-activated transcription factors that include
receptors for steroid hormones, thyroid hormone, vitamin D, retinoic
acid, and eicosanoids (1, 2, 3, 4, 5, 6). After diffusion into the cell, estradiol
binds to the ER, leading to ER dimerization followed by binding to a
conserved estrogen responsive element (ERE) in the regulatory region of
target genes. Amino acid sequence comparison of the ER with other
nuclear receptors has shown that the receptor is composed of conserved
functional regions. The N-terminal transactivating region (AF1) is able
to activate transcription in a hormone-independent manner, and this
region has been shown to be a target of the mitogen-activating protein
kinase-regulatory pathway (7). The DNA-binding domain enables
the receptor to bind to its specific DNA target, the ERE. The consensus
ERE consists of a palindrome of the half-site sequence 5'-GGTCA-3'
separated by 3 bp. The AF2 domain is overlapped by the hormone-binding
domain (HBD) and activates transcription in response to estrogen or
synthetic estrogen agonists (8).
The mechanism of transactivation by nuclear receptors has recently
achieved further complexity by the discovery of an increasing number of
coregulators. This group of coregulators can be subdivided into
coactivators, corepressors, and integrators. The coactivators were
initially biochemically identified as ERAP160 and 140 and RIP160, 140,
and 80 (9, 10) by their ability to specifically interact with the HBD
of the receptor in a ligand-dependent manner. Specifically for the ER,
this interaction was promoted by E2 but antiestrogens such
as 4-OH-tamoxifen were able to effectively block this interaction.
Yeast two-hybrid screening led to the molecular cloning of the steroid
receptor coactivator (SRC) 1, which when cotransfected with nuclear
receptors, including ER, was capable of augmenting ligand-dependent
transactivation (11). Subsequent cloning and sequence comparison of
transcriptional intermediary factor (TIF)2 and glucocorticoid
receptor interacting protein (GRIP)1 revealed GRIP1 to be the mouse
homolog of human TIF2. More recently, p300/CBP cointegrator protein
(p/CIP) [also receptor-associated coactivator (RAC)3 (12) and
activator for thyroid hormone and retinoid receptors (ACTR) (13)] were
shown to be new members of this family (14). Interestingly, this ER
coactivator was also identified as amplified in breast cancer (A1B)
(15). In addition, the phospho-CREB-binding protein CBP and the related
p300 have been demonstrated to be ER-associated proteins and involved
in ligand-dependent transactivation (16, 17). In contrast to the
coactivators mentioned above, these proteins are targets of signals
mediated by a variety of distinct pathways. Moreover, by interaction
with components of the basal transcription machinery, these proteins
are thought of as integrators of signals from these diverse pathways.
This increasing number of coregulatory factors has added immensely to
our understanding of how steroids such as E2 are able to
alter the expression of specific genes at the molecular level.
Recently, a new member of the nuclear receptor family with high
homology to ER was cloned from rat, mouse, and human and was termed
ERß (18, 19, 20). The homology to the rat ER protein (now ER
) was
shown to be 95% in the DNA-binding domain and 55% in the HBD (18).
In situ hybridization studies in rat revealed a prominent
expression of this novel receptor in the epithelial cells of the
secretory alveoli of the prostate and the granulosa cells of the
primary, secondary, and mature follicles of the ovary. ERß was found
to bind E2 with high affinity and in transient transfection
experiments ERß was capable of activating transcription of a reporter
gene in an estrogen-dependent manner.
Recently, a partial clone for an alternative splice variant of ERß2
has been described (21). Here we report the complete cloning and
functional analysis of this novel rat ERß isoform.
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RESULTS
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Cloning of ERß2, an Alternative Splice Variant of ERß
To obtain clones of ERß we screened a
gt11 rat prostate cDNA
library with two oligonucleotide probes derived from the published
ERß sequence, corresponding to the nucleotides 418477 and
12481307. Primary screening of
900,000 phage revealed four
positive plaques that were confirmed in a secondary screening and
isolated in a tertiary screening. Inserts of all independently isolated
plaques were then subcloned and subjected to nucleotide sequencing. The
full-length cDNA clone diverged from the previously published rat cDNA
at two positions (496 T to A; 729 C to G). These nucleotide changes
result in amino acid changes that are conserved in the published
sequence of human ERß. Therefore, it is likely that these result from
polymorphisms in the ER. More interestingly, three of four independent
clones revealed an insertion of 54 nucleotides at position 1374 of the
previously published rat cDNA (Fig. 1
).
This results in an in-frame insertion of 18 amino acids in the
predicted HBD of this receptor. We therefore termed this alternative
splice variant ERß2, ERß1 being the originally published sequence.
The amino acid sequence of this insert exhibits no homology to known
proteins or peptide motifs when computer database searches were
performed. Recently, the sequence of ERß2 was also reported as an
ERß splice variant (21).

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Figure 1. Structure of ERß1 and ERß2
The upper panel shows the functional domains of the
previously published rat ERß deduced from its homology to ER .
Numbers indicate amino acids as described (18 ). The
lower panel shows the amino acid sequence encoded by the
54-nucleotide insert found in ERß2.
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Expression of ERß1 and ERß2 in Different Human Cancer Cell
Lines
Since screening of the prostate cDNA library revealed that
the majority of clones (3/4) encode ERß2, we examined the expression
pattern of this nuclear receptor splice variant in a variety of human
cancer cell lines derived from breast, uterus, ovary, and prostate
tissue. We isolated RNA from these cells, subjected it to RT using
oligo dT primers, and performed PCR reactions with primers derived from
the human ERß sequence flanking the insertion site of the 18 amino
acids (aa) in the ERß sequence. ER
-specific primers and primers
from the ß2-microglobulin gene were used to control for the quality
of the cDNA. As shown in Fig. 2
, as
expected, all cDNAs revealed a product for the ubiquitously expressed
ß2-microglobulin (lower panel). The ER
transcript was
detectable in breast cancer cell lines previously described as
ER
-positive such as MCF7, T47D, and BT-20 as well as in the
endometrial cancer cell line ECC1 and in the ovarian cancer cell line
OVCAR-3. When the PCR reaction was performed with ERß-specific
primers, a band corresponding to the expected size for the previously
published ERß sequence was detectable in the breast cancer cell lines
BT-20, MDA-MB231, and T47D and in primary normal human mammary
epithelial cells (HMECs). Moreover, a transcript for ERß1 was
detectable in the ovarian cancer cell lines OVCAR-3 and UPN36T. Among
endometrium cancer cell lines tested, only the ER
-negative Ishikawa
cell line showed an ERß1 transcript. The human prostate cancer cell
lines, PC-3 and DU145, were also positive for ERß1 expression. To
confirm that the bands indeed correspond to ERß, we transferred the
PCR products onto nitrocellulose and subjected it to Southern blot
analysis with a radiolabeled nucleotide probe derived from the
previously published ERß sequence. As shown in Fig. 2
, the band of
340 bp was indeed reactive with this probe. Interestingly, using PCR
primers flanking the insertion in the rat cDNA also revealed a band
with the expected size for ERß2 in the human ovarian cancer cell line
OVCAR-3 and the osteosarcoma cell line U2OS. To confirm the origin of
this PCR product as ERß2, we probed duplicate blots with an
oligonucleotide probe corresponding to the unique 54 nucleotides of the
ERß2 sequence as shown in Fig. 2
, top panel. These data
indicate that although ERß1 is present in a variety of different
cells, the ERß2 transcript is restricted to a minority of these cell
lines, suggesting a specific mechanism regulating expression of these
splice variants.

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Figure 2. Expression of ER , ERß1, and ERß2 in Various
Human Cancer Cell Lines
RNA extracted from various human cancer cell lines was subjected to RT
using oligo dT primers. Cell lines used were the human ovarian cancer
cell lines SW626 (lane 1), OVCAR-3 (lane 2), CAOV-3 (lane 3), UPN36T
(lane 4), and the human breast cancer cell lines BT-20 (lane 5),
MDAMB231 (lane 6), T47D (lane 7), and MCF7 (lane 8), normal HMEC (lane
9), two human endometrium cancer cell lines ECC1 (lane 10) and Ishikawa
(lane 11), the human prostate cancer cell lines PC-3 (lane 12), Du145
(lane 13), and LnCAP (lane 14), the green monkey kidney cell line CV-1
(lane 15), and the human osteosarcoma cell line U2OS (lane 16).
Integrity of the cDNAs was verified by PCR using primers specific to
human ß2-microglobulin (ß2MG) cDNA (lower panel).
Expression of ER was analyzed using primers specific to the human
ER cDNA (second panel from bottom). To analyze
expression of ERß1 and ERß2, PCR was performed with primers
flanking the alternative splice site. Southern blot analysis on these
PCR products was performed using either the ERß2-specific insert as a
probe (top panel) or a probe to the common ERß region
(second panel from the top).
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ERß2 Binds Estradiol with Lower Affinity Than ERß1
Since insertion of the 18 aa in ERß2 occurs in the predicted
hormone-binding domain of this receptor, we first examined the binding
affinity of estradiol for ERß1 and ERß2. Therefore,
[3H]E2 was used to conduct competition
ligand-binding studies of ERß1 and ERß2. Using bacterial expressed
glutathione S-transferase (GST) fusion proteins containing
the HBDs of both receptors, dissociation constants (Kd) for
E2 were 1 nM for ERß1 but 8-fold higher
(Kd = 8 nM) for ERß2 (Fig. 3
). These data indicate that the 18-aa
insertion in the HBD of ERß2 lowers its affinity for
E2.

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Figure 3. Estradiol Binds to ERß1 and ERß2
E2 binding to GST fusion proteins of either ERß1
(open squares) or ERß2 (closed squares)
HBD was assayed as described in Materials and Methods.
Binding is expressed as the percentage of bound radiolabeled
E2 at a given competitor concentration compared with
binding of labeled E2 in the absence of competitor.
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ERß2 Binds to an ERE
It has been previously shown that ERß1 is capable of binding to
a consensus ERE with high affinity. To test whether the 54-nucleotide
insertion of ERß2 had an influence on DNA binding, we conducted
electrophoretic mobility shift assays (EMSAs). Receptors were expressed
in vitro to comparable levels (data not shown). As
demonstrated in Fig. 4
, both ERß1 and
ERß2 were able to bind specifically to the ERE probe in a
hormone-dependent manner, under the stringent conditions used (22).
Specificity of binding was confirmed by the fact that unlabeled ERE
could compete for binding of all receptors to the labeled probe (lanes
5), whereas competition with a mutated ERE (lanes 6), or an unrelated
AP-1 sequence (lanes 7), did not have an influence on binding.

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Figure 4. ER , ERß1, and ERß2 Bind in an
Estradiol-Dependent Manner to an ERE
To analyze DNA binding of the different ERs, EMSA was performed with
in vitro translated ER (left panel),
ERß1 (middle panel), and ERß2 (right
panel). Protein/DNA incubation was performed in the absence of
hormone (lane 1) or increasing concentrations of E2 (1
nM, 10 nM, 100 nM) (lanes 24).
Specificity of binding was confirmed by competition using an unlabeled
ERE (lanes 5), a mutated ERE (lanes 6), or the unrelated AP-1 site
(lanes 7) in the presence of 100 nM E2.
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When EMSA was performed with increasing E2 concentrations
(lanes 24), it was interesting to note that maximal binding of ER
(to the ERE) occurred at the lowest concentration of
E2 used (1 nM) (lane 2), whereas both ERß1
and ERß2 showed a dose response with maximal binding at 100
nM. Despite the apparent differences in hormone binding
affinity for ERß1 and ERß2, both receptors bound with similar
affinity to DNA.
Heterodimerization Occurs between ER
and Both ERß Isoforms
Since it has been demonstrated that ER
and ERß1 are capable
of forming heterodimers upon ligand binding, we set forth to
investigate whether the newly identified splice variant of ERß also
forms heterodimers with ER
. Therefore, we performed EMSA on a
labeled ERE probe using in vitro translated full-length
receptors in the presence of 100 nM E2 (Fig. 5
). When ER
(lanes 1 and 2), ERß1
(lanes 3 and 4), or ERß2 (lanes 5 and 6) were incubated with the
labeled ERE probe, antibodies specific for ER
or ERß were able to
supershift the homodimeric ER/DNA complexes (lanes 2, 4, and 6). When
in vitro translated ER
and ERß1 (lanes 710) or ER
and ERß2 (lanes 1114) were coincubated with the labeled probe, each
antibody was able to shift the protein/DNA complex to a different size
than the respective homodimeric receptor (lanes 8, 9 and 12, 13). When
both antibodies were coincubated either with ER
/ERß1 or with
ER
/ERß2, a supershifted band was detected, indicating that indeed
ER
forms heterodimers on DNA with ERß1 and ERß2 (lanes 10 and
14). Heterodimers could be detected at E2 concentrations as
low as 1 nM (data not shown).
Interaction of SRC1 with ER
and ERß1, but Not ERß2
The role of putative coactivators in the hormone-dependent
transcriptional regulation by ERß1 and ERß2 was addressed by
affinity purification of ERß1- and ERß2-associated proteins. The
HBD of these receptors was fused to GST and used to purify proteins
from the metabolically labeled human breast cancer cell line MCF-7
(Fig. 6
), the human breast cancer cell
line MDA-MB231, and the human osteosarcoma cell line U2OS (data not
shown). In the case of ER
(lanes 13), previously described
proteins bound GST-HBD-ER
in the presence of E2 (lane
2), but not in its absence (lane 1) nor in the presence of the ER
antagonist tamoxifen (lane 3). Using the GST-HBD-ERß1 fusion protein
as an affinity matrix (lanes 46), only the ERAP160/SRC1 family of
proteins could be detected (lane 5). Interestingly, when performing
this experiment with the same amount of GST-HBD-ERß2, we were unable
to detect associated proteins that specifically interact with ERß2
(lane 8). As ERß2 ligand-binding analysis had detected an 8-fold
lower affinity for E2, we conducted the same experiment
using increasing amounts of estradiol (1 µM and 10
µM). Again, using GST-HBD-ERß2 as an affinity matrix,
we were unable to detect proteins interacting with the receptor in an
estradiol-dependent manner (data not shown). To exclude the possibility
that other domains of ERß2 were required for coactivator interaction,
we performed GST pull-down experiments using full-length ERß1 and
ERß2 as GST fusion proteins and obtained similar results (data not
shown). To test whether the proteins in the 160-kDa range, detected in
this assay to bind to ER
and ERß1, include the cloned steroid
receptor coactivator SRC1, we performed Western blot analysis with an
anti-SRC1-specific antibody on proteins associating with the
ligand-binding domain of these receptors (Fig. 7A
). For this purpose we used whole-cell
extracts from MCF-7 cells. Cell lysates were incubated with the GST-HBD
affinity matrices of the different receptors in the absence or presence
of increasing amounts of E2 (1 µM and 10
µM) or 4-OH-Tamoxifen (1 µM).
Specifically bound proteins were resolved by SDS-PAGE and transferred
onto nitrocel-lulose. Immunoblotting with a monoclonal
antibody directed against human SRC1 revealed significant SRC1
binding only for GST-HBD-ER
and GST-HBD-ERß1 in the presence of
E2 but not for GST-HBD-ERß2 (Fig. 7A
). Negative control
samples consisting of glutathione-sepharose or GST immobilized on
glutathione sepharose did not show binding (data not shown). To
confirm these results, we tested the ability of recombinant SRC1 to
interact with ERß1 or ERß2. Again, in vitro synthesized
[35S] methionine-labeled SRC1 associated specifically in
an E2 dependent manner only with GST-HBD-ER
(lane 2) and
GST-HBD-ERß1 (lane 5) but not GST-HBD-ERß2 (lane 8) (Fig. 7B
).

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Figure 6. The HBD of ERß1, but Not ERß2, Interacts with a
Protein of 160 kDa in Vitro
Whole cell extracts of MCF-7 cells were metabolically labeled with
[35S]methionine and were bound to the GST-HBD fusion of
ER (lanes 13), ERß1 (lanes 46), and ERß2 (lanes 79). The
fusion proteins were previously immobilized on glutathione-Sepharose in
the absence (lanes 1, 4, and 7) or the presence (lanes 2, 5, and 8) of
1 mM17ß-estradiol (E) or 4-OH-tamoxifen (T) (lanes 3, 6,
and 9). Proteins were eluted in SDS/sample buffer and were resolved on
7.5% SDS/PAGE.
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Figure 7. SRC1 Associates with GST-HBD-ER and
GST-HBD-ERß1, but Not with GST-HBD-ERß2
A, Whole-cell extracts from the human breast cancer cell line MCF-7
were incubated with GST-HBD-ER (top panel),
GST-HBD-ERß1 (middle panel), and GST-HBD-ERß2
(bottom panel) immobilized on glutathione-Sepharose in
the absence (lane 1) or presence of 1 µM E2
(lane 2), 10 µM E2 (lane 3), or 1
µM 4-OH-tamoxifen (T) (lane 4). Specifically associated
proteins were recovered by boiling in SDS/sample buffer, resolved on
7.5% SDS/PAGE, and transferred to nitrocellulose. Filters were cut and
probed with an SRC1 monoclonal antibody, and specifically bound primary
antibody was detected with peroxidase-coupled secondary antibody and
chemiluminescence. B, Recombinant 35S-labeled SRC1 produced
by in vitro transcription/translation in a rabbit
reticulocyte lysate was bound to GST-HBD-ER (lanes 13),
GST-HBD-ERß1 (lanes 46), and GST-HBD-ERß2 (7 8 9 ) im-mobilized
on glutathione-linked Sepharose in the absence (lanes 1, 4, and 7) or
presence of 1 µM estradiol (lanes 2, 5, and 8) and 1
µM tamoxifen (lanes 3, 6, and 9). The translation product
(15 ml) was incubated with the different affinity matrices that had
been blocked with 3% BSA under the indicated conditions at 4 C for 30
min. After extensive washing, bound proteins were eluted in 20
mM reduced glutathione-containing elution buffer (120
mM NaCl/100 mM Tris-HCl, pH 8.0) and separated
by 7.5% SDS-PAGE. Gels were treated with a fluorophore, dried, and
visualized by autoradiography.
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Transcriptional Activity of ERß2 on an ERE
To investigate whether an ERE can mediate transcriptional activity
of ERß2, we used a luciferase reporter gene containing two copies of
an ERE and the viral thymidine kinase promoter, driving the expression
of the luciferase reporter gene for transactivation studies. We
performed cotransfection experiments in which U2OS cells were
transfected with either an expression plasmid for ER
, ERß1, or
ERß2 and the estrogen-responsive reporter gene construct.
E2 maximally stimulates ER
-mediated transactivation
through an ERE of approximately 6-fold at a doses as low as 0.1
nM (Fig. 8
). ERß1 was less
potent in stimulating transcription through an ERE with only 50% of
the maximal level seen with ER
, although transcriptional activation
occurs with a similar dose response. In contrast, when the same
experiment was performed with an expression plasmid for ERß2,
E2 failed to stimulate transactivation through the ERE at
0.1 nM or 1 nM E2 (Fig. 8
).
Increasing doses of E2 to 100 nM were able to
stimulate ERß2-mediated activation to a level comparable ERß1 (Fig. 8
). These data indicate that ERß2 requires 100- to 1000-fold
higher concentrations of E2 to stimulate transcription to
the same extent as ERß1.

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Figure 8. Transcriptional Activation of ER , ERß1, or
ERß2
U2OS cells were transiently cotransfected with the ERE reporter plasmid
and expression plasmids for either ER , ERß1, or ERß2. Cells were
left untreated or treated with 0.1 nM, 1 nM, 10
nM, and 100 nM estradiol. Data were normalized
as the ratio of raw light units to ß-gal-units and expressed as the
fold of induction relative to untreated controls. The data presented
are the mean of three independent experiments. Error
bars represent SEM.
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DISCUSSION
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Estradiol mediates its diverse biological effects by binding to
the ER, thereby allowing the receptor to bind to and activate
transcription through estrogen-responsive elements in the promoter
region of target genes. The phenotype of ER
knockout mice pointed to
the potential existence of an alternative mediator for E2
action (23). Recently, a new member of the nuclear receptor family with
very high homology to ER
was cloned and termed ERß (18). The
identification of ERß has added a new level of complexity to
E2 signaling.
In this report we describe the cloning of an alternative splice variant
of ERß, ERß2, which contains an 18-aa insert in the predicted
hormone binding domain of this nuclear receptor, adding yet another
level of complexity to E2 signaling. We have demonstrated
that this isoform is expressed in normal rat prostate as well as
various human cancer cell lines. The fact that in some cell lines one
isoform appears to be more prominent than the other, and that this
relative ratio varies from tissue to tissue examined, suggests a
specific mechanism regulating expression of one or the other splice
variant.
Interestingly, we demonstrate that both receptors coexist in certain
cells and can heterodimerize on the ERE. Further complexity is
achieved, since not only do ERß1 and ERß2 heterodimerize, but each
can also heterodimerize with ER
. Since we could not demonstrate that
ERß2 binds the coactivator SRC1 one could speculate that
heterodimerization of ER
or ERß1 with ERß2 regulates the
recruitment of this coactivator to the transcriptional complex.
Since previous studies have demonstrated that other transcriptional
cointegrators, namely p300 and the phospho-CREB binding protein (CBP)
appear to be rate limiting for active transcription (16), this
reduction of SRC1 recruitment might reduce transcriptional activity
through the ERE.
Another recent study has shown that while both ER
and ERß1
activate E2-mediated transcription through an ERE, they
exert opposite effects through an AP-1 site (24). While E2
stimulates ER
-mediated transcription through an AP-1 site,
E2 inhibits ERß1-mediated transcription through the same
response element. It still has to be demonstrated which effect ERß2
mediates through an AP-1 site, and which effect the different
heterodimers of these receptors mediate through this response element
in the presence of different ligands.
Interestingly, alignment of the ER
sequence with ERß, as compared
with the predicted structure of nuclear receptors, indicates that the
18-aa insert in ERß2 lies in helix 6 of this receptor. This
relatively nonconserved region among different nuclear receptors
follows immediately after the
-turn within the ligand binding domain
of the receptor (25). The addition of 18 aa in this region might
distort the correct conformation of this receptor for high-affinity
E2 binding as supported by our E2 binding data.
High physiological E2 concentrations achieved especially in
the ovary during pregnancy or the periovulatory phase might be
sufficient to activate ERß2. Another interesting possibility is that
this insertion creates a new conformational change required for
high-affinity binding of a yet unidentified ligand other than
E2.
With respect to the basic mechanisms by which nuclear receptors
initiate transcription of their target genes, much of recent research
in the field has focused on so-called coactivators of these proteins,
which bind the nuclear receptors in a ligand-dependent manner to
augment AF-2-mediated transactivation. The coactivators have been
identified by the in vitro interaction of the ligand-binding
domain of ER
fused to GST as an affinity matrix for proteins
interacting in an E2 dependent manner (9, 10). Using the
same approach we could demonstrate the interaction of the coactivator
of nuclear receptors, SRC1 with both ER
and ERß1. Interestingly,
GST fusion proteins of both the ligand-binding domain and the
full-length ERß2 failed to interact with SRC1 in a ligand-dependent
manner, despite the fact that both fusion proteins were able to bind
both E2 and an ERE. It is striking that this results
in a shift in the dose response of ERß2 to E2 but not the
maximal level of activation. This suggests the possibility that under
certain conditions ERß2 might act to dampen cellular responses to
estrogen.
We and others (29) have identified an alternative splice variant of
ERß termed ERß2. This protein exhibits interesting properties as a
mediator of estrogen action and provides new complexity to the spectrum
of potential cellular responses to estrogen.
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MATERIALS AND METHODS
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Library Screening and Plasmids
We screened a
gt11 rat prostate cDNA library (CLONTECH, Palo
Alto, CA), according to the manufacturers guidelines with two
radiolabeled oligonucleotides corresponding to nucleotides 418477 and
12481307 of the previously published rat ERß sequence. Labeling of
the probe was performed with
-32P-ATP (6000 mCi/mmol;
New England Nuclear, Boston, MA) in the presence of T4 polynucleotide
kinase according to standard procedures (26). Positive plaques from the
primary screening were isolated by secondary and tertiary screening,
and phage DNA was obtained by boiling plaques in 100 ml
H2O. Plaque DNA was then amplified by PCR using
ERß-specific primers corresponding to nucleotides 402419 and
18661885 of the ERß sequence. One plaque contained the full-length
ERß cDNA. The resulting PCR fragment was blunt ended using T4 DNA
polymerase and was subcloned into the EcoRV site of pcDNA3.0
(Invitrogen, San Diego, CA), resulting in pERßI and was completely
sequenced from both strands by automated sequence analysis (ABI 3000,
Molecular Biology Core Facility, Dana Farber Cancer Institute). DNA
from the remaining plaques yielded PCR products when a 5'-primer
corresponding to nucleotides 922941 of the ERß sequence was used
for amplification, indicating that these were partial cDNA clones.
Amplification of the predicted HBD from these different clones was
performed with primers
5'-CGTGGATCCGAGCAGGTACACTGCCTG-3'
5'-GATGAATTCTCACTGAGACTGTAGGTTC-3' and the resulting fragments were
BamHI/EcoRI digested and subcloned into the
corresponding sites of pGEX2TK (27) resulting in pGEX ERß1HBD and
pGEX ERß2HBD. These plasmids were also subjected to complete
nucleotide sequence analysis, revealing the alternative splice variant
in three of these clones. To obtain the full-length ERß2 cDNA we
liberated the ERß1 cDNA from pERßI by
EcoRI/NotI digest and subcloned the 1.4-kb insert
into the corresponding sites of pBluescript SK(-), resulting in pBS
ERß1. This subclone was confirmed by partial sequence analysis. The
3'-sequence of ERß2 was then liberated from the pGEX2TK ERß2
plasmid by EcoRI digest, blunt ending, and SmaI
digest and subcloned into NotI-digested, blunt ended, and
then SmaI-digested pBSERß1, resulting in pBSERß2.
Correct orientation of the 3'-end was confirmed by restriction analysis
and confirmed by partial sequence analysis, also revealing the presence
of the 54-nucleotide insert. To generate eukaryotic expression plasmids
for ERß1 and ERß2, the corresponding cDNAs were liberated from
pBSERß1 and ERß2 by SacII digest, blunt ending followed
by XhoI digest, and subcloned into NotI-digested,
blunt ended, and then XhoI-digested pcDNA 3.1(-) vector
(Invitrogen), resulting in pcDNA ERß1 and pcDNA ERß2. To obtain
full-length GST fusion proteins of ERß1 and ERß2 the corresponding
cDNAs were PCR amplified with primers
5'-GAAGATCTATGACATTCTACAGTCCTGC-3'
5'-GATGAATTCTCACTGAGACTGTAGGTTC-3' using pBSERß1 and ERß2 as
templates, BglII/EcoRI digested, and subcloned
into BamHI/EcoRI-digested pGEX2TK plasmid,
resulting in pGEX ERß1fl and pGEX ERß2fl. Both plasmids were
verified by complete nucleotide sequence analysis.
Cells and Cell Culture
Cell lines MCF-7, BT-20, MDAMB231, T47D, ECC1, Ishikawa, PC-3,
Du145, LnCAP, CV1, and U2OS were obtained from American Type Culture
Collection (Manassas, VA). The normal HMECs were purchased from
Clonetics (San Diego, CA). The human ovarian cancer cell lines Sw626,
OVCAR-3, CAOV-3, and UPN36T were a gift from Dr. S. Cannistra. Cells
were maintained in DMEM containing 10% FBS (vol/vol) (Sigma Chemical
Co., St. Louis, MO) at 37 C and 5% CO2/95% air.
RT-PCR and Southern Blot
RNA extraction was performed using the Ultraspec RNA isolation
system (Biotecx, Houston, TX) according to manufacturers guidelines.
RT was performed using oligo dT primers. Using the GIBCO BRL RT Kit
(GIBCO BRL, Grand Island, NY). Two micrograms of cDNA were used as a
template for PCR reactions using two microglobulin-specific primers
(CLONTECH, primers specific to human ER (5'-GGGAGCTGGTTCACATGATC-3'
and 5'-GTCCAGGACTCGGTGGAT-ATG-3') or primers specific to human ER
((5'-GCCTCCATGATGATGTCCCTG-3' and 5'-GATCATGGCCTTGACACAGAG-3'). PCR
cycling was performed using a touch down program: 1) 95 C, 1 min; 2) 95
C, 30 sec; 60 C, 30 sec (-0.5 C/cycle); 72 C, 2 min; 3) 72 C, 2 min, 4
C, for ever, 35 cycles, 60 C to 40 C. Resulting PCR products were
subjected to electrophoresis in 2% agarose gels. In case of ER,
products were transferred to nitrocellulose and probed with
end-labeled oligonucleotides either common to ERß1 and
ERß2.
(5'-TCCTCAGAAGACCCTCACTGGCCACGTTGCGCAG-ATGAAGAGTGCTGCCCCAAGG-3')
or specific for ERß2
(5'-GCCAAGAAGATTCCCGGCTTTGTGGAGCTCAGCCTG-TTCGACCAAGTGCGGCTCTTGGAG-3').
Both were washed three times in 1x saline sodium citrate, 0.1%
SDS at room temperature and for 30 min in buffer containing
0.1x saline sodium citrate, 0.1% SDS at 50 C.
Ligand Competition Analysis
For ligand competition studies GST HBD fusion proteins of ERß1
and ERß2 were diluted in HED buffer (20 mM HEPES, pH 7.4,
1 mM EDTA, 1 mM dithiothreitol) and incubated
with 1 nM [3H]17ß-estradiol and various
concentrations of unlabeled diethylstilbestrol. The bound and unbound
estrogens were separated using dextran-coated charcoal (28). The amount
of bound [3H]17ß-estradiol is presented as a percent of
total bound in the absence of diethylstilbestrol.
Gel Mobility Shift Assay
Recombinant ER
, ERß1, and ERß2 cDNAs were transcribed and
translated in vitro in TNT-T3 coupled rabbit
reticulocyte lysates (Promega, Madison, WI) from the T3
Promoter following the manufacturers guidelines. Typically 4 ml of
programmed lysate (or 4 ml of a 1:50 dilution of the ERß1 GST-fusion
protein for ERß1 and ERß2 heterodimers) were used in each binding
reaction. The binding reactions were carried out in binding buffer A100
(20 mM HEPES, pH 7.9, 20% glycerol, 100 mM
KCl, 1 mM dithiothreitol), 0.5 mg of poly
deoxyinosinic-deoxycytidylic acid, 20 mg BSA, 4 ml H2O, 7.5
mM MgCl2 (final concentration), and 4 ng of
probe that was labeled by end-filling with Klenow in the presence of
[32P]
-dGTP. Preincubations containing ligand,
antibody, and/or cold competitor as indicated were performed at room
temperature for 20 min. After the incubation step the probe was added
and binding was conducted for 15 min at room temperature. The entire
reaction of 17 ml was loaded onto a 4% gel, and electrophoresis was
carried out at 110 V for 2 h at room temperature. Gels were dried
and exposed for 25 h at -80 C. The following antibodies were used:
AER 314 (Neomarkers, Fremont, CA), mouse polyclonal serum for ERß1
and ERß2. We used the following oligonucleotides and their
compliments as probes and competitors:
ERE, 5'-GATCTCTTTGATCAGGTCACTGTGACCTGACT-TTG-3';
mtERE, 5'-GATCTCTTTGATCAGGACACAGTGTCCTGA-CTTTG-3';
AP1, 5'-GAATGGTGACTCATATTTGAACAAGCCTGCAA-TGCCCAGCAGA-3'.
Metabolic Labeling and Protein-Protein Interaction Assay
Before the metabolic labeling, MCF-7 cells were preincubated
with methionine-free DMEM for 1020 min. Confluent 150-mm diameter
dishes were labeled with 1 mCi (1 Ci = 37 GBq)
[35S]methionine (New England Nuclear, Boston, MA) for
4 h in methionine-free DMEM. After labeling cells were washed
extensively with ice-cold PBS and lysed in 1 ml of buffer A (150
mM NaCl, 50 mM Tris, pH 7.4, 5 mM
EDTA, 0.5% Nonidet P-40). After 30 min of rotation at 4 C, cell
extracts were clarified by centrifugation at 12,000 rpm, and the
supernatant was collected in a fresh tube. Lysates containing 2.5 x
107 cpm were then incubated with a GST fusion protein
containing the HBDs of either ER
, ERß1, or ERß2 (GST-HBD ER
,
ERß1, or ERß2) immobilized on 50 ml of glutathione-Sepharose beads
in the presence or absence of the appropriate ligand in buffer B (150
mM NaCl, 50 mM Tris, pH 7.4, 5 mM
EDTA) as previously described (9). After washing the beads three times
in 1 ml of buffer B and once in 1 ml of buffer A, proteins were eluted
in SDS/sample buffer and resolved on 7.5% SDS/PAGE. Gels were fixed in
35% methanol/10% glacial acetic acid, fluorographed in Enhance
solution (New England Nuclear), and dried before exposure to film.
Western Blotting
Protein-protein interaction assays were performed as described
above using unlabeled cell extracts from MCF-7 cells. Proteins were
resolved directly in SDS/polyacrylamide gels after boiling in SDS
sample buffer. Immunodetection was performed after blocking the
membranes overnight at 4 C in 20 mM Tris-HCl, pH 7.5, 137
mM NaCl, 0.05% Tween 20, and 5% powdered milk by
incubating membranes with an anti-SRC1 antibody for 2 h at room
temperature. Monoclonal antibody raised against GST-SRC1 was used for
immunoblot analysis in a dilution of 1:100. Specifically bound primary
antibody was detected with peroxidase-coupled secondary antibody and
chemiluminescence.
In Vitro Transcription and Translation
Recombinant SRC1 cDNA in pBluesript was transcribed and
translated in TNT-T3 coupled reticulocyte lysates (Promega,
Madison, WI) in the presence of [35S]methionine from the
T3 promoter following the manufacturers guidelines.
Transient Transfection and Luciferase Assay
For transient transfections U2OS cells were seeded in 24-well
plates in phenol red-free DMEM supplemented with 10% charcoal
dextran-treated FBS. Cells at a density of 40,000/well were transfected
with 100 ng of reporter plasmid, 10 ng of receptor expression vector,
10 ng ßActinßGal plasmid and 680 or 690 ng of salmon sperm DNA to a
total of 800 ng using the calcium phosphate/DNA precipitation method.
After 16 h, cells were washed once with PBS and were left either
untreated or treated with 0.1 nM, 1 nM, 10
nM, or 100 nM E2 for 16 h. For
luciferase assays, cells were lysed in potassium phosphate containing
1% Triton X-100. Light emission was detected using a luminometer after
addition of luciferin. ß-Gal activity was detected using the
Galacto-Star (Tropix, Bedford, MA).
 |
ACKNOWLEDGMENTS
|
---|
We thank S. Cannistra for the gift of the UPN36T cell line, J.
DiRenzo and J. DeCaprio for SRC1 antibodies, and J. Brüning for
helpful discussions and critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Myles Brown, M.D., Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115. E-mail:
myles-brown{at}dfci.harvard.edu
This work was supported by funds from a Deutsche Forschungsgemeinschaft
Fellowship (to B.H.) and by National Cancer Institute Grant CA-57374
(to M.B.).
1 Present address: Frauenklinik, Heinrich Heine Universität,
Postfach 10 10 07, D-40001 Düsseldorf, Germany. 
2 Present address: Robert Lurie Cancer Center, Northwestern University
School of Medicine, 745 North Fairbanks, Chicago, Illinois 60611. 
Received for publication January 29, 1998.
Revision received August 24, 1998.
Accepted for publication October 1, 1998.
 |
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