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Department of Adult Oncology Dana-Farber Cancer Institute Boston, Massachusetts 02115
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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gt11 rat prostate cDNA
library with two oligonucleotide probes derived from the published
ERß sequence, corresponding to the nucleotides 418–477 and
1248–1307. 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
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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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-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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). These data indicate that the 18-aa
insertion in the HBD of ERß2 lowers its affinity for
E2.
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, 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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(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 7–10) or ER
and ERß2 (lanes 11–14) 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).
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and ERß1, but Not ERß2), the human breast cancer cell
line MDA-MB231, and the human osteosarcoma cell line U2OS (data not
shown). In the case of ER (lanes 1–3), 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 4–6), 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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, 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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gt11 rat prostate cDNA library (CLONTECH, Palo
Alto, CA), according to the manufacturers guidelines with two
radiolabeled oligonucleotides corresponding to nucleotides 418–477 and
1248–1307 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 402–419 and
1866–1885 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 922–941 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 manufacturer’s 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 manufacturer’s 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 2–5 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 10–20 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 manufacturer’s 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).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß at AP1 sites [see comments]. Science 277:1508–1510This article has been cited by other articles:
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T. R. Pak, W. C. J. Chung, T. D. Lund, L. R. Hinds, C. M. Clay, and R. J. Handa The Androgen Metabolite, 5{alpha}-Androstane-3{beta}, 17{beta}-Diol, Is a Potent Modulator of Estrogen Receptor-{beta}1-Mediated Gene Transcription in Neuronal Cells Endocrinology, January 1, 2005; 146(1): 147 - 155. [Abstract] [Full Text] [PDF]
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 A Bardin, N Boulle, G Lazennec, F Vignon, and P Pujol Loss of ER{beta} expression as a common step in estrogen-dependent tumor progression Endocr. Relat. Cancer, September 1, 2004; 11(3): 537 - 551. [Abstract] [Full Text] [PDF]
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 S. Chu, Y. Nishi, T. Yanase, H. Nawata, and P. J. Fuller Transrepression of Estrogen Receptor {beta} Signaling by Nuclear Factor-{kappa}B in Ovarian Granulosa Cells Mol. Endocrinol., August 1, 2004; 18(8): 1919 - 1928. [Abstract] [Full Text] [PDF]
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L. Xu, Q. Pan-Hammarstrom, A. Forsti, K. Hemminki, L. Hammarstrom, D. Labuda, J.-A. Gustafsson, and K. Dahlman-Wright Human Estrogen Receptor {beta} 548 Is Not a Common Variant in Three Distinct Populations Endocrinology, August 1, 2003; 144(8): 3541 - 3546. [Abstract] [Full Text] [PDF]
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 S. B. Pillai, J. M. Jones, and R. D. Koos Treatment of Rats with 17{beta}-Estradiol or Relaxin Rapidly Inhibits Uterine Estrogen Receptor {beta}1 and {beta}2 Messenger Ribonucleic Acid Levels Biol Reprod, December 1, 2002; 67(6): 1919 - 1926. [Abstract] [Full Text] [PDF]
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M. J. Evans, K. Lai, L. J. Shaw, D. C. Harnish, and C. C. Chadwick Estrogen Receptor {alpha} Inhibits IL-1{beta} Induction of Gene Expression in the Mouse Liver Endocrinology, July 1, 2002; 143(7): 2559 - 2570. [Abstract] [Full Text] [PDF]
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H. A. LaVoie, D. C. DeSimone, C. Gillio-Meina, and Y. Y. Hui Cloning and Characterization of Porcine Ovarian Estrogen Receptor {beta} Isoforms Biol Reprod, March 1, 2002; 66(3): 616 - 623. [Abstract] [Full Text] [PDF]
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 S. Nilsson, S. Makela, E. Treuter, M. Tujague, J. Thomsen, G. Andersson, E. Enmark, K. Pettersson, M. Warner, and J.-A. Gustafsson Mechanisms of Estrogen Action Physiol Rev, October 1, 2001; 81(4): 1535 - 1565. [Abstract] [Full Text] [PDF]
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 K. Paradiso, J. Zhang, and J. H. Steinbach The C Terminus of the Human Nicotinic {alpha}4{beta}2 Receptor Forms a Binding Site Required for Potentiation by an Estrogenic Steroid J. Neurosci., September 1, 2001; 21(17): 6561 - 6568. [Abstract] [Full Text] [PDF]
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H. Cardenas, K.A. Burke, R.M. Bigsby, W.F. Pope, and K.P. Nephew Estrogen Receptor {beta} in the Sheep Ovary During the Estrous Cycle and Early Pregnancy Biol Reprod, July 1, 2001; 65(1): 128 - 134. [Abstract] [Full Text] [PDF]
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 S. F. Palter, A. B. Tavares, A. Hourvitz, J. D. Veldhuis, and E. Y. Adashi Are Estrogens of Import to Primate/Human Ovarian Folliculogenesis? Endocr. Rev., June 1, 2001; 22(3): 389 - 424. [Abstract] [Full Text] [PDF]
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R. H. Price Jr., C. A. Butler, P. Webb, R. Uht, P. Kushner, and R. J. Handa A Splice Variant of Estrogen Receptor {beta} Missing Exon 3 Displays Altered Subnuclear Localization and Capacity for Transcriptional Activation Endocrinology, May 1, 2001; 142(5): 2039 - 2049. [Abstract] [Full Text]
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