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Department of Cell Biology, Neurobiology, and Anatomy University of Cincinnati College of Medicine Cincinnati, Ohio 45267
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER) to region E in vivo. In this system, the
cDNAs corresponding to the A–D, E, E/F, A–E (
F), and full-length
(wtER) domains of the human ER were each cloned into the yeast
two-hybrid vectors GAL4 DB and GAL4 TA and expressed in different
combinations in yeast harboring a GAL1-lacZ reporter. The reporter was
used as a relative measure of the interaction between the ER domains,
through reconstitution of GAL4 activity. We found that the interaction
of E or E/F domains of the ER with full-length ER is estradiol
dependent and estrogen responsive element independent, as
measured by the reconstitution of GAL4 activity from GAL4-E
domain-containing fusion protein interactions. In the presence of F
domain, this activity is reduced 10-fold. The results suggest that
sequences in the F domain are inhibitory to the dimerization signal
that is present in the E region. We propose that the full-length ER
contains intrinsic dimerization restraints contributed by regions
outside domain E that are released upon binding hormone agonist. In
addition, we have demonstrated that coactivator RIP140 is able to
interact with the ER in vivo at the E domain of the
receptor in the presence of estrogen. Yeast two-hybrid analysis shows
that RIP140 does not homodimerize in the presence or absence of
estrogens. We present evidence showing that the ER has the inherent
ability to interact with RIP140 in the presence of antiestrogens, but
sequences inherent in the ER itself that are present outside of the E
domain compromise this ability. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER), a member of the steroid/nuclear
receptor superfamily, is a ligand-inducible transcription factor that
mediates the actions of estrogens in target cells. Estrogen action on
target cells involves a distinct pathway where estradiol freely
diffuses across the cell membrane and binds the ER. The ligand-bound ER
homodimerizes, binds specific upstream DNA sequences called
estrogen-responsive elements (EREs), and activates transcription of its
target genes by as yet unknown mechanisms. All of the steroid receptors possess a modular structure, with discrete regions of the protein (domains) responsible for transcriptional activation, DNA binding, nuclear localization, ligand binding, and dimerization (1, 2). The estrogen receptor can be divided into six functionally independent domains denoted from N- to C-terminal by the letters A to F. Dimerization properties of the estrogen receptor have been primarily localized to the E region (1, 3), a complex domain that integrates several functions including hormone binding and ligand-dependent transcriptional activation (AF-2) (4). The ER also possesses a ligand- independent transcriptional activation function in region A/B that is promoter and cell type dependent (AF-1) (5) and a possible third activation function near the N-terminal end of domain E (AF-2a) (6, 7). The DNA-binding domain (DBD or region C) also possesses a weak dimerization property that stabilizes binding to an isolated ERE in vitro (8) and has been suggested to restrain steroid receptor transcriptional synergy through the DBD-dimer interface (9). A role of the F domain has not been established in dimerization of the receptor, but it has been proposed that F has a specific modulatory function that affects the agonist/antagonist effectiveness of antiestrogens and the transcriptional activity of the liganded ER in cells (10).
Recently, a new concept of steroid hormone action has developed with the discovery of several novel coactivators that increase the ability of the receptors to activate transcription (reviewed in Refs. 11, 12). It is unknown whether the coactivators have a role in the dimerization of the steroid receptors, but it has been suggested that these cofactors may act as a bridging apparatus between the receptor and the transcriptional machinery. Among the coactivators of ER, RIP-140 associates in vitro and in vivo with the ER carboxyl terminus in the presence of estrogen, but not in the presence of antiestrogen (13, 14).
Several in vitro studies have demonstrated that the formation of the ER homodimer after ER-ERE binding is not dependent on estrogen (1, 15, 16, 17, 18). Others suggest roles for ligand in the interaction (1) and that salt conditions, ionic strength, and temperature influence binding (19, 20). In the absence of an ERE, we recently showed that dimerization of full-length ER is ligand dependent in vivo using the yeast two-hybrid system (21).
In the current study, the yeast two-hybrid system was used to localize the ligand-dependent dimerization domain of the receptor and to determine the domains that heterodimerize with the full-length receptor. Yeast has been used extensively to study the estrogen receptor and several other members of the nuclear receptor family of genes (22, 23, 24, 25), taking advantage of the absence of these factors to perform experiments without any cellular background. Here, we show that the E domain can interact strongly with the full-length receptor in the presence of ligand and that the F domain can decrease this interaction. We also confirm that the human ER (hER) forms a protein-protein interaction with RIP-140 and that the interaction is dependent on the presence of ligand. In addition, the presence of ER domain F somewhat disrupts interaction of the full-length receptor with RIP-140 and is a likely factor in the inability of antiestrogen-bound ER to interact with RIP-140.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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F)
domains (Fig. 1A
) of the human estrogen
receptor were each cloned into the yeast two-hybrid vectors GAL4 DB
(pPC62) and GAL4 TA (pPC86) and expressed in yeast harboring a
GAL1-lacZ reporter (26). These particular vectors were chosen to ensure
low expression levels to avoid artificial scenarios that often
complicate in vitro studies and others that involve protein
overexpression. In all of these studies, Western blotting was performed
to confirm correct and equivalent expression of GAL4 ER fusion protein
constructs (Fig. 1B). The lacZ reporter was used as a relative measure
of the interaction between the ER domains, through reconstitution of
GAL4 activity. Since the estrogen receptor contains three independent
transcriptional activation functions (AF-1, AF-2, and AF-2a), it was
initially unknown whether GAL4 fusions to A–D and F (containing
AF-1), or E, E/F, and F (containing AF-2 and AF-2a) themselves could
activate transcription of the GAL1-lacZ reporter gene in this system.
Therefore, each fusion construct was transformed into yeast alone and
ß-galactosidase activity was measured by 5-bromo-4-chloro-3-indoyl
ß-D-galactoside (X-gal) reaction. GAL4 DB-A-D and GAL4
TA-A-D alone, which contain AF-1 as well as a DBD, showed no activation
of the GAL1-lacZ reporter in the presence or absence of 17ß-estradiol
(Table 1, controls). The AF-2 and
AF-2a-containing constructs, represented by domains E, E/F, and F,
showed no activation of the reporter when expressed as fusions with
GAL4 DB or GAL4 TA (Table 1, controls), indicating these domains cannot
activate the reporter gene by themselves. This finding may be explained
by the fact that the AF-1 and AF-2 regions of hER have been found to
mediate transactivation by nonacidic amino acids (4), whereas GAL4
transactivation occurs through acidic residues. To address the
possibility that the GAL4 DB-wild-type (wt)ER fusion alone is able to
activate GAL1-lacZ transcription through the particular AFs present in
the estrogen receptor, we reasoned that coexpression of unfused
coactivator RIP140 with GAL4 DB-wtER should increase transcriptional
activity of the reporter contributed by ER and amplified by RIP140. Our
reasoning was based upon the fact that, depending on the
estrogen-responsive promoter and expression level, RIP140 enhances ER
transactivation between 1.5- to 4-fold (27) or 30- to 100-fold in yeast
(our unpublished observations). However, in the presence or absence of
estrogen, coexpression of GAL4 DB-wtER with RIP140 did not amplify
ß-galactosidase activity through GAL1-lacZ, suggesting that our
reporter in the yeast two-hybrid system is measuring relative
protein-protein interaction and not transcriptional activity
contributed by ER (data not shown). It is therefore reasonable to
conclude that AF-1, AF-2, or AF-2a of the ER do not activate GAL1-lacZ
transcription by themselves when these regions are fused to GAL4 DB or
GAL4 TA.
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) were coexpressed
as fusions with GAL4 DB and GAL4 TA in PCY2 yeast treated without and
with 17ß-estradiol. The ß-galactosidase activity was determined as
a relative measure of homodimerization of the individual domains of the
ER. Throughout all of our experiments, in yeast treated with vehicle
alone, none of the ER domains tested using two-hybrid analysis showed
any activation of ß-galactosidase activity. However, upon treating
PCY2 yeast with 17ß-estradiol, full-length ER (wtER) as well as ER
missing the F domain (F) both reconstituted ß-galactosidase
activity in individual homodimerization assays (Table 1 and Fig. 2A). Estrogen receptor missing the F
domain had almost twice the ability as full-length receptor in
reconstituting reporter gene activity. GAL4 fusions containing the
A–D, E, or E/F domains could not individually homodimerize in the
presence or absence of estrogen (Fig. 2A). This finding is in contrast
to in vitro evidence showing that the hormone-binding domain
(region E/F) homodimerizes in the absence of hormone (28). It is
possible that the in vitro data may be due to an artificial
scenario due to the greatly overexpressed estrogen receptor necessary
for in vitro studies and may be complicated by minute
concentrations of estrogen present in the media. In vivo,
however, our results clearly show that estrogen induces the formation
of a full-length estrogen receptor homodimer and demonstrate that an ER
dimer also forms when the receptor is missing domain F (Fig. 2A).
Furthermore, the F dimer formation results in twice the
ß-galactosidase activity as the wild-type full-length receptor.
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and Fig. 2B). These results are consistent with evidence that the dimerization
signal in region C is influenced or stabilized by the presence of an
ERE (8, 9) not present in PCY2 yeast, or that this dimerization signal
is masked by accessory factors. Individual expression of GAL4 TA
fusions to E, E/F, or F domains showed estrogen-dependent
interaction with GAL4 DB-wtER (Table 1 and Fig. 2B), indicating the
dimerization signal in region E is functional in yeast and activated by
estrogen. When the relative ß-galactosidase activities of the
constructs containing this signal are compared, in the presence of
estrogen, E domain interaction with wtER influences an 8-fold increase
in activity compared with levels of the full-length ER homodimer (Fig. 2B). When F domain is added back to E domain (indicated by GAL4
TA-E/F), the activity drops to similar GAL4-wtER homodimer levels.
Estrogen receptor missing domain F (F) reconstituted
ß-galactosidase activity at more than twice the level of the
full-length ER homodimer. The interactions of GAL4 DB-wt ER with GAL4
TA-wtER, GAL4 TA-E, or with GAL4 TA-E/F are all estradiol dose
dependent, with 1 nM concentrations of estradiol sufficient
to elicit a response (Fig. 3). Similar
estradiol dose dependency was observed between GAL4 DB-wtER and
GAL4TA-F, with the maximum ß-galactosidase activity at an
intermediate level (
25 U, data not shown). The data suggest that the
presence of F domain is inhibitory to the ligand-dependent dimerization
signal in the ligand-binding domain.
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),
indicating estrogen is required for the protein-protein interaction of
RIP140 with ER.
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), similar to
mouse ER hormone-binding domain/RIP 140 interaction using mammalian
two-hybrid assays (13). Strikingly, truncation of carboxy- and
amino-terminal ends of the ER to express the hormone-binding domain
fusion, GAL4 TA-E, resulted in extremely high ß-galactosidase
activity (Fig. 4A). Comparing relative interactions of RIP-140 with the
full-length ER shows that truncating ER to the E domain increased
ß-galactosidase activity 20-fold. Addition of the ER domain F
significantly decreased the interaction with RIP140 relative to E
domain alone (compare GAL4 TA-E with GAL4 TA-E/F, Fig. 4A), although
the activity is still 14 times higher relative to RIP140 interaction
with the full-length receptor. The interaction of RIP140 with E or E/F
domain is estrogen dose dependent, again showing the presence of the F
domain decreases the relative amount of interaction of RIP-140 with E
domain (Fig. 4B).
To determine whether RIP140 can form a homodimer, RIP140 fused to
GAL4DB and GAL4 TA was coexpressed in PCY2 yeast, and GAL1-lacZ
reporter gene activation was measured. In the presence or absence of
estradiol, RIP140 was unable to form a homodimer in vivo
(Fig. 4A).
In our assay, RIP-140 also associates with E domain in the presence of
the antiestrogens tamoxifen and ICI 182,780, and with E/F in the
presence of ICI only (Fig. 5). When the E
domain fusion of the ER is coexpressed with the RIP140 fusion in the
presence of antiestrogens, the ß-galactosidase activity is
significantly higher than in yeast treated with vehicle alone.
Treatment of yeast with ICI 182,780 evokes a 10-fold increase in
reporter activity relative to yeast treated with tamoxifen. However,
relative to yeast treated with estradiol, tamoxifen and ICI 182,780
decreased ß-galactosidase activity 100-fold and 10-fold,
respectively.
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).
In the presence of physiological concentrations of antiestrogens, no
interaction of RIP-140 with the full-length receptor could be detected
in our system. These data suggest an inherent ability of ER at the E
domain to interact with coactivator RIP140 in the presence of
antiestrogens and estrogens that is somehow repressed by ER sequences
outside the E domain. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In using this system to measure protein-protein interactions, the
levels of ß-galactosidase measured are relative levels of interaction
and do not represent the dissociation constant (Kd) of the
interaction of the two proteins in question (29). When the individual
GAL4 fusions with the domains of ER are transformed alone into yeast or
cotransformed with empty GAL4 vectors, no ß-galactosidase
transcription is measured (Table 1). This indicates that the ER
transcriptional activation functions (AF-1, AF-2, and AF-2a) do not
activate GAL1-lacZ transcription alone or that the assay is not
sensitive enough to detect the activation. However, it may be that, by
adding several functional AFs to the region of the promoter, the
reporter gene will be activated. In this case for example, to address
the possibility that the GAL4 DB-wtER fusion alone is able to activate
GAL1-lacZ transcription through the particular AFs present in the
estrogen receptor, we coexpressed unfused coactivator RIP140 with GAL4
DB-wtER, reasoning that we would see an increase in transcriptional
activity of the reporter contributed by ER and amplified by RIP140.
This reasoning was based on evidence demonstrating that RIP140 enhances
ER transcriptional activity in yeast between 4- to 100-fold, depending
on promoter context (Ref. 27 and our unpublished observations).
However, coexpression of GAL4 DB-wtER with RIP140 did not amplify
ß-galactosidase activity through GAL1-lacZ, suggesting that our
reporter in the yeast two-hybrid system is measuring relative
protein-protein interaction and not transcriptional activity
contributed by ER. It is important to note that ER AF-1 and AF-2
activity depend on cellular and promoter context (5), and in our
promoter context, RIP140 was unable to amplify any background activity
contributed by wtER on GAL1-lacZ. However, as in most in
vivo approaches, we cannot exclude the possibility of other
endogenous proteins contributing to the interactions. In any case, in
yeast two-hybrid analysis, protein-protein interaction is required to
bring GAL4 DB and GAL4 TA together to reconstitute the full activity
that switches on the reporter.
It is apparent that by truncating the full-length estrogen receptor to
only the E region, the dimerization or interaction potential of the
receptor is accentuated. With the addition of F domain, we observed
that the ability of E to dimerize with ER repressed to levels relative
to the full-length ER homodimer. Possibly sequences in the E domain
essential to dimerization are masked by intramolecular folding or
secondary structure contributed by the F domain. If this is the case,
crystal structure data of the ligand-binding domain (30) and DBD
(8) of the ER must be reexamined in the context of the full receptor.
Crystal data of the E domain dimer show that the structure is
essentially -helical, with a major repositioning of helix 12 (at the
carboxy terminus of E domain) as the receptor binds hormone. We propose
that the binding of hormone agonist to the receptor changes the
conformation in the ligand-binding domain so that the intrinsic
dimerization restraint is released. Lui et al. (9) have
suggested that the estrogen receptor contains a DBD dimerization
restraint that once released, allows AF-1 and AF-2 synergistic
activity. This release of the restraint could be hypothesized to be
facilitated by the binding of a coactivator such as RIP-140, which
would be expected to alter ER conformation upon binding. In this
respect, RIP140 differs in the fact that it contains two distinct sites
having a LXXLL motif that facilitates interaction with the estrogen
receptor (14, 31), while coactivators mSUG1 and TIF1 contain single
sites of interaction with nuclear receptors. This additional site of
interaction could be required to release the dimerization restraint
inherent in the full-length estrogen receptor.
Alternatively, sequences within the ER may interact with factors such as the heat shock proteins or other chaperones that direct the proper folding of the full-length receptor or play a role in the dimerization of the receptor. It is possible that putative ER corepressor proteins may also mask the dimerization signal in E, either directly or by altering the conformation of the receptor. The extreme C terminus of the progesterone receptor (PR) has been recently shown to contain a transcriptional repressor domain that functions through a putative corepressor (32).
In this report, we have shown GAL4 DB-wtER does not interact with GAL4
TA-A–D in the presence or absence of estrogen, suggesting the DBD of
ER (domain C) does not contribute to the dimerization of the receptor
in the absence of an ERE. In heterodimerization experiments, the
dimerization signal was localized to the E domain, which was repressed
when F domain was added back. It is possible that F domain protein
residues fold back onto residues in domain E that are essential to
dimerization, similar to the mechanism by which synthetic peptides have
been engineered as antiestrogens to disrupt ER dimerization by
interfering with phosphorylation site Tyr537 (33). Previous studies
have shown that domain F is not required for transcriptional response
to estrogen (2, 3), and that this region does not affect the turnover
rate of the ER in target cells (34). A role of domain F, however, has
been proposed in modulating the affects of agonist/antagonist
effectiveness of antiestrogens and the transcriptional activity of the
liganded ER in cells (10). In certain cells and promoter contexts,
antiestrogens, which stimulate transcription of ERE reporter constructs
with the full-length ER, were unable to stimulate transcription with
F ER. In this regard, our experiments show that F exhibits a
4-fold decrease in the ability to interact with coactivator RIP140
compared with full-length ER in the presence of agonistic hormone. It
appears that one mechanism that antiestrogens use in modifying gene
transcription is related to inducible changes in conformation of the
carboxy-terminal tail of the receptor, as has been the case with the
influence of RU486 on PR (35).
The effects of RIP140 and other coactivators and corepressors on modulating the conformational structure of nuclear receptors deserve further attention. In one model, coactivators could act as factors that remodel nuclear receptor protein structure, exposing dimerization signals and directing an optimal ER complex conformation to the transcriptional machinery. The ability of each coactivator to optimize receptor conformation could be directly related to the ability of the coactivator to enhance transcription, and the stochiometric amount present in each cell may play a significant role in transcriptional regulation of the tertiary ER complex. In this regard, RIP140 has been proposed to indirectly regulate AF-2 transcriptional activity by competing with SRC-1 for receptor binding (36).
With the discovery of several truncated ER variants in normal tissue
(37, 38, 39) or tumors (40, 41, 42, 43, 44), the effects of removing dimerization
restraints of the ER would give cells the ability to form ER dimers
even if coactivators were absent from the cell. The results of these
experiments may also have implications on the development of potential
dominant negative ERs to impair dimerization in the case of ER-positive
breast cancer. Comparison of the carboxy-terminal ends of ER
and the recently described ERß (45, 46) show that hERß F domain is
missing 16 amino acids at the extreme C terminus relative to ER
(Fig. 6). The remaining part of ERß is
not well conserved with ER, showing homology with only 5 of 27 amino
acid residues. Since the domain structures of the other human receptor
proteins for gonadal and adrenal hormones (PR-A, PR-B, glucocorticoid
receptor, mineralocorticoid receptor, and androgen receptor)
show the absence of F domain (47), it is possible that sequences in F
domain may play a unique role specific for ER and/or ERß.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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gal4 gal 80
URA3::GAL1-lacZ lys2–810amber his
3- 200 trp1-
63leu2ade2–101ochre) was used for all assays
(26). Yeast strains were grown in yeast extract/peptone/dextrose (YEPD)
or supplemented synthetic dextrose medium (-leu,-trp). Transformation
of yeast was carried out using dimethylsulfoxide/polyethylene
glycol/lithium acetate method with plasmid DNA (48).
cDNA and Constructs
Construction of GAL4-wtER fusion vectors was described (21).
Yeast two-hybrid vectors pPC62 (GAL4DB) and pPC86 (GAL4TA) were used to
allow expression of the various domains at a low level (26). To
subclone E domain into GAL4 DB (pPC62) and GAL4 TA (pPC86) (26), E
domain cDNA was amplified with PCR using oligonucleotides
5'-ACGCACGTCGACGAAGAAGAACAGCC-3' and 5'-GGGGGTTGAACTAGTGGGCGCATGTA-3'
containing SalI and SpeI sites, respectively.
After restriction enzyme digestion, the PCR product was directionally
cloned into GAL4 DB at the SalI/SpeI sites to
create GAL4DB-E. GAL4DB-E was digested with
SalI/SpeI to release domain E; the cDNA was
placed into GAL4 TA to create GAL4TA-E.
Previously, we subcloned the full-length hER cDNA digested with SalI into pBluescript II SK+ at the SalI site such that its transcription is dependent on T7 polymerase (T7-hER) (21). T7-hER domains E and F were amplified using T3 primer and the same SalI-containing oligonucleotide used for E domain cloning above. The PCR product was digested with SalI and NotI and placed into the DB fusion vector to create GAL4DB-E/F. GAL4DB-E/F was digested with SalI/NotI to release domains E–F; the cDNA was placed into GAL4TA to create GAL4TA-E/F.
Similarly, PCR was used to amplify the cDNA encoding A–D from pCMV (a gift from B. Katzenellenbogen), using primers 5'-AATCGTCGACAATGACCATGACCCTCC-3' (SalI) and 5'-GGACTAGTTAAGAGCGTTTGATCATGAG-3' (SpeI). The PCR product was digested with SalI/SpeI and placed into both GAL4 vectors to create GAL4DB-A–D and GAL4TA-A–D.
Human F domain cDNA was amplified using the
SalI-containing primer (used for A–D cloning) and the
SpeI-containing primer (used for E-domain cloning), and each
PCR product was directly cloned into GAL4 DB and GAL4 TA at the
SalI/SpeI site. Each GAL4 DB and GAL4 TA fusion
cDNA construct was sequenced to confirm correct reading frame before
transforming yeast.
RIP-140 cDNA in pEF-BOS [kindly provided by Dr. Malcolm Parker (13)] was amplified by PCR with primers 5'-GCGTCGACGCTTCTATTGAACATGACTCAT-3' (SalI) and 5'-GGACTAGTCCAAAACTGGATGGCAGGT-3' (SpeI). The PCR-amplified fragment was cloned into pBluescript II SK+ at SalI/SpeI restriction sites. The RIP-140 coding region was released from pBluescript II SK+ by SalI/SpeI and cloned into GAL4DB and GAL4TA vectors. Each GAL4 DB and GAL4 TA fusion cDNA construct was sequenced to confirm correct reading frame before transforming yeast.
Ligand Treatment and ß-Galactosidase Activity Assay
ß-Galactosidase activity in PCY2, which was the product of
LacZ driven by the GAL1, was used to indicate reconstitution of GAL4
transactivation activity via the interaction of the two fusion
proteins. Transformed yeast were selected and cultured in synthetic
medium. Yeast were grown in 1% ethanol and then transferred to 2%
glucose medium containing ligand. After treating with ligand (18–22
h), yeast cells were resuspended in Z-buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1
mM MgSO4) containing 0.03% SDS. The reaction
was started with the addition of 0.2 ml of 4 mg/ml
o-nitrophenol-ß-D-galactoside (ONPG) at 30 C
and stopped by adding 0.5 ml of 1 M
Na2CO3. ß-Galactosidase activity was
determined by measuring the values at A420 and
A550 using the following equation: U = 1000
x[(A420) - (1.75 x A550)]/[t x
v x A600] [(t = time of reaction (min); v
= volume of yeast culture used in reaction mixture (ml)].
For X-gal reaction, paper filter lifts of colonies were transferred to selection medium containing 1 µM 17ß-estradiol or 0.1% ethanol for 6 h, submerged in liquid nitrogen, and transferred to X-gal. The number of blue and/or white colonies were counted.
Immunoblotting
Yeast were collected by low-speed centrifugation, resuspended in
0.25 M NaOH and 1% ß-mercaptoethanol, and placed on ice
for 10 min. Then, 0.16 ml of trichloroacetic acid (50%) was added to 1
ml of suspension on ice, and yeast were pelleted. After washing with
acetone, the pellet was dried and resuspended in SDS-PAGE sample
buffer. Equal amounts of total protein were analyzed on 9% SDS-PAGE
and transferred to polyvinylidene difluoride membrane. After the
transfer, blots were stained with 0.5% Ponceau S Red to monitor
transfer efficiencies and subsequently probed with either ERC314 (Santa
Cruz Biotechnology, Santa Cruz, CA) or H222 (Abbott Diagnostics, North
Chicago, IL) antibody.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by National Aeronautics and Space Administration U95–002 Predoctoral Fellowship (to G.P.) and NIH Grant CA-72039 and American Cancer Society Grant CN-77110 (to S.K).
1 Present address: Department of Molecular Biology, NC-20, The Lerner
Research Institute, The Cleveland Clinic Foundation, 9500 Euclid
Avenue, Cleveland, Ohio 44195. 
Received for publication August 6, 1998. Revision received October 23, 1998. Accepted for publication November 9, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A. Tamrazi, K. E. Carlson, J. R. Daniels, K. M. Hurth, and J. A Katzenellenbogen Estrogen Receptor Dimerization: Ligand Binding Regulates Dimer Affinity and DimerDissociation Rate Mol. Endocrinol., December 1, 2002; 16(12): 2706 - 2719. [Abstract] [Full Text] [PDF]
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J. A. Schwartz, L. Zhong, S. Deighton-Collins, C. Zhao, and D. F. Skafar Mutations Targeted to a Predicted Helix in the Extreme Carboxyl-terminal Region of the Human Estrogen Receptor-alpha Alter Its Response to Estradiol and 4-Hydroxytamoxifen J. Biol. Chem., April 5, 2002; 277(15): 13202 - 13209. [Abstract] [Full Text] [PDF]
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R. Métivier, F. G. Petit, Y. Valotaire, and F. Pakdel Function of N-Terminal Transactivation Domain of the Estrogen Receptor Requires a Potential {alpha}-Helical Structure and Is Negatively Regulated by the A Domain Mol. Endocrinol., November 1, 2000; 14(11): 1849 - 1871. [Abstract] [Full Text]
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 J. Font de Mora and M. Brown AIB1 Is a Conduit for Kinase-Mediated Growth Factor Signaling to the Estrogen Receptor Mol. Cell. Biol., July 15, 2000; 20(14): 5041 - 5047. [Abstract] [Full Text]
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 J. F. Couse and K. S. Korach Estrogen Receptor Null Mice: What Have We Learned and Where Will They Lead Us? Endocr. Rev., June 1, 1999; 20(3): 358 - 417. [Abstract] [Full Text]
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A. Warnmark, T. Almlof, J. Leers, J.-A. Gustafsson, and E. Treuter Differential Recruitment of the Mammalian Mediator Subunit TRAP220 by Estrogen Receptors ERalpha and ERbeta J. Biol. Chem., June 22, 2001; 276(26): 23397 - 23404. [Abstract] [Full Text] [PDF]
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M. A. Loven, V. S. Likhite, I. Choi, and A. M. Nardulli Estrogen Response Elements Alter Coactivator Recruitment through Allosteric Modulation of Estrogen Receptor beta Conformation J. Biol. Chem., November 21, 2001; 276(48): 45282 - 45288. [Abstract] [Full Text] [PDF]
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