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The Department of Physiology University of Wisconsin-Madison Madison, Wisconsin 53706
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The level of steroid receptors in cells changes with varying physiological states. In most cases, the primary endocrine regulator is the ligand itself. In an autoregulatory feedback loop, estrogen induces a decline in both ER protein and mRNA (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Several mechanisms have been proposed to explain how estrogen controls ER levels, most of which focus on regulation at the level of RNA. Studies using Rat-1 fibroblasts stably transformed with ER (Rat 1+ER) (7) and MCF-7 breast cancer cells (11, 12) support both transcriptional and posttranscriptional mechanisms. The focus on transcriptional mechanisms is based on the assumption that decreased protein concentration is largely a consequence of decreased steady state levels of mRNA.
ER regulation by estrogen has been documented in a number of systems, yet little is known about mechanisms governing ER regulation in estrogen target tissues outside of breast cancer cell lines and uterus. To examine ER regulation in the pituitary, we took advantage of the recent derivation of a lactotrope cell line named PR1. The PR1 cell line was derived from an estrogen-induced lactotrope hyperplasia in F344 rats (14). It exhibits unique sensitivity to estrogen with a high affinity for estradiol [dissociation constant (Kd) = 10-11 M (15)]. Like MCF-7 cells and other model systems, we observed that estrogen induces a decrease in ER protein levels in PR1 cells. However, within the first 1–2 h, ER protein levels decrease without a concomitant decline in mRNA levels. The rapid loss of ER protein in the absence of changes in ER mRNA suggested that ER protein may be regulated independently of transcription. Utilizing a short time frame of estrogen exposure, we are able to isolate changes in ER protein levels away from changes in RNA. This permits the exploration of regulatory mechanisms directly controlling ER protein. Here we report that estrogen stimulates degradation of ER protein via a proteasome-mediated proteolytic mechanism.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, A and B) and dose
response (Fig. 1, C and D) experiments indicate that 10-10
M 17ß-estradiol (E2) is sufficient to elicit
an approximate 50% decrease in ER protein levels at 1 and 2 h.
These results were confirmed using primary anti-ER antibodies directed
against the hinge and C-terminal regions to demonstrate that the loss
of signal was not due to epitope masking (data not shown). Moreover,
the identical results with different antibodies suggest that this
decline is representative of total ER protein and not an
epitope-specific pool. To further verify that the decrease in protein
was specific to ER, blots were reprobed with antibody directed against
the ubiquitous protein, I
B
, which is not regulated by estrogen.
IB protein levels were unchanged in the presence and absence of
E2 and serve as a loading control for total protein content
(Fig. 1A, lower panel).
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, lanes 1
and 2). These results suggested that estrogen may induce a rapid
decrease in ER protein that is independent of ER synthesis. To test
this possibility, ER protein levels were examined in the presence of
inhibitors of protein synthesis and transcription. Figure 2A shows that halting de novo
protein synthesis with a series of inhibitors can decrease ER protein
levels relative to untreated controls. However, in no case was the
decline in ER levels equivalent to that induced by estrogen.
Furthermore, the addition of inhibitors of either protein synthesis
(Fig. 2A) or transcription (Fig. 2B) failed to prevent estrogen-induced
down-regulation. In Fig. 2B (left panel) and in subsequent
figures, ER appears as a doublet. The appearance of the doublet is
spurious from gel to gel but is only present in estrogen-treated
groups. The higher molecular weight form may, therefore, represent an
estrogen-dependent posttranslational modification such as
phosphorylation. Neither form of ER is preferentially lost in response
to estrogen. These data indicate that the mechanism employed to elicit
a rapid loss of receptor protein does not require de novo
protein synthesis or transcription.
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) indicates
that ER half-life in PR1 cells is greater than 3 h but is within a
similar time range as the half-life reported in breast and uterus. This
result supports the notion that decreased ER synthesis cannot account
for the rapid decline in ER protein steady state levels since
presynthesized ER levels do not change significantly within 2 h.
In addition, the data imply that estrogen may induce degradation of ER
protein. Figure 3 illustrates that the
half-life of liganded ER is approximately 1 h. Nonspecific bands
that coimmunoprecipitate with ER are not regulated by estrogen and
serve as an internal control. This 3-fold change in ER turnover rate is
greater than that reported in MCF-7 cells measured by density shift
technique and binding of [3H]tamoxifen aziridine
(16, 18) and contrasts with previous studies in uterine cells (17, 19).
These findings show that estrogen can directly control ER protein by
inducing proteolysis and further highlight the importance of receptor
protein regulation in lactotrope cells.
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). High
concentrations of each inhibitor were used intentionally to ensure that
any potential effects might be noticed. The multicatalytic proteasome
is responsible for degradation of the majority of cellular proteins.
Rock et al. (24) demonstrated that proteasome peptidase
activity could be blocked by exposure to peptide aldehyde inhibitors
including MG115 and N-acetyleucylleucylnorleucinal (ALLnL).
Pretreatment of PR1 cells with MG132, a more potent derivative of
MG115, and ALLnL prevented estrogen-induced loss of ER. In contrast,
calpeptin and the peptide inhibitor
N-acetylleucylleycylmethional (ALLM), which preferentially
block calpain activity, failed to prevent ER degradation. E64D and
NH4CL, which inhibit cysteine proteases and lysosomal
function, respectively, were also without effect. These results suggest
that proteasome activity may regulate ER response to estrogen. However,
protease inhibitors do not exhibit strict specificity. In the case of
the proteasome, the rank order of potency of peptide aldehyde
inhibitors directly reflects their specific activity against proteasome
function (24). Thus, the relative effectiveness of these inhibitors can
be used as a measure of specificity for the proteasome. Dose response
experiments were performed with MG132 and ALLnL to determine their
relative potencies (Fig. 4B). As a control, similar experiments were
conducted with the calpain inhibitor, calpeptin. Examination of data in
Table 1 indicates that MG132 was the most
effective, preventing ER degradation to a dose of 0.03
µM. ALLnL was less effective but blocked ER degradation
between 100 uM and 12.5 µM.
L-1-Tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a
serine protease inhibitor that weakly inhibits proteasome activity,
partially prevented ER proteolysis at a high dose of 12.5 µg/ml (Fig. 4A). Calpeptin was ineffective at all doses tested. The order of
potency exhibited by these inhibitors in our studies (MG132 >
ALLnL) is identical to that previously reported by Rock et
al. (24). In addition, the weak activity of TPCK against
proteasome function is reflected in its partial antagonist action at
high doses. These data support the conclusion that estrogen can
regulate ER protein directly through a proteasome-mediated pathway. In
addition, the ability of the MG132 and ALLnL to increase ER levels
relative to controls suggests that the proteasome may also be involved
in basal turnover of receptor.
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shows a representative autoradiogram
of a Northern blot hybridized with a radiolabeled probe of human ER
cDNA. To control for equivalent loading, blots were rehybridized with
probe for glyceraldehyde phosphate dehydrogenase (GAPDH). The
results of multiple Northern analyses are presented
quantitatively in Fig. 5B. Our data show that neither pretreatment with
proteasome inhibitor nor treatment with E2 alters steady
state levels of ER mRNA within the first 2 h of treatment. To
further verify that the action of the inhibitor was limited to ER
protein, pulse chase experiments were performed (Fig. 6). As shown in the more detailed time
course of ER half-life (Fig. 3), estrogen induces greater than 50%
loss of ER within 2 h. We, therefore, chose the 2-h time point to
examine the effect of preventing proteasome function on
estrogen-induced shortening of ER half-life. In confirmation of our
previous results, estrogen treatment causes a dramatic decrease in ER
half-life. Addition of proteasome inhibitor maintained receptor levels
comparable to those of controls in both the presence and absence of
estrogen. Collectively, these results show that inhibition of
proteasome function prevents estrogen-induced proteolysis of ER
protein.
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, 4, and 6). Whole-cell estrogen uptake
assays were performed to determine what effect this may have on
estrogen-binding activity. For comparison, parallel samples were
evaluated by both binding assay and Western analysis. Since binding
data requires incubation with [3H]estradiol, there can be
no measure of ER in the absence of estrogen. Therefore, ER in
unstimulated cells is represented by Western only. In accordance with
our previous data, Fig. 7A illustrates
that total ER protein content in cells pretreated with proteasome
inhibitor is qualitatively similar to those in nontreated cells. This
level is higher than those pretreated with solvent since
[3H]estradiol induces degradation of receptor protein.
Surprisingly, pretreatment with proteasome inhibitor increased specific
binding by 1.75-fold relative to those pretreated with
dimethylsulfoxide (DMSO) (Fig. 7B). In light of the Western analysis,
the binding activity in cells pretreated with ALLnL more closely
reflects the receptor content in unstimulated cells. This implies that
inclusion of a proteasome inhibitor increases estrogen-binding activity
not by increasing receptor number but by maintaining it at the level of
nonstimulated cells. These findings suggests that since ER protein is
down-regulated during the procedure time required to measure specific
binding, the resultant measurement may only account for receptor that
remains after rapid proteolysis has taken place. They further suggest
that binding measurements in the absence of proteasome inhibitors may
underestimate cellular content of ER.
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illustrates that pretreatment with
ALLnL prevented down-regulation of ER protein in response to estrogen
exposure. The demonstration that this mechanism functions in both PR1
cells and MCF-7 cells shows that proteolytic regulation of ER extends
beyond rat lactotrope cells to human breast cancer cells and suggests
that it may be a common regulatory mechanism used by estrogen to
control ER protein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Western analysis of steady state levels showed a rapid loss of total ER
protein content in response to estrogen (Fig. 1). We used pulse chase
analysis to directly demonstrate that estrogen induces degradation of
ER protein and shortens its half-life from greater than 3 h to
1 h. Previous studies that examine the effect of estrogen on ER
half-life report conflicting results. For example, while estrogen
shortens ER half-life in MCF-7 cells (4, 16), it has no effect in
primary uterine cells (17, 19) and lengthens ER half-life in COS cells
transfected with mouse ER (25). This variation may simply reflect
differences in model systems. It may also, however, reflect differences
in methodology. ER levels are commonly determined based on specific
estrogen binding. With the exception of studies by Dauvois et
al. (25), all reported measurements of ER half-life have been
based on binding assays. Comparison of estrogen-binding activity and
total ER levels detectable by Western blot (Fig. 7) shows a discrepancy
between the total number of ERs present in the cell and the number
determined by binding assay. Our studies indicate that within the
length of time necessary to measure specific estrogen binding (16), a
portion of ER protein is degraded. This is supported by early studies
of Horwitz and McGuire (6) who demonstrated that the number of
ligand-bound sites extracted from the nucleus drops rapidly after 30
min. Consequently, the ER level estimated by binding assays may not
take into account ER that is lost during incubation with radiolabeled
ligand. The underestimation of ER in the absence of estrogen could
account for the lack of measurable difference in ER half-life in the
presence of estrogen. The advantage of pulse chase is that
[35S]methionine labeling permits the direct examination
of a presynthesized pool of ER protein before and after treatment with
estrogen, and it does not require binding studies to assess receptor
levels.
Throughout these studies, we focused on total ER content by analyzing ER in whole-cell extracts. Although ER is a nuclear protein, when cellular fractions are prepared to distinguish cytoplasmic and nuclear components, ER can be artifactually extracted with cytoplasmic proteins. Estrogen induces undefined changes in the biochemical properties of ER that prevent extraction of ER with hypotonic buffers. This process, referred to as "transformation," can be characterized by an increase in ER in the nuclear fraction upon stimulation with estrogen. It is possible that changes in ER solubility may account for the increased half-life of liganded ER reported for mouse ER (25) since the analysis was performed on lysate extracted with high salt. Earlier studies of ER regulation consider "cytoplasmic" and nuclear ER separately. This confounds interpretation of the results in light of the knowledge that ER is exclusively nuclear (26). To simplify our interpretation, we chose to examine whole-cell lysate, which eliminates any changes in ER levels associated with transformation or the extraction process.
We provide evidence that estrogen induces proteolysis of ER via a
proteasome-mediated pathway in both the pituitary and MCF-7 breast
cancer cell lines. Proteolysis is an important regulatory strategy
governing a number of processes, including cell cycle regulation,
signal transduction, antigen presentation, and protein quality control.
In particular, the proteasome pathway has been implicated as a major
protease responsible for the turnover of most proteins in the cell
(24). Among those proteins are transcriptional regulators, including
p53 (27), MyoD (28, 29), cJun (30), yeast MAT2 (31), and the steroid
family corepressor N-CoR (32). In most cases, substrate recognition by
the proteasome requires the attachment of multiple ubiquitin moieties
to proteins targeted for degradation. Nirmala and Thampan (33)
demonstrated that ER is ubiquitinated in an estrogen-dependent manner
in normal goat uterus (33). Further, the ubiquitin-activating enzyme,
UBA, and ubiquitin-conjugating enzymes, UBCs, can promote in
vitro degradation of ER protein (34). Consistent with
ubiquitin-dependent proteasome function, ubiquitination would serve as
a targeting signal to direct ER to the proteasome. Once ER is within
the multicatalytic enzyme, it would then be degraded into small
peptides, which may account for the lack of observable protein
intermediates associated with ER processing (16, 17). This proteolytic
mechanism can account for the rapid loss of ER protein that precedes
changes in ER RNA.
The threshold nature of this response is illustrated by both dose
response and binding studies. Examination of the Western blots shown in
Fig. 7A shows that the addition of 200-fold excess of
diethylstilbestrol (DES) had no greater effect on ER levels beyond
E2 alone. Dose analysis indicates that significant
down-regulation of ER protein does not occur at doses lower than
10-10 M E2. This is a saturable
dose of E2 and is sufficient to occupy 100% of ER in the
PR-1 cells (15). Although it remains possible that the detection method
is not sensitive enough to measure loss of a small amount of receptor,
doses of E2 sufficient to occupy 50% of the receptors,
i.e. 10-11 M E2, failed
to evoke a corresponding decrease in ER protein. Furthermore, levels of
estrogen sufficient to occupy 100% of receptors do not result in the
total loss of ER protein. The lack of correlation between receptor
occupancy and amount of ER degraded suggests that ligand binding itself
does not target ER for degradation. Since proteasomes are estimated to
comprise 1% of the total soluble cellular protein, it is unlikely that
they are a limiting component (24). It is possible that downstream
events may be required to manifest this response.
Estrogen treatment cannot completely deplete cells of ER protein.
Approximately 40% of ER protein remains despite exposure to large
doses of estradiol. It is interesting to speculate that certain ER
molecules may be resistant to degradation. It is unlikely that the
remaining ER represents receptor in a subpopulation of cells since the
PR-1 cells do not proliferate significantly (15) and are most likely
synchronized when maintained in an estrogen-free environment for 3
days. Moreover, based on molecular weight and epitope recognition, the
remaining ER is not ERß (35) or truncated estrogen receptor products
(TERPs) (36), additional ER species reported to be present in
pituitary cells. The question remains what distinquishes ER that is
destined for degradation from ER that is not. In several cases of
proteasome-regulated proteolysis, alterations in the biochemical and
physical properties of proteins serve as signals to induce
ubiquitination and degradation. The antiestrogen ICI induces rapid
degradation of ER (25, 37). In PR1 cells, this appears to operate
through a proteasome-mediated mechanism similar to E2
(E. T. Alarid, unpublished observation). Dauvois et al.
(20) suggest that ICI disrupts nuclear-cytoplasmic shuttling and
promotes cytoplasmic accumulation in certain cells. Interestingly,
cytoplasmic ER did not appear to degrade, suggesting that nuclear
localization may be a requirement for the degradation process.
Regulation of MyoD protein, a skeletal muscle transcription factor, is
controlled in part by ubiquitin-proteasome-mediated degradation (28, 29). Abu Hatoum et al. (28) recently demonstrated that
binding of MyoD to its cognate DNA response element stabilizes MyoD
protein and generates a complex that is resistant to proteasome
degradation. DNA binding may likewise confer resistance to ER protein.
Degradation of many proteins is additionally regulated by
phosphorylation. Activation of the NF-B signaling cascade by several
extracellular stimuli requires phosphorylation-dependent degradation of
IB by the ubiqutin proteasome pathway (38). The functional role
of phosphorylation of ER is not as yet clearly defined. Specific
protein-protein interactions also influence susceptibility to
proteasome-mediated degradation. Work by Whitesell and Cook (39)
demonstrates that changes in composition of the protein complex
associated with GR in the cytoplasm can result in rapid turnover of the
GR by the proteasome. Involvement of the coding sequence in
down-regulation of ER has been demonstrated previously (7, 13). Further
evaluation of the sequence requirement for proteasome-mediated
degradation of ER may predicate posttranslational modifications, such
as cellular compart-mentalization, DNA binding, phosphorylation, or
specific protein interactions, that may contribute to ER fate.
We demonstrate that estrogen can regulate ER protein in the absence of transcription and protein synthesis in the pituitary. ER protein regulation can, therefore, be added to the growing number of estrogen actions that do not involve ER-mediated transcription. Predominant among those activities is the activation of signal transduction cascades. Estrogen has been shown to activate MAPK (40) and ERK activity (41) and to lead to the accumulation of second messenger molecules including cAMP (42), inositol phosphate (41, 43, 44), and calcium (45). It has been hypothesized that these nonclassical mechanisms of estrogen action may be mediated through putative membrane-bound receptors that are derived from the same coding transcript (41) and are recognized by the same antibodies as the nuclear ER (41, 46). In the case of ER protein regulation, estrogen’s action involves nuclear, not membrane, ERs. Membrane-bound ERs make up less than 3% of the protein product from nuclear ER transcript (41). Since a significant proportion of ER is down-regulated in response to estrogen, it is most likely that nuclear ER is responsible for mediating estrogen’s action. To our knowledge, proteasome-mediated proteolysis of ER is the first identification of a nongenomic mechanism of estrogen action that involves nuclear ERs. Identification of this novel mechanism of estrogen action introduces the possibility for further exploration of nuclear signaling events induced by estrogen that do not involve transcriptional activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Inhibitor and Estrogen Stimulation
Before treatment, cells were washed with PBS and cultured at 5%
CO2 in phenol red-free and estrogen-free Optimem media
(Life Technologies, Inc.) for a minimum of 3 days. While
identical results are observed in cells maintained in media with
charcoal-stripped serum, we choose to utilize this defined medium to
minimize variation that may be associated with the stripping protocol.
On the day of treatment, cells were washed with PBS and collected by
dispersion with PBS followed by centrifugation. Cell pellets were then
resuspended in Optimem that was preequilibrated at 37 C at 5%
CO2 and distributed into 1 ml aliquots of 106
cells per tube. In experiments utilizing inhibitors, samples were
pretreated with the designated inhibitors for 30 min at 37 C while
gently rotating. After pretreatment, cells were exposed to
17ß-estradiol (E2; Sigma Chemical Co.,
St. Louis, MO) at various doses and for varying lengths of time as
indicated in the figure legends. During treatment, the cells were kept
at 37 C and rotated continuously. Protease inhibitors tested included
ALLnL, ALLM, Calpeptin, TPCK, ethyl(+)-(2S,
3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)
butylcarbamoyl]-2-oxiranecarboxylate (E64D), MG132, and
NH4Cl. Controls consisted of pretreatment with DMSO
(Sigma Chemical Co.) and treatment with ethanol (EtOH),
the solvents for the inhibitors and estradiol, respectively. For
practical purposes, ALLnL was used as the preferential proteasome
inhibitor. In experiments using protein synthesis inhibitors
(cyclohexamide, puromycin, anisomycin, emetine) and transcription
inhibitor [5,6-dichloro-1-b-ribofuranosyl benzimidazole (DRB)], cells
were pretreated for 30 min as described above before a 2-h treatment
with 10 nM E2. All inhibitors were purchased
from Sigma Chemical Co. except MG132 and TPCK, which were
gifts from Dr. Shigeki Miyamoto.
Western Blot Analysis
Upon termination of experiments, the cells were pelleted by
centrifugation, washed with PBS, and lysed immediately in 2x SDS
sample buffer (120 mM Tris-base, 20% glycerol, 2% SDS,
2% ß-mercaptoethanol, bromophenol blue, pH 6.8) to yield whole-cell
extracts. Whole-cell extracts were boiled and electrophoresed in a
7.5% or 10% SDS-PAGE gel. Proteins were electrophoretically
transferred using a Trans-blot Cell (Bio-Rad Laboratories, Inc., Richmond, CA) to nylon membrane (Immobilon-P,
Millipore Corp., Bedford, MA) in a Tris-glycine transfer
buffer with 20% methanol. The membranes were preblocked in a solution
of 5% milk, 0.02% sodium azide, 0.2% Tween 20 in PBS. Membranes were
then incubated overnight in the same solution containing primary
antibody. The primary antibodies used to detect ER were an anti-ER
antibody no. 715 (47) directed against a peptide within the hinge
region (amino acids 270–284) of the rat ER and anti-ER antibodies
directed against the hinge (amino acids 287–300; SR1000), and
C-terminal (amino acids 582–595; SR1010) regions of the human ER
(Stressgen, Vancouver, British Columbia, Canada). Antibody to
IB (C21-Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) was used to visualize protein not regulated by estrogen and as a
loading control. Antibody dilution curves were performed with all
primary antibodies to ensure that saturating concentrations were used
in the first probe reaction. Blots were washed in PBS containing 0.2%
Tween (PBST) before incubation with secondary antibodies conjugated to
horseradish peroxidase (HRP) diluted in the identical solution without
sodium azide. HRP-conjugated secondary antibodies used were directed
against rabbit or mouse IgG (Amersham Pharmacia Biotech,
Arlington Heights, IL) as appropriate. After washing in PBST,
the signal was visualized using the enhanced chemiluminescence (ECL)
detection method (Amersham Pharmacia Biotech) and exposed
to x-ray film.
Northern Blot Analysis
Total RNA was isolated from cells using phenol-chloroform
extraction as described previously (48). Twenty micrograms of total RNA
were electrophoresed in a 1% agarose gel containing formaldehyde and
transferred to nylon membrane (Genescreen; NEN Life Science Products, Boston, MA) (49). The RNA was immobilized to
the membrane by UV cross-linking (Bio-Rad Laboratories, Inc.). Prehybridization and hybridization of the membranes were
performed in a hybridization oven (Robbins Scientific Corp., Sunnyvale,
CA) at 55 C in a 25% formamide solution. The blots were probed
with 32P-radiolabeled cDNA fragments of the human ER, and
mouse GAPDH. Blots were stripped between hybridizations in a boiling
solution containing 1% glycerol, 2 mM EDTA, and 0.5% SDS
for a minimum of 10 min. Signal was quantified with a PhosphoImager
using Imagequant software (Molecular Dynamics, Inc.,
Sunnyvale, CA). Expression level of ER mRNA was determined by
normalizing values to those of the loading control, GAPDH. Values
obtained for the untreated DMSO control were set at 1. The data are
presented as the mean ± SD of the ER mRNA level
relative to the DMSO control for three independent experiments.
Whole-Cell Estrogen Uptake Assay
PR-1 cells that were maintained in Optimem for a minimum of 3
days were aliquoted into microcentrifuge tubes at a concentration of
2 x 106/ml in fresh medium that was preequilibrated
to 37 C at 5% CO2. The cells were pretreated for 30 min
with either DMSO or ALLnL at 37 C while rotating. After pretreatment, 2
nM [3H]estradiol (New England Nuclear/Dupont,
Boston, MA)was added to all samples. To account for nonspecific
binding, 0.4 µM DES (Sigma Chemical Co.) was
added in addition to 2 nM [3H]estradiol in a
parallel set of samples. During the 2-h treatment period, cells were
kept at 37 C and were rotated continuously. All samples were controlled
for equivalent amounts of ethanol. Cells were harvested by
centrifugation and were washed two times with 1% BSA in PBS at 4 C.
The final pellet was resuspended in ethanol and counted in a
scintillation counter. Specific binding was calculated by the
subtraction of nonspecific from total binding. Samples within
individual experiments were performed in duplicate. The data are
presented as the mean + SE of three independent
experiments. To compare total ER protein content to ER binding,
whole-cell extract from a parallel set of samples was examined by
Western blot analysis as described above.
Pulse Chase
Estrogen-deprived PR1 cells were rinsed twice in RPMI media
lacking phenol red, methionine, and cysteine (RPMI-,
Life Technologies, Inc.). Cells were incubated for 45 min
in RPMI- supplemented with L-glutamine, sodium
pyruvate, nonessential amino acids, and 5% stripped serum (50) that
had been dialyzed overnight against 0.9% NaCl. Metabolic labeling with
[35S]methionine was conducted for 2 h at a
concentration of 1 mCi/107 cells. After labeling, cells
were washed with complete phenol red-free RPMI media containing 10%
stripped serum. ALLnL and E2 treatment was performed as
described above for 1, 2, or 3 h in complete phenol red-free RPMI
medium containing 10% stripped serum. Treated cells were lysed in a
solution consisting of 10 mM Tris, pH 7.5, 150
mM NaCl, 1 mM EDTA, and 0.4% NP40, and ER was
immunoprecipitated using antirat ER antibody and protein A sepharose
(Pharmacia Biotech, Piscataway, NJ).
Immunoprecipitate was analyzed by SDS-PAGE. 35S-labeled ER
was visualized by autoradiography, and relative values of ER protein
were determined with a PhosphoImager using Imagequant software
(Molecular Dynamics, Inc.). Data are presented as a
percentage of the DMSO control group before exposure to E2.
Values represent three independent experiments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grant K01 CA-79090 to E.T.A.
Received for publication March 4, 1999. Revision received May 10, 1999. Accepted for publication May 25, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 O. Lanvin, S. Bianco, N. Kersual, D. Chalbos, and J.-M. Vanacker Potentiation of ICI182,780 (Fulvestrant)-induced Estrogen Receptor-{alpha} Degradation by the Estrogen Receptor-related Receptor-{alpha} Inverse Agonist XCT790 J. Biol. Chem., September 28, 2007; 282(39): 28328 - 28334. [Abstract] [Full Text] [PDF]
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D. Nonclercq, F. Journe, I. Laios, C. Chaboteaux, R.-A. Toillon, G. Leclercq, and G. Laurent Effect of nuclear export inhibition on estrogen receptor regulation in breast cancer cells J. Mol. Endocrinol., August 1, 2007; 39(2): 105 - 118. [Abstract] [Full Text] [PDF]
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 V. Duong, N. Boulle, S. Daujat, J. Chauvet, S. Bonnet, H. Neel, and V. Cavailles Differential Regulation of Estrogen Receptor {alpha} Turnover and Transactivation by Mdm2 and Stress-Inducing Agents Cancer Res., June 1, 2007; 67(11): 5513 - 5521. [Abstract] [Full Text] [PDF]
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C. Zhao, J. Matthews, M. Tujague, J. Wan, A. Strom, G. Toresson, E. W-F. Lam, G. Cheng, J.-A. Gustafsson, and K. Dahlman-Wright Estrogen Receptor {beta}2 Negatively Regulates the Transactivation of Estrogen Receptor {alpha} in Human Breast Cancer Cells Cancer Res., April 15, 2007; 67(8): 3955 - 3962. [Abstract] [Full Text] [PDF]
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M. Lupien, M. Jeyakumar, E. Hebert, K. Hilmi, D. Cotnoir-White, C. Loch, A. Auger, G. Dayan, G.-A. Pinard, J.-M. Wurtz, et al. Raloxifene and ICI182,780 Increase Estrogen Receptor-{alpha} Association with a Nuclear Compartment via Overlapping Sets of Hydrophobic Amino Acids in Activation Function 2 Helix 12 Mol. Endocrinol., April 1, 2007; 21(4): 797 - 816. [Abstract] [Full Text] [PDF]
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 N. Vasudevan and D. W. Pfaff Membrane-Initiated Actions of Estrogens in Neuroendocrinology: Emerging Principles Endocr. Rev., February 1, 2007; 28(1): 1 - 19. [Abstract] [Full Text] [PDF]
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 P. Germain, B. Staels, C. Dacquet, M. Spedding, and V. Laudet Overview of Nomenclature of Nuclear Receptors Pharmacol. Rev., December 1, 2006; 58(4): 685 - 704. [Abstract] [Full Text] [PDF]
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G. Shyamala, Y.-C. Chou, R. D. Cardiff, and E. Vargis Effect of c-neu/ ErbB2 Expression Levels on Estrogen Receptor {alpha}-Dependent Proliferation in Mammary Epithelial Cells: Implications for Breast Cancer Biology Cancer Res., November 1, 2006; 66(21): 10391 - 10398. [Abstract] [Full Text] [PDF]
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E. T. Alarid Lives and Times of Nuclear Receptors Mol. Endocrinol., September 1, 2006; 20(9): 1972 - 1981. [Abstract] [Full Text] [PDF]
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 G. B Silberstein, K. Van Horn, E. Hrabeta-Robinson, and J. Compton Estrogen-triggered delays in mammary gland gene expression during the estrous cycle: evidence for a novel timing system. J. Endocrinol., August 1, 2006; 190(2): 225 - 239. [Abstract] [Full Text] [PDF]
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R. Mo, S. M. Rao, and Y.-J. Zhu Identification of the MLL2 Complex as a Coactivator for Estrogen Receptor {alpha} J. Biol. Chem., June 9, 2006; 281(23): 15714 - 15720. [Abstract] [Full Text] [PDF]
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 M. G. Rosenfeld, V. V. Lunyak, and C. K. Glass Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response Genes & Dev., June 1, 2006; 20(11): 1405 - 1428. [Abstract] [Full Text] [PDF]
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 M. R. Meyer, E. Haas, and M. Barton Gender Differences of Cardiovascular Disease: New Perspectives for Estrogen Receptor Signaling Hypertension, June 1, 2006; 47(6): 1019 - 1026. [Full Text] [PDF]
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X. Long and K. P. Nephew Fulvestrant (ICI 182,780)-dependent Interacting Proteins Mediate Immobilization and Degradation of Estrogen Receptor-{alpha} J. Biol. Chem., April 7, 2006; 281(14): 9607 - 9615. [Abstract] [Full Text] [PDF]
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A. M. Fowler, N. M. Solodin, C. C. Valley, and E. T. Alarid Altered Target Gene Regulation Controlled by Estrogen Receptor-{alpha} Concentration Mol. Endocrinol., February 1, 2006; 20(2): 291 - 301. [Abstract] [Full Text] [PDF]
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L. Li, Z. Li, P. M. Howley, and D. B. Sacks E6AP and Calmodulin Reciprocally Regulate Estrogen Receptor Stability J. Biol. Chem., January 27, 2006; 281(4): 1978 - 1985. [Abstract] [Full Text] [PDF]
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M. Fan, A. Park, and K. P. Nephew CHIP (Carboxyl Terminus of Hsc70-Interacting Protein) Promotes Basal and Geldanamycin-Induced Degradation of Estrogen Receptor-{alpha} Mol. Endocrinol., December 1, 2005; 19(12): 2901 - 2914. [Abstract] [Full Text] [PDF]
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 M. Schupp, M. Clemenz, R. Gineste, H. Witt, J. Janke, S. Helleboid, N. Hennuyer, P. Ruiz, T. Unger, B. Staels, et al. Molecular Characterization of New Selective Peroxisome Proliferator-Activated Receptor {gamma} Modulators With Angiotensin Receptor Blocking Activity Diabetes, December 1, 2005; 54(12): 3442 - 3452. [Abstract] [Full Text] [PDF]
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C. C. Valley, R. Metivier, N. M. Solodin, A. M. Fowler, M. T. Mashek, L. Hill, and E. T. Alarid Differential Regulation of Estrogen-Inducible Proteolysis and Transcription by the Estrogen Receptor {alpha} N Terminus Mol. Cell. Biol., July 1, 2005; 25(13): 5417 - 5428. [Abstract] [Full Text] [PDF]
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M. Callige, I. Kieffer, and H. Richard-Foy CSN5/Jab1 Is Involved in Ligand-Dependent Degradation of Estrogen Receptor {alpha} by the Proteasome Mol. Cell. Biol., June 1, 2005; 25(11): 4349 - 4358. [Abstract] [Full Text] [PDF]
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 T. A. Ishunina, D. F. Swaab, and D. F. Fischer Estrogen Receptor-{alpha} Splice Variants in the Medial Mamillary Nucleus of Alzheimer's Disease Patients: Identification of a Novel MB1 Isoform J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3757 - 3765. [Abstract] [Full Text] [PDF]
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J. Cho, D. Kim, S. Lee, and Y. Lee Cobalt Chloride-Induced Estrogen Receptor {alpha} Down-Regulation Involves Hypoxia-Inducible Factor-1{alpha} in MCF-7 Human Breast Cancer Cells Mol. Endocrinol., May 1, 2005; 19(5): 1191 - 1199. [Abstract] [Full Text] [PDF]
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 S. Durrer, K. Maerkel, M. Schlumpf, and W. Lichtensteiger Estrogen Target Gene Regulation and Coactivator Expression in Rat Uterus after Developmental Exposure to the Ultraviolet Filter 4-Methylbenzylidene Camphor Endocrinology, May 1, 2005; 146(5): 2130 - 2139. [Abstract] [Full Text] [PDF]
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J E Sanchez-Criado, J M. de las Mulas, C Bellido, R Aguilar, and J C Garrido-Gracia Gonadotrope oestrogen receptor-{alpha} and -{beta} and progesterone receptor immunoreactivity after ovariectomy and exposure to oestradiol benzoate, tamoxifen or raloxifene in the rat: correlation with LH secretion J. Endocrinol., January 1, 2005; 184(1): 59 - 68. [Abstract] [Full Text] [PDF]
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M. Fan, H. Nakshatri, and K. P. Nephew Inhibiting Proteasomal Proteolysis Sustains Estrogen Receptor-{alpha} Activation Mol. Endocrinol., November 1, 2004; 18(11): 2603 - 2615. [Abstract] [Full Text] [PDF]
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S. Suzuki and R. J. Handa Regulation of Estrogen Receptor-{beta} Expression in the Female Rat Hypothalamus: Differential Effects of Dexamethasone and Estradiol Endocrinology, August 1, 2004; 145(8): 3658 - 3670. [Abstract] [Full Text] [PDF]
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H.-W. Tsai, J. A. Katzenellenbogen, B. S. Katzenellenbogen, and M. A. Shupnik Protein Kinase A Activation of Estrogen Receptor {alpha} Transcription Does Not Require Proteasome Activity and Protects the Receptor from Ligand-Mediated Degradation Endocrinology, June 1, 2004; 145(6): 2730 - 2738. [Abstract] [Full Text] [PDF]
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F. Barchiesi, E. K. Jackson, B. Imthurn, J. Fingerle, D. G. Gillespie, and R. K. Dubey Differential Regulation of Estrogen Receptor Subtypes {alpha} and {beta} in Human Aortic Smooth Muscle Cells by Oligonucleotides and Estradiol J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2373 - 2381. [Abstract] [Full Text] [PDF]
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S. T. Pearce, H. Liu, I. Radhakrishnan, M. Abdelrahim, S. Safe, and V. C. Jordan Interaction of the Aryl Hydrocarbon Receptor Ligand 6-Methyl-1,3,8-trichlorodibenzofuran with Estrogen Receptor {alpha} Cancer Res., April 15, 2004; 64(8): 2889 - 2897. [Abstract] [Full Text] [PDF]
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H. Masuyama and Y. Hiramatsu Involvement of Suppressor for Gal 1 in the Ubiquitin/Proteasome-mediated Degradation of Estrogen Receptors J. Biol. Chem., March 26, 2004; 279(13): 12020 - 12026. [Abstract] [Full Text] [PDF]
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 A. M. FOWLER, N. SOLODIN, M. T. PREISLER-MASHEK, P. ZHANG, A. V. LEE, and E. T. ALARID Increases in estrogen receptor-{alpha} concentration in breast cancer cells promote serine 118/104/106-independent AF-1 transactivation and growth in the absence of estrogen FASEB J, January 1, 2004; 18(1): 81 - 93. [Abstract] [Full Text] [PDF]
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O. M. Tirado, D. M. Selva, N. Toran, C. A. Suarez-Quian, M. Jansen, D. P. McDonnell, J. Reventos, and F. Munell Increased Expression of Estrogen Receptor {beta} in Pachytene Spermatocytes After Short-Term Methoxyacetic Acid Administration J Androl, January 1, 2004; 25(1): 84 - 94. [Abstract] [Full Text] [PDF]
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C. Woodham, L. Birch, and G. S. Prins Neonatal Estrogen Down-Regulates Prostatic Androgen Receptor through a Proteosome-Mediated Protein Degradation Pathway Endocrinology, November 1, 2003; 144(11): 4841 - 4850. [Abstract] [Full Text] [PDF]
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V. Marsaud, A. Gougelet, S. Maillard, and J.-M. Renoir Various Phosphorylation Pathways, Depending on Agonist and Antagonist Binding to Endogenous Estrogen Receptor {alpha} (ER{alpha}), Differentially Affect ER{alpha} Extractability, Proteasome-Mediated Stability, and Transcriptional Activity in Human Breast Cancer Cells Mol. Endocrinol., October 1, 2003; 17(10): 2013 - 2027. [Abstract] [Full Text] [PDF]
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D. K. Lee and C. Chang Expression and Degradation of Androgen Receptor: Mechanism and Clinical Implication J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4043 - 4054. [Abstract] [Full Text] [PDF]
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L. M. Henrich, J. A. Smith, D. Kitt, T. M. Errington, B. Nguyen, A. M. Traish, and D. A. Lannigan Extracellular Signal-Regulated Kinase 7, a Regulator of Hormone-Dependent Estrogen Receptor Destruction Mol. Cell. Biol., September 1, 2003; 23(17): 5979 - 5988. [Abstract] [Full Text] [PDF]
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B. M. Wittmann, N. Wang, and M. M. Montano Identification of a Novel Inhibitor of Breast Cell Growth That Is Down-Regulated by Estrogens and Decreased in Breast Tumors Cancer Res., August 15, 2003; 63(16): 5151 - 5158. [Abstract] [Full Text] [PDF]
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E. T. Alarid, M. T. Preisler-Mashek, and N. M. Solodin Thyroid Hormone Is an Inhibitor of Estrogen-Induced Degradation of Estrogen Receptor-{alpha} Protein: Estrogen-Dependent Proteolysis Is Not Essential for Receptor Transactivation Function in the Pituitary Endocrinology, August 1, 2003; 144(8): 3469 - 3476. [Abstract] [Full Text] [PDF]
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F. Yan, X. Gao, D. M. Lonard, and Z. Nawaz Specific Ubiquitin-Conjugating Enzymes Promote Degradation of Specific Nuclear Receptor Coactivators Mol. Endocrinol., July 1, 2003; 17(7): 1315 - 1331. [Abstract] [Full Text] [PDF]
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W. Tschugguel, W. Dietrich, Z. Zhegu, F. Stonek, A. Kolbus, and J. C. Huber Differential Regulation of Proteasome-Dependent Estrogen Receptor {alpha} and {beta} Turnover in Cultured Human Uterine Artery Endothelial Cells J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2281 - 2287. [Abstract] [Full Text] [PDF]
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M. Qiu, A. Olsen, E. Faivre, K. B. Horwitz, and C. A. Lange Mitogen-Activated Protein Kinase Regulates Nuclear Association of Human Progesterone Receptors Mol. Endocrinol., April 1, 2003; 17(4): 628 - 642. [Abstract] [Full Text] [PDF]
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M. Wormke, M. Stoner, B. Saville, K. Walker, M. Abdelrahim, R. Burghardt, and S. Safe The Aryl Hydrocarbon Receptor Mediates Degradation of Estrogen Receptor {alpha} through Activation of Proteasomes Mol. Cell. Biol., March 15, 2003; 23(6): 1843 - 1855. [Abstract] [Full Text] [PDF]
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M. Fan, R. M. Bigsby, and K. P. Nephew The NEDD8 Pathway Is Required for Proteasome-Mediated Degradation of Human Estrogen Receptor (ER)-{alpha} and Essential for the Antiproliferative Activity of ICI 182,780 in ER{alpha}-Positive Breast Cancer Cells Mol. Endocrinol., March 1, 2003; 17(3): 356 - 365. [Abstract] [Full Text] [PDF]
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S. T. Pearce, H. Liu, and V. C. Jordan Modulation of Estrogen Receptor alpha Function and Stability by Tamoxifen and a Critical Amino Acid (Asp-538) in Helix 12 J. Biol. Chem., February 21, 2003; 278(9): 7630 - 7638. [Abstract] [Full Text] [PDF]
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 C. Stirone, S. P. Duckles, and D. N. Krause Multiple forms of estrogen receptor-alpha in cerebral blood vessels: regulation by estrogen Am J Physiol Endocrinol Metab, January 1, 2003; 284(1): E184 - E192. [Abstract] [Full Text] [PDF]
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D. A. Schreihofer, D. F. Rowe, E. F. Rissman, E. M. Scordalakes, J.-a. Gustafsson, and M. A. Shupnik Estrogen Receptor-{alpha} (ER{alpha}), But Not ER{beta}, Modulates Estrogen Stimulation of the ER{alpha}-Truncated Variant, TERP-1 Endocrinology, November 1, 2002; 143(11): 4196 - 4202. [Abstract] [Full Text] [PDF]
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M. Stoner, B. Saville, M. Wormke, D. Dean, R. Burghardt, and S. Safe Hypoxia Induces Proteasome-Dependent Degradation of Estrogen Receptor {alpha} in ZR-75 Breast Cancer Cells Mol. Endocrinol., October 1, 2002; 16(10): 2231 - 2242. [Abstract] [Full Text] [PDF]
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 M. S. Cotroneo, J. Wang, W. A. Fritz, I.-E. Eltoum, and C. A. Lamartiniere Genistein action in the prepubertal mammary gland in a chemoprevention model Carcinogenesis, September 1, 2002; 23(9): 1467 - 1474. [Abstract] [Full Text] [PDF]
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C. Qin, T. Nguyen, J. Stewart, I. Samudio, R. Burghardt, and S. Safe Estrogen Up-Regulation of p53 Gene Expression in MCF-7 Breast Cancer Cells Is Mediated by Calmodulin Kinase IV-Dependent Activation of a Nuclear Factor {kappa}B/CCAAT-Binding Transcription Factor-1 Complex Mol. Endocrinol., August 1, 2002; 16(8): 1793 - 1809. [Abstract] [Full Text] [PDF]
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M. J. Henderson, A. J. Russell, S. Hird, M. Munoz, J. L. Clancy, G. M. Lehrbach, S. T. Calanni, D. A. Jans, R. L. Sutherland, and C. K. W. Watts EDD, the Human Hyperplastic Discs Protein, Has a Role in Progesterone Receptor Coactivation and Potential Involvement in DNA Damage Response J. Biol. Chem., July 12, 2002; 277(29): 26468 - 26478. [Abstract] [Full Text] [PDF]
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C. L. Smith, D. G. DeVera, D. J. Lamb, Z. Nawaz, Y.-H. Jiang, A. L. Beaudet, and B. W. O'Malley Genetic Ablation of the Steroid Receptor Coactivator-Ubiquitin Ligase, E6-AP, Results in Tissue-Selective Steroid Hormone Resistance and Defects in Reproduction Mol. Cell. Biol., January 15, 2002; 22(2): 525 - 535. [Abstract] [Full Text] [PDF]
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A. D. Wallace and J. A. Cidlowski Proteasome-mediated Glucocorticoid Receptor Degradation Restricts Transcriptional Signaling by Glucocorticoids J. Biol. Chem., November 9, 2001; 276(46): 42714 - 42721. [Abstract] [Full Text] [PDF]
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B. He, N. T. Bowen, J. T. Minges, and E. M. Wilson Androgen-induced NH2- and COOH-terminal Interaction Inhibits p160 Coactivator Recruitment by Activation Function 2 J. Biol. Chem., November 2, 2001; 276(45): 42293 - 42301. [Abstract] [Full Text] [PDF]
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J. S. Jorgensen and J. H. Nilson AR Suppresses Transcription of the {alpha} Glycoprotein Hormone Subunit Gene Through Protein-Protein Interactions with cJun and Activation Transcription Factor 2 Mol. Endocrinol., September 1, 2001; 15(9): 1496 - 1504. [Abstract] [Full Text] [PDF]
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D. L. Osburn, G. Shao, H. M. Seidel, and I. G. Schulman Ligand-Dependent Degradation of Retinoid X Receptors Does Not Require Transcriptional Activity or Coactivator Interactions Mol. Cell. Biol., August 1, 2001; 21(15): 4909 - 4918. [Abstract] [Full Text] [PDF]
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D. A. Schreihofer, E. M. Resnick, V. Y. Lin, and M. A. Shupnik Ligand-Independent Activation of Pituitary ER: Dependence on PKA-Stimulated Pathways Endocrinology, August 1, 2001; 142(8): 3361 - 3368. [Abstract] [Full Text] [PDF]
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T. Osada, G. Watanabe, Y. Sakaki, and T. Takeuchi Puromycin-Sensitive Aminopeptidase Is Essential for the Maternal Recognition of Pregnancy in Mice Mol. Endocrinol., June 1, 2001; 15(6): 882 - 893. [Abstract] [Full Text] [PDF]
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A. McDougal, M. Wormke, J. Calvin, and S. Safe Tamoxifen-induced Antitumorigenic/Antiestrogenic Action Synergized by a Selective Aryl Hydrocarbon Receptor Modulator Cancer Res., May 1, 2001; 61(10): 3902 - 3907. [Abstract] [Full Text]
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C. T. Baumann, H. Ma, R. Wolford, J. C Reyes, P. Maruvada, C. Lim, P. M. Yen, M. R. Stallcup, and G. L. Hager The Glucocorticoid Receptor Interacting Protein 1 (GRIP1) Localizes in Discrete Nuclear Foci That Associate with ND10 Bodies and Are Enriched in Components of the 26S Proteasome Mol. Endocrinol., April 1, 2001; 15(4): 485 - 500. [Abstract] [Full Text]
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 M. POTIER, S. J. ELLIOT, I. TACK, O. LENZ, G. E. STRIKER, L. J. STRIKER, and M. KARL Expression and Regulation of Estrogen Receptors in Mesangial Cells: Influence on Matrix Metalloproteinase-9 J. Am. Soc. Nephrol., February 1, 2001; 12(2): 241 - 251. [Abstract] [Full Text]
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J. L. Turgeon and D. W. Waring Progesterone Regulation of the Progesterone Receptor in Rat Gonadotropes Endocrinology, September 1, 2000; 141(9): 3422 - 3429. [Abstract] [Full Text] [PDF]
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D. A. Schreihofer, M. H. Stoler, and M. A. Shupnik Differential Expression and Regulation of Estrogen Receptors (ERs) in Rat Pituitary and Cell Lines: Estrogen Decreases ER{alpha} Protein and Estrogen Responsiveness Endocrinology, June 1, 2000; 141(6): 2174 - 2184. [Abstract] [Full Text] [PDF]
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S. Oesterreich, Q. Zhang, T. Hopp, S. A. W. Fuqua, M. Michaelis, H. H. Zhao, J. R. Davie, C. K. Osborne, and A. V. Lee Tamoxifen-Bound Estrogen Receptor (ER) Strongly Interacts with the Nuclear Matrix Protein HET/SAF-B, a Novel Inhibitor of ER-Mediated Transactivation Mol. Endocrinol., March 1, 2000; 14(3): 369 - 381. [Abstract] [Full Text]
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S. Hauser, G. Adelmant, P. Sarraf, H. M. Wright, E. Mueller, and B. M. Spiegelman Degradation of the Peroxisome Proliferator-activated Receptor gamma Is Linked to Ligand-dependent Activation J. Biol. Chem., June 9, 2000; 275(24): 18527 - 18533. [Abstract] [Full Text] [PDF]
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C. T. Baumann, P. Maruvada, G. L. Hager, and P. M. Yen Nuclear Cytoplasmic Shuttling by Thyroid Hormone Receptors. MULTIPLE PROTEIN INTERACTIONS ARE REQUIRED FOR NUCLEAR RETENTION J. Biol. Chem., March 30, 2001; 276(14): 11237 - 11245. [Abstract] [Full Text] [PDF]
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A. L. Wijayaratne and D. P. McDonnell The Human Estrogen Receptor-alpha Is a Ubiquitinated Protein Whose Stability Is Affected Differentially by Agonists, Antagonists, and Selective Estrogen Receptor Modulators J. Biol. Chem., September 14, 2001; 276(38): 35684 - 35692. [Abstract] [Full Text] [PDF]
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M. T. Preisler-Mashek, N. Solodin, B. L. Stark, M. K. Tyriver, and E. T. Alarid Ligand-specific regulation of proteasome-mediated proteolysis of estrogen receptor-alpha Am J Physiol Endocrinol Metab, April 1, 2002; 282(4): E891 - E898. [Abstract] [Full Text] [PDF]
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