Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706
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ABSTRACT |
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Proteasome-mediated proteolysis modulates
the cellular concentration of estrogen receptor- (ER
) and is
induced by treatment of cells with 17
-estradiol. Herein, we show
that multiple receptor agonists, including 17
-estradiol and estriol
as well as the antagonist ICI-182780, stimulate proteasome-dependent
proteolysis of ER
in a process that requires ligand binding to the
receptor. Proteolysis of receptor depends on ligand concentration, and
there exists a direct correlation between ligand-binding affinity and
the half-maximal dose of ligand required to stimulate receptor
degradation. Furthermore, introduction of a point mutation into the
receptor ligand-binding pocket yields a stable receptor resistant to
proteolysis. Interestingly, although all ligands stimulate receptor
degradation, the extent to which overall ER levels are affected varies
with each ligand and is not related to ligand-binding affinity or
activation of transcription. These results demonstrate ligand-specific
regulation of ER
proteolysis, and they introduce the concept that
cellular receptor concentration is governed not only at the level of
induction of proteolysis but also by the efficiency with which the
receptor is degraded.
steroid; nuclear receptor; antagonist; estriol; pituitary
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INTRODUCTION |
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CELLULAR ESTROGEN RECEPTOR
LEVELS are dynamic and are particularly sensitive to changes in
circulating levels of 17-estradiol. It has been demonstrated
through a number of studies that the decline in estrogen receptor-
(ER
) upon exposure to 17
-estradiol results from a combination of
mechanisms that control both receptor synthesis and degradation
through transcriptional, posttranscriptional, and
posttranslational mechanisms (20, 27, 32, 33, 35, 36). The
most rapid of these regulatory mechanisms is the direct loss of ER
protein brought about by the induction of proteasome-mediated proteolysis (1, 21, 28).
Regulated proteolysis by proteasomes accounts for the turnover of most
short-lived proteins, including many nuclear receptors (9, 16,
23, 29, 41, 43, 45). Through a series of three enzymatic
reactions, ubiquitin moieties are attached to a protein substrate,
which targets it to the 26S proteasome. The molecular events that
direct ER into this pathway have not been clearly established.
However, earlier studies that examine changes in receptor-binding
capacity have shown that receptor levels can be controlled by both
receptor agonists and antagonists, suggesting the possibility that
receptor occupation by ligand may provide specificity (3, 4, 15,
18, 22, 33).
In our original report of ER protein regulation by proteolysis, we
utilized a pituitary lactotrope model system, the PR1 cell line. The
lactotrope cell population of the anterior pituitary is a major target
of estrogen action. Animals that lack ER
show a decrease in
lactotrope cell density and prolactin expression (37). In
contrast, animals treated with estrogen for prolonged periods develop
hyperplastic pituitaries comprised primarily of lactotrope cells. The
F344 rat model is a classic system for the study of estrogen regulation
of lactotrope growth and function (11, 44). The PR1 cell
line is an in vitro correlate of this model that was derived from an
estrogen-induced pituitary hyperplasia in F344 rats (30).
Using this lactotrope cell line, we showed that estrogen shortened the
half-life of ER
protein in the pituitary from >3 h to 1 h,
which translated into a decrease in steady-state levels of ER
protein within 2 h of ligand exposure (1). During this time frame, there was no measurable change in ER
mRNA levels. We have capitalized on the identification of a time window in which
proteolysis alone is responsible for changes in ER protein to further
examine the regulation of proteasome-dependent degradation of
endogenous ER
.
Herein, we demonstrate that proteasome-dependent proteolysis of ER
can be induced by multiple estrogens, including short-acting estrogens
and the pure antiestrogen ICI-182780 (ICI). This response requires the
direct binding of the various ligands to receptor, since introduction
of a point mutation in a critical residue of the ER
ligand-binding
pocket yields a receptor resistant to degradation. In addition, we find
that binding affinity of ligand for receptor correlates with the
potency with which ligand can induce proteolysis, further supporting a
critical role for ligand binding in regulating receptor proteolysis.
Interestingly, through quantitative assessment of changes in receptor
level at varying concentrations of ligand, we observed that the overall
decline in receptor levels achieved by proteolysis varies depending on
the ligand that occupies the receptor. These results indicate that
ligands can differentially regulate receptor proteolysis, and they
suggest the possibility that cellular ER concentration is controlled by
not only the induction of proteolysis but also its efficiency.
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EXPERIMENTAL PROCEDURES |
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Cell culture. PR1 lactotrope and HEK 293 cells were maintained under standard conditions of humidity and temperature in high-glucose DMEM medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD).
Steroid stimulation.
For experiments involving steroid treatment, cells were grown in either
Optimem medium (Life Technologies) or phenol red-free DMEM with 10%
charcoal-stripped serum (34) and antibiotics. Cells were
maintained in medium lacking steroid for a minimum of 3 days before
treatment. Steroid stimulation proceeded for 2 h as previously
described (1) unless otherwise indicated. The final
concentration of ethanol (EtOH) in control and treated samples was
0.1%. 17-Estradiol and 4-hydroxytamoxifen (4-OHTam) were purchased
from Sigma Chemical (St. Louis, MO). 17
-Estradiol and estriol were
purchased from Steraloids (Newport, RI). The antiestrogen ICI was a
gift from Dr. Jack Gorski. In experiments utilizing the proteasome
inhibitor ALLnL (Calbiochem, La Jolla, CA), samples were pretreated for
30 min with inhibitor before the addition of steroid.
Western blot analysis.
Western blot analysis was performed on whole cell extracts that were
obtained by either direct dissolution of cell pellets in 2× sample
buffer (125 mM Tris-base, 20% glycerol, 4% SDS, 10% -mercaptoethanol, and bromphenol blue, pH 6.8) or by extraction in
Totex buffer (19) followed by Bradford assay
(5). Proteins were separated on a 7.5% acrylamide gel.
Immunoblotting was performed as previously described (1),
utilizing anti-rat ER (13) or anti-human ER (Santa Cruz
Biotechnology, Santa Cruz, CA) antibodies as appropriate. Saturating
concentrations of antibody were used in all cases to allow quantitative
analysis of relative protein levels. Equivalent loading of lanes was
verified by reprobing blots with antibody against
-tubulin
(Calbiochem) or
-actin (Santa Cruz Biotechnology). Secondary
antibodies were conjugated either to horseradish peroxidase or
125I for visualization and quantification, respectively.
Receptor levels were determined by phosphoimager analysis by use of
Imagequant software (Molecular Dynamics, Sunnyvale, CA) or laser
densitometry. Where densitometry was employed, an internal standard
curve was generated for each blot. The EtOH-treated control was
arbitrarily set at 100, and a series of dilutions of this control were
run in parallel to generate the standard curve for the determination of
receptor levels relative to the EtOH-treated control. Each experiment
was repeated a minimum of three times to ensure reproducibility. The
EC50 for the downregulation response for each ligand was
calculated on the basis of nonlinear regression analysis (Prism 3.0;
GraphPad Software, San Diego, CA). Statistical significance of
differences between treatment groups was determined by one-way ANOVA
followed by t-test analysis, with a 95% confidence interval
(Microcal Origin, Microcal Software, Northampton, MA).
Transient transfection.
Transfections were performed utilizing Fugene reagent (Roche Molecular
Biochemicals, Indianapolis, IN) or calcium phosphate precipitation in
pituitary and 293 cells, respectively. To assess transcriptional
activity of ER, cells were transfected with either an ERE-tk-Luc
reporter, which encodes a multimerized vitellogenin estrogen-response-element (ERE) enhancer fused to a thymidine kinase
(tk) promoter (42), or a Prl-luc construct bearing 2.5 kb
of the Prl regulatory region. The Prl-Luc construct was obtained from
Dr. Richard Maurer. In addition, cells were cotransfected with a
-galactosidase reporter (CMV-
gal) to control for transfection efficiency. Cells were treated 24 h after transfection with EtOH or the indicated estrogen and were harvested after an additional 24 h. Assays for
-galactosidase (Tropix, Bedford, MA) and
luciferase activity (Promega, Madison, WI) were performed as directed
by the manufacturers. Luciferase activity was normalized to
-galactosidase activity, and degree of activation was determined
relative to the ethanol control.
Generation of cell lines expressing wild-type and mutant estrogen
receptors.
Stable 293 cell lines were generated using calcium phosphate
transfection of retroviral expression vector LHL-CA (26)
containing wild-type (WT) and mutant ERs. The G521R-ER was generated
by PCR mutagenesis, in which primers encoding the mutation
(5'-AGTAACAAACGCATG-GAGCATCTGTACAGC-3') were used in two
reactions, generating ER fragments between the XbaI site and
the mutation and between the mutation and a region downstream of the ER
stop codon that contained a BstEII site. The two reactions
were used as a template in a third reaction to create a final fragment
that was then digested with XbaI and BstEII and
subcloned back into an ER-encoding vector. The resultant mutant was
verified by sequencing and then subcloned into the LHL-CA expression
vector. This vector contains a Moloney murine leukemia viral
LTR driving the expression of a gene-encoding hygromycin resistance.
Expression of WT and G521R ER is controlled by a CMV enhancer and actin
promoter. The LHL-CA vector contains no viral coding regions. Selection
of cell colonies with hygromycin B (200 µg/ml) began 24 h after
transfection. Colonies were isolated and screened for ER expression by
Western blot analysis by use of anti-human ER. All results obtained
after experimental treatment were confirmed in multiple clones.
Binding assay.
Whole cell binding assays were performed as previously described
(1) by use of 10 pM [3H]estradiol (New
England Nuclear/Du Pont, Boston, MA) on the basis of the reported
disassociation constant of ER in PR1 cells (7). All assays were performed in the presence of ALLnL to prevent the
proteolysis of receptor that occurs during establishment of equilibrium
conditions. For competition, cells were incubated with increasing
concentrations of 17
-estradiol, 17
-estradiol, estriol, or ICI
that ranged from 1 pM to 1 µM in log increments. Steroid binding
proceeded for 2 h. Assays were performed with duplicate samples
and were repeated twice. (Interassay variation was neglible and is
included in Fig. 4 but is hidden by the data symbols.) EC50
values were determined by nonlinear regression analysis based on
single-site competition (Prism 3.0; GraphPad Software).
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RESULTS |
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Initial experiments were performed to assess the specificity of
inducible proteolysis of ER. PR1 cells were exposed for a brief
period of 2 h to various steroids, including testosterone, progesterone, cortisol, and estrogens (17
-estradiol,
17
-estradiol, and estriol). In addition, cells were treated with
the receptor antagonists 4-OHTam and ICI. After treatment, changes in
ER
levels were evaluated by Western blot analysis of whole cell
lysates. Shown in Fig. 1, treatment of
cells with 17
-estradiol resulted in a decline in steady-state levels
of ER
protein (10). The acute loss of receptor protein
could also be induced by treatment with other receptor ligands,
including 17
-estradiol and estriol, but not by steroids such as
testosterone, progesterone, and cortisol that do not bind specifically
to ER
(Fig. 1A and Table
1). Unlike agonists, ER
antagonists
showed differential activity in regulating receptor protein levels. ICI
was similar to estrogens in its ability to decrease receptor levels
(Fig. 1B and Table 1). However, the partial antagonist
4-OHTam was without effect. These results indicate that downregulation
of ER
may be restricted to specific ligands of the receptor (Fig.
1A).
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The involvement of proteasomes in the degradation of ER induced by
17
-estradiol, estriol, and ICI was tested using an inhibitor of the
proteasome pathway, ALLnL. Proteasome activity was blocked by a 30-min
preincubation of cells with proteasome inhibitor. In parallel, a
control group was pretreated with DMSO, the inhibitor solvent. After
inactivation of proteasomes, cells were treated with estrogens and ICI
as before. As shown previously, treatment of cells with estrogen and
ICI resulted in a significant decline in receptor level. The loss of
receptor was effectively inhibited by ALLnL (Fig.
2A) and a second inhibitor,
lactacystin (data not shown). Examination of the relative receptor
levels illustrated in Fig. 2C demonstrates that ALLnL
stabilized the receptor level to that of control cells and completely
prevented the rapid loss of receptor due to steroid treatment. ALLnL
was also effective at blocking the actions of 17
-estradiol,
17
-estradiol, and estriol (Fig. 2B). In Fig.
2B, it can be observed that inclusion of the proteasome
inhibitor alone can slightly increase basal levels of the receptor.
However, upon quantification of relative ER levels by phosphoimager
analysis, this apparent increase was not statistically significant.
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The limitation of this proteolytic response to ligands of ER
suggested that ligand binding to receptor is an essential first step in
directing the receptor to the proteasome pathway. To directly test this
possibility, stable cell lines were generated that express either a
WT-ER
or an ER
ligand-binding point mutant (G521R-ER
). HEK 293 cells were transfected with a retroviral expression vector that encodes
hygromycin resistance and either WT-ER
or G521R-ER
. Stable
colonies were selected after maintenance in hygromycin-containing medium and screened for the expression of ER
by Western blot. Multiple clones were identified for each line, and a representative line was selected for further analysis. The receptor transcriptional activity in the WT and G521R cell lines was confirmed by transient transfection of an estrogen-responsive reporter construct (ERE-tk-LUC). Shown in Fig. 3A, cells that
express WT-ER
exhibit increases in reporter gene activity in
response to 17
-estradiol, 17
-estradiol, and estriol. In line with
its antagonistic activity, ICI inhibited transcription, confirming the
specificity of the transcriptional response. In contrast, cells
expressing G521R-ER
did not exhibit ligand-dependent changes in
transcription activity (Fig. 3B). These results are
consistent with the original characterizations of this mutation in
mouse and human ER
(12). To examine the effect of
introduction of this mutation on estrogen-induced proteolysis, cells
were treated with 17
-estradiol, 17
-estradiol, estriol, or ICI for
2 h and analyzed for changes in ER
protein levels. Figure
3C shows that cells expressing WT-ER
respond to estrogens like PR1 cells by decreasing steady-state levels of ER
protein. Thus
estrogen-induced proteolysis is faithfully reconstituted in this
heterologous system. These cells, however, show a heightened sensitivity to ICI such that, within 2 h, WT-ER
is barely
detectable. G521R-ER
levels, however, were unchanged by stimulation
with either agonists or ICI (Fig. 3). These results demonstrate that ligand binding is essential in the induction of ER
proteolysis.
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17-Estradiol, 17
-estradiol, estriol, and ICI bind to ER
with
varying affinities. To further examine the relationship between ligand
binding and induction of proteolysis, PR1 cells were treated with
different doses of estrogens ranging from 10
12 to
10
6 M (Fig. 4,
top). Changes in ER levels were determined by quantification of receptor levels relative to an internal standard curve generated by
dilution of extract from ethanol-treated control cells. For direct
comparison, competitive binding assays were performed within the
identical dose range (Fig. 4, bottom). Nonlinear regression analysis was used to calculate an EC50 for both the
degradation response and receptor binding in PR1 cells. The rank order
of potency indicates that 17
-estradiol is the most potent agonist and possesses approximately one order of magnitude greater activity in
both binding and ability to induce degradation than estriol, 17
-estradiol, and ICI. Comparison of the EC50 values for
binding and degradation for individual ligands (Table
2) clearly shows a direct correlation
between ligand affinity for receptor and the ability of individual
ligands to induce receptor proteolysis. Thus ligands stimulate
proteolysis at concentrations consistent with their binding affinity,
providing further evidence that ligand binding triggers the events that
lead to receptor proteolysis by proteasomes. It also points out that
proteolysis can be modulated by hormone concentration.
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Interestingly, although all ligands induce proteolysis, the maximum
change in receptor level affected by these ligands varies. Examination
of the dose curves illustrated in Fig. 4A shows that the
plateau level of receptor achieved by stimulation with estriol and
17-estradiol is higher than that reached by 17
-estradiol and ICI.
This is also supported by data in Table 1, which shows that at a
saturable dose for all ligands (10 nM), significant differences exist
between the relative receptor levels. 17
-Estradiol and ICI are
similar in their ability to degrade the receptor. Both induce a loss of
~50% of receptor. 17
-Estradiol and estriol, however, affect
smaller declines in the receptor that are significantly different from
17
-estradiol and ICI. This ligand-specific variation cannot be
accounted for by differences in binding, since all of the ligands
tested are capable of occupying 100% of receptor at the 10 nM dose.
Furthermore, 17
-estradiol, estriol, and ICI share similar binding
affinities for the receptor, yet show different capacities to alter ER levels.
Recent studies have suggested that the transcriptional activation of
receptor correlates with receptor proteolysis (24). However, in WT-ER stable cells, 17
-estradiol, estriol, and
17
-estradiol appear to activate transcription equivalently despite
exhibiting differences in modulating receptor proteolysis (Fig.
2A). To directly compare the ability of agonists to modulate
proteolysis and activate gene expression, ER
transcriptional
activity was assessed in PR1 cells over a range of doses (Fig.
5). Cells were transfected with a
reporter construct representative of a natural estrogen-regulated gene
in lactotropes (Prl-Luc) and treated with varying doses of 17
-estradiol, 17
-estradiol, estriol, and ICI. Consistent with its
pure antiestrogenic activity, ICI inhibited the transcriptional activity of ER
in PR1 cells (data not shown). Examination of the
transcriptional response at different doses shows that ligand ability
to activate transcription parallels the ligand-binding affinity. The
EC50 values for transcriptional activation are ~0.09 nM,
1 nM, and 0.4 nM for 17
-estradiol, 17
-estradiol, and estriol, respectively. Interestingly, however, differences in transactivation capacity between these ligands are abolished at doses of 1 nM and
higher. Thus it appears that, although ligand binding is required to
induce both receptor transcriptional activity and proteolysis, ligand-specific differences in the proteolytic response are not reflected in ligand capacity to activate ER
transcriptional
function.
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DISCUSSION |
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Proteasome-mediated proteolysis has recently garnered increased
attention as a conserved mechanism responsible for the control of
steroid receptor protein stability. Because of its potential importance
to the regulation of receptor function, much interest has been
generated in determining the mechanism through which steroids target
the receptor for proteolysis. Although it has been demonstrated that
estradiol treatment of cells can stimulate proteolysis, the molecular
signals that engage the receptor in the proteasome pathway have not
been delineated. In this study, we demonstrate that proteolysis of
ER is a specific response that is induced by ligand binding to
receptor. ER
ligands, however, show varying capacity to limit
intracellular ER
concentration through this pathway. These results
demonstrate the existence of ligand-specific regulation of receptor
proteolysis by proteasomes.
Multiple ligands stimulate the destruction of ER, including potent
agonists such as 17
-estradiol as well as the weak or short-acting
agonists estriol and 17
-estradiol. Ligand binding induces a
"transformation" of ER
in which the receptor becomes tightly
associated with components of the nucleus. Release of ER
from this
complex(es) requires high salt (0.4 M) extraction. In studies with
GFP-ER
fusion protein, estradiol stimulation results in a formation
of nuclear foci, providing a visual representation of the strong
interactions of ER
with nuclear factors (39). Weak
agonists, like estriol and 17
-estradiol, are so called because of
their ability to activate some but not all of ER
responses, depending on the length of time of exposure. For example, a single administration of estriol can induce short-term estrogenic responses, such as water imbibition of the uterus, but not long-term growth responses (17). This selective regulation of receptor
activity by estriol is not correlated with its ability to activate
transcription, because estriol and estradiol are equally efficient at
stimulating transcription in vitro (25), and under chronic
stimulation, estriol functionally mimics estradiol (Fig. 5 and Ref.
2). Rather, it is more closely associated with the
inability of estriol-bound receptor to sustain tight nuclear
interactions (2, 8). This led to the concept that factors
that form complexes with ER
in the nucleus may have a role in the
selective regulation of the receptor by estriol and other weak
agonists. Our studies show that estriol and 17
-estradiol are less
efficient at lowering receptor levels than 17
-estradiol or ICI,
suggesting that fewer receptors are targeted to the proteasome pathway
by these ligands despite 100% receptor occupancy. These data suggest
that, like receptor transformation, proteolysis may be sensitive to the
weak or short-term interactions between receptor and other nuclear factors. This notion is supported by a recent study showing that coactivator interaction with ER
is necessary for
17
-estradiol-induced downregulation (24). Our
data, however, do not agree with the further conclusion suggested in
the latter report, that receptor turnover is linked to the efficiency
of receptor transcriptional activity. We show instead that estriol and
17
-estradiol both activate transcription to comparable levels as
17
-estradiol (Fig. 5), yet they have diminished capacity to decrease
receptor concentration (Table 1). Our data, thus, better support the
hypothesis that interactions within the nucleus may have a role in
providing specificity to the proteolytic response.
We demonstrate that ICI induces proteasome-dependent degradation of
ER in lactotrope cells. Early studies by Gibson et al. (14) demonstrated that ICI-164384 could elicit a rapid
decrease in uterine ER
, and it was suggested that the mechanism
through which ICI operated involved proteolytic processing of the
receptor (14). Dauvois et al. (10) also
demonstrated that ICI-164384 shortened the half-life of mouse ER
,
supporting the direct action of ICI on protein stability. We show that
ICI targets the receptor to proteasomes in lactotrope cells, in
agreement with studies by Stenoien et al. (40). In the PR1
cells, however, 17
-estradiol and ICI are equally effective, and
dose-response studies indicate that higher doses of ICI are required to
elicit an effect equivalent to that of 17
-estradiol. This differs
from the response in MCF-7 cells (40), in which ICI
appears to induce a complete loss of receptor and is more effective
than estradiol when saturating concentrations of ligand are used.
Differences in the effectiveness of ICI were also apparent in 293 stable cell lines expressing the wild-type receptor (Fig. 3), in which
ICI stimulation resulted in a greater reduction in receptor levels,
similar to what is seen in MCF-7 cells. These results suggest the
possibility that regulation of proteolysis of ER
displays cell-type
specificity in addition to ligand dependency.
Demonstration that ligand-induced proteolysis is abolished by a point
mutant in the ligand-binding pocket provides direct evidence that
ligand binding is required to target receptor to proteasomes. This is
also supported by deletion of the entire ligand-binding domain
(24), and by the correlation between ligand-binding affinity and potency of ligand to induce proteolysis. Ligand binding alone, however, is insufficient to induce proteolysis, since ligands of
the triphenyltheylene class, such as tamoxifen, are incapable of
downregulating ER (see Fig. 1 and Ref. 4). Structural
studies of the ER
ligand-binding domain demonstrate that tamoxifen
binding disrupts the organization of critical helix 12 of ER
and
prevents interaction of the receptor with coactivators (6,
38). It is unlikely, however, that tamoxifen's activity in
disrupting coactivator receptor interaction is responsible for its lack
of ability to induce proteolysis of receptor, because ICI binding produces a similar structural occlusion to the coactivator interface yet is a potent stimulator of receptor degradation (31).
The demonstration that not all receptor ligands can target receptor to
proteasomes, however, highlights the observation that, although ligand
binding is required to induce proteolysis, signals downstream of
binding are also necessary to direct receptor into the proteasome pathway.
Varying doses of ligand were utilized in this study to allow for the
quantitative analysis of changes in ER levels due to proteolysis.
Only a few studies have directly examined proteasome-dependent proteolysis of ER
, and commonly, saturating concentrations of ligand
are utilized to induce proteolysis. In addition, changes in receptor
level are assessed by qualitative determination of relative receptor
protein levels. Although these other studies provide important insight
relevant to the induction of proteolysis, they overlook ligand-specific
differences in the regulation of ER
degradation that might provide
insight into underlying regulatory mechanisms. Our results indicate
that proteolysis can be modulated by ligand concentration. They also
demonstrate that ligands that induce proteolysis can differentially
control the numbers of receptors that are degraded. These findings
illustrate an important concept, that regulation of receptor stability
is governed not only at the level of proteolysis induction but also by
the efficiency with which receptor is degraded. As such, small changes
in receptor stability may be misinterpreted as an absence of
proteasome-dependent proteolysis, when instead, they may simply reflect
changes in efficiency of receptor processing. Our results highlight the
importance of the quantitative assessment of changes in receptor levels
and provide evidence for ligand-specific regulation of
proteasome-dependent proteolysis of ER
.
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ACKNOWLEDGEMENTS |
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We thank Drs. Jack Gorski, Jyoti Watters, Fern Murdoch, Mike Fritsch, and Shigeki Miyamoto for helpful discussion throughout the course of this investigation and for critical reading of the manuscript. In addition, we thank Dr. Richard Maurer for the gift of the Prl-Luc reporter gene construct.
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FOOTNOTES |
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This work was supported by National Cancer Institute Grant K01 CA-79090 to E. T. Alarid.
Address for reprint requests and other correspondence: E. T. Alarid, Dept. of Physiology, 120 Service Memorial Institute, 1300 University Ave., Madison, WI 53706 (E-mail: alarid{at}physiology.wisc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00353.2001
Received 3 August 2001; accepted in final form 14 December 2001.
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