From the Robert H. Lurie Comprehensive Cancer Center, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
Received for publication, October 30, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Estrogen receptor Estrogen receptor The ER is an important therapeutic target for the treatment and
prevention of breast cancer. Selective estrogen receptor modulators (SERMs) are compounds that bind to the ER and exert tissue-specific effects. Tamoxifen was the first SERM approved clinically for the
treatment and prevention of breast cancer. Tamoxifen acts as an
antiestrogen in the breast but has estrogenic properties in that it
maintains bone density (6), lowers circulating cholesterol (7) and
causes an increased risk of endometrial cancer in women over 50 (8).
Raloxifene is a chemically related SERM that is used for the prevention
of osteoporosis but also lowers cholesterol and reduces the risk of
both breast cancer and endometrial cancer (9). ICI 182,780 is
considered to be a pure antiestrogen in that it displays no agonist
activity at the ER (10). This occurs because ICI 182,780 interferes
with receptor dimerization (11) and increases ER protein turnover
(12).
Analysis of the crystal structure of the ligand·ER complex has been
instrumental in understanding ER conformation at the molecular level
and highlights the importance of helix 12 in modulating estrogenic and
antiestrogenic actions. Helix 12 is located in the LBD of the ER, but
the composition and orientation of helix 12 differs depending on the
ligand bound to the ER (13). When the ER LBD is complexed with the ER
agonists estrogen (E2) or diethylstilbestrol (DES), helix
12 is positioned over the ligand binding pocket (see Fig.
2A) (13, 14). This proper positioning generates AF2 and
forms a surface for the recruitment of coactivators. However, when
4-hydroxytamoxifen (4-OHT, the active metabolite of tamoxifen) or
raloxifene is bound to the ER LBD, the antiestrogenic side chain
displaces helix 12 from its normal position, thereby preventing the
formation of a functional AF2 (Fig. 2B) (13, 14). Having
excluded AF2, the reported partial agonist activity of 4-OHT can only
be mediated by AF1 (15). In a previous study, a binding site
responsible for the estrogen-like action of 4-OHT was defined that is
referred to as AF2b (16). This site contains two critical components:
Asp-351 and a portion of helix 12 (Asp-538, Glu-542, and Asp-545). AF2b
is proposed to be a docking site for coactivators or corepressors that
modulate the estrogenicity of the 4-OHT or raloxifene ER complex
(17-19). Therefore, different ligands induce different receptor
conformations, and the positioning of helix 12 is the key event that
permits discrimination between ER agonists and antagonists by
influencing the interaction of the ER with coregulators.
The estrogenic or antiestrogenic action of ligands at the ER depends on
the subtle changes in ER shape that programs the ER to form an active
or inactive transcription complex or to be degraded by the proteasome.
The amount of available ER in the cell is controlled by a balance
between synthesis and degradation. ER stability is influenced by the
nature of the bound ligand such that ligand-induced conformational
changes modulate the ability of the ER to interact with proteins
involved in the degradation process (20). The transcriptional activity
of the resulting ER pool is also influenced by the ligands present. The
ER is activated if the ligand is estrogenic, and the established
estrogens can be classified as class I or class II (21). Class I
estrogens, such as DES or E2, are planar compounds that use
the AF2 site for optimal action. Class II estrogens, represented by
angular triphenylethylene compounds such as 4-OHT and fixed ring
4-hydroxy triphenyl pentene, utilize AF2b for activity. However,
ligands such as SERMs or pure antiestrogens can block the activity of
the ER by creating a ligand·ER complex that is inactive. Overall, the
complex decision-making network depends upon the protein recognition
sequences exposed on the external surface of the relevant SERM·ER
complex in response to ligand binding.
Analysis of the helix 12 region of AF2b using the 3m mutation
(D538A/E542A/D545A) yielded important insight into mechanism of 4-OHT
agonism. The transforming growth factor Amino acid L540 is a nearby amino acid of interest on the underside of
helix 12 when it is sealing estrogen in the hydrophilic pocket of the
LBD. The L540Q mutation was initially generated by random chemical
mutagenesis and is a dominant negative ER mutant (28-31). Previous
studies in MDA-MB-231 breast cancer cells have shown that an ERE-CAT
reporter is activated by 4-OHT and ICI 164,384, but not by
E2, in the presence of the L540Q mutant (27). Therefore, the L540Q mutation reverses the pharmacology of E2 and ICI
182,780 that is normally observed at the wild type ER in MDA-MB-231 cells.
We have stably transfected individual mutant ER cDNAs into
MDA-MB-231 human breast cancer cells to create an in vitro
model to address the contribution of specific amino acids in helix 12 (D538A, E542A, D545A, and L540Q) to the agonist activity of 4-OHT at
the AF2b site. We have found that Asp-538 is the critical amino acid in
helix 12 that not only reduces the estrogen-like actions of 4-OHT but
also enhances the degradation of the ER upon 4-OHT treatment.
Cell Culture and Reagents--
Stable cell lines were maintained
in phenol red-free minimum essential media supplemented with 5%
calf serum treated 3× with dextran-coated charcoal, 0.5 mg/ml G418
(Geneticin, Invitrogen, Carlsbad, CA), 2 mM
L-glutamine, 0.1 mM non-essential amino acids, 100 units/ml penicillin, 100 µg/ml streptomycin, and 6 µg/ml
insulin. This media is referred to as stripped media, indicating that
it is free of E2.
S30 cells are MDA-MB-231 human breast cancer cells stably transfected
with wild type ER
4-OHT and E2 were purchased from Sigma (St. Louis, MO). ICI
182,780 was obtained from AstraZeneca (Macclesfield, England). Raloxifene was a generous gift from Eli Lilly and Co. (Indianapolis, IN). All drugs were dissolved in ethanol and stored at Mutagenesis--
Site-directed mutagenesis was performed using
the QuikChange site-directed mutagenesis kit according to the
manufacturer's instructions (Stratagene, La Jolla, CA). The ER Generation of Stable Transfectants--
MDA-MB-231 (clone 10A)
cells (24) were grown in stripped media for 3-4 days prior to
transfection. The cells were transfected with 10 µg of the ER mutant
in the pIRESneo2 plasmid. 5 × 106 cells were
electroporated in a 0.4-cm cuvette (Bio-Rad, Hercules, CA) at a voltage
of 0.250 kV and a high capacitance of 0.95 microfarad in phenol
red-free minimal essential media with no additives. The cells were
transferred to a 10-cm plate and incubated overnight in 10 ml of
stripped media without G418, and the media was changed the next day.
The following day, media containing 0.5 mg/ml G418 was added, and the
cells were subsequently maintained in this media. Individual colonies
appeared ~1 month after transfection, and these were isolated and
screened for stable expression of the ER by Western blotting.
Transient Transfections and Luciferase Assays--
The cells
were transfected with 1 µg of the ERE3-luciferase plasmid (33) and 1 µg of the mutant or wild type ER
The cells were washed once with cold PBS, and 100 µl of extraction
buffer (0.1 M potassium phosphate (pH 7.5), 1% Triton
X-100, 100 µg/ml BSA, 2.5 mM phenylmethylsulfonyl
fluoride, and 1 mM dithiothreitol) was added to each well.
The cells were incubated on ice for 2 min, dislodged from the plates,
and transferred to an Eppendorf tube. The lysate was centrifuged for 2 min at top speed in a microcentrifuge, and the supernatant was
used for the assay. 50 µl of the lysate was mixed with 350 µl of
reaction buffer (160 mM MgCl2, 75 mM glycylglycine (pH 7.8), 0.5 mg/ml BSA, 19 mg/ml ATP, and
15 mM Tris-HCl (pH 7.5)) and 100 µl of luciferin (0.4 mg/ml). Luminescence was measured in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) for 10 s. Northern Blots--
Stable transfectants were treated with
compounds for 24 h. Total RNA was isolated using TRIzol reagent
(Invitrogen, Carlsbad, CA) according to the manufacturer's
instructions. 20 µg of RNA was loaded per lane in a 1% agarose/0.66
M formaldehyde gel. The RNA was transferred to a MagnaGraph
nylon transfer membrane (Osmonics, Minnetonka, MN) overnight in 10×
SSPE buffer (20× SSPE is 3.6 M NaCl/0.2 M
NaH2PO4/0.02 M EDTA (pH 7.4)). The
RNA was fixed to the membrane by UV-cross-linking. The membrane was
prehybridized in hybridization solution (0.5 M sodium
phosphate, 10 mM EDTA, 1% BSA, 7% SDS (pH 7.2)) for a
minimum of 2 h at 60 °C. The TGF Protein Isolation and Western Blots--
Cells were treated for
24 h with compound. To harvest protein, cells were washed once
with PBS, scraped using a cell scraper into 10 ml of PBS, and
transferred to a 15-ml conical tube. The cells were pelleted, and the
supernatant was aspirated. The cell pellet was resuspended in 100 µl
of extraction buffer (50 mM HEPES, 150 mM NaCl,
1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.5%
Nonidet P-40, 10 mM
20 µg of cell lysate were separated on a 7.5% SDS-PAGE gel and
transferred to nitrocellulose. The blot was blocked in blotto (2.5% dry milk/0.05% Tween/0.5× PBS) for 1 h to overnight.
Blots were probed with polyclonal ER Quantitation and Statistics--
Western and Northern blots were
quantitated using the gel plot feature in Scion Image version 4.0.2. The results were statistically analyzed using SPSS 9.0.
Previous results in our laboratory showed that a triple mutation
in helix 12 of the ER (3m, D538A/E542A/D545A) caused a change in ER
stability and eliminated 4-OHT agonist activity (18). To analyze the
role of individual amino acids in helix 12, multiple mutations were
generated. These mutations include single point mutations of the 3m
mutation (D538A/E542A/D545A) and the
L540Q mutation (Figs. 1 and 2).
(ER) is a ligand-activated
transcription factor implicated in breast cancer growth. Selective
estrogen receptor modulators (SERMs), such as tamoxifen (4-OHT), bind
to the ER and affect the position of helix 12, thereby influencing coregulator binding and ER transcriptional activation. Previous studies
have shown that a triple mutation in helix 12 (3m;
D538A/E542A/D545A) caused a change in ER stability and
obliterated 4-OHT action (Liu, H., Lee, E. S., de los Reyes,
A., Zapf, J. W., and Jordan, V. C. (2001) Cancer
Res. 61, 3632-3639). Two approaches were taken to determine the
role of individual mutants (D538A, L540Q, E542A, and D545A) on the
activity and stability of the 4-OHT·ER complex. First, mutants
were evaluated using transient transfection into ER-negative T47D:C4:2
cells with an ERE3-luciferase reporter, and second, transforming growth
factor
(TGF
) mRNA was used as a gene target in
situ for stable transfectants of MDA-MB-231 cells.
Transcriptional activity occurred in the presence of estrogen in all of
the mutants, although a decreased response was observed in the L540Q,
3m, and D538A cells. The 3m and D538A mutants lacked any estrogenic
responsiveness to 4-OHT, whereas the other mutations retained
estrogen-like activity with 4-OHT. Unlike the other mutants, the ER was
degraded in the D538A mutant with 4-OHT treatment. However, increasing
the protein levels of the mutant with the proteasome inhibitor MG132
did not restore the ability of 4-OHT to induce TGF
mRNA. We
suggest that Asp-538 is a critical amino acid in helix 12 that not only
reduces the estrogen-like actions of 4-OHT but also facilitates the
degradation of the 4-OHT·D538A complex. These data further illustrate
the complex role of specific surface amino acids in the modulation of
the concentration and the estrogenicity of the 4-OHT·ER complex.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(ER)1 is a member of the
steroid hormone superfamily of nuclear receptors, which are gene
regulatory transcription factors. Similar structural domains,
designated A-F, are shared between the nuclear receptors (for review,
see Refs. 1 and 2). Two transcriptional activation functions,
activation function 1 (AF1) and activation function 2 (AF2), are
present in the ER (see Fig. 1). AF1 is a constitutive activation
function located in the A/B region, and AF2 is a
ligand-dependent activation function in the E region or
ligand binding domain (LBD). The activity of AF1 and AF2 is largely
mediated by the cell and promoter context (3, 4) and can be independent
or synergistic (5).
(TGF
) gene is recognized
as a target of estrogen action and is involved in cell growth
stimulation by estrogen (22, 23), so the biological activity of the
4-OHT·ER complex can be assessed using Northern blotting for TGF
mRNA. Expression of TGF
mRNA is normally induced by
E2 and 4-OHT treatment in MDA-MB-231 human breast cancer
cells stably transfected with the wild type ER (S30 cells) (24). The 3m
mutation resulted in a decreased induction of TGF
in response to
E2 and no response to 4-OHT (18). Therefore, the 3m
mutation abolished the agonist activity of 4-OHT and decreased the
agonist activity of E2. In addition, a slight degradation
of the ER was observed when the 3m mutant stable cell line was treated
with E2, 4-OHT, and ICI (18). This is in contrast to stable
cell lines containing the wild type ER, which displayed a large
down-regulation of the ER in the presence of E2 and ICI,
but an increase in ER protein with 4-OHT treatment. Although the effect
of E2 on the 3m mutation and the three individual amino
acids comprising the 3m mutation has been studied using ERE-luciferase
assays (4, 25-27), the majority of the studies were not performed in
breast cancer cell lines and in a comprehensive manner. In addition, the precise interaction between 4-OHT and the individual mutations is
not known.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(24) and are referred to as wild type cells. These
cells are grown in stripped media. T47D:C4:2 cells are ER
-negative
human breast cancer cells (32) that were propagated in phenol red-free
RPMI media containing 10% fetal serum calf serum treated 3× with
dextran-coated charcoal, as well as the concentrations of amino acids,
penicillin, streptomycin, and insulin described above.
ER
represents a G418-resistant clone that is
ER-negative. The 3m stable cell line (18) containing the triple
mutation (D538A/E542A/D545A) was also grown in stripped media.
20 °C. MG132 was dissolved in Me2SO and obtained from Calbiochem
(San Diego, CA).
PSG5
plasmid (HEGO, kindly provided by P. Chambon) was used as a template
for PCR. Primers used were as follows: D538A (5'-GCA TCT CCA GCA GCA
GGG CAT AGA GGG GCA CCA CG-3' and 5'-CGT GGT GCC CCT CTA TGC CCT GCT GCT GGA GAT GC-3'), L540Q (5'-GGC GTC CAG CAT CTC CAG CTG CAG GTC ATA
GAG GGG-3' and 5'-CCC CTC TAT GAC CTG CAG CTG GAG ATG CTG GAC GCC-3'),
E542A (5'-GGG CGT CCA GCA TCG CCA GCA GCA GGT C-3' and 5'-GAC CTG CTG
CTG GCG ATG CTG GAC GCC C-3'), and D545A (5'-GTA GGC GGT GGG CGG CCA
GCA TCT CCA GC-3' and 5'-GCT GGA GAT GCT GGC CGC CCA CCG CCT AC-3').
Miniprep DNA was isolated from the resulting bacterial colonies using
the QIAprep Spin Miniprep kit (Qiagen, Valencia, CA) according to the
manufacturer's instructions. The Miniprep DNA was sequenced for the
presence of the mutation. A larger scale DNA preparation was made from
a chosen mutant using the Qiagen Maxiprep kit, and the entire ER DNA
was sequenced. Each mutant was then cloned into the pIRESneo2 plasmid
(Clontech, Palo Alto, CA) using the
EcoRI site flanking the ER cDNA.
PSG5 plasmid. To normalize for
transfection efficiency, 0.2 µg of the PCMV
plasmid
(Clontech, Palo Alto, CA) were also transfected.
5 × 106 cells were electroporated in a 0.4-cm cuvette
(Bio-Rad, Hercules, CA) at a voltage of 0.320 kV and a high capacitance
of 0.95 microfarad in serum-free media. The cells were transferred to
12-well plates and incubated overnight. The next day, the cells were
treated with the appropriate compound for 24 h.
-Galactosidase activity was measured using 10 µl of each sample and the Galacto-Light Plus detection system (Applied Biosystems, Bedford, MA). Data are reported as relative light units, which is the
luciferase reading divided by the
-galactosidase reading.
probe (a gift from Dr. R. Derynck, Genentech, South San Francisco, CA) or the ER
probe (the
EcoRI fragment from the ER
PSG5 plasmid) was labeled with
[32P]dCTP using the Megaprime DNA labeling system
(Amersham Biosciences, Piscataway, NJ), and the labeled probe was
separated from free 32P using Microspin columns (Amersham
Biosciences) according to the manufacturer's instructions. The probe
was heated at 95 °C for 5 min, added to the hybridization buffer,
and incubated at 60 °C overnight. The next day, the membrane was
washed for 30 min at 60 °C with 1× SSPE/0.1% SDS, 30 min with
0.5× SSPE/0.05% SDS, and 2 × 15 min with 0.1× SSPE/0.1% SDS.
To visualize TGF
, the membrane was exposed to film overnight. Equal
loading of samples was verified by stripping the membrane and reprobing
with
-actin.
-glycerophosphate (pH 8), containing
a 1:100 dilution of a freshly added protease inhibitor mixture (Sigma
P8340, St. Louis, MO)), passed through a 22-gauge needle, and
incubated on ice for 30 min. The cell lysate was centrifuged at
10,000 × g for 10 min at 4 °C, and the supernatant
was transferred to a new tube. Samples were quantitated using the
Bio-Rad protein assay kit.
antibodies at 1:200 (G20, Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal
-actin antibodies at 1:20,000 (Sigma A5441, St. Louis, MO) for 1 h at room
temperature. The membrane was then washed 3 × 5 min with wash
buffer (0.5× PBS/0.05% Tween). The blot was incubated in a 1:3000
dilution of horseradish peroxidase-conjugated secondary antibodies
(Santa Cruz Biotechnology) for 1 h. The membrane was washed 3 × 5 min with wash buffer, and bands were visualized using
chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Wild type and mutant ER helix 12 constructs. The human wild type ER is a 595-amino acid protein
consisting of domains A-F. The locations of activation
function 1 (AF1) and activation function 2 (AF2)
are indicated. The 3m mutation is a triple mutation composed of the
D538A, E542A, and D545A mutations. The individual mutations D538A,
E542A, and D545A were also constructed. The L540Q mutant is another
mutation located within helix 12. The location of each mutation within
the ER is denoted with an asterisk. Stable transfectants of
each mutant were generated in MDA-MB-231 cells.
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Fig. 2.
Crystal structures of ER·ligand complexes
show the position of helix 12 mutations. The estradiol·ER
complex (A) (1ERE) (14) and the 4-OHT·ER
(B) (3ERT) (13) complex are depicted. Helix 12 (amino acids
536-547 for E2 and 536-551 for 4-OHT) is
shaded in yellow, the ligand is blue,
Asp-351 is green, and Asp-538, Leu-540, Glu-542, and Asp-545
are red. Leu-540 is not visible in the 4-OHT·ER structure,
because it is in the interior of the complex. In the
diethylstilbestrol·ER structure, helix 12 comprises residues
538-546, whereas in the 4-OHT structure, it comprises residues
536-544 (13). For clarity, modified amino acids in helix 12 are
shaded. The published crystal structures were obtained from
the RCSB Protein Data Bank and are colored using Insight
II.
The transcriptional activity of the ER mutants was first tested by
transient transfection of ER-negative T47D:C4:2 cells with the mutant
ER cDNA and an ERE3-luciferase reporter (33). Transfection with the
empty vector PSG5 showed no induction of luciferase activity with any
of the treatments used (Fig. 3). In
addition, none of the mutants exhibited any response to treatment with
the vehicle control ethanol (EtOH). E2 treatment resulted
in a statistically significant induction of luciferase activity with
the wild type ER and all of the mutants when compared with the EtOH
control. The greatest induction was observed with the E542A mutant. The wild type, D538A, D545A, and 3m mutants displayed an intermediate level, and the L540Q mutant displayed the smallest induction. The L540Q
mutant was the only mutant that showed a slight induction of luciferase
activity during ICI 182,780 treatment, but this was not statistically
significant. In addition, the wild type, E542A, and D545A ERs displayed
an induction of luciferase activity upon 4-OHT treatment, whereas the
D538A, L540Q, and 3m mutants did not. When wild type, 3m, and D538A
cells were treated with E2 plus 4-OHT, the response of the
cells was the same as that observed with 4-OHT alone, indicating that
4-OHT acts as a complete antiestrogen in these cells (data not shown).
Therefore, of the three mutations present in the 3m mutant, the D538A
mutation is responsible for the elimination of the agonist activity of
4-OHT at an ERE in T47D:C4:2 cells.
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To further evaluate these mutants in a reproducible manner, stable
transfectants were generated in ER-negative MDA-MB-231 cells. At least
five clones were obtained representing each mutation, and the clones
were screened for the presence of the ER using Western blotting. Two
clones harboring each mutation were initially screened using Northern
blotting for TGF mRNA levels. Both of the clones studied showed
similar TGF
levels in response to various treatments, so a single
representative clone was chosen for further analysis. Each of the
stable clones was screened to ensure that the proper mutation was
present using reverse transcription-PCR and sequencing.
ER protein levels were compared between each of the stable cell lines
(Fig. 4). All of the cell lines contained
similar levels of ER protein, so the characteristics observed in each
cell line were not a result of varying ER levels. A clone that was
stably transfected but ER-negative by Western blot analysis was used as
a control and designated ER.
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The transcriptional activity of the ER mutants was also analyzed using
Northern blot analysis of TGF mRNA. The advantage of this assay
is that the TGF
gene is an endogenous gene in MDA-MB-231 cells, and
induction of TGF
mRNA levels reflects a process that is inherent
to these cells. Cells from the ER
clone were treated with
EtOH, E2, 4-OHT, and E2 plus 4-OHT, and no
induction of TGF
mRNA was observed (data not shown). Wild type
cells showed an induction of TGF
mRNA in response to
E2 and 4-OHT, but 4-OHT did not act as an antiestrogen in
these cells, because it was not able to significantly block the
E2 response (Fig. 5). A
similar pattern of mRNA expression was observed in the E542A and
D545A mutants. Although the 3m and D538A mutants showed an increase in
TGF
mRNA in response to E2 treatment, the level of
induction was less than that observed for the other mutants. In
addition, no induction occurred with the 4-OHT treatment. This is in
agreement with ERE-luciferase assay results and suggests that Asp-538
is the single amino acid within the 3m mutation required for the
agonist activity of 4-OHT at the ER. The L540Q mutant produced an
induction of TGF
mRNA with ICI 182,780 and 4-OHT treatment but
not with E2 treatment. In addition, raloxifene (Ral) treatment had no effect on TGF
mRNA levels with any of the
transfected stable cell lines (Fig. 5).
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Because the expression of E2-induced TGF mRNA was
lower in the D538A and 3m stable transfectants compared with all other transfectants, E2 concentration response curves were
completed to ensure that an E2 response was present (Fig.
6). Wild type cells displayed an
induction of TGF
mRNA at the lowest concentration of
E2 (10
10 M), and the amount of
TGF
mRNA continued to increase up to the highest concentration
of E2 (10
6 M). An
EC50 value of 7 × 10
10 M
was calculated for the wild type cells. A similar profile was observed
in the D545A mutant, in that a 10
10 M
concentration of E2 induced TGF
mRNA, and the
increase continued up to 10
6 M
E2. This is consistent with an EC50 of 2 × 10
10 M. The 3m and D538A mutants behaved
similarly in that they first displayed an increase in TGF
mRNA
at a 10
9 M concentration of E2,
and the induction continued with increasing concentrations of
E2. Therefore, the 3m and D538A mutants required higher
concentrations of E2 before TGF
mRNA was
transcribed, when compared with the wild type and D545A cells. The
EC50 of the 3m and D538A cells were 2 × 10
9 M and 4 × 10
9
M, respectively, which are ~10 times higher than the wild
type and D545A cells. In addition, TGF
mRNA levels in the D538A
mutant were lower at each concentration of E2 tested in
comparison to the other mutants.
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In general, a SERM can negatively affect ER activity using two
different mechanisms. First, the SERM could cause the degradation of
the ER by creating a SERM·ER complex that is targeted for
destruction. Second, a SERM·ER complex may not be degraded but be
present and have no intrinsic activity. To distinguish between these
possibilities, wild type and mutant ER protein levels were analyzed in
the presence of ICI 182,780, E2, and 4-OHT (Fig.
7A). ICI 182,780 degraded the
wild type ER at all concentrations tested, and the D545A and D538A
mutants were also degraded by ICI 182,780 (Fig. 7B). In contrast, the L540Q mutant exhibited no changes in ER protein levels at
any ICI 182,780 concentration. The 3m and E542A mutants displayed
intermediate ER levels after ICI 182,780 treatment.
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When wild type cells were treated with E2, less ER protein was observed by Western blot (Fig. 7C). E2 treatment also resulted in a down-regulation of ER levels in the D538A and D545A mutants, whereas ER levels were stable in the 3m, L540Q, and E542A mutants. In the presence of 4-OHT, ER levels increased in the wild type cells and remained stable in the L540Q, E542A, and D545A mutants. Importantly, 4-OHT treatment reduced ER protein levels in the D538A and 3m stables. The stable cell lines that showed an induction of ER transcriptional activity upon 4-OHT treatment (wild type, E542A, and D545A) all contained ER protein levels that were maintained or increased when treated with 4-OHT. In contrast, the D538A and 3m mutants exhibited no transcriptional activity in response to 4-OHT, and they were the only mutants that showed a decrease in ER levels with 4-OHT treatment. This observation prompted us to explore the possibility that the decreased transcriptional activity of the D538A mutant was a result of decreased ER protein levels in response to 4-OHT.
In the D538A mutant, the degradation of ER protein by 4-OHT could be a
result of 4-OHT-induced transcriptional down-regulation of the ER
message, or 4-OHT-induced post-translational degradation. To
distinguish between these possibilities, Northern blot analyses for
ER were performed in wild type and D538A cells that were treated
with EtOH or 4-OHT (Fig. 7D). A comparison of ER mRNA and protein levels showed that 4-OHT up-regulated both ER mRNA and
protein in the wild type cells. In contrast, 4-OHT treatment did not
change ER mRNA levels and decreased ER protein levels in the D538A
cells by 70%. This indicated that the 4-OHT-mediated decrease in ER
protein levels in the D538A mutant was not a result of decreased ER
mRNA stability.
ER protein levels were measured in all of the stably transfected cell
lines to establish whether the proteasome is involved in the
degradation of the ER. The cells were treated with the proteasome
inhibitor MG132 before the addition of ligand, and a Western blot was
performed to analyze ER levels (Fig. 8).
Preincubation with MG132 elevated the amount of ER protein present,
indicating that the ligand-induced degradation of the ER could be
mediated by the proteasome. However, subtle differences were observed, depending on the mutation and the ligand evaluated, especially in the
case of ICI 182,780. MG132 was able to prevent the ICI 182,780-mediated
degradation in D538A and E542A cells, but smaller increases were
detected in wild type and D545A cells. It is possible that MG132 was
not able to restore ER levels in the ICI 182,780-treated cells to
control levels, because a relatively high concentration of ICI 182,780 (106 M) was used. Experiments using lower
concentrations of ICI 182,780 (10
6-10
8
M) in combination with MG132 were performed in the wild
type cells (data not shown), but MG132 treatment did not restore ER levels in the ICI 182,780-treated cells to control levels. In addition,
MCF-7 cells were treated under the same conditions as described in Fig.
8, and the results were essentially the same (data not shown) in that
MG132 treatment could not fully abrogate ICI 182,780 treatment.
Therefore, MG132 was unable to restore ER protein to control levels in
wild type, D545A, and MCF-7 cells treated with ICI 182,780. This is in
contrast to MG132 treatment in E2-treated cells, where the
ER levels are greater than control cells. L540Q cells were not included
in this experiment, because the ER levels in these cells remained the
same after treatment with E2, ICI 182,780, and 4-OHT (Fig.
7). These data indicate that the degradation of the ER in the stably
transfected cell lines occurs through the proteasome.
|
When D538A cells were treated with MG132 and 4-OHT, ER protein levels
were increased 2.3-fold, compared with the decreased levels of the
receptor normally observed with 4-OHT treatment alone (Fig. 8). The
D538A stable cells were treated with a range of MG132 concentrations
and 4-OHT or E2 to determine whether preventing the
degradation of the 4-OHT·D538A complex could restore 4-OHT agonist
activity (Fig. 9). TGF mRNA was
induced with E2 treatment, but no induction of TGF
mRNA levels was observed with MG132 treatment alone or MG132 in
combination with 4-OHT. Therefore, increasing the amount of D538A ER
that would normally be available in the presence of 4-OHT did not
restore the agonist activity of 4-OHT. Therefore, adjusting the levels
of the 4-OHT·D538A complex did not have an effect, because the
complex has no intrinsic activity.
|
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DISCUSSION |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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The finding that Asp-538 is an essential amino acid that modulates estrogen action with 4-OHT supports and extends the idea that helix 12 plays a vital role in the mechanics of estrogen action (13, 14). Asp-538 appears to be a central control mechanism for both the intrinsic activity of the 4-OHT·ER complex and the processing and degradation of the complex by the proteasome. These observations introduce a new dimension for consideration with SERMs as modulators of estrogen responsive genes.
Proteolysis is involved in the regulation of a variety of cellular functions such as cell cycle progression, oncogenesis, transcription, development, tissue growth, elimination of abnormal proteins, and antigen processing (34). Degradation of proteins can occur through three major pathways, which include mechanisms mediated through lysosomes, calpains (calcium-dependent cysteine proteases), and the proteasome. It has been shown previously that the ER is a ubiquitinated protein and that ubiquitination targets the protein to the proteasome, which causes ER degradation (35-37). Our data indicate that the proteasome is responsible for the degradation of ER protein observed in all of the stably transfected cell lines (Fig. 8).
The stability of the ER complex has been shown to be influenced by the bound ligand. E2 and ICI 182,780 decrease ER levels, and 4-OHT increases the accumulation of the ER in MCF-7 human breast cancer cells (38) and pituitary lactotrope PR1 cells (20). A similar situation occurs when MDA-MB-231 cells are stably transfected with the wild type ER (Fig. 7). However, the typical pattern of ER stability is changed by mutation of residues in helix 12. For example, treatment of cells with E2 did not down-regulate the ER in the 3m, L540Q, and E542A mutants, whereas down-regulation is observed in the D538A and D545A mutants. ICI 182,780 degraded the ER in all of the stable cell lines except for the L540Q cells, and ICI 182,780 had a reduced affect in the 3m and E542A cells. 4-OHT either increased or did not affect ER levels in L540Q, E542A, and D545A cells but had a dramatic effect on the degradation of the ER in 3m and D538A cells. This suggests that the presence of an aspartic acid at 538 prevents degradation of the ER when liganded by 4-OHT.
Alterations in other amino acids in helix 12 are reported to result in changes in the stability of the ER. The protein levels of the L539A/L540A double mutant remained constant upon E2 treatment, whereas 4-OHT induced degradation of the ER (38). The mouse ER mutants L543A/L544A (L539A/L540A in human) and M547A/L548A (M543A/L544A in human) (26) as well as the human ER mutants L540Q, E542A/D545A, and L540Q/E542A/D545A (27) are not degraded by ICI 182,780. This indicates that helix 12 is important for maintaining the proper regulation of the ER protein in response to ligands, especially ICI 182,780.
The signal that ultimately targets the ER for ubiquitination and subsequent degradation has not been definitively established. Several possibilities have been proposed that modify the shape of a protein so that is it recognized by the E3 ubiquitin protein ligase, such as ER phosphorylation, binding of ancillary proteins to the ER, or binding of ligand. Modulation of the ubiquitination machinery or masking of a degradation signal are also possibilities (for review, see Refs. 39-42). It is likely that a combination of these mechanisms could contribute to the ER degradation observed in the stable cell lines.
In addition to degradation, another consequence of changing the shape
or charge distribution of the external surface of the ligand·ER
complex is the modulation of ER transcriptional activity. Transcriptional activity was measured initially using transient transfection of the ER mutant and an ERE3-luciferase reporter into
T47D:C4:2 cells (Fig. 3), but a further study of TGF mRNA in the
stably transfected MDA-MB-231 cells extended our observations (Figs. 5
and 6). The results of both of these assay approaches were consistent.
In wild type cells, E2 and 4-OHT induced transcriptional activity but ICI 182,780 did not. The 3m stable cells exhibited no response to ICI 182,780 and 4-OHT and showed a decreased response to
E2 compared with the wild type cells. The E542A and D545A
cells showed essentially the same transcriptional activity as wild type cells. Little transcription occurred with E2 treatment in
the L540Q mutant, but activation was observed upon ICI 182,780 and 4-OHT treatment. Remarkably, ICI 182,780 can produce a biological effect when the receptor is stable. The crystal structure of a pure
antiestrogen and ER
, but not ER
, is available. Although the
ER
:ICI 164,384 (a pure antiestrogen related to ICI 182,780) crystal
structure has been solved, helix 12 is invisible in the experimental
electron density maps (43), so structure/function speculations are not
yet possible.
The D538A mutant exhibits decreased transcription in the presence of E2 and no transcription in the presence of ICI 182,780 and 4-OHT. This indicates that the D538A mutation is the single mutation responsible for the decreased activity of the 3m triple mutation. These data are important, because it is now possible to redefine the components of AF2b as Asp-351 and Asp-538, which must interact with AF1, because only these regions are required for the estrogen-like activity of 4-OHT. By analogy with our approach to defining the precise amino acids on helix 12, we are currently addressing the question of the one or more critical amino acids in AF-1 that may be required for SERM activity.
The external surface of the ER affects protein stability and
transcriptional activity, but the amount of the ligand·ER complex does not necessarily correlate with activity. Because 4-OHT treatment results in rapid degradation of the D538A protein with an associated lack of transcriptional activity, we increased the level of the 4-OHT·D538A ER complex using an inhibitor of the proteasome. However, activity of the D538A mutant was not restored in the TGF assay (Fig.
9), indicating that the 4-OHT·D538A complex has no intrinsic activity. An interesting point is that the amount of TGF
mRNA was reduced when the proteasome inhibitor was combined with
E2 in the D538A cells. This observation is in agreement
with a study by Lonard et al. (44), which suggests that
proteasomal degradation is required for E2-mediated ER
transcription and that coactivator binding is required for
ligand-mediated degradation of the ER.
Our studies and the known crystal structures of the ligand·ER complex emphasize the idea that the amino acids present in helix 12 are located in unique positions to influence the interaction of coregulators with the ER. In the E2·ER complex, helix 12 is positioned over the ligand binding pocket and the charged residues Asp-538, Glu-542, and Asp-545 are on the outside of the complex (Fig. 2A) (14). Leu-540 is positioned more toward the inside of the ligand binding pocket. Glu-542 is uniquely positioned in the DES·ER crystal structure as an N-terminal capping amino acid that stabilizes the conformation of the coactivator GRIP1 peptide when this peptide is bound to the ER (13, 45). Contacts are also made to the GRIP1 peptide in the DES·ER structure by Asp-538 (13). In the 4-OHT·ER structure, the side chain of 4-OHT repositions helix 12 so that it binds to and blocks the GRIP1 coactivator binding surface (Fig. 2B) (13). In fact, the side chains of Leu-540, Met-543, and Leu-544 on the inner hydrophobic surface of helix 12 mimic the interactions made by the coactivator's nuclear receptor binding motif LXXLL (45). None of the helix 12 mutations utilized in our study are residues that contact the ligand or Asp-351, which also modulates the estrogenicity of 4-OHT (16, 18, 46).
An active transcription complex contains coactivators that enhance the transcriptional activity of the ER. An inactive complex contains corepressor or is in a conformation that is unable to bind coactivators (47). Much of the transcriptional activity of the 4-OHT·D538A complex could be explained by the differential binding of coregulators. For example, the D538A mutation results in decreased transcriptional activity in the presence of E2. Because Asp-538 contacts a coactivator, the decreased agonist activity of E2 in the D538A mutant (Figs. 3, 5, and 6) could be a result of a decreased ability to recruit a coactivator. The agonist activity of 4-OHT in the wild type cells is mediated by constitutive AF1 activity, because the AF2 coactivator binding site has been disrupted by the side chain of 4-OHT. The D538A mutant eliminates the agonist activity of 4-OHT, suggesting that the D538A mutation in AF2 has allosterically affected AF1. The charge alterations produced by this mutation could favor the recruitment of a corepressor, because it has been demonstrated that 4-OHT can induce the formation of a ER·corepressor complex on the promoter (48, 49).
The dominant negative activity of the L540Q mutant occurs as a result of competition for ERE binding, formation of inactive heterodimers with the wild type receptor, and transcriptional silencing (31). In addition, the L540Q mutant recruits a coregulator protein called repressor of estrogen receptor activity (REA) (50). These mechanisms contribute to the activity of the L540Q mutant in vitro, but activity is also observed in vivo. The L540Q mutant was introduced into T47D human breast cancer cells using adenoviral infection, and when these cells were injected into athymic mice, tumor formation was inhibited (51). Injection of adenoviruses encoding the L540Q mutation into pre-existing T47D tumors resulted in tumor regression.
In summary, ER action is a complex and tightly regulated system
involving interactions between the ligand, the receptor, and effectors
that are all coordinated to modulate the appropriate action (52). Helix
12, more specifically Asp-538, is central to these diverse
interactions. The model we propose illustrates an unusually dramatic
regulation of ER degradation and efficacy of the SERM·ER complex. The
SERM 4-OHT normally causes an accumulation of the ER complex that is
promiscuous and can induce estrogen-like action at the TGF target
gene. 4-OHT degrades the ER complex if a specific amino acid (Asp-538)
is mutated, but if the 4-OHT·D538A complex is prevented from being
destroyed by the proteasome, the complex is not estrogen-like. We
believe that the modulation of the estrogenic and antiestrogenic
properties of the SERM·ER complex occur through the multiple
dimensions of ER destruction and the interaction of the ER with other
coregulatory proteins. The finding that Asp-538 in helix 12 can control
both ER stability and the intrinsic activity of the 4-OHT·ER complex
may not only provide new opportunities in drug design but also provide
a new insight into the regulation of ER protein concentrations within
the cell.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Ishwar Radhakrishnan (Northwestern University) for help in generating the crystal structure figures.
![]() |
FOOTNOTES |
---|
* This work was supported by the Training Program in Signal Transduction and Cancer (Grant T32-CA70085), the Lynn Sage Cancer Research Foundation, the Avon Foundation, and the National Institutes of Health Special Program for Research Excellence in Breast Cancer (Grant CA-89018-02).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.
To whom correspondence should be addressed: Robert H. Lurie
Comprehensive Cancer Center, The Feinberg School of Medicine, Olson
Pavilion Rm. 8258, Northwestern University, 303 E. Chicago Ave.,
Chicago, IL 60611. Tel.: 312-908-4148; Fax: 312-908-1372; E-mail:
vcjordan@northwestern.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211129200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ER, estrogen
receptor ;
AF1 and -2, activation functions 1 and 2;
LBD, ligand
binding domain;
SERM, selective estrogen receptor modulator;
E2, estrogen;
DES, diethylstilbestrol;
4-OHT, 4-hydroxytamoxifen;
TGF
, transforming growth factor
;
Ral, raloxifene;
PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;
ERE, estrogen response element.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
MacGregor, J. I.,
and Jordan, V. C.
(1998)
Pharmacol. Rev.
50,
151-196 |
2. |
Nilsson, S.,
Makela, S.,
Treuter, E.,
Tujague, M.,
Thomsen, J.,
Andersson, G.,
Enmark, E.,
Pettersson, K.,
Warner, M.,
and Gustafsson, J. A.
(2001)
Physiol. Rev.
81,
1535-1565 |
3. | Tora, L., White, J., Brou, C., Tasset, D., Webster, N., Scheer, E., and Chambon, P. (1989) Cell 59, 477-487[Medline] [Order article via Infotrieve] |
4. | Tzukerman, M. T., Esty, A., Santiso-Mere, D., Danielian, P., Parker, M. G., Stein, R. B., Pike, J. W., and McDonnell, D. P. (1994) Mol. Endocrinol. 8, 21-30[Abstract] |
5. | Kraus, W. L., McInerney, E. M., and Katzenellenbogen, B. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12314-12318[Abstract] |
6. | Love, R. R., Mazess, R. B., Barden, H. S., Epstein, S., Newcomb, P. A., Jordan, V. C., Carbone, P. P., and DeMets, D. L. (1992) N. Engl. J. Med. 326, 852-856[Abstract] |
7. | Love, R. R., Wiebe, D. A., Newcomb, P. A., Cameron, L., Leventhal, H., Jordan, V. C., Feyzi, J., and DeMets, D. L. (1991) Ann. Intern. Med. 115, 860-864[Medline] [Order article via Infotrieve] |
8. |
Fisher, B.,
Costantino, J. P.,
Wickerham, D. L.,
Redmond, C. K.,
Kavanah, M.,
Cronin, W. M.,
Vogel, V.,
Robidoux, A.,
Dimitrov, N.,
Atkins, J.,
Daly, M.,
Wieand, S.,
Tan-Chiu, E.,
Ford, L.,
and Wolmark, N.
(1998)
J. Natl. Cancer Inst.
90,
1371-1388 |
9. |
Cummings, S. R.,
Eckert, S.,
Krueger, K. A.,
Grady, D.,
Powles, T. J.,
Cauley, J. A.,
Norton, L.,
Nickelsen, T.,
Bjarnason, N. H.,
Morrow, M.,
Lippman, M. E.,
Black, D.,
Glusman, J. E.,
Costa, A.,
and Jordan, V. C.
(1999)
JAMA
281,
2189-2197 |
10. | Wakeling, A. E., Dukes, M., and Bowler, J. (1991) Cancer Res. 51, 3867-3873[Abstract] |
11. | Fawell, S. E., White, R., Hoare, S., Sydenham, M., Page, M., and Parker, M. G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6883-6887[Abstract] |
12. | Dauvois, S., Danielian, P. S., White, R., and Parker, M. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4037-4041[Abstract] |
13. | Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937[Medline] [Order article via Infotrieve] |
14. | Brzozowski, A. M., Pike, A. C., Dauter, Z., Hubbard, R. E., Bonn, T., Engstrom, O., Ohman, L., Greene, G. L., Gustafsson, J. A., and Carlquist, M. (1997) Nature 389, 753-758[CrossRef][Medline] [Order article via Infotrieve] |
15. | Berry, M., Metzger, D., and Chambon, P. (1990) EMBO J. 9, 2811-2818[Abstract] |
16. |
MacGregor Schafer, J.,
Liu, H.,
Bentrem, D. J.,
Zapf, J. W.,
and Jordan, V. C.
(2000)
Cancer Res.
60,
5097-5105 |
17. |
Yamamoto, Y.,
Wada, O.,
Suzawa, M.,
Yogiashi, Y.,
Yano, T.,
Kato, S.,
and Yanagisawa, J.
(2001)
J. Biol. Chem.
276,
42684-42691 |
18. |
Liu, H.,
Lee, E. S.,
de los Reyes, A.,
Zapf, J. W.,
and Jordan, V. C.
(2001)
Cancer Res.
61,
3632-3639 |
19. |
Liu, H.,
Park, W. C.,
Bentrem, D. J.,
McKian, K. P.,
de los Reyes, A.,
Loweth, J. A.,
Schafer, J. M.,
Zapf, J. W.,
and Jordan, V. C.
(2002)
J. Biol. Chem.
277,
9189-9198 |
20. | Preisler-Mashek, M. T., Solodin, N., Stark, B. L., Tyriver, M. K., and Alarid, E. T. (2002) Am. J. Physiol. 282, E891-E898[Medline] [Order article via Infotrieve] |
21. |
Jordan, V. C.,
Schafer, J. M.,
Levenson, A. S.,
Liu, H.,
Pease, K. M.,
Simons, L. A.,
and Zapf, J. W.
(2001)
Cancer Res.
61,
6619-6623 |
22. | Bates, S. E., Davidson, N. E., Valverius, E. M., Freter, C. E., Dickson, R. B., Tam, J. P., Kudlow, J. E., Lippman, M. E., and Salomon, D. S. (1988) Mol. Endocrinol. 2, 543-555[Abstract] |
23. | Lee, D. C., Fenton, S. E., Berkowitz, E. A., and Hissong, M. A. (1995) Pharmacol. Rev. 47, 51-85[Medline] [Order article via Infotrieve] |
24. | Jiang, S. Y., and Jordan, V. C. (1992) J. Natl. Cancer Inst. 84, 580-591[Abstract] |
25. | Danielian, P. S., White, R., Lees, J. A., and Parker, M. G. (1992) EMBO J. 11, 1025-1033[Abstract] |
26. | Mahfoudi, A., Roulet, E., Dauvois, S., Parker, M. G., and Wahli, W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4206-4210[Abstract] |
27. | Montano, M. M., Ekena, K., Krueger, K. D., Keller, A. L., and Katzenellenbogen, B. S. (1996) Mol. Endocrinol. 10, 230-242[Abstract] |
28. |
Ince, B. A.,
Zhuang, Y.,
Wrenn, C. K.,
Shapiro, D. J.,
and Katzenellenbogen, B. S.
(1993)
J. Biol. Chem.
268,
14026-14032 |
29. | Ince, B. A., Montano, M. M., and Katzenellenbogen, B. S. (1994) Mol. Endocrinol. 8, 1397-1406[Abstract] |
30. | Ince, B. A., Schodin, D. J., Shapiro, D. J., and Katzenellenbogen, B. S. (1995) Endocrinology 136, 3194-3199[Abstract] |
31. |
Schodin, D. J.,
Zhuang, Y.,
Shapiro, D. J.,
and Katzenellenbogen, B. S.
(1995)
J. Biol. Chem.
270,
31163-31171 |
32. | Pink, J. J., Bilimoria, M. M., Assikis, J., and Jordan, V. C. (1996) Br. J. Cancer 74, 1227-1236[Medline] [Order article via Infotrieve] |
33. | Catherino, W. H., and Jordan, V. C. (1995) Cancer Lett 92, 39-47[CrossRef][Medline] [Order article via Infotrieve] |
34. |
DeMartino, G. N.,
and Slaughter, C. A.
(1999)
J. Biol. Chem.
274,
22123-22126 |
35. |
Nawaz, Z.,
Lonard, D. M.,
Dennis, A. P.,
Smith, C. L.,
and O'Malley, B. W.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1858-1862 |
36. | El Khissiin, A., and Leclercq, G. (1999) FEBS Lett. 448, 160-166[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Alarid, E. T.,
Bakopoulos, N.,
and Solodin, N.
(1999)
Mol. Endocrinol.
13,
1522-1534 |
38. |
Wijayaratne, A. L.,
and McDonnell, D. P.
(2001)
J. Biol. Chem.
276,
35684-35692 |
39. | Ciechanover, A., Orian, A., and Schwartz, A. L. (2000) Bioessays 22, 442-451[CrossRef][Medline] [Order article via Infotrieve] |
40. | Ciechanover, A., Orian, A., and Schwartz, A. L. (2000) J. Cell. Biochem. Suppl. 34, 40-51[Medline] [Order article via Infotrieve] |
41. | Hirsch, C., and Ploegh, H. L. (2000) Trends Cell Biol. 10, 268-272[CrossRef][Medline] [Order article via Infotrieve] |
42. | Verma, R., and Deshaies, R. J. (2000) Cell 101, 341-344[Medline] [Order article via Infotrieve] |
43. | Pike, A. C., Brzozowski, A. M., Walton, J., Hubbard, R. E., Thorsell, A. G., Li, Y. L., Gustafsson, J. A., and Carlquist, M. (2001) Structure (Camb.) 9, 145-153[Medline] [Order article via Infotrieve] |
44. | Lonard, D. M., Nawaz, Z., Smith, C. L., and O'Malley, B. W. (2000) Mol. Cell 5, 939-948[Medline] [Order article via Infotrieve] |
45. | Pike, A. C., Brzozowski, A. M., and Hubbard, R. E. (2000) J. Steroid Biochem. Mol. Biol. 74, 261-268[CrossRef][Medline] [Order article via Infotrieve] |
46. | Levenson, A. S., Tonetti, D. A., and Jordan, V. C. (1998) Br. J. Cancer 77, 1812-1819[Medline] [Order article via Infotrieve] |
47. |
McKenna, N. J.,
Lanz, R. B.,
and O'Malley, B. W.
(1999)
Endocr. Rev.
20,
321-344 |
48. | Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A., and Brown, M. (2000) Cell 103, 843-852[Medline] [Order article via Infotrieve] |
49. |
Shang, Y.,
and Brown, M.
(2002)
Science
295,
2465-2468 |
50. |
Montano, M. M.,
Ekena, K.,
Delage-Mourroux, R.,
Chang, W.,
Martini, P.,
and Katzenellenbogen, B. S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6947-6952 |
51. | Lee, E. J., Jakacka, M., Duan, W. R., Chien, P. Y., Martinson, F., Gehm, B. D., and Jameson, J. L. (2001) Mol. Med. 7, 773-782[Medline] [Order article via Infotrieve] |
52. | Katzenellenbogen, B. S., Montano, M. M., Ediger, T. R., Sun, J., Ekena, K., Lazennec, G., Martini, P. G., McInerney, E. M., Delage-Mourroux, R., Weis, K., and Katzenellenbogen, J. A. (2000) Recent Prog. Horm. Res. 55, 163-193[Medline] [Order article via Infotrieve] |