From the Departments of Pharmaceutical Chemistry and Cellular Molecular Pharmacology, University of California, San Francisco, California 94143-0446
Received for publication, June 21, 2000, and in revised form, October 31, 2000
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ABSTRACT |
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The two subtypes of human estrogen receptor, The estrogen receptors (ER One explanation for the different effects is that a ligand may elicit
different responses when the receptor acts through different effector
sites (4). The estrogen receptor regulates transcription through
binding to estrogen response elements (EREs) in the upstream promoter
regions of target genes as well as through interactions with a growing
number of "nonclassical" response sites (5, 6). These nonclassical
sites do not necessarily require DNA-protein interactions between the
receptor and the promoter element, but instead regulate transcription
through protein-protein interactions between the receptor and other
transcription factors such as AP-1 and Sp-1. By receptor interactions
with different response elements, the same ligand could then cause both
activation and repression of different sets of genes.
The estrogen receptor exists in two different forms, ER Plasmids--
The construction of the expression vectors for
both hER
Chimera
To make the N-terminal deletion mutant
The F region chimera
The F region chimera
The F domain mutant Tissue Culture, Transfection, and Luciferase
Assays--
HeLa cells were grown in 0.1 µm filtered Dulbecco's
modified Eagle's medium supplemented with 4.5 g/liter glucose,
0.876 g/liter glutamine, 100 mg/liter streptomycin sulfate, 100 units/ml penicillin G and 10% newborn calf serum. Cells were
grown to a density of not more that 5 × 104
cells/cm2. For transient transfection assays, cells were
suspended in 0.5 ml of electroporation buffer in 0.4-cm gap
electroporation cuvettes at ~1.5 × 106
cells/cuvette with 5 µg of the reporter plasmid and the optimal amount of the receptor expression vector. The optimal receptor plasmid
concentration for maximal ligand activation was determined for each
mutant and was found to be 5 µg of plasmid per transfection except
for the
After 48 h of incubation at 37 °C, the cells were lysed by
first removing the medium from the wells, washing with PBS, and then adding 0.2 ml of lysis buffer consisting of 100 mM
potassium phosphate (pH 7.5), 0.2% Triton X-100, and 1 mM
dithiothreitol. The plates were frozen at Ligand Binding Assay and Data Normalization--
Transfection
efficiency and receptor expression levels were tested using a whole
cell ligand binding assay (16). After each transfection with the
reporter and expression plasmids described above, a portion of the
transfected cell suspension was plated into four wells of a 24-well
plate at a density of 105 cells/well. The cells were grown
as described above for ~12 h, then the medium was removed, the
cells were washed once with PBS and then treated with 200 µl of
medium minus the newborn calf serum and containing 20 nM [2,4,6,7,16,17-3H]estradiol. To two of the
four wells, diethylstilbestrol was added to a concentration of 10 µM. The cells were then incubated at 37 °C for
approximately 1 h. The medium was removed, the cells were
washed three times with 0.5 ml of ice-cold PBS and then extracted twice
with ethanol. The ethanol extractions were than diluted in
scintillation fluid and counted for activity. Specific binding to the
receptor was calculated by subtracting nonspecific binding (measured
from the cells treated with diethylstilbestrol) from total
binding (measured from the cells not treated with diethylstilbestrol). This assay has been repeated with each construct at least five times,
and the specific binding has differed less than 15% between experiments.
A normalization factor for each transfection was then determined by
dividing the amount of specific ligand binding for each transfect by
the specific ligand binding of a transfection standard performed with
every set of transfections using 5 µg of HE0 expression plasmid. The
relative light unit values and errors determined from the
luminescence experiments performed on each transfection were then
divided by the normalization factor to give the final, normalized
luminescence data.
The N-terminal Domain Was Not Required at ERE-tk Promoter in HeLa
Cells--
Deletional and chimeric mutants of ER
Chimeras and deletions of the N-terminal domain of both subtypes were
constructed (Fig. 1a) and
tested in a transient transfection reporter gene assay with a reporter
construct consisting of a luciferase gene driven by a classical ERE
(5). To account for potential differences in the expression levels of
each chimeric receptor, a whole cell estradiol binding assay was also
performed with every transfection (16). The estradiol binding data were used to normalize the luciferase reporter activity. This normalization corrects for differences in transfection efficiency and also serves as
a control to assay ligand binding activity of the various mutant receptors used in the study. All of the chimeric and deletion mutants
showed less than a 2-fold variation in the amount of specific tritiated
estradiol binding except for the ER
In HeLa cells, swapping or deleting the N-terminal domain causes some
changes in the magnitude of activation, but all of the mutants were
still capable of activating transcription in response to estradiol at
an ERE-tk promoter in HeLa cells (Fig.
2). Truncations in amino acids 21-96 of
the N-terminal domain of ER The C-terminal Tail Was Not Required at an ERE Site in HeLa
Cells--
The other region of the receptor that shows great
differences between ER Differences between ER
The F region chimeras and deletion mutants were also tested at an AP-1
site. As was seen with the N-terminal domain mutants, ER It has been previously shown that ER Of the three structural domains, the function of the N-terminal domain
is the least understood, but is generally believed to play a role in
transactivation and repression (19-26), The activation region known as
activation function 1 (AF-1) resides in the N-terminal domain of ER.
The AF-1 region has been shown to contribute to ligand-independent
activation and to synergistic enhancements of
ligand-dependent activation with activation function 2 (AF-2), located in the ligand binding domain. Both the constitutive and synergistic effects attributed to the AF-1 region are highly dependent on cell context (20, 23, 26). While ER Comparisons of the role of the N-terminal domain of the two ER subtypes
on subtype-selective responses have shown that the N-terminal domains
of both ER Subtype-selective responses are also seen with the AP-1 promoter, but
the domain requirements for these selective responses are more
complicated than with any subtype-selective response reported so far.
Estradiol causes transcriptional activation at an AP-1 site with ER There is also a subtype-selective response to raloxifene at an AP-1
site; ER In an attempt to identify the specific residues in the N-terminal
domain responsible for the raloxifene activation with ER Deletion mutants were also used to investigate the suppression of
raloxifene activation at the AP-1 site by the N-terminal domain of
ER Both ER The exact function of the F region is not known. Although it has been
shown to be unnecessary for transcriptional activation (29, 30) or
receptor half-life regulation (31), it has been shown to be important
in modulating the estrogen and anti-estrogen response in some cell
types by modulating both AF-1 and AF-2 (32). In addition, experiments
with ER fusion proteins suggest that the F region possess different
structural orientations depending on whether an agonist or an
antagonist binds the receptor (33).
The ER From these chimera and deletion mutant studies, it is clear that the
two subtypes of the estrogen receptor use different mechanisms to
respond to different ligands at an AP-1 site. Such a marked signaling
difference between two subtypes of a receptor suggests that the
receptors may be designed by nature to have different roles in
signaling from AP-1 sites. In contrast to other subtype-selective responses (6, 10, 11, 28), differences in the responses of ER In the case of the F region, it is known that the receptor undergoes a
major structural change upon ligand binding that results in the
reorganization of helix 12 in the LBD (34-36). Because the F region is
attached to helix 12, it is likely that the F region is susceptible to
structural perturbations that could put it in contact with other
regions of the receptor. Disturbing this interaction apparently
disrupts activation by ER Differences in the mechanism of ER (hER
) and
(hER
), regulate transcription at an AP-1 response
element differently in response to estradiol and the anti-estrogens
tamoxifen and raloxifene. To better understand the protein determinants
of these differences, chimeric and deletional mutants of the N-terminal domain and the F region of ER
and ER
were made and tested in transient transfection assays at the classical estrogen response element (ERE) site as well as at an AP-1 site. Although the same regions on each receptor subtype appeared to be primarily responsible for estradiol activation at an ERE and in HeLa cells, major differences between ER
and ER
mutants were seen in the estrogen and
anti-estrogen responses at an AP-1 site. This differential ligand
response maps to the N-terminal domain and the F region. These results
suggest that different estrogenic and anti-estrogenic ligands use
different mechanisms of activation and inhibition at the AP-1 site. In
contrast to previous studies, this work also shows that many of
subtype-specific responses are not transferred to the other subtype by
swapping the N-terminal domain of the receptor. This implies that there are other unique surfaces presented by each subtype outside of the
N-terminal domain, and these surfaces can play a role in
subtype-selective signaling. Together, these data suggest a complex
interface between ligand, response element, and receptor that underlies
ligand activation in estrogen signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
ER
)1 are members of a
large family of nuclear receptors that activate or repress the
transcription of hormone-regulated genes upon binding to a ligand (1).
One feature of the nuclear receptor family is that a receptor can both
activate and repress different sets of genes in response to the same
ligand, but the mechanisms behind these differential effects are still
not well understood. Estrogen receptor is unusual among the nuclear
receptors, because its differential regulatory effects manifest
themselves as tissue-specific responses to a given ligand (2). For
example, tamoxifen functions as an anti-estrogen in breast tissue, but
acts as an estrogen in the uterus and bone. Controlling these
tissue-specific effects is the ultimate goal in the design and study of
selective estrogen receptor modulators for the treatment of
diseases such as breast cancer and osteoporosis (3).
and ER
(7). It has since been shown that the response to both estrogens and
anti-estrogens at an AP-1 site depends on the subtype of the receptor
(8); estradiol elicits transcriptional activation with ER
, but
transcriptional repression with ER
. The two ER subtypes also respond
differently to the selective estrogen receptor modulator raloxifene at
an AP-1 site; ER
shows much stronger activation in response to
raloxifene than ER
. Subtype-selective activities have also been seen
at other classical and nonclassical estrogen response elements (6,
9-14). In an effort to understand the mechanism of estrogen receptor
action at the AP-1 site and nuclear receptor action at nonclassical
response elements in general, this study describes efforts to identify
regions in ER
and ER
that can account for the differential ligand
activation at an AP-1 site. We report that different polypeptide
regions on ER
and ER
in both the N- and C-terminal regions are
important for the differential ligand response.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(HE0) and hER
as well as the AP-1-regulated luciferase
construct (Coll73-luc) have been described previously (5, 8).
The ERE-driven luciferase reporter gene consists of two repeats of the
upstream region of the vitellogenin ERE promoter from
331 to
289,
followed by region
109 to +45 of the thymidine kinase upstream region
and the luciferase gene. The chimeric ER constructs were made with
overlap extension PCR and inserted into SG-5 expression vectors. While
a point mutant of ER
that has a lower hormone-independent response
was used in these studies (HE0), constructs were also made using
wild-type ER
(HEG0), and no significant difference in the activation
profiles was seen (data not shown). The N-terminal chimera
,
replacing amino acids 1-178 of the ER point mutant HE0 with amino
acids 1-96 of hER
, was made by amplifying a fragment of
corresponding to amino acids 179-476 of HE0 using primers with the
sequences 5'-AGTCAGTGAGCGAGGAAGCG (this primer will hereby be known as
primer RW1) and 5'-CTATGCTTCAGGATATCATTATGGAGTC. The fragment was then
digested and inserted into the EcoRV and BamHI sites in the pSG5-hER
expression vector.
, replacing amino acids 1-96 of hER
with amino
acids 1-178 of HE0, was made by PCR overlap extension. A
SacII site in the N-terminal domain of HE0 was eliminated by
first amplifying one fragment with primers with the sequence
5'-GATCCCGCGGATGACCATGACCCTCCACACC (primer RW2) and
5'-CGCGTTGGCGGCGGCCGCCGCGTTGAACTCGTAG and the other fragment with
primers with the sequence 5'-CTACGAGTTCAACGCGGCGGCCGCCGCCAACGCG and 5'-GATCGATATCCTGAAGCATAGTCATTGCACAC (primer RW3). The overlap extension was then performed using primers RW2 and RW3 and then digesting and inserting the resulting fragment into the
SacII and EcoRV sites of the pSG5-hERb expression
vector. The
chimera deletion mutant
(
129-178)
,
which deletes amino acids 130-177 in the N-terminal domain of
, was made using the same procedure but using the plasmid
129-178 (15) as the PCR template. The
chimera deletion
mutants
(
109)
and
(
117)
, which deletes amino
acids 2-108 and amino acids 2-116, respectively, were made by
using primer RW2 and RW3 with the templates n109 and n117, respectively
(15), and inserting the fragments into the SacII and
EcoRV sites in the pSG5 hER
expression vector.
, which deletes amino
acids 2-96 of hER
, a PCR fragment of ER
was generated using the
primer RW1 and a primer with the sequence
5'-AGGGATCCGCGGATGTGCGCTGTCTGCAGCG. The PCR product was then digested
and inserted into the SacII and BamHI sites of
the pSG5-hER
construct. Five other N-terminal ER
deletion
mutants,
9
,
17
,
21
,
28
, and
53
, were made in the same fashion with primer RW1 and the
primer containing the appropriate truncation.
F
, swapping amino acids 451-477 of hER
with amino acids 553-595 of HE0, was made by PCR overlap extension. One fragment was amplified from HE0 using the RW1 primer and a primer
with the sequence 5'-AGCCGTGGAGGGGCAT. The other fragment was amplified
from ER
using primers with the sequence 5'-AAGAGCTGCCAGGCCTGCCG (primer RW4) and 5'-ATGCCCCTCCACGGCTCTTGCACCCGCGAAG. The overlap extension was then performed using primers RW1 and RW4 and then digesting and inserting the resulting fragment into the SacI
and BamHI sites of the pSG5-hER
expression vector.
F
, swapping amino acids 553-595 of HE0 for
amino acids 451-477 of hER
, was made by first inserting a silent
mutation to insert a SpeI restriction site into HE0 via Quikchange mutagenesis (Stratagene, La Jolla, CA) using primers with the sequence 5'-GCGCCCACTAGTCGTGGAG and 5'-CTCCACGACTAGTGGGCGC. A
PCR fragment was then amplified from ER
using the RW1 primer and a
primer with the sequence 5'-GAACTAGTTCCATCACGGGGTC. The fragment was
then digested and inserted into the BamHI site and the newly
generated SpeI site of HE0.
F, deleting amino acids 553-595 of HE0, was
made by PCR amplification of HE0 using primer RW4 and a primer with the
sequence 5'-ATGGGATCCTCAAGTGGGCGCATGTAGGC. The fragment was digested
and inserted into the HindIII and BamHI sites of
the HE0 expression vector. The other deletion mutant,
F, deleting
amino acids 451-477 of hER
, was made by PCR amplification of ER
with primer RW4 and a primer with the sequence
5'-ATGGGATCCTCACTTGCACCCGCGAAG. The fragment was then digested and
inserted into the SacI and BamHI sites of the
pSG5-hER
expression vector.
and
chimera, which required only 1 µg
expression plasmid per transfection. The electroporation buffer
consisted of 0.2 µm filtered PBS, 0.1% glucose, and 0.001%
Biobrene detergent. Cells were transfected by electroporation at
a potential of 0.25 kV and a capacitance of 960 microfarads. The
transfected cells were immediately resuspended in growth medium
supplemented as described above with the exception that the newborn
calf serum had been treated with charcoal. Cells were plated into
six-well dishes at 2 ml/well at a density ~1 × 105
cells/well. After 2 h of incubation at 37 °C, hormones were
added in 2 µl of ethanol (8).
80 °C, thawed,
and scraped with a rubber policeman to loosen the cell fragments. The
lysate was centrifuged for 5 min, and 0.1 ml of the supernatant was
combined with 0.3 ml of the luciferase assay buffer consisting of 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 15 mM potassium phosphate (pH 7.8)
with the addition to a final concentration of 1 mM
dithiothreitol, 2 mM ATP, and 0.2 mM
luciferin. Luminescence was measured for 10 s with a
Monolight 3010 luminometer (Analytical Luminescence Laboratory,
San Diego, CA). Each hormone dose was performed in triplicate, and the
relative error was determined by calculating the S.E. of the mean. Each
construct was tested at least five times, and no significant
differences in the results were observed between experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ER
were
made to determine the regions of the ER responsible for the different ligand effects at an AP-1 site. Polypeptide regions that had the highest sequence variability between the two subtypes were chosen. The
estrogen receptor, like all nuclear receptors, has three structural domains, an N-terminal domain, a DNA binding domain (DBD) and a ligand
binding domain (LBD). Of these three domains, only the N-terminal
domain shows significant differences between ER
and ER
in both
sequence identity and length. There have been reports of longer forms
of ER
that extend the N terminus of the receptor (17). These longer
forms of ER
also possess very low sequence similarity with ER
in
the N-terminal domain and show no difference in activity at the AP-1
site (15).
construct containing the
N-terminal domain of ER
(
), which showed 8-fold increased binding activity (Table I). All the
mutant receptors also showed maximal ligand activation in the reporter
gene assay at the same receptor expression plasmid concentration except
for the
and
chimeric receptors, which had an
optimal concentration 5-fold lower than the others.
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Fig. 1.
Schematic drawing of chimera and deletion
mutants of the N-terminal domain (a) and F-region
(b) that were used in this study.
Relative estradiol binding ability of estrogen receptor mutants
. Differences in relative binding differ
less than 15% between multiple transfections.
or in different regions in the
N-terminal domain of the
chimera also had no effect on
estradiol activation at an ERE-driven promoter (data not shown). As
expected, little or no activation is seen with tamoxifen or raloxifene
at any of the full-length or mutant receptors. The lack of tamoxifen
activation at this ERE in HeLa cells is consistent with other reports
(5, 18-20).
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Fig. 2.
Transient transfection assay with N-terminal
deletion and chimera mutant receptors and the vitellogenin A2 ERE-tk
driven luciferase reporter gene. All ligand doses were 1 µM. Relative light units were normalized for each
construct using estradiol binding capacity as described under
"Experimental Procedures."
and ER
is the C-terminal tail (also known
as the F region). The F regions of ER
and ER
share relatively low
sequence identity (23%) compared with the rest of the LBD and DBD. In
addition, ER
also has a longer F region than ER
(42 residues for
ER
versus 26 for ER
). Chimeras and deletion mutants
were also made for the two subtypes (Fig. 1b) and tested in
the same transient transfection assays described above. Although there
are some changes in the overall magnitude of the activation (Fig.
3), each of the mutants behaved like
full-length ER
and ER
in their response to ligands: estradiol
activated transcription, whereas the anti-estrogens raloxifene and
tamoxifen did not. A higher level of hormone-independent activation was
also seen with the F deletion mutants, but the underlying cause for
this increase is still unknown.
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Fig. 3.
Transient transfection assay with C-terminal
tail deletion and chimera mutant receptors and the vitellogenin A2
ERE-tk-driven luciferase reporter gene. All ligand doses were 1 µM. Relative light units were normalized for each
construct using estradiol binding capacity as described under
"Experimental Procedures."
and ER
at an AP-1 Site--
When the
chimera and deletion mutants were tested at an AP-1 site, significant
differences in ligand activation were seen. Deleting the N-terminal
domain of ER
(
) or replacing it with the N-terminal domain
of ER
(
) resulted in receptor that showed no activation by
estradiol, a weak activation by tamoxifen, and a much stronger
activation response to raloxifene than that of full-length ER
(Fig.
4). On the other hand, deletion of the
N-terminal domain in ER
(
) abolished all ligand activation
at an AP-1 site. Deleting the first 53, 28, or 21 amino acids in the
N-terminal domain of ER
abolished all ligand activation at the AP-1
site as well (Fig. 5). The chimera of
ER
with the N-terminal domain of ER
(
) showed
transcriptional activation in response to only tamoxifen at a level
3-fold higher than the hormone-independent activation but showed no
activation by raloxifene compared with the 10-fold activation seen with
full-length ER
. Deleting either the first 109 or 117 amino acids of
eliminated any tamoxifen activation but resulted in a
2.5-fold activation by raloxifene (Fig.
6). An internal deletion between amino
acids 129 and 178 in the N-terminal domain of
also showed no
activation by tamoxifen and a slight activation by raloxifene.
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Fig. 4.
Transient transfection assay with N-terminal
deletion and chimera mutant receptors and the AP-1-driven luciferase
reporter gene. All ligand doses were 1 µM. Relative
light units were normalized for each construct using estradiol binding
capacity as described under "Experimental Procedures."
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Fig. 5.
Transient transfection assay with N-terminal
deletion mutant receptors of ER and the
AP-1-driven luciferase reporter gene. The numbered scheme refers
to the form of ER
with 96 amino acids in the N-terminal domain. All
ligand doses were 1 µM. Relative light units were
normalized for each construct using estradiol binding capacity as
described under "Experimental Procedures."
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Fig. 6.
Transient transfection assay with N-terminal
deletion receptors of the
chimera and the AP-1-driven luciferase reporter gene. All
ligand doses were 1 µM. Relative light units were
normalized for each construct using estradiol binding capacity as
described under "Experimental Procedures."
and ER
appear to function differently (Fig. 7).
Deleting the F region of ER
(
F) or replacing it with the F
region of ER
(
F
) does not affect the ligand response of the
receptor. However, removing the F region of ER
(
F) or
replacing the F region with that from ER
(
F
) results in
receptors that show little or no activity in response to estradiol at
an AP-1 site but still allows a significant tamoxifen activation. The
deletion mutant (
F) also shows significant activation by
raloxifene in contrast to the full-length ER
receptor.
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Fig. 7.
Transient transfection assay with C-terminal
tail deletion and chimera mutant receptors and the AP-1-driven
luciferase reporter gene. All ligand doses were 1 µM. Relative light units were normalized for each
construct using estradiol binding capacity as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ER
have different
ligand activation properties at an AP-1 site (8). Both estrogens and
anti-estrogens stimulate transcriptional activation with ER
whereas
with ER
, anti-estrogens promote transcriptional activation, while
estrogens promote repression. The aim of this study was to define the
elements of each ER that are responsible for the differential responses
at AP-1. We focused on the N-terminal (A/B) domain and the C-terminal F
region of ER
and ER
, since these regions are the most dissimilar
in terms of length and sequence between the two ERs.
possesses this AF-1 region
between amino acid 41 and amino acids 120-150 depending on the cell
type, the equivalent AF-1 region in ER
is either extremely weak or
absent entirely (27, 28).
and ER
are important for signaling depending on the
response element and cell type. Tamoxifen activates transcription in an
ER
-selective manner at certain ERE sites in a number of cell types
(though not in HeLa cells, which was also confirmed here) (14, 20,
22-24, 26). The importance of the N-terminal domain in this response
has been demonstrated by the elimination of the tamoxifen activation
through deletion of the N-terminal domain. The tamoxifen activation
could also be transferred to ER
by making a chimera of ER
that
contained the N-terminal domain of ER
(11). The ER
selective
response to estradiol at a Sp-1 site was also found to be sensitive to deletion of the N-terminal domain of ER
and the activation could be
transferable to ER
by swapping the N-terminal domain (6, 10). An
ER
-selective response to anti-estrogens was also seen with the
RAR
-1 promoter and this response could be significantly reduced by
deleting the N-terminal domain of ER
. In addition, the anti-estrogen
activation of the RAR
1 promoter could also be
transferred to ER
by swapping the N-terminal domain (10).
and transcriptional repression with ER
. This estradiol activation by
ER
requires the presence of the N-terminal domain as demonstrated by
the absence of activation in the N-terminal deletion mutant
(
). This would correlate with a previous report that suggests
the estradiol activation at AP-1 is dependent on AF-1 (15). The
chimeras which swapped the N-terminal domains of ER
and ER
(
and
) showed no activation to estradiol. The loss
of estradiol activation with the
construct could be
rationalized by the absence of a strong AF-1 in the new chimeric
receptor, but the absence of estradiol activation with
was
not expected. The
chimera does contain the AF-1 region from
ER
, yet still does not activate transcription in the presence of
estradiol. This suggests that there is a fundamental difference between
ER
and ER
ligand activation at an AP-1 site that involves protein
determinants other than or in addition to the N-terminal domain.
is activated much more strongly than ER
in response to
raloxifene. Previous work has suggested that the AF-1 in the N-terminal
domain of ER
suppresses the raloxifene activation at the AP-1 site
and that the raloxifene activity requires only the DNA binding domain
and ligand binding domain (15). Deletion of the N-terminal domain of
ER
converts a weak raloxifene activation into a very strong
activation, the ER
chimera with the N-terminal domain of ER
(
) also shows strong activation to raloxifene and the ER
chimera with the N-terminal domain of ER
(
) has a
significantly lowered raloxifene activation. However, raloxifene activation at an AP-1 site by ER
is eliminated when the N-terminal domain is deleted (
). This indicates that raloxifene
activation of ER
at AP-1 requires a unique activation function
located in the N-terminal domain.
and
raloxifene suppression with ER
, deletion mutants of ER
and
were constructed. Deletion of the first 21, 25 or 53 amino acids of the N-terminal domain of ER
had no significant effect on
estradiol activation at an ERE site, but it resulted in abolishment of
activation by all ligands at an AP-1 driven promoter. This indicates
that an activation region resides in the first 21 amino acids of the
N-terminal domain. Attempts to precisely define this activation region
were unsuccessful as deletions within the first 21 N-terminal amino
acids led to transcriptionally inactive mutants which were also unable
to bind estradiol.
. In a previous report, deleting the N-terminal 109 amino acids of
ER
, cutting into the middle of AF-1, caused the receptor to activate
transcription in response to raloxifene at an AP-1 site (15). This is
also seen here with the
(
109)
mutant, suggesting that the
AF-1 region of ER
can suppress raloxifene activation by ER
as
well. Deleting between amino acids 129 and 178 in
, which
corresponds in ER
to a flanking region outside of AF-1 known as
iAF-1B (15), showed reduced ligand activation as well, further
emphasizing the importance of the AF-1 region in raloxifene repression.
and ER
activate transcription in response to tamoxifen at
the AP-1 site in HeLa cells, which is also seen with the regulation of
the human quinone reductase gene (12), but the work reported here
suggests the two subtypes activate transcription by different
mechanisms. Previous work indicated that the tamoxifen activation of
ER
at an AP-1 site was slightly repressed by AF-1 but required a
region in the N-terminal domain outside of the AF-1 region for full
activation (15). The relatively small tamoxifen activation with the
N-terminal domain deletion mutant of ER
(
) seen here is
consistent with this hypothesis. Also consistent with this hypothesis
is the tamoxifen activation seen with the ER
chimera containing the
N-terminal domain of ER
(
). The
chimera is
particularly interesting compared with ER
and ER
because it is
only activated by tamoxifen and not by estradiol or raloxifene. This is
the first evidence for a mechanism for tamoxifen activation at an AP-1
site that is different from activation by estradiol. The loss of
tamoxifen activation by the N-terminal deletion mutant of ER
(
) suggests that a similar activation function in the
N-terminal domain of ER
exists that is necessary for tamoxifen
activation. This activation function in the N-terminal domain of ER
is not interchangeable with the N-terminal activation function in ER
as evidenced by the lack of tamoxifen activation by the
chimera. This is further emphasized by the
deletion mutants.
Deleting the first 109 amino acids in ER
removes over half the AF-1
region, but the receptor still shows tamoxifen activation at an AP-1
site (15). In contrast, the
(
109)
mutant shows no tamoxifen
activation at AP-1. This implies that there are mechanistic differences
in the response to tamoxifen between ER
and ER
at AP-1 sites.
-selective estradiol activation depends on the presence of the
F region as demonstrated by the diminished estradiol activation in the
F deletion mutant relative to the tamoxifen activation. The ER
chimera that contained the F region from ER
(
F
) also showed
lowered estradiol activation. Consistent with previous studies on
classical EREs (32), the drop in estradiol activity with the
F
construct can be explained by a decrease in the AF-1 and AF-2 activity
of the receptor caused by the deletion of the F-region. However, the
loss of activity with
F
was not expected. The
F
construct
strongly activates transcription in the presence of estradiol at an
ERE-tk site, demonstrating that this mutant is functional in terms of
ligand binding and transactivation from a classical ERE. It is unclear
whether the F-region of ER
contains a specific region necessary for
the estradiol activation at an AP-1 site or if the AP-1 response is
simply more sensitive than an ERE response to F-region attenuation in
the AF-1 and AF-2 activity. As was seen with the
chimera,
tamoxifen activation by the
F and
F
mutants is independent
of estradiol activation, further emphasizing a difference between the
mechanisms of tamoxifen and estradiol activation by ER
at the AP-1
site. At the AP-1 site, the F-regions of ER
and ER
do not appear
to have unique roles in ER
-selective raloxifene activation or
tamoxifen activation by either receptor, since deleting the F-region of
either ER
or ER
has no effect on tamoxifen activation.
and
ER
can not be explained solely by the presence of an AF-1 region in
ER
that is absent in ER
because swapping N-terminal domains does
not entirely swap the ligand response profile. It appears that there is
a unique region in the N-terminal domain of ER
that is necessary for
activation at an AP-1 site by anti-estrogens and a region in another
part of the ER
protein that is responsible for preventing activation
by estrogens at an AP-1 site.
in response to estrogens at both an ERE
and an AP-1 site. The chemical extensions of anti-estrogens such as
tamoxifen and raloxifene disrupt the reorganization of helix 12 and
cause it to interact with a different part of the ligand binding domain
(1, 35, 36). In this alternate conformation, changing the F-region by
mutation or deletion apparently does disrupt activation by tamoxifen at
an ERE-tk site but does not affect activation by anti-estrogens at an
AP-1 site.
and ER
activity and
differences in the response of ER
to estrogens and anti-estrogens have also been suggested by peptide blocking studies using phage display (37, 38). Peptides were identified that could specifically block estradiol activation at an ERE site by one ER subtype and not the
other. Peptides that could specifically block ER
activation by
estradiol or by tamoxifen were also reported. The peptides that were
specific for blocking either estradiol or tamoxifen activation by ER
at an ERE site also blocked activation at an AP-1 site. Interestingly
all of those peptides bound to regions in the ligand binding domain of
the receptor. Perhaps some of these selective peptides are binding to
surfaces on the LBD that communicate with other regions of the
receptor, for example the N-terminal domain. Although direct
protein-protein interactions between the N-terminal domain and the LBD
have not been detected using two hybrid
systems,2 unique
surfaces on the LBD of each subtype could interact indirectly through
accessory proteins with surfaces on the N-terminal domain that are
unique to each subtype. Regardless of mechanisms, this suggests a
subtle and complex program of transcriptional regulation by the
estrogen receptors.
![]() |
ACKNOWLEDGEMENTS |
---|
Expression vectors HE0, hER, ER
,
129-178, n109, n117, and the ERE and AP-1-driven luciferase
reporter genes were gifts from Paul Webb and Peter Kushner. Kolja Paech
synthesized raloxifene.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant DK 57574 from the National Institutes of Health.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.
Supported by a postdoctoral fellowship from the American Cancer
Society, California Division.
§ To whom correspondence should be addressed: Dept. of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446. E-mail: scanlan@cgl.ucsf.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M005414200
2 P. Webb and P. Kushner, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ER, estrogen receptor; LBD, ligand binding domain; DBD, DNA binding domain; ERE, estrogen response element; AF-1, activation function 1; AF-2, activation function 2; tk, thymidine kinase; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.
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