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Receptor Chimeras1
Departments of Molecular and Integrative Physiology (E.M.M., K.E.W., J.S., B.S.K.) and Cell and Structural Biology (B.S.K.), University of Illinois, Urbana, Illinois 61801; and the Target Discovery Unit, N.V. Organon (S.M.), Oss, The Netherlands
Address all correspondence and requests for reprints to: Dr. Benita Katzenellenbogen, Department of Molecular and Integrative Physiology, University of Illinois, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801-3704. E-mail: katzenel{at}uiuc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
ERß, and chimeric constructs with ER and ERß to examine the
bioactivities of these receptors and their responses to estrogen and
antiestrogen ligands. Transcriptional activity of ERß is highly
dependent on cell/promoter context and on the nature of the ligand.
ERß activated significant levels of transcription in response to
estrogens in certain cell types, but showed only moderate activity
compared with ER in others. Antiestrogens such as tamoxifen and
2-phenylbenzofuran, which show some agonistic activity with ER,
exhibit no agonistic activity with ERß. Alteration of the
amino-terminal A/B receptor domain can result in a dramatic change in
cell type- and ligand-specific transcriptional activity of ERß. Upon
replacing the A/B domain of ERß with the A/B domain of ER, this
receptor chimera not only exhibits an improved transcriptional response
to estrogens, but also is now able to activate transcription upon
treatment with these antiestrogens. As antiestrogen agonism was lacking
in ERß and the ERß/ chimera containing the amino-terminal A/B
domain of ERß fused to domains C through F of ER, but was restored
in an ER/ß chimera containing the A/B domain of ER,
antiestrogen agonism was shown to depend on the A/B domain (activation
function-1-containing region) of ER. Together, these results
indicate that the differences in the amino-terminal regions of ER
and ERß contribute to the cell- and promoter-specific differences in
transcriptional activity of these receptors, and their ability to
respond to different ligands, thus providing a mechanism for
differentially regulated transcription by these two ERs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transcription of estrogen-responsive genes by ER can be antagonized by antiestrogens, such as trans-hydroxytamoxifen (TOT) and ICI 164,384 (17, 18). Although these antiestrogens promote DNA binding by ER (19, 20), it is thought that antiestrogens such as TOT activate transcription only poorly because they are unable to effectively stimulate AF-2 activity (7, 17). However, antiestrogens such as TOT have been shown to have partial agonistic activity in certain cells, such as chicken embryo fibroblasts, MDA-231 human breast cancer cells, and human endometrial cancer cells (21). In addition, it has been suggested that certain antiestrogens can activate transcription in these cell types, because in them AF-1 acts as a strong transcriptional activator (8, 17, 21). Together, these reports have implied that antiestrogen agonism is AF-1 dependent.
Since the cloning of the ER about 10 yr ago (22, 23), there has been
the general acceptance that only one ER existed. This contrasted with
other members of the nuclear receptor superfamily, for which multiple
forms have been reported (e.g. thyroid receptor and ß
and retinoic acid receptor , ß, and 
) (1, 24). Recently,
however, a novel ER has been cloned and characterized (25, 26, 27, 28); it has
been termed ERß to distinguish it from the previously identified ER,
now called ER. There is currently intense interest in understanding
its role in estrogen action and how its activity compares and contrasts
with that of ER. ERß, which is encoded by a different gene, has an
overlapping, but nonidentical, tissue distribution as ER (27).
ERß, like ER, binds estrogens and antiestrogens and has been shown
to modulate the transcription of estrogen-responsive reporter genes in
Chinese hamster ovary (CHO) cells (25, 26). However, it should be noted
that in this one cell type examined, the transcriptional activity of
ERß was significantly less (
50% of ER) than the activity
observed with ER.
Human ERß is highly homologous to ER. The DNA-binding domain is
highly conserved (96% identity) between ERß and ER, and the
hormone-binding domain is also relatively well conserved (58%
identity) between these two receptors (25). It is of note, however,
that the AF-2 core at the C-terminus of the hormone-binding domain of
these receptors differs by one amino acid. Sequence alignment of ER
and ERß reveals a highly conserved region from amino acids 537–547
in ER and 488–498 in ERß, with the only exception being amino
acid D545 in ER (amino acid N496 in ERß). In addition, the A/B
domains of ERß and ER are poorly conserved (only 20%),
suggesting that their AF-1 activities might be different and possibly
that different coactivators interact with this region.
We were interested in examining the transcriptional activity of ERß
and determining how the activity of ERß is influenced by cell and
promoter context, and we postulated that the different transcriptional
activity of ERß compared with that of ER might be attributable to
the low conservation of the A/B domain. We also examined the importance
of the nonconserved residue within the AF-2 core. In the studies
presented, we demonstrate that the ability of ERß to function as an
estrogen-dependent transcriptional activator is highly dependent on
cell and promoter context. We also found that in certain contexts where
antiestrogens are able to activate ER, ERß displays no
transcriptional response to antiestrogens. However, upon replacing the
A/B domain of ERß with the A/B domain of ER, this chimeric
receptor not only shows a significantly greater transcriptional
response to estrogens, but also exhibits a transcriptional response to
antiestrogens. Our findings suggest that despite great similarity
between the DNA-binding domains of these two receptor subtypes, the
transcriptional responses of ERß to different ligands are distinctly
different and are strongly influenced both by the nature of the
receptor N-terminal A/B domain and by the cellular context.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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monoclonal
antibodies H222 and H226 were provided by Dr. G. Greene (University of
Chicago, Chicago, IL). The antiestrogen TOT was provided by Dr. A.
Wakeling, Zeneca Pharmaceuticals (Macclesfield, UK), and
the 2-phenylbenzofuran (BF) antiestrogen was provided by Dr. E. von
Angerer, University of Regensburg (Regensburg, Germany) (29).
The estrogen P1496 was described previously (21).
Plasmid constructions
The human ER expression vector for ER (pCMV5-hER) was
constructed as previously described (15, 30). The expression vector
pCMV5-ERß was constructed by inserting the full-length complementary
DNA encoding the human ERß (530 residues, pNGV1-ERß), described
previously (25), and including the additional 53 N-terminal amino acids
shown in Fig. 1A
(same as GenBank
accession no. AF051427) into the BamHI site of pCMV5. The
expression vector for the chimeric ER/ß receptor was constructed
to contain the A/B domain of ER and domains C, D, E, and F of ERß.
First, an MluI site was created at residue 145 of
pCMV5-ERß by site-directed mutagenesis (31) to create
pCMV5-ERß-(Mlu145). The MluI fragment of
pCMV5-ERß-(Mlu145) was replaced by a PCR-generated insert
of the A/B domain of ER containing an MluI site created
at residue 181 of ER. The ERß(N496D) point mutant was created by
site-directed mutagenesis by replacing nucleotides AAT, representing
amino acid 496 of pCMV5-ERß, with nucleotides GAT. The expression
vector for the chimeric receptor ERß/ was constructed to contain
the A/B domain of ERß and domains C, D, E, and F of ER. Using
site-directed mutagenesis, we incorporated an EcoRV site at
amino acid 195 of ER. Because of high sequence homology between
ER and ERß, there is an EcoRV site in ERß at amino
acid 158 that is in the equivalent location and reading frame as the
EcoRV site created in ER. Consequently, the A/B domain of
ER was excised by EcoRV digestion and replaced by the
EcoRV fragment containing the A/B domain of ERß. The
sequences of all mutants were confirmed by dideoxy sequencing methods
to assure accuracy.
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/ß: forward oligo, 5'-CG CGT CCA CGG ACC ATG GAC TAC AAG
GAC GAC GAT GAC AAG TCT AGA AT-3'; and reverse oligo, 5'-C GAT TCT AGA
CTT GTC ATC GTC GTC CTT GTA GTC CAT GGT CCG TGG A-3'. The Flag
epitope-tagged receptors contain 29 additional amino acids at the
amino-terminus of ER/ß and 28 additional amino acids at the
amino-terminus of ERß. The estrogen response element (ERE)-containing reporter plasmids were (ERE)3-pS2-chloramphenicol acetyltransferase (CAT), constructed as described previously (15), and (ERE)2-TATA-CAT (32), provided by D. J. Shapiro of the University of Illinois (Urbana, IL). The plasmid pCH110 (Pharmacia Biotech, Piscataway, NJ) or pCMVß (Clontech, Palo Alto, CA), which contains the ß-galactosidase gene, or pSV40-luciferase was used as an internal control for transfection efficiency.
Cell culture and transient transfections
ER-negative CHO cells were maintained in phenol red-free
DMEM-Ham’s F-12 tissue culture medium supplemented with 5% charcoal
dextran-treated calf serum (CDCS), 100 U/ml penicillin, and 100 µg/ml
streptomycin. Cells were plated at 1.8 x 105
cells/60-mm plate, maintained in phenol red-free DMEM-Ham’s F-12
medium with 5% CDCS, and given fresh medium 5 h before
transfection. Cells were transiently transfected by the
CaPO4 coprecipitation method (33), and all cells for
transfection were maintained at 37 C in a humidified CO2
atmosphere. Cells were given 400 µl precipitate containing 2 µg
(ERE)2-TATA-CAT reporter plasmid, 0.3 µg pCH110 internal
control ß-galactosidase plasmid, 6 µg pTZ19R carrier DNA, and 10 ng
ER expression vector, or 400 µl precipitate containing 0.5 µg
(ERE)3-pS2-CAT reporter plasmid, 0.5 µg pSV40-LUC
internal control plasmid, 10 ng ER expression plasmid, and 7 µg
pTZ19R carrier DNA, as indicated. After 12–16 h, cells were shocked
with 20% glycerol-HBSS for 1.5 min and rinsed with HBSS. Cells were
then given fresh medium and hormone treatment as indicated.
Human endometrial cancer (HEC-1) cells were maintained in MEM plus phenol red (21) supplemented with 5% calf serum and 5% FCS. MDA-MB-231 human breast cancer (231) cells were maintained in Leibovitz’s L-15 medium (21) supplemented with 5% calf serum. MDA-231 cells or HEC-1 cells were grown in MEM plus phenol red supplemented with 5% CDCS for 2 days before transfection. Cells were plated at a density of 3 x 106 cells/100-mm dish in phenol red-free improved MEM and 5% CDCS and were given fresh medium 24 h before transfection. One milliliter of precipitate contained 0.8 µg pCMVß as an internal control, 5 µg of an ERE-containing reporter plasmid (ERE)3-pS2-CAT, 200 ng ER expression vector, and pTZ19R carrier DNA to a total of 15 µg DNA. Cells remained in contact with the precipitate for 4 h and were then subjected to a 2.5-min glycerol shock (20% in transfection medium). Cells were rinsed with HBSS and given fresh medium with hormone treatment as indicated.
After transfection, cells were harvested 24 h after glycerol shock and hormone treatment, and extracts were prepared in 200 µl 250 mM Tris, pH 7.5, using three freeze-thaw cycles. ß-Galactosidase activity was measured to normalize for transfection efficiency in all experiments, and CAT assays were performed as previously described (34).
Immunoblots
Protein expression for the receptors and receptor chimeras and
for Flag epitope-tagged receptors and receptor chimeras was monitored
in CHO, 231, and HEC-1 cells after transfection with each
cytomegalovirus promoter-based expression plasmid. Cells were
transfected in 100-mm dishes with 10 µg ER expression plasmid and 5
µg pTZ19R using either the calcium phosphate method (30, 35, 36) or
the transferrin-lipofectin method (37). The transferrin-lipofectin
method gave increased transfection efficiency, but similar findings
were observed using both methods. For the transferrin-lipofectin
method, we used 250 µg transferrin (Sigma) in 1 ml HBSS complexed
with 45 µg lipofectin reagent (Life Technologies). After
15-min incubation, 15 µg DNA were added to the mixture and incubated
for an additional 15 min before dispensing to the 100-mm plate of
cells. Whole cell extracts were prepared, and immunoblots were
performed as previously described (35, 36), using ER monoclonal
antibody H222 or H226 (2 µg/ml) or anti-Flag M2 monoclonal antibody
(4 µg/ml; IBI).
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Results |
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, and ER chimeras in CHO
cells. In addition,
although the DNA-binding and hormone-binding domains show considerable
homology between ER and ERß, the A/B domains of these two
receptors are poorly conserved (Fig. 1A
). To determine whether the
reduction in transcriptional activity of ERß (entry 2, Fig. 1B)
compared with that of ER (entry 1, Fig. 1B) was due to the
differences in the amino-terminal A/B domain, we replaced the A/B
domain of ERß with the A/B domain of ER to generate a chimeric
receptor ER/ß (entry 4, Fig. 1B). We also created an ERß point
mutant, ERß(N496D), containing an N to D amino acid substitution at
residue 496 to determine whether the differences in transcriptional
activity of ER compared with ERß could be partially attributed to
this nonconserved residue in the AF-2 activation helix (entry 3, Fig. 1B). We also made a chimeric receptor, ERß/, containing the
amino-terminal A and B domains of ERß and the carboxyl-terminal
domains C, D, E, and F of ER (entry 5, Fig. 1B). The functional
domains (A/B, C, D, E, and F) and activation functions (AF-1 and AF-2)
of ER are shown at the top of Fig. 1B along with
schematics for ERß and the receptors we constructed and tested.
We first examined the E2-dependent transcriptional activity
of ER and ERß over a range of expression vector amounts in CHO
cells. Maximal transcriptional activity for both ER and ERß was
obtained in response to E2 within the range of 1.5–15 ng
expression vector (Fig. 2). In these
cells, the trans-activation by ERß was only about half of
that achieved by ER regardless of the amount of expression vector
employed. In human endometrial cancer (HEC-1) cells and MDA-231 human
breast cancer (231) cells, 50–500 ng expression vector resulted in
maximal E2-stimulated activity for ER, ERß, and the
chimeric receptors (data not shown). Therefore, for our studies to be
within this range of maximal trans-activation for the
receptors, we used 10 ng ER expression vectors for studies in CHO cells
and 200 ng for studies in HEC-1 and MDA-231 cells.
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, we examined the
transcriptional responses of ER, ERß, and the chimeric ER/ß
or ERß/ to increasing concentrations of E2. In the CHO
cells, ER maximal activity was stimulated approximately 10-fold at
10-8 M E2. ERß activated maximal
transcription that was about 50% of that seen with ER, consistent
with previous reports (25, 26) as well as with the data in Fig. 2.
ERß(N496D) showed no change in transcriptional activity from that
observed with the wild-type ERß in CHO cells or in another cell type
examined [note the point labeled ERß(N496D) in Figs. 3A and 7A],
suggesting that although this is the only single amino acid difference
in the AF-2 region between ER and ERß, it does not play a critical
role in the activity of ERß. In addition, replacing the A/B domain of
ERß with the A/B domain of ER (ER/ß) resulted in a modest,
but significant, increase in E2-dependent activity compared
with that of the wild-type ERß. Interestingly, ERß/ showed
transcriptional activity similar to that of ER.
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). Both ER subtypes activated transcription in response to
P1496 in a dose-dependent manner, reaching maximum levels at
10-8 M. Again, as was the case with
E2, the highest activity of ERß with P1496 was only
approximately 40% the activity of ER. In addition, replacing the
A/B domain of ERß with the ER A/B domain (ER/ß) resulted in a
slight increase in the P1496-dependent activity compared with that of
wild-type ERß. No significant transcriptional activity was observed
for any of these receptors upon treatment with the antiestrogens TOT or
BF in this cell context [with either the (ERE)2-TATA-CAT
or (ERE)3-pS2-CAT promoter reporter gene constructs; data
not shown]. Together, these results demonstrate that ERß is
transcriptionally active in CHO cells in response to estrogens, albeit
to a lesser extent than ER, and that the presence of the C-terminal
or N-terminal region of ER improves trans-activation
by ERß in this cell context.
We also examined the responses of ER, ERß, and the receptor
chimeras using a different estrogen-responsive promoter construct in
CHO cells (Fig. 4). The general pattern
of activity observed for the receptor constructs was similar to that
seen with the more simple TATA promoter. ERß activity was about 40%
of that seen with ER, and the presence of either the C- or
N-terminal region of ER improved trans-activation by
ERß.
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(21).
We first characterized the transcriptional response to E2
for ER, ERß, and the chimeric receptors ER/ß and ERß/
(Fig. 5).
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evoked maximal levels of transcription at 10-9
M E2 in HEC-1 cells. Although ERß was
transcriptionally active, its E2-dependent activity was
significantly less than that of ER, as observed in CHO cells, and
using greater amounts of ERß expression plasmid did not increase this
activity further (data not shown). ER/ß activity in the presence
of E2 was greater than the activity of the wild-type ERß.
Therefore, the patterns of E2 response of ER, ERß, and
ER/ß in the HEC-1 cells were similar to the pattern of activity
observed in CHO cells. Interestingly, however, ERß/, which showed
very good transcriptional activity in CHO cells, was a poorer
transcriptional activator in HEC-1 cells (Fig. 5).
Of note, ERß was not activated by the antiestrogens TOT or BF,
although ER and the ER/ß chimera showed a substantial response
to these antiestrogen ligands (Fig. 6).
In fact, the ER/ß chimera displayed a relatively strong
transcriptional response to these antiestrogens, which was about
50–60% of the ER response. Therefore, replacement of the A/B
domain of ERß with the A/B domain of ER allows this chimeric
receptor to activate transcription upon antiestrogen binding. That the
activation function-1-mediated agonism of antiestrogens works through
ER activation function-1 is supported by the observations presented
in Fig. 6 that in the ERß/ chimera, the ERß activation
function-1 region (domains A and B) is ineffective in supporting
antiestrogen agonism. These findings highlight the fact that the A/B
domain of ER plays an important role in antiestrogen-dependent
transcription, and that the nature of the A/B domain can determine the
ligand specificity of transcription activation by ERß.
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), ER reached maximal activity at
10-8 M E2; however, ERß appeared
to be almost transcriptionally inactive, even at high
(10-7 M) or very high (1 x
10-6 or 3 x 10-6 M; not
shown) levels of E2. In addition, almost no activity was
observed with ERß when using higher amounts of ERß, up to 1 µg
ERß expression plasmid (data not shown). Likewise, the ERß point
mutant (N496D) and the chimeric ERß/ containing domains A and B
from ERß and domains C–F of ER showed no transcriptional response
to E2. Results similar to those seen with E2
were obtained with the estrogen compound P1496 (Fig. 7B). With P1496,
ER activated a high level of transcription, yet no stimulation of
activity was observed with transfected ERß (even with
10-5 M P1496; not shown).
As the transcriptional activity of AF-1 in the A/B domain of ER is
known to vary in different cell contexts (7, 21), we examined the
effects of replacing the A/B domain of ERß with the A/B domain of
ER. Interestingly, this ER/ß chimera showed a dramatic increase
in transcriptional response to E2 (Fig. 7A) to nearly 60%
of ER activity in the 231 cells. Similarly, with the estrogen P1496,
the chimeric ER/ß activated transcription to approximately 50% of
that observed with ER (Fig. 7B).
Using the same cell type, we examined the transcriptional response of
these receptors to the antiestrogen TOT and the antiestrogen BF. In the
231 cells, these antiestrogens are known to function as partial
agonists, activating transcription by ER to levels approximately
25% of that achieved by ER with E2 (21).
The transcriptional response of ER to the antiestrogen TOT reached
maximal levels at 10-9 M in 231 cells (Fig. 8A). In these cells, where ERß appeared
transcriptionally inactive in response to estrogens, ERß also
exhibited no transcriptional response to the antiestrogen TOT. However,
when the A/B domain of ERß was replaced with the A/B domain of ER,
this chimeric receptor (ER/ß) activated some transcription upon
treatment with TOT, to a level about 20% of that observed with
ER.
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). ER, but not ERß, transfected cells
showed a transcriptional response to BF. The chimeric ER/ß was
able to activate significant levels of transcription upon treatment
with BF (60% of ER). Interestingly, we observed a large
difference in the magnitude of transcriptional activity of ER/ß
with these two antiestrogens, as ER/ß exhibited a significantly
greater response to the antiestrogen BF than to TOT.
Expression of ER, ERß, and ER chimeric proteins in the three
cell types
ER and ERß and the ER/ß and ERß/ chimeric
constructs were expressed in the three cell types (CHO cells, HEC-1
cells, and human breast cancer MDA-231 cells) after transient
transfection. The immunoblot data in Fig. 9 show that proteins of the correct size
were produced in the cells: approximately 66 kDa for ER, 58 kDa for
ERß, and chimeric receptor proteins of about 62 kDa. (Note that the
Flag epitope adds an additional 3 kDa to the epitope-tagged
constructs.) As ER antibodies do not detect ERß (27) and
antibodies are not currently available that detect human ERß, we
detected these proteins in two different ways. Flag epitope-tagged
versions of these proteins were produced and detected with a Flag
monoclonal M2 antibody, and chimeric proteins were detected with
monoclonal antibodies to human ER using either H222 that recognizes
an epitope in the carboxyl-terminal domain E of ER or H226 that
recognizes an epitope in the amino-terminal B domain of ER. These
studies revealed that all receptors were expressed at high and
approximately similar levels (Fig. 9 and data not shown), except in the
case of ERß and ERß/ in MDA-231 cells, where only low levels of
these two receptors were observed. Hence, the very poor transcriptional
response to ERß and ERß/ in these cells may in part be due to
the low level of these proteins in the transfected 231 cells. In
studies not shown, we also compared the transcriptional activity of
ER and ERß with the activity of Flag-ER and Flag-ERß and
found their activities to be very similar, indicating that the epitope
tag did not alter the character of the ER or ERß protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß and their two major
activation functions (AF-1 and AF-2) in transcriptional effectiveness.
Intriguingly, we found that the transcriptional activity of ERß and
that of ERß and ER receptor chimeras were highly dependent on cell
type. In addition, changing the amino-terminal A/B domain of the
receptor can result in a dramatic change in both cell type- and
ligand-specific transcriptional activity. Upon replacing the A/B domain
of ERß with the A/B domain of ER, this receptor not only exhibited
an improved transcriptional response in certain cell types, but was
able to activate transcription upon treatment with tamoxifen and
phenylbenzofuran antiestrogens.
In the cell and promoter contexts studied, ER showed the greatest
transcriptional effectiveness, but the degree to which receptor
activity depended on the type of AF-1 or AF-2 (i.e. an ER
or ERß type), as studied in our chimeras, differed in different cells
and with the different ligands. This is summarized in Table 1. In CHO cells, the activity observed
with E2 with the TATA promoter was determined by the type
of AF-2, being maximal with an ER-type AF-2 and only approximately
half-maximal with ERß-type AF-2. As long as the AF-2 was of the ER
type, either an ER or ERß AF-1 gave maximal activity. The activity
dependence on the AFs seen in CHO cells with the TATA promoter appeared
to be largely maintained in CHO cells with the more complex pS2
promoter. A somewhat different pattern was seen in HEC-1 cells with the
pS2 promoter. Maximal activity with E2 required both an
ER-type AF-1 and AF-2. If either or both AFs were of the ERß type,
activity was reduced.
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(17, 21), it is known that mixed
agonist/antagonist antiestrogens, such as tamoxifen, derive their
agonistic activity principally through the AF-1 of ER. Consistent
with this, we have found the agonistic activity of TOT and BF to have
an absolute requirement for an ER-type AF-1. AF-2 appears to play an
enhancing role, in that activity levels of TOT and BF were twice as
high with both -type AFs than in the /ß chimera containing a
ß-type AF-2. Thus, the activities of different ligand classes show
distinctly different reliance on the AF types.
The AF-1/AF-2 dependence of different ligands in the MDA-231 cell/pS2
promoter context appeared quite similar to that observed in the
HEC-1/pS2 system as far as we could determine in these experiments.
Maximal activity for all ligands was observed with ER-type AF-1 and
AF-2, with the activities of E2, P1496, and BF reduced to
approximately 50% with an ERß-type AF-2. For TOT, the decrease in
agonism with the ERß-type AF-2 was even greater. Interpretation of
the transcriptional effectiveness of ERß and the ß/ chimera, the
two species with a ß-type AF-1, is unfortunately limited by the low
expression levels observed for these proteins in 231 cells. The role of
an ERß-type AF-1 could thus not be unequivocally determined in these
cells. Still, as was the case in HEC-1 cells, we saw in 231 cells that
the agonism of all four compounds with an ER-type AF-1 is enhanced
by an ER-type AF-2.
A biologically important finding was that antiestrogens are not
agonists with human ERß. Similar findings have been reported recently
for mouse ERß (38), suggesting that the inability of antiestrogens to
activate ERß may be general for ERßs of different species. Our
studies further amplify these observations by showing that antiestrogen
agonism via the ER is largely mediated via the A/B region of ER and
is not supported by the AF-1 (A/B domain) of ERß. We previously
identified a distinct region of 24 residues within the A/B domain of
ER that is required for antiestrogen agonism, but not for
transcription stimulated by E2 (21). Interestingly, this
region is not found within the A/B domain of ERß, and ERß exhibits
no transcriptional activity with antiestrogens. Our findings suggest
that the differences in sequence between the amino-terminal domains of
ER and ERß contribute to the cell- and promoter-specific
transcriptional activity of these receptors and their ability to
respond to different ligands, thus providing a mechanism for
differentially regulated transcription by these two ERs.
In contrast to the major effect that the nonconserved N-terminal A/B
domains of the ER and ERß receptors have on the transcriptional
activity, the single, nonconserved amino acid in the AF-2 activation
helix region [ER (D545) vs. ERß(N496)] does not
appear to account for any of these differences. Indeed, it is likely
that other regions in the ER ligand-binding domain contribute to
forming a composite protein surface important for ligand binding and
coregulator interactions (12, 16, 39, 40, 41).
The cell/promoter differences in the activities of our ER constructs
are intriguing. In each of these cell types, we performed plasmid
titration experiments to ensure that our studies used receptor levels
at which we obtained maximal, i.e. plateau,
trans-activation activity levels (as shown in Fig. 2), and
transfection of higher amounts of expression vector failed to increase
transcriptional activity. We therefore believe that we are observing
the intrinsic trans-activation activities of the different
ER subtypes and chimeras. However, in MDA-231 cells, in which the
expression levels of ERß and the ERß/ chimera appeared to be
very low, the lack of response could be due in part to deficient
protein levels as well as to the low intrinsic activity of these
receptors in these cells. However, it is intriguing that we found ERß
RNA levels to be similar in CHO, HEC-1 and MDA-231 cells after
transfection (data not shown), indicating that the ERß expression
plasmid is transcribed well in all of these cells. This aspect merits
further examination.
The cell type dependence of the transcriptional activity of ERß and
ER chimeras may be related to cell-specific differences in
phosphorylation patterns of the different ER subtypes and/or to
differences in the levels of various coregulator proteins. It is known
that the hormone-dependent phosphorylation of ER enhances receptor
activity (42, 43, 44). Significantly, the major sites of phosphorylation of
ER are located in the A/B domain and appear to be critical for full
ER transcriptional activity (43, 44). In view of the differences in
the A/B domain between ER and ERß as well as the different kinase
pathways that are activated in different cell types, it is perhaps not
surprising that ERß exhibits variable transcriptional activity in
different cell backgrounds. There is also now substantial evidence for
multiple coregulators that interact with both the A/B and E domains of
nuclear receptors (3, 16, 39, 45, 46, 47, 48) and promote a transcriptionally
functional association of AF-1 and AF-2 (15), required for high levels
of transcription activation. It will be of interest to determine
whether the cell- and promoter-specific factors required for ERß
function are similar to or distinct from those used by ER. Continued
analysis of the functional domains and coactivator interactions of
these two ERs will be important for a better understanding of ERß
function and how it compares with ER in the diverse target cells
known to be responsive to estrogens.
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Acknowledgments |
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Footnotes |
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Received March 18, 1998.
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and ß. Endocrinology 138:863–870This article has been cited by other articles:
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K. W. Gaido, L. S. Leonard, S. C. Maness, J. M. Hall, D. P. McDonnell, B. Saville, and S. Safe Differential Interaction of the Methoxychlor Metabolite 2,2-Bis-(p-Hydroxyphenyl)-1,1,1-Trichloroethane with Estrogen Receptors {alpha} and {beta} Endocrinology, December 1, 1999; 140(12): 5746 - 5753. [Abstract] [Full Text]
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