Cloning, Chromosomal Localization, and Functional Analysis of the Murine Estrogen Receptor ß
Gilles B. Tremblay1,
André Tremblay1,
Neal G. Copeland,
Debra J. Gilbert,
Nancy A. Jenkins,
Fernand Labrie and
Vincent Giguère
Molecular Oncology Group (G.B.T., A.T., V.G.) Royal Victoria
Hospital Montréal, Québec, Canada H3A 1A1
Departments of Biochemistry, Medicine, and Oncology (V.G.)
McGill University Montréal, Québec, Canada
Mammalian Genetics Laboratory (N.G.C., D.J.G., N.A.J.)
ABL-Basic Research Program National Cancer Institute-Frederick
Cancer Research and Development Center Frederick, Maryland
21702
Laboratory of Molecular Endocrinology (F.L.) Centre
Hospitalier de lUniversité Laval Research Center Ste-Foy,
Québec, Canada G1V 4G2
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ABSTRACT
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Estrogen receptor ß (ERß) is a novel steroid
receptor that is expressed in rat prostate and ovary. We have cloned
the mouse homolog of ERß and mapped the gene, designated
Estrb, to the central region of chromosome 12. The cDNA
encodes a protein of 485 amino acids that shares, respectively, 97%
and 60% identity with the DNA- and ligand-binding domains of mouse (m)
ER
. Mouse ERß binds to an inverted repeat spaced by three
nucleotides in a gel mobility shift assay and transactivates promoters
containing synthetic or natural estrogen response elements in an
estradiol (E2)-dependent manner. Scatchard
analysis indicates that mERß has slightly lower affinity for
E2 [dissociation constant
(Kd) = 0.5 nM] when
compared with mER
(Kd = 0.2
nM). Antiestrogens, including
4-hydroxytamoxifen (OHT), ICI 182,780, and a novel compound, EM-800,
inhibit E2-dependent transactivation
efficiently. However, while OHT displays partial agonistic activity
with ER
on a basal promoter linked to estrogen response elements in
Cos-1 cells, this effect is not observed with mERß. Cotransfection of
mERß and H-RasV12 causes enhanced activation
in the presence of E2. Mutagenesis of a serine
residue (position 60), located within a mitogen-activated protein
kinase consensus phosphorylation site abolishes the stimulatory effect
of Ras, suggesting that the activity of mERß is also regulated by the
mitogen-activated protein kinase pathway. Surprisingly, the coactivator
SRC-1 up-regulates mERß transactivation both in the absence and
presence of E2, and in vitro
interaction between SRC-1 and the ERß ligand-binding domain is
enhanced by E2. Moreover, the
ligand-independent stimulatory effect of SRC-1 on ERß transcriptional
activity is abolished by ICI 182,780, but not by OHT. Our results
demonstrate that while ERß shares many of the functional
characteristics of ER
, the molecular mechanisms regulating the
transcriptional activity of mERß may be distinct from those of ER
.
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INTRODUCTION
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Estrogens regulate female reproductive functions through a nuclear
receptor that belongs to a superfamily of ligand-activated
transcription factors that includes receptors for steroids, thyroid
hormone, retinoids, prostanoids, and vitamin D (1, 2). Like other
members of the superfamily, the estrogen receptor (ER) has a modular
structure consisting of distinct functional domains (3). The
DNA-binding domain (DBD) enables the receptor to bind to its cognate
target site consisting of an inverted repeat of two half-sites with the
consensus motif AGGTCA (or closely related sequences) spaced by 3 bp
and referred to as an estrogen response element (ERE). The
ligand-binding domain (LBD) also harbors a nuclear localization signal
as well as sequences necessary for dimerization and transcriptional
activation (AF-2). A second activation function, AF-1, is present in
the amino-terminal domain of the receptor. Although transcriptional
activation is mediated through both AF-1 and AF-2, only activation by
AF-2 requires hormone binding (4, 5). Studies using the estrogen
antagonists 4-hydroxytamoxifen (OHT) and ICI 164,384 indicated that
although both compounds blocked estrogen effects, their mode of action
differed: the mixed agonist/antagonist OHT inhibited only AF-2 function
while the pure antiestrogen ICI 164,384 inhibited activation by both
AF-1 and AF-2 (6).
Additional signals that modulate nuclear receptor function have
been described and commonly involve phosphorylation. Steroid receptors,
including the progesterone and glucocorticoid receptors, have an
increased state of phosphorylation upon ligand binding (7). Similar
results have been described for ER where treatment of cells with
peptide growth factors or agents that alter cellular cAMP levels result
in up-regulation of the receptor coupled with an increase in its
phosphorylation state (8, 9). These reports, as well as subsequent
studies (10), demonstrated that antiestrogens also caused an increase
in receptor phosphorylation albeit to a lesser extent than estradiol
(E2), even though they inhibited transactivation.
Deletion mapping and mutagenesis of human ER revealed that
phosphorylation at Ser118 was required for full activity of
AF-1 (10). Furthermore, this residue was shown to be a direct substrate
for mitogen-activated protein (MAP) kinase, providing an in
vivo link between estrogen action and the Ras-MAP kinase signaling
cascade (11, 12). These results have begun to shed light on the
molecular events responsible for regulation and proliferation of
different cell types by ER in response to estrogens and growth
factors.
Transactivation by nuclear receptors has recently been shown to be
modulated by a growing family of coregulators (13). These include
corepressors such as N-CoR and silencing mediator for retinoid and
thyroid hormone receptors (SMRT) (14, 15, 16), which participate in the
ligand-independent silencing functions of TR and retinoic acid
receptor, and several coactivators: TRIP1/SUG-1 (17, 18), ERAP-140
(19), RIP-140 and RIP-160 (20, 21), TIF1 (22), and the related TIF2
(23), GRIP1 (24), SRC-1, and ERAP-160 (19, 25, 26). The LBD of ER was
demonstrated to specifically interact with many of these coactivators,
including steroid receptor coactivator-1 (SRC-1), ERAP-160, and
RIP-140, and the strength of interaction was increased by
E2 but not by the antiestrogens OHT and ICI 164,384 (20, 25, 27). The significance of these coactivators in nuclear receptor
function is underscored by recent results demonstrating that
SRC-related proteins interact with the nuclear integrators, CBP and
p300, to augment nuclear receptor transactivation in mammalian cells
(26, 28, 29). The activation domain of CBP can interact with TFIIB
(30), thus providing evidence for a link between nuclear receptors and
the basal transcription machinery. This growing network of interacting
factors has increased our understanding of how steroids such as
E2 are able to alter the expression of specific genes at
the molecular level.
Although human and mouse ER cDNAs were cloned several years ago (31, 32), RT-PCR of rat prostate mRNA has revealed the presence of a second
ER, referred to as ERß (33). This novel receptor was found to bind
E2 with relatively high affinity and to be capable of
activating transcription of a reporter gene in the presence of this
ligand. In situ hybridization of rat tissues indicated that
ERß is expressed in female animals in primary, secondary, and mature
follicles as well as granulosa cells in the ovary and in the prostate
of male rat. As an initial step toward elucidating the physiological
role of this second ER and understanding its functional relationship
with ER
, we report the cloning of the murine homolog of ERß. We
show that mERß is able to bind to an ERE in electrophoretic mobility
shift assays as well as to transactivate, in an
E2-dependent manner, promoters containing either synthetic
or natural EREs in Cos-1 and HeLa cells. In addition,
E2-induced mERß activity is inhibited by several
antiestrogens that have been previously shown to be selective for
ER
. We also demonstrate that SRC-1 interacts in vitro
with the mERß LBD in a ligand-dependent manner. However, SRC-1
modulates mERß transcriptional activity in intact cells both in the
presence and absence of ligand. Finally, we show that the
E2-induced transcriptional activity of mERß is enhanced
by cotransfection with Ras. The functional differences observed between
the two ER isoforms are discussed.
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RESULTS
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The Mouse ERß cDNA
The mERß cDNA contains an open reading frame of 1455
nucleotides encoding a protein of 485 amino acids (Fig. 1
). Alignment of mERß with mER
indicates that
the receptors share 97% and 60% amino acid similarity in the DBD and
the LBD, respectively (Fig. 2
). The AF-2 core sequences
in each receptor are virtually identical; however, no significant
regions of homology could be detected between the two amino-terminal
domains, a region of the protein that contains the AF-1 in ER
. Two
potential MAP kinase phosphorylation sites (Ser60 and
Ser94) are present in the amino-terminal region of the
mERß.

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Figure 1. Nucleotide Sequence of Murine ERß
Amino acid numbers are indicated on the left. The
potential MAP kinase phosphorylation sites are
underlined. GenBank accession number of the mERß
sequence is U81451.
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Figure 2. Amino Acid Sequence Comparison between Murine ERß
with ER
The alignment begins with the first Cys residue of the respective DBDs.
Identities are indicated with vertical bars,
biochemically similar residues by vertical dots, and
gaps by dashed lines. The stop codon in ERß is
indicated by an asterisk. The AF-2 domain is
underlined.
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Chromosomal Localization
The mouse chromosomal location of ERß (Estrb) was
determined by interspecific backcross analysis using progeny derived
from matings of [(C57BL/6J x Mus spretus)F1 x
C57BL/6J] mice. This interspecific backcross mapping panel has been
typed for more than 2200 loci that are well distributed among all the
autosomes as well as the X chromosome (34). C57BL/6J x M.
spretus DNAs were digested with several enzymes and analyzed by
Southern blot hybridization for informative restriction fragment
length polymorphisms (RFLPs) using a mouse cDNA ERß probe (positions
816-1361). The 8.4- and 5.1-kb EcoRI M. spretus
RFLPs (see Materials and Methods) were used to follow the
segregation of the Estrb locus in backcross mice. The
mapping results indicated that Estrb is located in the
central region of mouse chromosome 12 linked to Sos2,
Spnb1, and Fos. Although 165 mice were analyzed
for every marker and are shown in the segregation analysis (Fig. 3
), up to 184 mice were typed for some pairs of markers.
Each locus was analyzed in pairwise combinations for recombination
frequencies using the additional data. The ratios of the total number
of mice exhibiting recombinant chromosomes to the total number of mice
analyzed for each pair of loci and the most likely gene order are:
centromere - Sos2 - 7/184 - Spnb1 - 0/181 -
Estrb 7/173 - Fos. The recombination frequencies
[expressed as genetic distances in centiMorgans (cM) ± the
SE] are - Sos2 - 3.8 ± 1.4 -
[Spnb1, Estrb] - 4.1 ± 1.5 -
Fos. No recombinants were detected between Spnb1
and Estrb in 181 animals typed in common, suggesting that
the two loci are within 1.7 cM of each other (upper 95% confidence
limit).

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Figure 3. Chromosomal Localization of the ERß Gene
The ERß gene, Estrb, maps to the central region of
mouse chromosome 12. Estrb was placed on mouse
chromosome 12 by interspecific backcross analysis. The segregation
patterns of Estrb and flanking genes in 165 backcross
animals that were typed for all the loci are shown at the
top of the figure. For individual pairs of loci, more
than 165 animals were typed (see text). Each column represents the
chromosome identified in the backcross progeny that was inherited from
the (C57BL/6J x M. spretus) F1 parent.
The shaded boxes represent the presence of C57BL/6J
allele, and open boxes represent the presence of a
M. spretus allele. The number of offspring inheriting
each type of chromosome is listed at the bottom of each
column. A partial chromosome 12 linkage map showing the location of
Estrb in relation to linked genes is shown at the
bottom of the figure. Recombination distances between
loci in centimorgans are shown to the left of the
chromosome, and the positions of loci in human chromosomes, where
known, are shown to the right. References for the human
map positions of loci cited in this study can be obtained from GDB
(Genome Data Base), a computerized database of human linkage
information maintained by The William H. Welch Medical Library of The
Johns Hopkins University (Baltimore, MD).
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The mERß Gene Expresses Several Transcripts
A Northern analysis was performed to determine the size of the
message encoding mERß using the 550-bp PCR fragment that spans the
LBD of mERß as a probe. As shown in Fig. 4
, mouse
ovary expressed at least four predominant transcripts of approximately
3.5, 4.6, 7.2, and 9.5 kb and a weaker message of about 4.9 kb. In
contrast, only one transcript could be detected in ovary mRNA when
probed with the mER
cDNA. Analysis of total RNA from several other
mouse tissues including liver, heart, kidney, skeletal muscle, thymus,
spleen, and brain were all negative for presence of the ERß
transcripts indicating that expression of ERß was below the level of
detection by Northern blotting in these tissues (data not shown).

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Figure 4. Northern Blot Analysis of ERß mRNA
Five micrograms of mouse ovary poly-A+ RNA were probed with
a 550-bp cDNA fragment encoding the mERß LBD and a full-length mER
cDNA. The blot was hybridized with the mERß cDNA fragment, stripped,
and subsequently probed with the mER cDNA. Exposure was carried out
for the same length of time in each case. The position of 18S and 28S
rRNAs are indicated on the left.
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Mouse ERß Binds to an ERE
The homology between the DBDs of mER
and ß indicated that the
receptors most likely bind to the same type of response element, namely
an inverted repeat spaced by three nucleotides (IR-3). We tested this
by conducting electrophoretic mobility shift assays (EMSA) with both
mouse receptors. Figure 5
demonstrates that both ER
isoforms produced in vitro by rabbit reticulocyte lysates
were able to bind specifically to a vitellogenin A2 (vitA2)-ERE (35)
probe. It was noted that the mERß did not bind as strongly to the
element as mER
(compare lanes 25 with lanes 710) even though
both receptors were present at roughly the same level in the crude
lysates as shown by [35S]methionine-labeled proteins
(data not shown). In addition, both receptors were able to bind to the
pS2-ERE (36) but less efficiently than to the vitA2-ERE (data not
shown), which can most likely be accounted for by the fact that the
pS2-ERE contains an imperfect consensus sequence in the second
half-site.

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Figure 5. Binding of mERß and mER to an ERE
Each receptor produced by rabbit reticulocyte lysates (RRL) was
incubated at room temperature with approximately 0.1 ng of labeled
vitA2-ERE in the absence (lanes 2 and 7) and presence of 100
nM E2 (lanes 3 and 8), 100 nM OHT
(lanes 4 and 9), and 100 nM ICI 182,780 (lanes 5 and 10).
Unprogrammed RRL was used as a negative control (lane 1) and
specificity was determined in the presence of 100x unlabeled vitA2-ERE
(compare lanes 6 and 11).
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Addition of E2 to the binding reactions did not have an
effect on binding under the conditions used (Fig. 5
, lanes 3 and 8) nor
was there any effect on binding upon addition of specific antagonists
such as OHT (lanes 4 and 9) and ICI 182,780 (lanes 5 and 10), although
the migration rate of the protein/DNA complexes were differentially
altered by each ligand. However, as previously observed with ER
(37), a severe decrease in binding by ERß was observed when the
preincubation step was conducted at room temperature, 37 C, or for
shorter periods of time. The formation of the ERß/DNA complexes could
be partially restored in the presence of E2 at these
temperatures (data not shown).
Transcriptional Activity of mERß on Synthetic and Natural
Promoters
To test whether the vitA2-ERE could mediate mERß transcriptional
activity, we linked one copy of the ERE to either a basal
promoter containing a TATA box (BLuc) or to the more complex viral
thymidine kinase promoter (TKLuc) driving the expression of the
luciferase reporter gene for transactivation studies. In Cos-1 cells,
mERß induced a 5- to 10-fold response on all the ERE-containing
reporters tested when 10 nM E2 was added to the
medium (Fig. 6A
). mER
produced a slightly larger fold
induction of the ERE-containing reporters when studied under the same
transfection conditions (Fig. 6B
). In addition,
E2-dependent transcriptional activity of mERß was also
observed in HeLa cells (Fig. 6C
), suggesting a cell type- independent
effect. It is noteworthy that, as already observed in Cos-1 cells, the
levels of E2-induced activation in HeLa cells were somewhat
higher when mER
was used as compared with mERß (Fig. 6D
), although
it is not known whether similar levels of ER proteins were present in
these transient transfections.

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Figure 6. E2-Dependent Transcriptional Activities
of mERß vs. mER
A, Cos-1 cells were transfected with 2 µg of the various reporter
plasmids carrying either one copy of the vitA2-ERE (ERE-BLuc and
ERE-TKLuc) or vector only (BLuc and TKLuc), along with 1 µg
pCMX-mERß. Cells were treated with or without 10 nM
E2 for 12 h before being assayed for luciferase
activity. B, Transfection conditions were as in panel A, except that a
pCMX-mER expression vector was used. C and D, Transfection
conditions were as in panels A and B, respectively, except that HeLa
cells were used. E, mERß transactivates the pS2 promoter. HeLa cells
were transfected with 500 ng pCMX-mERß and 1 µg pS2Luc (pS2) or
pS2 ERELuc (pS2 ERE) reporter plasmids and incubated with or
without 10 nM E2 in the presence or absence of
100 nM of the indicated antagonists, 4-hydroxytamoxifen
(OHT) and ICI 182,780 (ICI).
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We next investigated whether mERß could modulate the
transcriptional activity of an ERE-containing promoter in its natural
context. The pS2 promoter has been shown to respond to hER
(36). As
shown in Fig. 6E
, mERß was able to transactivate the pS2 promoter in
HeLa cells with a 6-fold induction when E2 was added. In
addition, OHT and ICI 182,780 efficiently blocked the
E2-induced effect of mERß on pS2. This interaction is
mediated through the ERE since its deletion abolished the response to
E2 (Fig. 6E
).
Ligand Binding
To ascertain whether there was a dose dependency of E2
for mERß and mER
, we tested the activity of both receptors in the
presence of increasing E2 concentrations using the
vitA2-ERE-TKLuc reporter in Cos-1 cells. Comparison of the
dose-response curves of Fig. 7A
shows a shift of
approximately 4-fold of E2 concentration required to
achieve half of the maximal level of induction between the two
receptors. These results suggested that mERß may have lower affinity
for E2 than measured for mER
. To verify if the
difference in E2 responsiveness was due to a difference in
ligand binding, we performed a binding analysis on both mERß and
mER
. [3H[E2 was used to conduct binding
studies with mERß, and results were plotted by the method of
Scatchard. As shown in Fig. 7B
, this analysis yielded an average
dissociation constant (Kd) of 0.5 nM for
E2 when performed on receptor prepared from rabbit
reticulocyte lysates. This value is comparable to that obtained for the
rat ERß, which was reported to be 0.6 nM (33). We
obtained an average Kd of 0.2 nM for mER
(Fig. 7C
), which is well within the range of previously published
determinations for the cloned human receptor (38). Therefore, this
slightly reduced affinity of mERß for E2 may provide an
explanation for the shift in E2 responsiveness indicated by
the dose-response curves (Fig. 7A
).

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Figure 7. Dose-Response and Binding Properties of mERß
A, Cos-1 cells were transfected with 500 ng mERß (open
circles) or mER (closed circles) expression
vectors and 1 µg vitA2-ERE-TKLuc and then incubated for 12 h
with increasing concentrations of E2 as indicated. B,
Specific binding of [2,4,6,7-3H]-17ß-estradiol
([3H]E2) to mERß was determined using
receptors generated from rabbit reticulocyte lysates as described in
Materials and Methods. Binding was determined over a
concentration range of 0.013 nM
[3H]E2 in the absence or presence of a
200-fold excess of unlabeled E2. The saturation plot is
shown in the inset, and results were plotted by the
method of Scatchard. Each point was determined in triplicate in each
experiment, and the above results are representative of at least two
separate experiments. C, Specific binding to mER using the
conditions described in panel B.
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Estrogen Antagonists Block the E2-Dependent
Activity of mERß
Estrogen antagonists such as OHT and ICI 164,384 are known
to interact with hER
by blocking its transcriptional activity. More
precisely, OHT is a mixed agonist-antagonist and blocks the activity of
AF-2 but not of AF-1, whereas ICI 164,384 and 182,780 are pure
antagonists that block both AF-1 and AF-2 activities (Refs. 6 and 39
and see below). We tested the effects of OHT and ICI 182,780 along with
other antagonist compounds on mERß transactivation, and the results
are shown in Fig. 8
. All antagonists tested, including
OHT, ICI 182,780, hydroxy-toremifen, raloxifene, and EM-652 (the active
derivative of the novel nonsteroidal antiestrogen EM-800), effectively
inhibited E2 activity (Fig. 8A
). In contrast to its
stimulatory effect on mER
, OHT did not display an agonistic activity
on mERß when tested with a vitA2-ERE-BLuc reporter in either Cos-1
(Fig. 8B
) and HeLa cells (data not shown).

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Figure 8. Effect of Antagonists on mERß-Mediated
Transactivation
A, Cos-1 cells cotransfected with pCMX-mERß and vitA2-ERE-TKLuc
plasmids were incubated for 12 h in the presence or absence of 10
nM E2 or 100 nM of the indicated
antagonists: 4-hydroxitamoxifen (OHT), ICI 182,780 (ICI),
hydroxy-toremifen (OH-tor), raloxifen (RLX), and EM652. Results are
expressed as fold response over the mERß basal level in the absence
of E2; this value was set arbitrarily to 1. B, Cos-1 cells
cotransfected with pCMX-mERß or pCMX-mER and ERE-BLuc plasmids
were incubated for 12 h in the presence or absence of 100
nM OHT. C, Dose response of OHT and ICI in the presence of
10 nM E2 on mERß activity with the ERE-TKLuc
in Cos-1 cells. The maximal induction by E2 alone was
arbitrarily set at 100%. The untreated mERß basal level is also
shown. Compounds within a panel are differentiated by open
squares (OHT) and circles (ICI). D, Comparative
panel of the dose responses to OHT in the presence of 10 nM
E2 between mERß (open squares) and mER
(filled squares). Results are expressed as in panel B.
E, Comparative panel of the dose responses to ICI in the presence of 10
nM E2 between mERß (open
circles) and mER (filled circles).
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We further evaluated the specificity and potency of OHT and ICI 182,780
on both ER isoforms. Increasing concentrations of ICI 182,780 and OHT
led to a complete inhibition of the E2-induced mERß
activity in Cos-1 cells (Fig. 8C
). Furthermore, when added at 10
nM and above, ICI 182,780 and OHT lowered mERß activity
even below its basal level (compared with untreated in Fig. 8C
). We
observed a similar dose response for OHT and ICI 182,780 using either
receptor (Fig. 8
, D and E). However, for OHT, there was an apparent
shift in the dose response toward the lower concentrations for mERß
as compared with mER
(Fig. 8D
).
Ras Enhances E2-Induced Transcriptional
Activity of mERß
Phosphorylation of serine residues, in particular
Ser118, has been shown to be necessary for maximal activity
of AF-1 in the hER
and to mediate the effect of the
Ras-Raf-1-mitogen-activated protein kinase (MAPK) kinase and MAPK
pathway on the transcriptional activity of the ER (10, 11, 12, 40, 41). In
an attempt to investigate the effect of the activation of this pathway
on mERß activity, we used H-RasV12, a dominant active
form of H-Ras, in transactivation studies. As shown in Fig. 9A
, H-RasV12 acted to further increase by a
factor of 3 the E2-induced activation of mERß using the
vitA2-ERE-TKLuc reporter in Cos-1 cells. ICI 182,780, but not OHT,
completely abolished the E2-dependent induction of mERß
(Fig. 9A
) and mER
(Fig. 9B
) by Ras, suggesting that the effect of
Ras on mERß activity is mediated by a putative AF-1 present in the
amino-terminal domain. These results also show that ICI 182,780
suppresses transactivation mediated by both AF-1 and AF-2.

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Figure 9. Effects of H-Ras on E2-Induced mERß
Transcriptional Activity
A, Cos-1 cells were cotransfected with 1 µg ERE-TKLuc, 500 ng
pCMX-mERß, and with or without H-RasV12 expression
plasmid. The cells were then grown in the presence or absence of 10
nM E2 or 100 nM of the indicated
antagonists, 4-hydroxytamoxifen (OHT), and ICI 182,780 (ICI). B,
Similar to panel A except that mER was used. C, Potential MAP kinase
phosphorylation sites in the mouse ER and ß. D, Mutation of
Ser60 abolishes the stimulatory effect of
H-RasV12 on E2-induced mERß transcriptional
activity. Transfection conditions were as in panel A, except that
mERß mutants were used as indicated.
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Two potential serines at positions 60 and 94 in the mERß
amino-terminal domain matched the consensus MAPK phosphorylation site
(42) (Fig. 9C
). To determine whether one or both serine residues were
involved in the Ras-mediated induction, we mutated Ser60 to
Ala (S60A) and Ser94 to Ala (S94A) in mERß and used both
mutants in transactivation studies. Figure 9D
shows that H-ras had no
inducing effect on the S60A mutant in the presence of E2
while the S94A mutant retained its responsiveness to H-ras as compared
with the wild type mERß (Fig. 9A
). This suggests that
Ser60 in mERß is a potential target for phosphorylation
by the Ras-Raf-1-MAPK kinase-MAPK pathway.
SRC-1 Interacts with and Augments Transcriptional Activity of
mERß
The contribution of the AF-2 domain to mERß activity was
investigated by examining the effect of the coactivator SRC-1, which
has been shown to interact with a number of nuclear receptors including
hER
(25, 43). Glutathione-S-transferase (GST) fusion proteins were
generated with both the mERß and mER
LBDs (Fig. 10A
) and tested in a GST-pull down experiment (Fig. 10B
). GST-mERßEF and GST-mER
DEF were expressed in
Escherichia coli, purified with GST-Sepharose, and incubated
with [35S]methionine-labeled SRC-1. As shown in Fig. 10B
, the LBD of mERß interacted weakly with SRC-1 in the absence of
E2 (lane 3), whereas addition of E2 caused an
increase in interaction between the two proteins (lane 4). As expected,
estrogen antagonists that affect the AF-2 of ER, namely OHT (lane 5)
and ICI 182,780 (lane 6), do not promote a ligand-dependent SRC-1
interaction. The E2-dependent interaction with SRC-1 was
also efficiently blocked in the presence of 10-fold higher
concentration of the various antagonists (data not shown).
Protein-protein interactions between SRC-1 and the mER
LBD
paralleled those observed with mERß (see Fig. 10B
, lanes 812).

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Figure 10. SRC-1 Interacts with mERß and Induces Its
Transcriptional Activity
A, Structure of the GST fusion proteins used in the analysis. B, GST
pull-down experiments. The purified fusion proteins were incubated with
labeled SRC-1 in the absence (lanes 3 and 9) or presence of 10
nM E2 (lanes 4 and 10), 10 nM each
of OHT (lanes 5 and 11) and ICI 182,780 (ICI) (lanes 6 and 12). The
input lane represents 10% of the total amount of labeled SRC-1 used in
each binding reaction. An equivalent amount of protein extract was used
in the samples containing only GST (lanes 2 and 9). C, Cos-1 cells were
cotransfected with mERß and vitA2-ERE-TKLuc in the presence or
absence of 1 µg CMX-SRC-1. Cells were incubated with or without 10
nM E2 or 100 nM antagonists as
indicated. Separate experiments were also done with mER as shown. D,
Similar to panel A except that pS2Luc was used as the reporter and HeLa
cells were transfected.
|
|
The contribution of SRC-1 on mERß activity was analyzed in
vivo by transfection studies. When SRC-1 was cotransfected with
mERß and vitA2-ERE-TKLuc in Cos-1 cells, a 2- to 3-fold induction
over that of E2 alone was observed (Fig. 10C
). A similar
response was also seen with mER
in the presence of E2
(Fig. 10C
). However, SRC-1 alone efficiently increased the basal level
of transcriptional activity by mERß, suggesting a ligand-independent
effect of SRC-1 on mERß (Fig. 10C
). The basal transcriptional level
of mER
was not affected by the addition of SRC-1. We also studied
the effect of SRC-1 on the pS2 promoter in HeLa cells and found a
similar ligand-independent activation of mERß by SRC-1 as well as a
potentiation of the E2-induced activity (Fig. 10D
). These
results suggest that SRC-1 does not act on mERß in a promoter- and
cell-specific manner. Furthermore, ICI 182,780, but not OHT, was able
to abrogate the ligand-independent effect of SRC-1 on mERß
transcriptional activity (Fig. 10
, C and D).
 |
DISCUSSION
|
---|
The physiological actions of E2 are mediated through a
member of the steroid hormone receptor family, ER
, which, for many
years, has remained the only nuclear receptor known to have
E2 as a ligand. Recently, it has been shown that a second
receptor, ERß, is also able to respond to this hormone. ERß was
cloned by degenerative PCR from rat prostate (33) and more recently
from human testes (44), but its physiological role remains to be
elucidated. As a first step toward investigating the role of ERß in
development and homeostasis, we report the cloning of the murine
homolog. The availability of the murine cDNA provides us with the means
to characterize this receptor utilizing both biochemical and genetic
methodologies and will allow us to study its relationship, if any, with
ER
in normal reproductive physiology.
We first used a fragment of the LBD of the mouse ERß cDNA to
establish the localization of the gene to chromosome 12. We have
compared our interspecific map of chromosome 12 with a composite mouse
linkage map location of many uncloned mouse mutations (provided from
Mouse Genome Database, computerized database maintained at The Jackson
Laboratory, Bar Harbor, ME). Estrb mapped in a region of the
composite map that lacks mouse mutations with a phenotype that might be
expected for an alteration in this locus (data not shown). The central
region of mouse chromosome 12 shares a region of homology with human
chromosome 14q, suggesting that Estrb will reside on 14q as
well.
The mERß cDNA was used in a Northern analysis on murine ovary mRNA
that revealed that the mERß gene generates several transcripts as
compared with a single message for mER
in this tissue. Partial cDNAs
obtained from an ovary library all contained a 3'-untranslated region
(UTR) of 657 bp followed by a polyA+ tail, suggesting that
the different transcripts seen in the Northern analysis may differ at
their 5'-ends or in the coding region. Although we were unable to
detect any messages in the total RNA from other mouse tissues, the
hERß homolog appears to be expressed in mRNA isolated from testes,
spleen, and thymus in addition to ovary (44).
The two mouse ER isoforms are closely related to each other in their
LBD and DBD, indicating that these receptors may regulate common gene
networks and respond to similar ligands. Using EMSA, a ligand-binding
assay, and transient transfection experiments, we showed that mERß
has slightly lower affinities than mER
for ERE and E2
binding in vitro, but that mERß can transactivate reporter
genes driven by synthetic and natural E2-responsive
promoters in vivo as efficiently as mER
in both Cos-1 and
HeLa cells. However, the two ERs display no sequence homology in their
amino-terminal domains, suggesting that each receptor may possess
distinct transactivation functions. We made use of the differential
mode of action of the estrogen antagonists OHT and ICI 182,780 and
activation of receptor activity by the Ras-Raf-1-MAPK pathway to
demonstrate functional similarities and differences between mERß and
mER
. We first showed that, when assayed in Cos-1 cells with an
E2-responsive reporter gene driven by a basal promoter, OHT
does not display an agonistic activity with ERß. It has recently been
shown that a specific region of the mER
AF-1 is required for OHT
agonism (45): the absence of a corresponding domain in mERß may
explain the present observation. We demonstrated that mERß, as
previously shown for ER
(11, 12), can be activated by the
Ras-Raf-1-MAPKK-MAPK pathway. We have identified a serine residue in
mERß that could be the target of phosphorylation upon cotransfection
with Ras. This was demonstrated by the fact that a mutation at
Ser60 in the amino terminus of mERß eliminates the effect
seen with Ras. Although another serine residue at position 94 also
matches the MAP kinase consensus, mutation at this position has no
effect on Ras-mediated activation. Moreover, alignment of the mouse,
rat, and human amino acid sequences in this region indicates that
Ser60 is conserved in all species whereas Ser94
is replaced by a glycine in the human ERß. Finally, we showed that
the induction of the E2-dependent activation of mERß by
Ras can be abolished by ICI 182,780, but not OHT, an observation that
provides further evidence of the involvement of an AF-1-like domain in
regulating mERß functions.
Antiestrogens play an important role in the treatment of breast cancer.
We therefore monitored the efficacy of a series of antiestrogens
previously shown to be selective for ER
on mERß and found that all
compounds tested, including OHT, ICI 182,780, hydroxy-toremifen,
raloxifen, and the novel nonsteroidal antiestrogen EM-800, inhibited
E2-dependent activation by mERß. We showed that OHT is
also AF-2 selective on mERß, and that ICI 182,780 inhibits both
activation functions, displays no estrogenic activity, and thus can be
considered as a pure antagonist of mERß activity. However, in
dose-response studies, OHT proved to be a more potent inhibitor of
mERß than of mER
. In addition, we show that the mode of action of
ICI 182,780, as previously observed for ICI 164,384, involves the
inhibition of both AF-1 and AF-2 activity of ER
.
A growing network of coactivators that interact with ER
has now been
cloned. These include RIP140 (20), TIF2 (23), and SRC-1 (25). However,
only SRC-1 has been shown to up-regulate ER-stimulated transcription
(25, 43). mERß transcriptional activity could also be stimulated by
SRC-1 in a ligand-dependent manner in cotransfection assays as well as
in vitro where we observed a very strong ligand-dependent
interaction with a GST-mERß LBD fusion protein. Surprisingly, we also
observed ligand-independent SRC-1 enhancement of mERß transcriptional
activity. These results suggest that SRC-1 may interact with a region,
other than the AF-2, of the mERß protein. McInerney et al.
(43) have shown that constructs expressing the ABCD (no LBD) and EF
(LBD only) domains of hER
separately are able to transactivate in
the presence of E2, and that SRC-1 can enhance the level of
activation under these conditions. These results led them to suggest
that SRC-1 may act as an adapter to promote AF-1 and AF-2 receptor
activities and are in agreement with our suggestion that SRC-1 may
interact with several regions of nuclear receptor proteins.
The identification of a second ER in mammals is very exciting, and
these and previous studies have only begun to define the putative role
that it plays in the mediation of estrogen action. Clearly, further
studies into the mechanism of action of ERß are required if we are to
understand how it functions in vivo. The results we present
in this paper indicate that ERß and
may have both redundant and
distinct functions, as exemplified by the manner with which they
respond to OHT, Ras, and SRC-1, and suggest that ERß plays an unique
role in the physiological actions of natural estrogens.
 |
MATERIALS AND METHODS
|
---|
Cloning of the Mouse ERß cDNA
A combination of PCR and cDNA library screening was used to
obtain the full-length cDNA encoding the murine ERß. All
oligonucleotides used in this study were synthesized at the Sheldon
Biotechnology Center, McGill University. Total RNA was prepared from
several mouse ovaries and used to isolate poly-A+ RNA over
two oligo-dT columns (Pharmacia, Piscataway, NJ). An initial 550-bp
fragment was amplified from 100 ng poly-A+ RNA using
degenerate primers specific for the rat ERß LBD (33). The 5'- and
3'-sequence of these primers spanned amino acids 269277 (KKIPGVE) and
443450 (YDLLLEML, noncoding strand), respectively. The reaction was
carried out for 40 cycles using Pfu polymerase (Stratagene, La Jolla,
CA) at an annealing temperature of 54 C and for an extension time of 1
min. The PCR product was separated on low melt agarose (Life
Technologies, Gaithersburg, MD) and subcloned into pBluescriptKSII
(Stratagene). The sequence was determined on both strands using the T3
and T7 primers and found to encode an open reading frame that was
highly homologous to the rat ERß. This 550-bp fragment served as a
probe to screen a mouse ovary cDNA library constructed using the
Superscript cDNA Synthesis System (Life Technologies). Three strong
positives were plaque purified, subcloned into pBluescriptKSII, and
sequenced on both strands. Each of these clones started in the LBD and
ended with a poly-A tail. A PCR primer was designed based on the
sequence of the 3'-UTR of the partial mouse ERß cDNA and used to
synthesize first-strand cDNA from mouse ovary poly-A+ RNA.
The cDNA served as a template in a PCR reaction using a primer that
spans the first 21 bases of the 5'-UTR of rat ERß ending with the
putative initiator codon and a second specific mouse ERß 3'-UTR
primer. This reaction yielded a single product of 2.1 kb that was
subcloned into pBluescriptKSII directionally using SalI and
EcoRI sites that were designed in the 5'- and 3'-PCR
primers, respectively, and sequenced on both strands. Sequencing was
conducted at the sequencing facility at the Sheldon Biotechnology
Center, McGill University.
Chromosomal Localization of the ERß Gene
Interspecific backcross progeny were generated by mating
(C57BL/6J x M. spretus) F1 females and C57BL/6J males
as described (34). A total of 205 N2 mice were used to map
the Estrb locus (see text for details). DNA isolation,
restriction enzyme digestion, agarose gel electrophoresis, Southern
blot transfer, and hybridization were performed essentially as
described (46). All blots were prepared with Hybond N+
nylon membrane (Amersham). The probe, a 550-bp
HindIII/EcoRI fragment of the mouse cDNA, was
labeled with [32P]-
-dCTP using a random-primed
labeling kit (Stratagene); washing was done to a final stringency of
0.8x SSCP, 0.1% SDS, at 65 C. Fragments of 13.5 and 5.5 kb were
detected in EcoRI-digested C57BL/6J DNA, and fragments of
8.4 and 5.1 kb were detected in EcoRI-digested M.
spretus DNA. The presence or absence of the 8.4- and 5.1-kb
EcoRI M. spretus-specific fragments, which
cosegregated, was followed in the backcross mice.
A description of the probes and RFLPs for the loci linked to
Estrb including Sos2, Spnb1, and
Fos has been reported previously (47). Recombination
distances were calculated as described (48) using the computer program
SPRETUS MADNESS. Gene order was determined by minimizing the number of
recombination events required to explain the allele distribution
patterns.
Northern Analysis
Five micrograms of mouse ovary poly-A+ RNA were
electrophoresed on a 1% formaldehyde-agarose gel and blotted onto
Hybond-N+. The entire mouse ER
cDNA (49) and the 550-bp
PCR product encoding mERß were radiolabeled with
[32P]-
-dCTP (Amersham, Arlington Heights, IL) by
random priming (Pharmacia). Prehybridization was carried out for 4
h in 50% formamide, 5xSSPE, 5x Denhardts solution, 1% glycine,
and 100 µg/ml denatured salmon sperm DNA at 42 C. Hybridization was
conducted overnight at the same temperature in 50% formamide, 5x
SSPE, 1x Denhardts solution, 10% dextran sulfate, 0.3% SDS, 100
µg/ml denatured salmon sperm DNA, and 2 x 106
cpm/ml denatured probe. The membrane was washed to high stringency
(0.1x SSC, 0.1% SDS at 65 C for 30 min) and exposed to X-OMAT AR film
(Kodak) overnight at -85 C. The positions of the 18S and 28S rRNAs
were determined by the electrophoresis of total mouse ovary RNA on the
same gel.
Plasmids
The expression vector pCMX-mER
was constructed by ligating a
1.9-kb NaeI-EcoRI fragment of mER
(49) into
the appropriate sites of the eukaryotic expression vector, pCMX (50).
pCMX-mERß was constructed in a similar fashion by subcloning the
2.1-kb SalI-EcoRI fragment described above into
pCMX. Fusion proteins were generated between the mERß and mER
LBDs
and GST by subcloning fragments of the mouse cDNAs into the pGEX-2T
expression vector (Pharmacia). Briefly, a unique PvuII site
located at position 678 and an EcoRI site in the vector were
used to excise the LBD (domains E and F) from the full-length mERß
cDNA. The fragment was then subcloned into the SmaI site of
pGEX to produce pGST-mERßEF. For mER
, the hinge region and the LBD
(domains D, E, and F) were amplified from the full length mER
cDNA
using PCR and subcloned directionally into the BamHI and the
blunted EcoRI sites of the pGEX vector (pGST-mER
DEF) (see
Fig. 9A
). Both constructs were verified to be in-frame with GST by
sequencing. vitA2-ERE-BLuc and vitA2-ERE-TKLuc were constructed by
ligating the vitA2-ERE oligonucleotide (see below for sequence) into
SalI-BamHI-digested TKLuc vector. pS2-ERELuc
contains the
1050-bp pS2 promoter (36) preceding the luciferase
reporter of pGL3 (Promega, Madison, WI). The pS2
ERELuc, in which the
ERE was replaced by sequences encoding
EcoRI-EcoRV sites, was generated by PCR
mutagenesis using the ExSite kit from Stratagene as described by the
manufacturer. The serine to alanine mutants at positions 60 and 94 of
mERß were also generated by PCR mutagenesis. The oligonucleotides
used were: S60A,
5'-CTCTATGCAGAACCTCAAAAGGCTCCTTGGTGTGAAGC-3'; S94A,
5'-GGTTGTGCCAGCCCTGTTACTGCTCCAAGCGCCAAGAGG-3'. The
H-RasV12 expression plasmid was a generous gift from Dr.
Morag Park, McGill University.
Chemicals
E2 was obtained from Sigma Chemical Co. (St. Louis,
MO). EM-800, EM-652, ICI-182780, and OH-toremifene were synthesized in
the medicinal chemistry division of the Laboratory of Molecular
Endocrinology, CHUL Research Center, Québec, Canada. OHT was
kindly provided by Dr. D. Salin-Drouin, Besins-Iscovesco, Paris,
France.
EMSA
mERß and mER
proteins were synthesized by in
vitro transcription-translation using rabbit reticulocyte lysates
(Promega) and pCMX-mERß and pCMX-mER
, respectively, as templates.
Typically 5 µl of programmed lysate were used in each binding
reaction. DNA-binding reactions were carried out in binding buffer (5
mM Tris, pH 8.0, 40 mM KCl, 6% glycerol, 1
mM dithiothreitol, 0.05% Nonidet P-40), 2 µg of
poly(deoxyinosinic-deoxycytidylic)acid, 0.1 µg of denatured salmon
sperm DNA, and 10 µg of BSA with 0.1 ng probe that was labeled by
end-filling with Klenow in the presence of
[32P]-
-dCTP. Preincubations containing ligand and/or
cold competitor as indicated were conducted on ice for 30 min, after
which the probe was added and allowed to bind for 30 min at room
temperature. The entire reaction (20 µl) was loaded onto a 4%
polyacrylamide gel and electrophoresed at 150 V at room temperature.
Gels were dried and exposed overnight at -85 C. The following
oligonucleotides and their compliments were used as probes and
competitors: vitA2-ERE,
5'-TCGACAAAGTCAGGTCACAG-TGACCTGATCAAG-3' (51);
pS2-ERE,
5'TCGACCCTGCAAGGTCACGGTGGCCA-CCCCGTG-3' (36);
IR3,
5'-TCGACGTGTAGGTCA-CAGTGACCTCTTCA-3'.
Scatchard Analysis
Ligand binding studies were conducted essentially as previously
described (52) with the following modifications. mERß and mER
were
produced using rabbit reticulocyte lysates, diluted 12-fold in TEG
buffer (10 mM Tris, pH 7.5, 1.5 mM EDTA, 10%
glycerol) and kept on ice until use. One hundred microliters of this
dilution were used in each binding reaction at 4 C overnight containing
[2,4,6,7-3H]17ß-estradiol concentrations ranging from
0.013 nM. Nonspecific binding was assessed by including
200-fold excess E2 in a parallel set of samples. Unbound
steroids were removed with dextran-coated charcoal and counts per min
were determined by liquid scintillation counting.
Cell Culture, DNA Transfection, and Luciferase Assay
For transfection, Cos-1 and HeLa cells were seeded in six-well
plates in phenol red-free DMEM (GIBCO BRL, Gaithersburg, MD)
supplemented with 10% charcoal dextran-treated FBS, and 100 µg/ml
penicillin and 100 µg/ml streptomycin. At 5060% confluency, cells
were transfected with 12 µg of reporter plasmid, 0.51 µg
receptor expression vector, 1 µg CMX-ßgal or RSV-ßgal, and 67.5
µg pBluescriptKSII, using the calcium phosphate/DNA precipitation
method (53). After 816 h, cells were washed and typically 10
nM E2 or 100 nM antiestrogens,
unless otherwise stated, were added to the growth medium for 16 h.
For luciferase assay, cells were lysed in potassium phosphate buffer
containing 1% Triton X-100, and light emission was detected using a
luminometer after addition of luciferin. Values are expressed as
arbitrary light units normalized to the ß-galactosidase activity of
each sample.
In Vitro Protein-Protein Interactions
Fusion proteins were expressed in E. coli DH5
as
follows. An overnight culture was diluted 10-fold in 500 ml prewarmed
LB containing 100 µg/ml ampicillin and incubated for 1 h at 37
C. Isopropyl-ß-D-thiogalactopyranoside was added to a
final concentration of 1 mM to induce expression, and the
culture was allowed to grow for a further 3 h. Cells were cooled
on ice for 10 min and centrifuged at 2500 x g for 20
min. The pellet was resuspended in 12 ml ice-cold PBS and sonicated.
After the addition of one tenth volume of 10% triton, the extract was
centrifuged at 12,000 x g for 20 min at 4 C. Clarified
extracts were aliquoted in 1-ml samples and frozen at -85 C.
The 3.3-kb SRC-1 cDNA encoding the predicted 1061 amino acid open
reading frame as originally cloned (25) was digested with
XbaI and SalI, blunt ended with Klenow, ligated
into pCMX, and labeled with [35S]methionine (Amersham)
in vitro using rabbit reticulocyte lysates as described
above. Approximately 300 µg of total protein from extracts containing
the GST fusion proteins were loaded onto glutathione-Sepharose 4B
(Pharmacia) for 30 min at 4 C with gentle agitation. After a short
centrifugation, the beads were resuspended in 150 µl IPAB buffer (20)
containing 10 nM ligand or antagonists and 4 µl of
labeled SRC-1 crude lysate and allowed to bind for 90 min at 4 C. Beads
were washed twice in the presence of IPAB and twice with IPAB without
BSA, dried briefly, and resuspended in 30 µl loading buffer. Bound
proteins were analyzed by SDS-PAGE. The gel was treated with Amplify
(Amersham), dried, and exposed at -85 C.
 |
ACKNOWLEDGMENTS
|
---|
We thank M. Parker for the gift of mouse ER
and human SRC-1
cDNAs and M. Park for the gift of the H-RasV12 expression
vector. We are grateful to L. McBroom and R. Sladek for their
suggestions and comments on the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Vincent Giguère, Molecular Oncology Group, Room H5.21, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1.
Financial support was provided by the Medical Research Council of
Canada, the National Cancer Institute of Canada, and the Cancer
Research Society, Inc., to V. Giguère. G. B. Tremblay is a
Postdoctoral fellow and V. Giguère a Scientist of the Medical
Research Council of Canada. This work was also supported by
EndoRecherche Inc.
1 Co-first authors. 
Received for publication December 6, 1996.
Revision received December 23, 1996.
Accepted for publication December 30, 1996.
 |
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