From the
Department of Physiology, Wayne State
University School of Medicine, ¶Barbara Ann
Karmanos Cancer Institute, Detroit, Michigan 48201 and the
Department of Biochemistry and Molecular
Biology, University of Chicago, Chicago, Illinois 60637
Received for publication, April 11, 2003 , and in revised form, May 6, 2003.
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ABSTRACT |
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INTRODUCTION |
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Drugs that antagonize estrogen action, such as tamoxifen, raloxifene, and ICI 182,780, are used to treat and prevent breast cancer (18, 19). Because tamoxifen and raloxifene show tissue-specific agonist, as well as antagonist, effects, they are referred to as selective estrogen receptor modulators, or SERMs. The agonist activity of SERMs is due at least in part to AF-1 (20, 21). To develop compounds with desirable profiles of agonist and antagonist activity, particularly for use as breast cancer prevention agents that need to be taken for extended periods of time, it is necessary to fully understand the mechanisms through which these compounds exert their tissue-specific agonist and antagonist activity.
Crystallographic studies of the 4OHT- and raloxifene-bound LBD show that
the extended side chains of these ligands displace helix 12, which then
occupies the coactivator binding groove and blocks the binding of coactivators
to AF2
(1315).
The orientation of helix 12 is, therefore, a critical determinant of the
activity of the ligand-bound hER. Interestingly, the position at which
helix 12 initiates is different in the agonist- and antagonist-bound
structures; helix 12 starts at Asp-538 in the agonist-bound structure and at
Leu-536 in the antagonist-bound structures
(1315).
Particular motifs termed "capping motifs" have frequently been
found to stabilize the start of helices in model peptides and in
proteins. These motifs, which are characterized by the dihedral angles of the
peptide backbone as well as by the pattern of hydrophilic and hydrophobic
residues at the start of the helix, use hydrogen-bonding and hydrophobic
interactions to stabilize the start of
helices (reviewed in Ref.
22). Leu-536 has previously
been suggested to be part of a capping motif at the start of helix 12 in the
agonist-bound ER
; it could participate in a hydrophobic interaction
with the hydrophobic residues Leu-540 and/or Leu-541 within the helix
(23). Examination of the
crystallographic structures shows that, in the diethylstilbestrol-bound
ER
LBD (Protein Data Bank code 3ERD
[PDB]
, the side chain of Leu-536 is
oriented toward the ligand and the core of the LBD in one molecule, possibly
interacting with the side chains of Leu-540 and Leu-541, but it projects away
into the solvent in the other. On the other hand, no side chain of Leu-536 is
visible in the estradiol-bound ER
LBD (PDB code 1ERE
[PDB]
(13)). In the 4-OHT-bound
complex (PDB code 3ERT
[PDB]
(14)),
Leu-536 points toward the core of the LBD. In the complex with raloxifene (PDB
code 1ERR
[PDB]
(13)), the side
chain of Leu-536 is in direct contact with the ligand and so is predicted to
play a role in sensing the nature of the bound ligand
(Fig. 1)
(13,
14). We therefore wanted to
determine the role of hydrophobic interactions involving Leu-536 in the
E2-bound and the tamoxifen-bound states by examining the effects of
mutating Leu-536 on the activity and conformation of ER
.
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EXPERIMENTAL PROCEDURES |
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Construction of VectorsThe expression vector containing the
estrogen receptor cDNA, HEGO in pSG5, was the generous gift of Drs.
Pierre Chambon and Hinrich Gronemeyer. Mutants at Leu-536 were constructed by
site-directed mutagenesis using the Gene Editor kit (Promega). The mutations
converted leucine 536 to alanine (L536A), glutamic acid (L536E), glycine
(L536G), isoleucine (L536I), lysine (L536K), or asparagine (L536N).
The pAP1-luc reporter vector was constructed by cloning a DNA fragment corresponding to the AP-1 consensus sequence from the promoter region of human collagenase 1 (-73 to -52) into the HindIII and XhoI restriction sites of the multicloning region of pLuc-MCS vector, which has a simple TATA box between the multicloning site and the firefly luciferase coding region (Stratagene). The single strands of the AP1 site (sense: AGCTTATGAGTCAGACACCTCTGGCTTC and antisense: tcgagaagccagaggtgtctgactcata) were synthesized by Operon and were annealed into double strands for cloning.
The p2ERE-luc reporter vector was constructed by cloning a DNA fragment containing two estrogen response elements corresponding to the consensus vitellogenin ERE into the HindIII and XhoI restriction sites in the vector pLuc-MCS. The single strands (sense: AGCTTCTAGAGGATCCAGGTCACAGTGACCTGGGCCCGGATCCGGGCCCAGGTCACAGTGACCTGGCCC and antisense: tcgagggccaggtcactgtgacctgggcccggatccgggcccaggtcactgtgacctggatcctctaga) were synthesized by Operon and were annealed prior to cloning. The nature of each mutant ER and the constructed reporter vectors was confirmed by DNA sequencing. The internal control vector pRL-SV40 was purchased from Promega.
The expression vectors for mammalian two hybrid assay, which will produce
the Gal4-ER LBD and VP16-SRC1 fusion proteins, were generated by
cloning ER
LBD amino acids 264595 into pBind-CMV and SRC1 amino
acids 190400 into pAct-CMV (Promega). The Gal4-ER LBD fusion protein
contains a yeast Gal4 DNA-binding domain, which will bind to the Gal4 binding
sites of the pG5luc reporter vector (Promega). The VP16-SRC1 fusion protein
possesses the VP16 activation domain. In addition, the pBind-CMV vector
expresses Renilla luciferase for normalizing transfection efficiency.
For detecting the binding of estradiol to the wt and mutated ER
, the
cDNA encoding Ser-282 to Val-595 of the receptor was inserted into the
multicloning site of pET-42b(+) (Novagen).
Construction of Yeast Two-hybrid VectorsYeast strains
EGY48, MAT his3 trp1 ura3
leu2::6LexAop-LEU2, and RFY206, MATa his3
200
leu23 lys2
201 trp1
::hisG
ura352, have been described
(24,
25) and were purchased from
Origene. The plasmids for B42-monobody, B42-SRC-1 fusions, and
pEGER
297595 have been described
(26). Variants of
pEGER
297595 with Leu-536 mutations were constructed by
subcloning the NcoI-BamHI fragment (BamHI digestion
was followed by Klenow treatment) of pSG-hER-Leu-536 mutants into the
NcoI-XhoI segment (XhoI digestion followed by
Klenow treatment) of pEGER
297595.
Cell Transfection and Luciferase AssaysHeLa cells were
maintained in Dulbecco's modified Eagle's medium/F-12 with 1%
penicillin/streptomycin and 10% dextran-coated charcoal-treated fetal calf
serum without phenol red. Cells (3 x 105/well) were seeded in
six-well dishes and 20 h later cotransfected by 0.8 µg of ER
expression vector (wild-type or mutant), 50 ng of pRL-SV40 with either 2 µg
of p2ERE-luc or pAP1-luc reporter plasmid using SuperFect as carrier. After 4
h, the transfection medium was removed and replaced with culture medium
containing ethanol (vehicle control), 17-
estradiol (0.1, 1, 10, or 100
nM), or 4-hydroxytamoxifen (0.1, 1, 10, or 100 nM and 1
µM). After 48 h, the cells were harvested and the luciferase
activity was measured with the Dual Luciferase Assay kit on a Turner 20/20
luminometer. The activity of firefly luciferase reporter was normalized to the
activity of Renilla luciferase expressed from the internal control
vector pRL-SV40, and the results are expressed as relative luciferase units
(RLUs).
Mammalian Two-hybrid AssayFor determination of the
interactions between SRC1 and the wt or mutated ER, HeLa cells were
transfected with 1 µg of pBind-ER
(wt or mutant), 1.2 µg of
pAct-SRC1, and 1.5 µg of pG5luc using SuperFect. The empty vectors, either
1.0 µg of pBind-CMV or 1.2 µg pAct-CMV, were also transfected into
parallel cultures of HeLa cells as negative controls. After 3 h, the
transfection medium was removed, and cells were washed three times with
phosphate-buffered saline and culture medium containing ethanol (vehicle
control) or 17
estradiol (100 nM) and then maintained for an
additional 24 h. The cells were then harvested, and luciferase activity was
measured with the Dual Luciferase Assay kit on a Turner 20/20 luminometer. The
activity of firefly luciferase was normalized to the activity of the
Renilla luciferase.
Western BlottingHeLa cells were transfected with vectors
expressing the wt or mutated ER and the EGFP vector, from which
enhanced green fluorescent protein was translated and used as internal
control. The whole cell extracts were prepared, and SDS-gel electrophoresis
was carried out. The proteins were transferred onto a polyvinylidene
difluoride membrane. The membrane was immunoblotted with ER
-specific
Ab-1, and with EGFP-specific Color Antibody. Bands were visualized using the
ECLTM system.
Binding of Estradiol to the wt and Mutant
ERBL21(DE3)pLysS (Novagen) was used as host for
expressing a GST-tagged ER
(S282-V595) fusion protein. After
transformation with pET-42b(+)-hER
, a single colony was cultured in 4
ml of LB broth with glucose (1%), chloramphenicol (35 µg/ml), and kanamycin
(35 µg/ml) until the A600 was between 0.6 and 1. Two
milliliters of the culture was transferred to 25 ml of the same medium and
cultured to a density of 0.6 of A600 at 37 °C with
shaking. 20 ml of the culture was induced by
isopropyl-1-thio-
-D-galactopyranoside at 0.4 mM
for 4 h at room temperature (about 25 °C), and the cells were collected by
centrifugation. Cell extracts were prepared following procedures similar to
those described in Ref. 27,
with the exceptions that cells were lysed using a French press, and cell
debris was pelleted by centrifugation at 27,000 x g for 30 min.
Aliquots (200 µl) of the cell extracts containing wild-type or mutant
ER
s were incubated with 50 nM [3H]estradiol for 1
h at 0 °C. The nonspecific binding was determined in a parallel set of
incubations containing a 200-fold molar excess of unlabeled estradiol. Free
and bound steroids were separated by dextran/charcoal assay. The concentration
of protein in the cell extracts was measured using the Bradford protein
assay.
Yeast Two-hybrid AssayYeast was grown in YPD media or YC
dropout media following instructions from Origene and Invitrogen. Quantitative
assays were performed using the RFY206 strain containing all plasmids as
described previously for the interactions of SRC-1, monobodies (small binding
proteins) E3#6, E2#23, and the hER mutants
(26). To measure interactions
of monobodies OHT#1 and OHT#33 with the Leu-536 mutants, EGY48 harboring the
monobody plasmid and RFY206 harboring the hER
-EF plasmid and
pSH1834 were mated, and then
-galactosidase assays were performed
on the diploid cells. Assays using haploid and diploid cells, respectively,
yielded essentially the same results (not shown).
Data AnalysisTo analyze the activity of the wt and mutated ER on the ERE-driven and AP-1-driven reporters and in the mammalian two-hybrid assay (Figs. 2, 3, 4, 5, 6), the data were first subjected to a log transformation. Random effects-generalized linear models were used to assess the statistical significance of differences in response; observations from experiments run on the same day were assumed to be correlated. The first model fitted included indicators for mutants, for ligand concentrations, and for their interaction. Ligand concentrations were included in the model as indicator variables to avoid constraining the shape of the relationship between concentration and response. If the simultaneous test for interaction terms was not significant, a reduced model was fitted in which the interaction terms were omitted. Finally, an even simpler model was fitted in which ligand was parameterized as presence or absence, collapsed over different concentrations. If there was significant interaction in the full model, separate models were fitted for each mutant in which ligands were compared. In the case of significant interaction, separate models for each ligand were also fitted, and the mutants were compared. Holm's stepdown procedure was used to control type I errors when making multiple tests among coefficients. Model goodness of fit was assessed graphically.
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RESULTS |
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Activity of the Leu-536 Mutants at an ERE-driven
PromoterMutation of Leu-536 alters the response of ER to
E2 (p = 0.03). The wt ER
and the L536I mutant
exhibited 18- and 6-fold higher transcriptional activity in the presence of
E2 than their basal activity, respectively
(Fig. 2). Remarkably, all other
mutants have lost the E2 dependence of their transcription activity
(Fig. 2). Furthermore, the
activity of the L536A, L536E, L536I, and L536K mutants in the presence of
E2 is less than the activity of the E2-stimulated wt ER
(p = 0.05 for L536E; all others p < 0.001). No consistent
differences in the level of expression of the wt and mutant proteins were
detected by Western immunoblotting (Fig.
2). We tested whether the loss of response to E2 was
due to an inability of receptors having mutations at Leu-536 to bind hormone.
We expressed GST-tagged ER
(S282-V595) having the wt or mutated ER
sequence in E. coli and measured the binding of
[3H]estradiol (50 nM) in cell extracts. All
GST-ER(S282-V595) fusion proteins bound estradiol in vitro, at levels
ranging from 10.6 ± 2.5 pmol of [3H]E2 bound/mg
of protein for the non-mutated ER sequence, up to 76 ± 20 pmol of
[3H]E2 bound/mg of protein for the L536G mutation
(n = 3). Therefore, the lack of effect of E2 on
transcription activation was not due to a loss of hormonebinding activity.
Moreover, ERs having mutations at Leu-536 exhibit estradiol-dependent activity
in yeast and mammalian two-hybrid assays, which provides additional support
for their ability to bind E2 (see below). These results show that
the presence of a large hydrophobic residue, either leucine or isoleucine, at
position 536 of the hER
is critical for the ability of E2 to
stimulate activity on an ERE-driven promoter.
In addition to the effect of mutations at Leu-536 on hormonestimulated
transcription, the constitutive activity of the L536N mutant is significantly
greater than that of the wt ER (p = 0.04)
(Fig. 2). Although the median
constitutive activity of the L536A, L536E, L536G, and L536K mutants is greater
than that of the wt protein, the differences are not statistically significant
because of the variability in these experiments
(Fig. 2). Overall, our results
are consistent with the previous report that substitution of Leu-536 with
proline led to a receptor having increased constitutive activity
(28). They are also consistent
with the idea that position 536 of the hER is involved in maintaining
the receptor in an inactive state in the absence of ligand.
Response of the Mutated ERs to 4-OHT and ICI 182,780 on an ERE-driven
PromoterTo determine whether antagonists of hER modulate
the activity of the mutant ERs, we tested the effects of 4OHT and ICI 182,780
on the transcriptional activity of the wt and mutant hER
s on an
ERE-driven promoter using the same transient cotransfection assay
(Fig. 3). As reported by others
(16,
17), we found that 4OHT could
cause a significant increase in transcriptional activity of the wt ER
(p<0.001), but this increase is much weaker than that stimulated by
estradiol (compare Fig. 2 with
Fig. 3). By contrast, 4-OHT
reduced the transcriptional activity of the L536G, L536K, and L536N mutants by
one-half to two-thirds relative to their basal activity (p = 0.04 or
less) and had no effect on the activity of the L536A, L536E, and L536I mutants
(Fig. 3). Interestingly,
although the presence of ICI 182,780 alone had, as expected, no effect on the
activity of the wt ER
(Fig.
3), it reduced the activity of the L536A, L536E, L536G, L536K, and
L536N mutants (p = 0.003 or less); the effect on the L536I mutant was
not statistically significant (Fig.
3). Thus, mutations at Leu-536 either eliminate or
"invert" the activity of 4-OHT on an ERE-driven reporter and cause
the strong antagonist ICI 182,780 to reduce the basal activity of the protein.
The ability of mutations at Leu-536 to convert 4-OHT from a weak agonist to an
"inverse" agonist is reminiscent of the effects of the L540Q
mutation on the activity of ER
(29).
Activity of the Leu-536 Mutants on an AP1-driven PromoterWe
next investigated the effect of these same mutations on the activity of
ER on an AP-1-driven promoter
(16,
17,
30). The constitutive activity
of the L536A, L536G, L536K, and L536N mutants was greater than the
constitutive activity of the wt hER
on the AP1-responsive reporter
(p = 0.03 or less) (Fig.
4). It is especially striking that the L536K and L536N mutants
exhibit constitutive activities equal to (L536K) or even greater than (L536N,
p < 0.001) the 4-OHT-stimulated activity of the wild-type
ER
.
When compared with the activity of the wt ER in the presence of 4-OHT, the
activity of two mutants was greater than the wt protein (L536K and L536N;
p < 0.001); the activity of the remaining mutants (L536A, L536E,
L536G, and L536I) was indistinguishable from the wt. Because of the increased
constitutive activity of the mutated receptors, although 4-OHT increased the
activity of the mutated receptors overall (p = 0.007), the degree of
stimulation was substantially reduced, from 6.5-fold in the wild-type
ER
to
3-fold for the L536E and L536I mutants, and even less for
the others (Fig. 4). Comparing
Figs. 2 and
4 also shows that the effects
of mutations at Leu-536 depend on the promoter: the L536K and L536N mutants
display similar levels of activity on the AP-1-driven reporter (p =
0.08, difference not significant), whereas the L536N mutant exerts greater
activity on an ERE-driven reporter than does the L536K mutant (p =
0.05). Overall, these data show that Leu-536 in ER
is a critical
position for an AP1-regulated promoter.
Response of the Leu-536 Mutants to E2 and
ICI 182,780 on an AP-1-driven PromoterBecause E2 is
also an agonist for ER at AP-1 sites, we investigated the responses of
the wt and mutant hER
s to E2 on the AP-1-driven reporter. We
also wanted to determine whether ICI 182,780 exerts antagonist effects on
ER
acting through AP-1 sites. E2 increased transcription
activation by the wild-type ER
(p = 0.004), whereas as
expected ICI 182,780 by itself had no effect
(Fig. 5). E2 exerted
a marginally significant stimulatory effect on the L536E mutant (p =
0.05). By contrast, E2 reduced the activity of the L536A, L536G,
and L536N mutants (p = 0.003 or less)
(Fig. 5). E2 exerted
no significant effect on the activity of the L536I and L536K mutants
(Fig. 5). ICI 182,780 reduced
the activity of the L536A, L536G, L536K, and L536N mutants (p = 0.002
or less); it had no effect on the wt ER, the L536E, or the L536I mutant
(Fig. 5). These results show
that mutations at Leu-536 greatly reduce, and even eliminate or invert, the
agonist activity of E2 at an AP-1-driven promoter.
Interaction of the Leu-536 Mutants with SRC1 in a Mammalian Two-hybrid
AssayA critical step in the activation of transcription by ER is
the binding of coactivator proteins, such as SRC-1, by the receptor
(11,
12). We wanted to determine
whether the observed alterations in ER function, which are a
consequence of mutations of Leu-536, result from alterations in its
interaction with coactivators such as SRC-1. We used a mammalian two-hybrid
assay system to probe the interaction of the wt and mutated ER
LBDs
with endogenous coactivators as well as their interaction with the nuclear
receptor interacting domain of SRC-1.
We constructed vectors to express Gal4DBD-ER (amino acids
264595) and VP16-SRC1 (amino acids 190400) fusion proteins. The
Gal4DBD targets the DEF domains of ER
, which include the LBD, to the
Gal4 promoter on the firefly luciferase reporter vector; this vector also
constitutively expresses the Renilla luciferase, which serves as a
transfection control. The VP16-SRC1 (amino acids 190400) fusion protein
contains the receptor interacting domain (RID) of SRC-1, including three
LXXLL boxes. When the two fusion proteins associate with each other
through the ligand-dependent interaction between the hER
LBD and the
SRC1 RID, they recruit the basal transcriptional machinery to the Gal4
promoter, resulting in the production of firefly luciferase. The luciferase
activity, therefore, reflects the interaction between the hER
LBD and
the SRC1 RID, or, because the interaction between the LBD and the coactivator
is estrogen-dependent, the ability of estrogens to alter the conformation of
the hER
LBD to one that can associate with the SRC1 RID. When the assay
is carried out in the absence of the SRC-1 fusion vector, upon estrogen
binding the Gal4-ER
fusion protein will associate with the endogenous
coactivators and again stimulate transcription; this provides an indication of
the ability of the mutated ER
to interact with endogenous, full-length
coactivators. In both cases, the activity of the firefly luciferase was
normalized to the activity of the Renilla luciferase and is expressed
in relative luciferase units (RLUs).
In marked contrast to the inability of estradiol to stimulate the activity
of the mutated receptors in the transient cotransfection assays, the addition
of estradiol (100 nM) increased the activity of the wt and mutated
receptors in both the absence and presence of SRC-1 (p < 0.01,
Fig. 6). These results show
that the wt and mutated Gal4-hER LBDs could interact with endogenous
coactivators in the HeLa cells to activate reporter gene transcription in an
estradiol-stimulated manner. However, although the presence of SRC-1 increased
estradiol-stimulated transcription driven by the wt receptor (p <
0.001), it had no effect on the activity driven by the mutated receptors
(p = 0.06 for L536G; p > 0.40 for L536A, L536E, L536I,
L536K, and L536N). The 2-fold stimulation of transcription driven by the
unmutated Gal4-hER LBD by the SRC-1 RID is similar to the stimulation of
transcription driven by the full-length wt hER when full-length SRC-1
is overexpressed in HeLa cells (data not shown).
Overall, these findings demonstrate that estradiol binding to the wt and
mutated hER LBDs increases the association between the receptor and
endogenous coactivators and that the mutations introduced at Leu-536 greatly
impair the association of the hER
LBD with the SRC-1 RID in the
mammalian two-hybrid system. Again, the ability of estradiol to stimulate the
interaction between the mutated hER
LBDs and endogenous coactivators
contrasts with the inability of E2 to stimulate transcription on an
ERE-driven reporter (Fig.
2).
Probing the Conformational Changes of the Leu-536 Mutants in Yeast
Cells Using Monobodies Recognizing Specific Ligand-bound Structures of the
hER LBDAlthough the previous results demonstrate
that mutations at Leu-536 have profound effects on the activity of ER
and its response to ligands on two different promoters, they provide no direct
evidence regarding the effect(s) of these mutations on the conformation of the
receptor. To address this question, we used a number of small binding
proteins, "monobodies," that can probe ligand-induced
conformational changes of the hER
-LBD
(26). Two of the monobodies
react with the agonist-bound, wt, ER
LBD; two react with the
4-OHT-bound, wt, ER
LBD
(26). As an additional probe
for the conformation of ER
, we used a fragment of SRC-1 that contains
three NR-boxes (residues 190400) and binds specifically to the
agonist-bound form of hER
LBD
(11,
12). Note that this is the
same SRC-1 fragment used in the mammalian two-hybrid experiments. We examined
the interactions of these conformation-specific probes with the ligand-binding
domain of the wt or mutated ER
s in the absence and presence of selected
ligands using a semi-quantitative
-galactosidase assay in the yeast
two-hybrid system.
We first tested the interaction between the mutated ERs and probes
that specifically recognize the agonist-bound conformation of the wild-type
ER
, the SRC-1 fragment, monobody E2#23, and monobody E2#6. As reported
previously (26), these
reagents interacted with the wild-type receptor in the presence of
E2 but not in the presence of 1 µM concentrations of
ICI 182,780, raloxifene, 4-OHT, and progesterone or in the absence of a ligand
(Fig. 7, AC,
top panels). Like the wt ER
, the interactions of the mutants
with the SRC-1 fragment and monobody E3#6 were increased in the presence of
E2 (Fig. 7, A and
B, lower panels). By contrast, marked
interactions between the mutated receptors (except for L536I), and either the
SRC-1 fragment or monobody E3#6 were detected in the absence of ligand and in
the presence of ICI 182,780, raloxifene, and progesterone but not in the
presence of 4OHT. Thus, mutating Leu-536 increased the constitutive
interaction with probes that recognize the agonist-bound conformation of
ER
, and the increased basal reactivity was blocked by 4OHT but not
raloxifene or ICI 182,780. The enhanced SRC-1 interaction of these mutants in
the absence of ligand is consistent with the increased constitutive activity
of certain mutants on the ERE-driven and AP-1-driven reporters (Figs.
2 and
4). However, the interaction of
the mutated ERs with the SRC-1 RID in the presence of 1 µM
E2 observed in this system contrasts with the inability of the
mutated ERs to interact with SRC-1 in the presence of 100 nM
E2 in the mammalian two-hybrid system. Differences in the activity
of a mutated ER in yeast and mammalian cells have previously been reported;
for example, the S554fs mutant is a potent mediator of
E2-stimulated transactivation in yeast, yet its activity in CHO
cells is markedly impaired
(31).
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Monobody E3#23 is also specific for the
hER·LBD·agonist complex. As was observed for SRC-1
fragment and monobody E3#6, the interaction between this reagent and the wt
and mutant LBDs is increased in the presence of E2
(Fig. 7C). However,
the interaction of the mutant hER
s with this monobody
(Fig. 7C) did not show
the increased reactivity in the absence of ligand and in the presence of other
ligands observed with the SRC-1 fragment and monobody E3#6
(Fig. 7, A and
B). These results indicate that the interactions between
hER and a probe depend on the nature of a probe, its affinity for the complex,
and the particular site of the interaction. The observation that all the
mutants interacted with these probes more strongly in the presence of
E2 than in its absence is also consistent with the results of the
mammalian two-hybrid assay, and provides additional evidence that these
hER
mutants retain the ability to bind E2. However, unlike
the mammalian interaction assay, the yeast interaction assay did not show any
impairment of SRC1-LBD interaction by the mutants.
Taken together, the results provide strong evidence that the mutant
hER LBDs can assume the canonical "agonist" conformation in
the presence of 1 µM E2. The increased reactivity of
the mutants lacking a large, hydrophobic side chain at Leu-536 with agonist
conformation-specific probes in the absence of ligand suggests that removing
that side chain facilitates adoption of the agonist-bound conformation by the
hormone-free ER
.
We also found drastic changes in the interaction profile when we probed the
mutants with monobodies that recognize the 4OHT-bound conformation of
ER, OHT#1 and OHT#33 (Fig.
8). Monobody OHT#1 is highly specific to the wild-type
hER·OHT complex (Fig.
8A, top panel). Unlike the results with the
agonist complex-specific reagents above, no increase in reactivity of the
mutants with monobody OHT#1 was observed in the absence of ligand. Rather, the
mutants, except for L536I, interacted more strongly with OHT#1 in the presence
of raloxifene than in the presence of 4OHT. These results suggest that
mutating Leu-536 altered the conformation of the 4OHT-bound ER
, leading
to a reduced interaction with OHT#1; at the same time, mutating Leu-536
altered the conformation of the raloxifene-bound ER
to more closely
resemble that of the 4OHT-bound, wild-type ER
. Thus, the greatest
effect of the mutations at Leu-536 on the SERM-bound ER
is on the
actual conformation of the receptor, rather than a reduction of a barrier
between different conformations.
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When probed with monobody OHT#33, the mutations in general increased the
reactivity of the receptor to the probe in the presence of either OHT or ICI
182,780 (Fig. 8B). No
increase in reactivity was observed in the absence of ligand. However, unlike
the results with OHT#1 (Fig.
8A), no significant interaction was found between OHT#33
and the mutants in the presence of raloxifene. As was observed with the
agonistspecific monobodies above, these results indicate that the interactions
between hER and a probe depend on the nature of a probe, its affinity
for the complex, or the particular site to which it binds. We have not yet
identified the binding sites of these monobodies on hER.
Collectively, the results using the yeast two-hybrid conformation probes
indicate that these mutations do not significantly alter the conformation of
ER when bound to E2 and that they do increase the ability of
the ligand-free receptor to adopt the agonist-bound conformation. In addition,
these results show that mutations at Leu-536 alter the conformation of
hER
when complexed with 4OHT, raloxifene, and ICI 182,780.
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DISCUSSION |
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The L536N mutant exhibits the highest constitutive activity of all the
studied proteins on both AP1- and ERE-driven reporters; indeed, its
constitutive activity on the AP-1 reporter is greater than that of the
4OHT-stimulated activity of the wt ER. Most of the mutants, however,
exhibit differences in activity on the two promoters (Figs.
2 and
4). It is especially striking
that the L536K mutant, like the L536N mutant, exhibits constitutive activity
greater than or equal to the 4-OHT-stimulated activity of the wild-type
ER
on the AP-1-driven reporter, yet its activity on the ERE-driven
reporter remains less than the activity of the L536N. This mutation could
therefore be a useful tool for the dissection of ERE-driven and AP-1-driven
pathways.
The mutant ER LBDs are impaired in their interaction with SRC1 in the
mammalian two-hybrid system but not the yeast two-hybrid system, and they
exhibit greater reactivity with agonist conformation-specific reagents in the
absence of ligand in the yeast system than in the mammalian system.
Differences in the transcriptional activity of mutated ERs in yeast and
mammalian cells have previously been reported
(31). The mammalian two-hybrid
system has a smaller dynamic range (2-fold) than the yeast two-hybrid
system because of the strong activity in the absence of SRC-1. Also, in the
mammalian two-hybrid system, SRC-1 is in direct competition with endogenous
nuclear receptor coactivators. Thus, the reduced interaction of the mutant
receptors with the SRC-1 RID may be caused by a reduction of the affinity of
the receptor for the SRC-1 RID relative to endogenous coactivators.
In contrast, the yeast two-hybrid system does not have endogenous
coactivators, and, in the absence of competing coactivators, it may not be as
sensitive to small changes in the affinity of the receptor to SRC-1.
The observed difference in the results between the two systems could also suggest that either 1) the agonist-bound conformation of ER is easier for the mutants to adopt in the yeast environment than in the mammalian cell environment or 2) lower affinity interactions between proteins are more easily detected in the yeast system than the mammalian system. This in turn could result from the differences in other proteins within the two cellular environments, differences in the concentration of E2 used, 100 nM in the mammalian system versus 1 µM in the yeast system, or simply the lower temperature used in the yeast system than in the mammalian system, 30 °C versus 37 °C. The results obtained in both systems, however, are fully consistent with the idea that mutating Leu-536 alters the coupling between the binding of ligand and the conformation of the receptor.
The full-length, mutated, ER lost sensitivity to ligand stimulation
in a transient cotransfection assay; by contrast, the mutated LBD, when
analyzed in either the mammalian or the yeast two-hybrid systems, can be
stimulated by ligands. We suggest the difference between the two types of
experiments is due at least in part to the presence or absence of AF1 of
ER
, as well as the presence or absence of other receptor-interacting
regions besides the three NR boxes of SRC1. AF1 and AF2 of the hER
functionally interact to drive transcription on ERE- and AP1-driven reporters
(12). An alteration in AF2
would change the coordination between AF1 and AF2 of the hER
in the
interaction with cellular coregulators and thereby alter transactivation of
the target genes. We suggest that the increased constitutive activity of AF2
in the mutants suffices to interact with AF1 and, thereby, drive transcription
at relatively high levels in the absence of ligand. The involvement of AF2 is
supported by the ability of 4OHT to reduce the activity of several mutants on
an ERE, as well as to block the interaction of the mutants with the agonist
conformation-specific probes in the yeast two-hybrid system.
Mutations at Leu-536 caused tamoxifen to repress the activity of ER
on an ERE, as well as causing E2 to repress the activity of the
hER
on an AP-1-driven reporter (Figs.
3 and
5). We also observed that
mutations at Leu-536 greatly reduced binding to SRC1 by ER
in the
mammalian two-hybrid assay (Fig.
6). The loss or inversion of activity of 4OHT and E2 on
specific promoters is reminiscent of the effects of deleting the C-terminal 58
amino acids of ER
, including helix 12
(32). This deletion did not
eliminate the ability of ER
to bind ligand and DNA but caused the
hER
to repress ERE-dependent transaction in a E2- and
tamoxifen-dependent manner, to constitutively interact with silencing mediator
for retinoic acid and thyroid hormone receptors (SMRT), and to eliminate the
binding to SRC1 (32).
Similarly, the L540Q mutant of ER
binds E2 yet responds to
ligands in an inverse manner and does not interact with SRC1, but does react
with a corepressor (29). The
similarities in the phenotypes of the mutants are consistent with a structural
model predicting a hydrophobic interaction between Leu-536, Leu-540, and/or
Leu-541, so that mutation of these residues would impair formation of the
active ER conformation. However, this is contradicted by the striking increase
in the constitutive reactivity of the mutated receptors with either SRC1 or
the agonist-conformation-specific monobodies in the yeast two-hybrid system,
which shows that formation of the active ER
conformation in the absence
of ligand is increased by the mutations at Leu-536. Thus, additional studies
are necessary to specifically determine the effects of the mutations on the
inactive state of ER
, the active state of ER
, and the transition
between the inactive and active states in the absence and presence of
E2.
Additional support for the idea that residues near the start of helix 12
are important in the function of ER comes from recent studies of
Asp-538, another residue located near the start of helix 12 in the structure
of the agonist-bound ER
. This residue, although not part of the
coactivator interface formed by helices 12, 3, and 5, has been shown to affect
the function, stability, and interaction of ER
with coactivators and
corepressors (33). Asp-538,
which is necessary for the agonist activity of 4OHT, has been defined as part
of a transcription activation region termed "AF2b"
(33). It has also been
suggested that Asp-538 interacts with AF1
(33). Certain mutations of
another neighboring residue, Tyr-537, alter the affinity and kinetics of the
interaction between estradiol and the hER
(27,
34). It will be interesting to
determine the effects of mutations at Leu-536 on the affinity and kinetics of
hormone binding to ER
.
Finally, the effects of mutations at Leu-536 on the conformation of the
SERM-bound ER are strikingly revealed in the experiments with the
monobody probes that recognize the OHT-bound wt ER
. When we studied the
interaction between monobodies OHT#1 and OHT#33 with the LBD having mutations
at Leu-536, the results demonstrated that the structures of the mutated,
ligand-bound LBDs had changed substantially. These experiments strongly
support the importance of Leu-536 in maintaining particular conformations of
the SERM-bound hER
; in other words, Leu-536 is "reading"
the extended side chain of raloxifene, 4-hydroxytamoxifen, and ICI
182,780.
Overall, our findings provide strong support for the idea that Leu-536 is
critical in coupling ligand binding with the conformational changes of
ER not only in the agonist-bound ER
but in the SERM-bound
ER
as well. Additional studies of this interesting region of the
protein are ongoing.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Dept. of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. Tel.: 313-577-1550; Fax: 313-577-5494; E-mail: dskafar{at}med.wayne.edu.
1 The abbreviations used are: hER, human estrogen receptor-
;
E2, 17
-estradiol; 4OHT, 4-hydroxytamoxifen; DBD, DNA-binding
domain; LBD, ligand-binding domain; ERE, estrogen response element; SERMs,
selective estrogen receptor modulators; EGFP, enhanced green fluorescent
protein; CMV, cytomegalovirus; RLU, relative luciferase unit(s); wt, wild
type; GST, glutathione S-transferase; aa, amino acid(s); RID,
receptor interacting domain; DES, diethylstilbestrol.
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REFERENCES |
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