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and Estrogen Receptor-ß: Correlations with Biological Character and Distinct Differences among SRC Coactivator Family Members1
Departments of Molecular and Integrative Physiology (D.M.K., J.S., B.S.K.), Cell and Structural Biology (B.S.K.), and Chemistry (J.A.K.), University of Illinois and College of Medicine, Urbana, Illinois 61801
Address all correspondence and requests for reprints to: Dr. Benita S. Katzenellenbogen, Department of Molecular and Integrative Physiology, 524 Burrill Hall, 407 South Goodwin Avenue, University of Illinois, Urbana, Illinois 61801-3704. E-mail: katzenel{at}life.uiuc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or ERß would be
useful in elucidating the biology of these two receptors and might
assist in the development of estrogen pharmaceuticals with improved
tissue selectivity. In this study, we examine three compounds of novel
structure that act as ER subtype-selective ligands. These are a propyl
pyrazole triol (PPT), which is a potent agonist on ER but is
inactive on ERß, and a pair of substituted tetrahydrochrysenes (THC),
one enantiomer of which (S,S-THC) is an agonist on both ER and
ERß, the other (R,R-THC) being an agonist on ER but an antagonist
on ERß. To investigate the molecular mechanisms underlying the ER
subtype-selective actions of these compounds, we have determined the
conformational changes induced in ER and ERß by these ligands
using protease digestion sensitivity, and we have tested the ability of
these ligands to promote the recruitment of representatives of the
three SRC/p160 coactivator protein family members (SRC-1, GRIP-1, ACTR,
respectively) to ER and ERß using yeast two-hybrid and
glutathione-S-transferase (GST) pull-down assays. We find that the
ligand-ER protease digestion pattern is distinctly different for
stimulatory and inhibitory ligands, and that this assay, as well as
coactivator recruitment, are excellent indicators of their
agonist/antagonist character. Interestingly however, compared with
estradiol, the novel agonist ligands show some quantitative differences
in their ability to recruit SRC-1, -2, and -3. This implies that while
generally similar to estradiol, these ligands induce ER conformations
that differ somewhat from that induced by estradiol, differences that
are illustrative of the nature of their biological character. The
application of methods to characterize the conformations induced in ER
subtypes by novel ligands, as done in this study, enables a greater
understanding of how ligand-receptor conformations relate to estrogen
agonist or antagonist behavior. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (1, 2, 3, 4, 5), has added another
layer of complexity to the selectivity issue and has revitalized the
research effort to find estrogen pharmaceuticals having tissue- and
cell-selective activity (6, 7, 8, 9). ER and ERß are quite
different in their ligand-binding domains, having only 56% identity,
suggesting that they might bind some ligands with different affinity
and that these ligands might also have different agonist or antagonist
character. Subtype-selective ligands for other members of the nuclear
receptor superfamily, PPAR (10, 11, 12, 13), RAR
(14, 15, 16, 17, 18), and TR (19) have proved to be very
important.
The pharmacology of estrogen receptors is tripartite. Transcriptional
activities of ER and ERß are influenced not only by ligands, but
also by coregulator proteins with which they associate
(20, 21, 22). One group of related p160 coactivator proteins,
encoded by three distinct genes, seems to be particularly important in
enhancing the transcriptional activity of steroid hormone receptors.
This includes SRC-1 (23), SRC-2 [also known as TIF2
(24) or GRIP-1 (25)], and SRC-3 [also known
as ACTR (26), AIB1 (27), p/CIP
(28), RAC3 (29), and TRAM-1
(30)]. The recruitment of coactivator proteins by
estrogen receptors and other steroid hormone receptors complexed with
their respective ligands seems to be a good indicator of their
transcriptional activity, and thus is also thought to reflect the
conformation induced in these receptors by their ligands.
Crystallographic studies of the ligand-binding domains of ER
(31, 32) and ERß (33) have revealed that
both have a similar overall structure, although the conformation of
portions of this domain changes in response to ligands having different
biological character (i.e. agonist vs. antagonist
activity). When bound by the agonist estradiol or diethylstilbestrol,
ER adopts a conformation that positions helix H12, the AF-2 core
helix, in a manner that completes the formation of a hydrophobic groove
involving helices H3, H5, H6, and H12. This hydrophobic groove has been
shown to be important for binding an NR box (LXXLL) motif found in p160
coactivator proteins (32, 34, 35, 36). By contrast, when ER
is complexed with an antagonist, raloxifene or hydroxytamoxifen, helix
H12 is displaced from this position and becomes repositioned so that it
occupies the hydrophobic coactivator binding groove, precluding
coactivator binding. In most respects, the corresponding ERß
complexes have similar conformations (33). Thus, the
proper repositioning of helix H12 by agonist ligands seems to be a good
indicator of the ability of nuclear receptors to bind coactivator
proteins and appears to be a strong determinant of the magnitude of
transcriptional activity of the receptor. Consistent with these
findings, recent data obtained through the use of phage-displayed
peptide libraries have suggested that various ER ligands induce
distinct conformational changes in ER and ERß that correlate with
the ability of these receptor subtypes to bind the NR box motif in
coactivator proteins (37, 38, 39).
Recently, we identified tetrahydrochrysene (THC) enantiomers that act
as ER subtype-selective ligands. The S,S-enantiomer (S,S-THC) is an
agonist on both ER and ERß, whereas the R,R-enantiomer (R,R-THC)
is agonistic on ER but antagonistic on ERß (40, 41).
In addition we describe here a propyl pyrazole triol (PPT; 4-propyl-1,
3, 5-Tris (4-hydroxyphenyl) pyrazole), which is a potent agonist on
ER and is inactive on ERß, becoming an ERß antagonist only at
very high concentrations. The unusual structures of these
subtype-selective ligands raise interesting questions concerning the
relationships between ligand structure, ER conformation, coactivator
recruitment and biological activity that we explore in this report.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids
The expression vectors for human ER (pCMV5-hER) and human
ERß 1–530 (pCMV5-ERß) were constructed as described (41, 42). The estrogen-responsive reporter plasmid
(ERE)3-pS2-CAT (43) was previously
described. The complement C3-Luc reporter plasmid, kindly provided by
Donald McDonnell (Duke University, Durham, NC), was recently described
(44). The expression vector for human ER
(pBSIISK+ ER) for in vitro
transcription and translation was described (45). The
expression vector for human ERß (pCR 2.1 ERß) for in
vitro transcription and translation was prepared by inserting the
complementary DNA for hERß (1–530) into pCR 2.1
(Invitrogen, Carlsbad, CA). The yeast two-hybrid
expression plasmid pBD-GAL4 ER was recently described
(46). pBD-GAL4 ERß was constructed by subcloning ERß
(229–530>) into the
SalI/SmaI-digested pBD-GAL4
(Stratagene, La Jolla, CA). pGAD-424 SRC-1 full-length
(47) and pGAD-424 GRIP-1 full-length (25)
were provided by Michael Stallcup (USC, Los Angeles, CA). pGAD-10 ACTR
A16 (26) was obtained from Ron Evans (The Salk Institute,
La Jolla, CA). The GST fusion protein expression plasmid pGEX-2TK ER
(48), which contains the human ER spanning 282–595,
was kindly provided by Myles Brown (Harvard Medical School, Boston,
MA). The GST fusion protein expression plasmid pGEX 4T-1 ERß, which
contains the human ERß spanning 243–530, was prepared by inserting
the complementary DNA for ERß (243–530) into the
BamHI site of pGEX 4T-1 (Pharmacia & Upjohn, Piscataway, NJ). The expression plasmids for
in vitro transcription and translation, pBK-CMV SRC-1
(23), pSG5 GRIP-1 (47), and pCMX ACTR
(26) were provided by Ming Tsai and Bert O’Malley
(Baylor, Houston, TX), Michael Stallcup, and Ron Evans (The Salk
Institute), respectively.
Ligand binding assays
Ligand binding affinities were determined by competitive
radiometric binding assays as previously described (49).
Briefly, these assays used 10 nM
[3H] estradiol as tracer, purified preparations
of baculovirus-expressed human ER (1–595) and ERß (1–477) from
Panvera (Madison, WI), and hydroxylapatite to adsorb bound
receptor-ligand complex. Incubations were done at 0 C for 18
h.
Cell culture and transient transfections
Human endometrial cancer (HEC-1) cells were maintained in
culture and transfected as described (50). Briefly,
transfection of HEC-1 cells in 60-mm dishes used 0.4 ml of a calcium
phosphate precipitate containing 0.5 µg of pCMV ß-Gal as internal
control, 2 µg of the reporter gene plasmid, 100 ng of ER expression
vector, and carrier DNA to a total of 5 µg DNA. CAT or luciferase
activity, normalized for the internal control ß-galactosidase
activity, was assayed as described (51).
Protease digestion assays
The protease digestion assay was performed as described by
Lazennec et al. (52), with minor modifications.
Briefly, ER (1–595) and ERß (1–530) were generated in
vitro as radiolabeled proteins using the TNT-coupled
transcription-translation system according to the manufacturer
(Promega Corp., Madison, WI). Aliquots (50 µl) of the
[35S]-labeled proteins were incubated with
control (0.1%) ethanol vehicle or ligand at a final concentration of
10-5 M for 20 min at 22 C.
Aliquots (5 µl) of the ligand-treated receptor were incubated without
trypsin or with trypsin at a final concentration of 1.25, 2.50, 3.75,
5, 10, 15, or 20 µg/ml (Worthington Biochemical Corp.,
Freehold, NJ). After a 10-min incubation at 22 C, the digestions were
halted with 20 µl of Laemmli buffer. The samples were analyzed on a
12% SDS-PAGE gel and visualized by autoradiography.
Yeast two-hybrid transformation and ß-galactosidase assays
The yeast strain YRG-2 (Stratagene), made competent
with lithium acetate, was cotransformed with ER (pBD-GAL4 ER),
ERß (pBD-GAL4 ERß), or pBD-GAL4 and SRC-1 (pGAD-424 SRC-1), GRIP-1
(pGAD-424 GRIP-1), ACTR (pGAD-10 ACTR), pGAD-424, or pGAD-10.
Transformants were plated on media lacking leucine and tryptophan
(-leu-trp) and were grown for 3 days at 30 C to select for yeast that
had acquired both plasmids. Triplicate independent colonies from each
plate were grown overnight in 2 ml of (-leu-trp) liquid media with
0.1% ethanol vehicle or increasing concentrations
(10-8, 10-7,
10-6, 10-5 M)
of estradiol, R-R, THC, S,S-THC, or PPT. Cells were harvested and
assayed for ß-galactosidase activity as described
(53).
In vitro protein interaction assays
SRC-1, GRIP-1, and ACTR were generated in vitro as
radiolabeled proteins using the TNT-coupled transcription-translation
system according to the manufacturer (Promega Corp.,
Madison, WI). GST, GST-ER, and GST-ERß were individually expressed
in the BL21 (DE3) strain of Escherichia coli (Novagen,
Madison, WI) and each was purified to homogeneity by
glutathione-agarose affinity chromatography. GST, GST-ER, or
GST-ERß was bound to glutathione-agarose and equilibrated with
GST-binding buffer (1x GBB: 20 mM Tris, pH 7.6,
50 mM NaCl, 1 mM
dithiothreitol, 0.2% NP-40, and protease inhibitors: 4.0 µg/ml
aprotinin, 2.0 µg/ml leupeptin, 1.0 µg/ml pepstatin A, and 0.2
mM phenylmethylsulfonyl fluoride) and with 0.1%
ethanol vehicle or increasing concentrations
(10-8, 10-7,
10-6, 10-5
M) of estradiol, R,R-THC, S,S-THC, or PPT.
[35S] methionine-labeled proteins were
incubated with the immobilized GST fusion proteins in 100 µl of 1x
GBB for 1 h at 4 C. The beads were washed three times with 1x GBB
(0.5 ml) and twice with 50 mM Tris, pH 8.0 (0.5
ml) buffer. Bound proteins were eluted with 10 mM
reduced glutathione in 50 mM Tris buffer. Eluted
proteins were resolved by SDS-PAGE and visualized by autoradiography.
Images were quantitated using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the distinct
transcriptional activities of the three ER subtype-selective ligands,
R,R-THC, S,S-THC, and PPT. Human endometrial cancer (HEC-1) cells were
transfected with an expression plasmid for ER or ERß, together
with an estrogen-responsive reporter gene construct
[(ERE)3-pS2-CAT)], and were treated with
increasing concentrations of the three ligands, or with estradiol
(E2) for comparison. Interestingly, PPT behaved
as a potent ER agonist but failed to activate ERß, even at very
high concentrations. Similar findings were observed for PPT using
several different estrogen-responsive reporter gene constructs with
consensus, nonconsensus, and composite response elements in several
different cell types (data not presented). The S,S-THC was an agonist
on both ER and ERß, whereas R,R-THC was also an agonist on ER
but was a complete antagonist on ERß. PPT has a high relative binding
affinity for ER (49% ± 12% that of estradiol) but very low
binding affinity for ERß (0.12% ± 0.04% that of estradiol). The
good binding affinity of PPT for ER is reflected in the PPT
dose-response curve for transactivation of ER. PPT fails to activate
ERß even at very high concentrations (Fig. 1, and also at 10
µM, not shown). Its low affinity for ERß is evident by
its weak antagonist activity that is first seen at 1 µM
(and becomes more complete at 10 µM, not shown). Although
the two THC compounds are 6- to 15-fold ERß selective in their
binding affinities (40), these differences do not define
whether they are an agonist or an antagonist.
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and ERß reflected by differences in their proteolytic digestion
patterns and ERß
in the presence or absence of these novel ER ligands to evaluate the
ability of these ligands to induce active or inactive conformations in
the ERs. Such an approach has been used previously by us and others in
ER, where it was shown that agonists and antagonists stabilized
different size core ligand binding domains (52, 54, 55, 56).
These proteolytic digestion patterns of [35S]
methionine-labeled ER or ERß, complexed with the different
compounds, were analyzed by denaturing gel electrophoresis after
exposure to different concentrations of trypsin. These analyses are
shown for ER in Fig. 2 and for ERß
in Fig. 3.
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), ER is highly sensitive to
trypsin and gives a proteolytic digestion pattern in which the fragment
sizes decrease with increasing concentrations of protease (Fig. 2A).
The full-length, unliganded ER (arrow a), approximately
66 kDa, is converted to two proteolytic cleavage products of
approximately 28 kDa (arrow b) and 25 kDa (arrow
c), which are characteristic of the receptor being in the inactive
conformation. When the receptor is occupied with estradiol, only the
upper band is strongly stabilized (Fig. 2B, arrow b),
suggesting that the receptor in the active conformation is protected
from further cleavage by trypsin. Interestingly, ER when bound by
the antagonist ICI 182,780 (Fig. 2C) shows a cleavage pattern similar
to that of the unliganded, inactive ER (Fig. 2A). When ER is
liganded with either R,R-THC (Fig. 2D), S,S-THC (Fig. 2E), or PPT (Fig. 2F), each complex shows a degradation pattern similar to the
estradiol-bound, active conformation (Fig. 2B), each ligand strongly
stabilizing the upper 28 kDa band (arrow b). Stabilization
of an active ER conformation by these three ligands is consistent
with the observation that all three are ER agonists.
With ERß, agonist ligands also stabilize a larger form than do
antagonist ligands, but the sizes of these forms are somewhat smaller
than those with ER (ERß 26 and 24 kDa, Fig. 3, arrows b
and c, vs. ER 28 and 25 kDa, Fig. 2, arrows b and c). Most interestingly, the agonist
or antagonist character of the three novel ligands on ERß was clearly
reflected in their trypsin digestion patterns. Only the ERß agonist
ligand, S,S-THC, stabilizes the higher molecular weight form
characteristic of the active, estradiol-bound form (Fig. 3, B and E),
whereas the ERß antagonists, R,R-THC and PPT (Fig. 3, D and F),
generate this higher, as well as the lower molecular weight form,
characteristic of unliganded (Fig. 3A) or ICI 182,780 bound ERß (Fig. 3C).
Aside from these characteristic agonist vs. antagonist
ligand-induced differences in proteolysis of each receptor form, there
is a basic difference in the ultimate degradation product of the two ER
subtypes. At the highest concentrations of trypsin tested, ER in
both the inactive and active conformations is eventually degraded to a
band of approximately 17 kDa (Fig. 2, A–F, arrow d),
whereas both active and inactive conformations of ERß are not
degraded beyond the 26- and 24-kDa forms. Even in the presence of
higher concentrations (80 µg/ml) of trypsin, or when the time of
digestion was increased (20 min), ERß remained resistant to
proteolytic cleavage beyond the 24 kDa species (data not shown).
Subtype-selective ER ligands induce distinct patterns of
coactivator protein interaction with ER and ERß
Because the interaction of ER with coactivators is believed to
determine the magnitude of transcriptional activity of the receptor, we
examined the three selective ER ligands for their ability to recruit
coactivator proteins to ER and ERß, using yeast two-hybrid and
GST-pulldown assays. Interactions were monitored with a representative
member of the three different classes of the p160 nuclear receptor
coactivator proteins, namely, SRC-1, SRC-2, and SRC-3.
In the yeast two-hybrid system, concentration-dependent increases in
the interaction between ER and SRC-1 were observed with estradiol,
R,R-THC, S,S-THC, and PPT (Fig. 4A), all
of which are agonists on ER. Interestingly, when these ligands were
examined with ERß (Fig. 4B),
concentration-dependent increases in the interaction between ERß and
SRC-1 were observed with the ERß agonists, estradiol, and S,S-THC.
However, essentially no interaction was detected between ERß and
SRC-1 in the presence of R,R-THC or PPT, even at the highest
concentrations tested (Fig. 4B), consistent with the antagonist
character of these ligands on ERß. Similar patterns were observed for
the interaction of ER and ERß with GRIP-1 (also known as TIF2 or
SRC-2) (Fig. 4, C and D) and with ACTR (also known as AIB-1, p/CIP,
RAC3, TRAM-1, or SRC-3) (Fig. 4, E and F) in the presence of estradiol,
R,R-THC, S,S-THC, or PPT.
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and ERß, complexed
with estradiol, R,R-THC, S,S-THC, or PPT, with the three different
classes of p160 coactivator proteins, by in vitro
glutathione S-transferase (GST) pull-down assays (Figs. 5, 6, and 7). The findings by this in
vitro assay were consistent with those of the two-hybrid assay and
were good predictors or whether a ligand was an agonist or an
antagonist on ER or ERß.
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, depicting interactions of
[35S]-labeled SRC-1, strong interaction with
ER and ERß is seen in the presence of estradiol (lanes 4–7 and
23–26), but not in its absence (lanes 3 and 22). No interaction was
observed with purified GST alone (lanes 2 and 21). PPT, which is an
agonist on ER and is inactive on ERß, showed concentration-
dependent interaction with SRC-1 on ER (lanes 16–19), but no
interaction on ERß (lanes 35–38). Similarly, the ER agonist,
ERß antagonist, R,R-THC, showed interaction with SRC-1 through ER
(lanes 8–11), but not ERß (lanes 27–30). S,S-THC, which is an
agonist on both ER subtypes, recruited SRC-1 to both ER (lanes
12–15) and ERß (lanes 31–34). Patterns of SRC-2 (Fig. 6) and SRC-3
(Fig. 7) recruitment by these three ligands to ER and ERß were
qualitatively similar to those observed with SRC-1, that is,
recruitment was observed only with ligand-receptor combinations that
were agonistic.
Both of the coactivator recruitment assays gave consistent results that
correlated with the biological character of the ligand-receptor
combinations. However, some distinct quantitative differences were
observed. The magnitude of coactivator recruitment in both assays is
summarized in Table 1. PPT was equivalent
to estradiol in recruiting all three SRCs to ER. However, the two
THC ligands were always less effective than estradiol in recruitment of
coactivators, and on ER, the order of efficacy was SRC-1 >
SRC-2 > SRC-3. In particular, recruitment of ACTR (SRC-3 family)
to ER by both THCs was only about half that observed for SRC-1. This
suggests that the THC ligands put ER into a conformation that is
somewhat different from that of estradiol, resulting in differences in
efficacy in the recruitment of the three different p160 coactivators.
In contrast, the ERß agonist S,S-THC showed a similar magnitude of
recruitment of SRC-1, SRC-2, and SRC-3 to ERß, but this magnitude of
recruitment was slightly lower (70–80%) than that of estradiol.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies comparing the activity of novel ligands for ER and
ERß, however, indicate that the relationship between ligand
structure, ER conformation, and biological activity may be more
complex. In particular, we have found that certain tetrahydrochrysene
(THC) ligands are agonists on ER but antagonists on ERß, even
though they lack the bulky substituents characteristic of ER
antagonists. In addition, we find that another nonsteroidal ligand,
propyl pyrazole triol (PPT), has vastly higher affinity for ER than
for ERß, and is able to activate the former ER but not the
latter.
We have shown in this report that regardless of the structure of the ER subtype-selective ligand, those combinations of ligand and ER subtype that activate transcription have an agonist-like conformation of the ligand binding domain (as determined by protease sensitivity) and are able to effectively recruit the three p160 coactivators, whereas those combinations that are unable to activate transcription have an antagonist-like ER conformation that fails to recruit the coactivators. There are, however, significant differences in the levels and dose-response of coactivator recruitment shown by some of these ligands that are illustrative of particular quantitative differences in their agonist activity.
Conformations induced in ER and ERß by subtype-selective
ligands are reflective of their biological character
Protease sensitivity has proved to be a reliable method for
characterizing the ligand-induced conformations of the ER ligand
binding domain (52, 54, 55, 56). This is the first study to
examine the protease sensitivity of ER and ERß complexed with ER
subtype-selective ligands. Full-length 66 kDa ER is rapidly cleaved
by trypsin to a 28- kDa ligand binding domain core. Subsequent cleavage
to a 25-kDa species occurs more rapidly in ER complexes with
agonists than with antagonists. It is of note that the ligands in
this study, R,R- and S,S-THC and PPT, which are all ER agonists,
are as effective as estradiol in stabilizing the 28 kDa form of ER,
indicating that they induce an agonist-like conformation. With respect
to agonist/antagonist conformation discrimination, proteolysis of ERß
is similar to ER. Again, trypsin exposure reveals a somewhat smaller
stable core structure of 26 kDa, and further cleavage to a 24-kDa form
occurs more rapidly in the antagonist than in the agonist complexes.
What is significant for this study is that the two new subtype specific
ligands, R,R-THC and PPT, that are agonists on ER but antagonists on
ERß, induce agonist conformations in ER but antagonist
conformations in ERß, as probed by trypsin sensitivity. Thus, in
every case, the protease sensitivity of each combination of ligand and
ER subtype that activates transcription shows an agonist-like
conformation, whereas those combinations that do not activate
transcription have an antagonist-like structure.
Despite the similarity between ER and ERß in terms of the trypsin
cleavage patterns of the agonist vs. antagonist
ligand-induced conformations (28 to 25 kDa and 26 to 24 kDa forms,
respectively), there is a notable difference between ER and ERß
with respect to the further cleavage of these forms. Continued exposure
of ER to trypsin results in formation of smaller forms
[approximately 17 and 6–9 kDa (56, 57)]. This is the
result of an internal cut at K467, which is in an accessible loop in
ER between helices H9 and H10 (57). By contrast,
continued exposure of ERß to trypsin does not result in further
cleavage. This observation is consistent with findings recently
reported by Van Den Bemd et al. (58), and
with the fact that the helix H9/H10 loop in ERß is smaller and
does not contain either a lysine or arginine residue.
Coactivator interaction assays of ER and ERß faithfully
reflect the biological character of the ER subtype- selective
ligands
Coactivator recruitment/interaction assays provide a
rich context for examining the molecular pharmacology of different
nuclear hormone receptor complexes. Our studies show that the
agonist/antagonist character of the ligand is reflected in the ability
of the receptor complex to recruit different members of the p160
family. In general, in both the yeast two-hybrid assay and the GST
pull-down experiments, the pattern of coactivator recruitment by ER
and ERß was that which was expected on the basis of the
agonist/antagonist character of the ligands with which they were
complexed: the three novel ligands, as well as estradiol, recruited all
three p160 coactivators to ER, but only E2 and
S,S-THC recruited these coactivators to ERß. However, there were some
notable differences in the magnitude and dose response of the
recruitment.
On ER, PPT was as good as E2 in
recruitment of the three coactivators, SRC-1, SRC-2, and SRC-3,
although somewhat higher concentrations of PPT than
E2 were required for equivalent recruitment of
these coactivators. ER complexes with R,R-THC and S,S-THC were
somewhat less effective in the extent of recruitment of all of the
coactivators, and the dose response for coactivator recruitment by
these ligands was markedly shifted to the right, compared with
estradiol. These THC ligands were notably less effective in the
recruitment of ACTR to ER, as observed in both the two-hybrid
and GST pulldown assays. These differences may underlie the fact
that these THC ligands are agonistic, but not quite full agonists
on ER (as observed in transactivation assays, see Fig. 1).
Two of the ligands, R,R-THC and PPT, are not agonists on ERß, and
consistent with this, they fail to recruit any of the p160 coactivators
to ERß. By contrast, S,S-THC, which has substantial agonist activity
on ERß, does recruit these coactivators, but with reduced magnitude
and a right-shifted dose response compared with
E2. The ability of S,S-THC to recruit
coactivators to ERß is similar to its ability to recruit them to
ER, consistent with its similar level of agonist transactivation
activity on both ER subtypes.
Although it is now widely accepted that the p160 coactivator proteins
bind to the AF-2 domain of nuclear receptors through NR box (LXXLL)
motifs within each coactivator protein, accumulating evidence suggests
that there are distinct differences between each class of coactivators.
In fact, recent data have suggested that nuclear receptors display both
coactivator and NR box preferences (47) and that different
classes of coactivators recognize distinct but overlapping binding
sites on the ER ligand binding domain (59). In our
coactivator interaction assays, we found differences in the relative
strength of interaction of ER and ERß with SRC-1, SRC-2, and SRC-3
in the presence of agonist ligands. These differences may prove to be
physiologically important and contribute to selective activities,
depending on the expression levels of each of these coregulators in a
particular cell type or tissue.
Protease sensitivity and coactivator recruitment provide different
levels of discrimination of ligand-induced conformations of ER
subtypes
It is of interest that the differences in the pharmacology of
these new ER subtype selective ligands are quite accurately reflected
in the conformations induced in the ER subtypes, as probed by protease
digestion patterns, as well as in their distinct patterns of
coactivator recruitment. Protease digestion with trypsin was able to
clearly discriminate between those ligand/receptor complexes that were
agonist-like vs. unoccupied/antagonist-like, but this
methodology did not distinguish between the full agonist
E2 and the partial agonists R,R- and S,S-THC on
ER and the S,S-THC on ERß.
Although protease digestion can provide useful information on protein
conformation, it can only do so at sites where the protein provides a
sequence that is cleavable by the particular protease. In the case of
both ER and ERß, trypsin is particularly useful, because there are
two lysine residues at the C-terminus of helix-11 of the hormone
binding domain (K529/531 in ER and K480/482 in ERß). From x-ray
structures of ER, it is known that in the agonist
(E2 and DES) complexes these sites are in a
structured region (hence limited in protease access), whereas they are
more disordered in the antagonist complexes (raloxifene and
hydroxytamoxifen) (31, 35). It is presumed that these two
lysines are also in a disordered region in the unoccupied ERs, as well
as in ERs occupied by the full antagonist ICI 182,780, although this
crystallographic information is not yet available.
By contrast, the two-hybrid assay and GST pull-down experiments, which
provide mutually confirmatory information on coactivator recruitment by
ER and ERß complexed with the various ligands studied in this
report, gave results that were more discriminating of ER conformations
than protease sensitivity. In fact, from the coactivator interaction
assays, we were able to see differences in the magnitude and dose
response of the recruitment of the three p160 class coactivators by the
subtype selective ligands. In particular, we were able to discriminate
between those ligands that were nearly full agonists (vs.
the full agonist E2) and those that had different
affinities for the different ER subtypes. These differences suggest
that although these subtype-selective ligands produce considerable
transcriptional activation and promote the binding of coactivator
proteins, they probably do not reposition helix H12 (the AF-2 domain)
in precisely the same way that estradiol does.
Continued investigation of our novel subtype-selective ER ligands and
their ability to promote transactivation and coactivator recruitment
should increase our understanding of the contribution of ligands,
receptors, and coregulators in the regulation of ER target genes.
Compounds with differing ER subtype potency and efficacy
(agonist/antagonist character) must almost certainly have interesting
differences in the detailed interaction between ligand and ER. These
differences will need to be probed at a greater level of detail by
x-ray crystallographic analysis and computational modeling. The
solution of the crystal structures of the ER and ERß ligand
binding domains with these ligands, particularly R,R-THC and PPT, would
be of great assistance in further understanding the molecular
determinants of the subtype-selective activity of these compounds.
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Acknowledgments |
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Footnotes |
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Received April 26, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Biochemistry 38:185–190[CrossRef][Medline]
and PPAR 
ligands produce distinct biological
effects. J Biol Chem 274:6718–6725-selective antagonists. J Med Chem 40:2445–2451[CrossRef][Medline]
. Biochem Biophys Res Commun 186:977–983[CrossRef][Medline]
or ß- ligands. Mol Pharmacol 40:556–562[Abstract]
and ß. Mol Cell Biol 19:8226–8239 and ER ß. Proc Natl Acad Sci USA 96:3999–4004 or
estrogen receptor-ß. Endocrinology 140:800–804 and
ß by both the antiestrogens ICI 164,384 and ICI 182,780 leading to
opposite effects on receptor stability. Biochem Biophys Res Commun 261:1–5[CrossRef][Medline]
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|
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|
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|
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|
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|
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|
|
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 |
|
 D. P. McDonnell, C. E. Connor, A. Wijayaratne, C.-y. Chang, and J. D. Norris Definition of the Molecular and Cellular Mechanisms Underlying the Tissue-selective Agonist/Antagonist Activities of Selective Estrogen Receptor Modulators Recent Prog. Horm. Res., January 1, 2002; 57(1): 295 - 316. [Abstract] [Full Text] [PDF]
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