(Received for publication, September 20, 1996)
From the Department of Molecular and Integrative
Physiology and the § Department of Chemistry, University
of Illinois, Urbana, Illinois 61801
We have previously examined, by
alanine scanning mutagenesis, amino acids 515-535 of the estrogen
receptor (ER) ligand binding domain to determine which of these
residues are important in estradiol binding. Mutation at four sites
that potentially lie along one face of an -helix,
Gly521, His524, Leu525, and
Met528, all significantly impaired estradiol binding by the
ER (Ekena, K., Weis, K. E., Katzenellenbogen, J. A., and
Katzenellenbogen, B. S. (1996) J. Biol. Chem. 271, 20053-20059). In this report, we compare the pattern of residues that
are important in the recognition of several structurally diverse
estrogen agonists and antagonists (the synthetic nonsteroidal agonist
hexestrol, an agonist derived from the mold metabolite zearalenone,
P1496, and the partial agonist-antagonist trans-hydroxytamoxifen) with those that are predicted to
contact estradiol in the receptor-ligand complex. Although there are
some similarities in the pattern of residue recognition among all four ligands, each ligand showed distinct differences as well.
Interestingly, alanine substitution at only one residue, the leucine at
position 525, was found to inhibit binding of all the ligands tested.
Another residue, His524, was found to be important in the
recognition of three different agonists but not
trans-hydroxytamoxifen (the only ligand lacking a second
hydroxyl group). The recognition of estradiol and another agonist,
P1496, was impaired by the G521A mutation, whereas ligand-induced activity by the two compounds that lack B- and C-rings, hexestrol and
trans-hydroxytamoxifen, was unaffected. Our findings
demonstrate that these ligands fit into the ER ligand binding pocket
differently and that each contacts a distinct set of amino acids. The
smaller ligands (estradiol and hexestrol) have a narrower footprint of interacting residues than the larger ligands (P1496 and
trans-hydroxytamoxifen). This pattern of interaction is
most consistent with the amino acids within this region being in
contact with the portion of these ligands that corresponds to the
D-ring end of estradiol. The interplay between the shape of an ER
ligand and the residues that support its binding to ER may potentially
underlie the selective actions of different ER ligands in various cell
and promoter contexts.
Estrogens strongly influence the growth, differentiation, and functioning of the reproductive system, including the mammary gland and the uterus (1, 2). The effect of estrogen on the mammary gland is of particular interest because of its link to breast cancer; the proliferation and metastatic activity of nearly 40% of breast cancers are stimulated by estrogens (3).
Estrogens exert their effects by acting through a ligand-activated transcription factor, the estrogen receptor (ER).1 A member of a superfamily of nuclear receptors, the estrogen receptor contains a highly conserved DNA-binding domain, domain C, and a highly conserved ligand-binding domain, domain E. In addition to its ligand binding activity, the E domain also possesses dimerization activity and a hormone-dependent activation function (AF-2). A hormone-independent activation function (AF-1) is located within the receptor's A/B domain. In the presence of ligand, the ER bound to an estrogen response element (ERE) can either activate or suppress transcription of a downstream target gene in a cell- and promoter-specific manner (4-11).
Estrogen antagonists such as tamoxifen have been successfully used in the treatment of ER-positive breast cancers (3, 12, 13). These antagonists compete with estradiol for binding to the receptor but fail to elicit transcriptional activity. Unfortunately, tamoxifen treatment has several drawbacks. First, tamoxifen is not a complete antagonist and has agonistic effects in certain cell and promoter contexts. Second, breast cancer cells often become resistant to this treatment (14). Finally, despite the need to inhibit ER activity in breast cancer cells, the retention of some ER activity is important in a number of other biological processes, including bone maintenance, the cardiovascular system, and liver metabolism (2). Thus, it becomes important to understand how the ER recognizes and interprets ligands of different structure, that is, which amino acids are involved in their binding, and thereby how these ligands might result in different activities in various cell types. Such information could then be used to design new estrogen agonists and antagonists with target cell- and response-selective biological effects.
We and others have identified regions of the ER involved in recognition
of ligand (15-18). We have found amino acids near Cys530
to be important in estradiol binding, whereas residues C-terminal to
position 535 have been shown to contain AF-2 activity (19-21), and
residues 507-514 may contain dimerization activity (20, 22). Recently,
we used alanine scanning mutagenesis to identify those amino acids in
the 515-535 region of the ER ligand binding domain important in
estradiol binding (23). Mutation of the amino acids Gly521,
His524, Leu525, and Met528 to
alanine affected the ability of the ER to bind estradiol and had a
parallel effect on estrogen-dependent transcription without affecting the overall activity of the receptor. These results, taken
together with structural predictions based on crystal structures of
other ligand-bound nuclear receptors, indicated that these four amino
acids of the ER may lie along one face of an -helix and are presumed
to be in contact with the bound estradiol.
In this report, we extend those studies to include four other estrogen agonists and antagonists with varied structures: the synthetic nonsteroidal agonist meso-hexestrol; P1496, an agonist derived from the mold metabolite zearalenone; the partial agonist-antagonist trans-hydroxytamoxifen; and the pure antagonist ICI. Interestingly, we observe that different amino acids in the 515-535 region of the ER are important in the recognition of these different ligands. Only one amino acid (Leu525) was found to be important in binding all of the ligands. By comparing the amino acids important in binding each of the ligands and comparing the ER amino acid sequence with other nuclear receptors and published crystal structures, we suggest which portion of these ligands are in contact with this region of the ER ligand binding pocket.
The plasmids
(ERE)2-pS2-CAT (24), pCMV5hER (19), pCMV (Clonetech,
Palo Alto, CA), and pTZ19R (25) have been described. Plasmid DNA used
for transfection was purified either by CsCl gradient centrifugation
(26) or by Qiagen plasmid preparation kit according to the
manufacturer's instructions (Qiagen, Chatsworth, CA). Ligands were
from the following sources: meso-hexestrol and 17
-estradiol (Sigma); P1496 (kindly provided by IMC
Corp., Terre Haute, IN); and trans-hydroxytamoxifen and ICI
(kindly provided by Dr. Alan Wakeling and Zeneca Pharmaceuticals,
Macclesfield, UL). Cell culture medium, calf serum, and other reagents
for cell culture were purchased from Life Technologies, Inc. and Sigma.
All transfections were
performed using the ER-negative human breast cancer cell line
MDA-MB-231. Cells were maintained as described previously (10) and were
transfected using the calcium phosphate method (27). Cells were
incubated in 5% CO2 for 40-48 h prior to transfection
with 0.1 µg of ER expression plasmid, 2.0 or 5.0 µg of
(ERE)2-pS2-CAT reporter plasmid, 0.8 µg of pCMV internal control
-galactosidase plasmid, and pTZ19R to 15 µg of
total DNA/100-mm plate. After incubation of cells with DNA for 4 h, cells were glycerol shocked for 2.5 min with 20% glycerol in growth
medium and washed with Hanks' balanced salt solution for 2.5 min.
Cells were then treated with ligand in growth medium, harvested after
24 h, and lysed by cycles of freezing on dry ice and thawing at
37 °C. ER activity was determined by CAT assay of whole cell lysates
and was normalized to
-galactosidase activity as described
previously (28).
The mutant
receptors used in this study, which consist of individual alanine
substitutions from amino acid 515 to amino acid 535 of the ER ligand
binding domain, were described by us (23) in a study in which their
ability to bind estradiol and activate transcription in response to
estradiol was analyzed. The responsiveness of these mutant receptors to
two different estrogen agonists, hexestrol and P1496, the partial
agonist/antagonist, trans-hydroxytamoxifen (TOT), and a pure
antagonist, ICI, was investigated in this study. The structures of
these ligands are shown in Fig. 1. The ability of these
ligands, all of which have high affinity for the ER (29-31), to induce
or antagonize transcriptional activity of the mutant receptors was
monitored in the ER-negative breast cancer cell line MDA-MB-231 using
an estrogen-responsive reporter gene construct, (ERE)2-pS2-CAT, in transient cotransfection experiments. As
expected for wild type ER, both hexestrol and P1496 fully induced
transactivation to near estradiol-induced levels (Fig.
2A). TOT induced activity to only 10-15%
that of estradiol, and it antagonized estradiol-induced activity down
to its own level of agonist activity (~10-15%; Fig. 2). ICI, as
expected, behaved as a pure antagonist (Fig. 2B).
To assess the importance of the individual amino acids from positions 515-535 in the recognition of these receptor ligands, the mutant ERs were initially screened at a single concentration of each ligand that resulted in near maximal stimulation of wild type (WT) ER. Mutants whose activity differed significantly from wild type at this concentration were then subsequently tested over a range of ligand concentrations to determine whether the mutant receptors were dose-shifted in their response to ligand, suggesting a decreased affinity for ligand, or if they were defective in the ability to achieve maximal transcriptional activity. For all of these receptors, activity was low in the absence of ligand, as reported previously (23).
HexestrolThe structure of the synthetic nonsteroidal
estrogen hexestrol (Fig. 1) differs from estradiol in that it is
symmetrical and lacks a formal B- or C-ring. The mutant ERs were
initially screened at 1 × 1010 M
hexestrol, which gave 75% of maximal activity with WT ER. As shown in
Fig. 3A, alanine substitutions at only two
positions, His524 and Leu525, resulted in large
reductions in hexestrol-induced transactivation (<5% of WT activity).
The R515A, M528A, and P535A receptors exhibited modest reductions in
activity (60-80% of WT). Dose response curves for the WT, H524A,
L525A, and P535A receptors using 1 × 10
12 to 1 × 10
6 M hexestrol, are shown in Fig.
3B. The most impaired mutant, H524A, was dose-shifted
1000-fold, whereas L525A and P535A were shifted 100× and 10×,
respectively. The R515A and M528A receptors were also dose-shifted
slightly, requiring slightly higher levels of hexestrol to reach wild
type maximal activity (data not shown). Though His524 and
Leu525 appear to be important in both estradiol and
hexestrol binding by ER (Table I), alanine substitution
at position 524 had a much greater effect on hexestrol-induced
activation. Interestingly, the G521A mutation, which was found to
significantly affect estradiol binding (Table I), had very little
effect on hexestrol-induced transactivation (Fig. 3C).
|
P1496, a derivative of the estrogenic mold metabolite
zearalenone, has a 14-membered ring resorcylic acid lactone structure that is significantly larger than either estradiol or hexestrol (Fig.
1). Nevertheless, it acts as a potent inducer of ER activity, stimulating ER transactivation to nearly the same level as estradiol (Fig. 2). Like hexestrol, 1 × 1010 M
P1496 gave near maximal (80%) stimulation of ER and was therefore used
to screen the 21 alanine-substituted ER mutants. Although mutation to
alanine at only two positions markedly impaired hexestrol-induced activity (Fig. 3A), half of the ER mutants were
substantially impaired for P1496-induced activity (Fig.
4A). Using higher concentrations of ligand
demonstrated that all of these mutant receptors could reach wild type
maximal transactivation at higher concentrations of ligand (Fig. 4,
B and C, and data not shown). Like estradiol induction, the positions where mutation to alanine most greatly affected P1496-induced transactivation were Gly521,
His524, Leu525, and Met528.
However, in addition, mutation at residues 515, 522, 526, and 531-535
also had a significant effect on P1496-induced activity. Fig. 4
(B and C) shows the P1496 dose response curves
for the R515A, G521A, M522A, H524A, L525A, and M528A receptors. Of
particular note is the observation that the M528A mutation affects
receptor response to P1496 more than this mutation affects response to any of the other tested ligands; 100-fold shift for P1496
versus <10-fold shift for all other ligands.
trans-Hydroxytamoxifen
TOT is an estrogen antagonist with
partial agonistic activity in certain cell types, including MDA-MB-231
cells (Fig. 2). To investigate the ability of the mutant ERs to
interpret TOT as an agonist, transient transfections were performed as
with hexestrol and P1496, except that more reporter plasmid, 5 µg
rather than 2 µg, was used in order to enhance the agonistic effects. The results of screening the mutant receptors with 1 × 109 M TOT are shown in Fig.
5A and show that like P1496, about half of
the mutant receptors were impaired for activity. However, unlike the
P1496-induced response, TOT dose response analysis revealed two
different classes of mutants. As observed before, many of the mutants
that were impaired for activity at the initial concentration of TOT
were able to reach wild type levels of activity in the presence of
higher concentrations of ligand (Fig. 5B and data not
shown). However, another class of mutants, which consisted of K520A,
M522A, N532A, and P535A (and indicated with light shading in
Fig. 5A), all exhibited reduced maximal activity and did not appear to be dose-shifted (Fig. 5C and data not shown).
Rather, the decreases in activity observed at 1 × 10
9 M TOT correlated with their reduced
maximal activities. Western blot analysis of these receptors
demonstrated that they were present at levels at least as great as that
of the wild type protein (data not shown), indicating that their
reduced activity was not attributable to reduced levels of these
receptors.
Antagonists
The antagonistic affects of two compounds, TOT
and ICI, were examined to determine whether any of the
alanine-substituted receptors failed to recognize these ER ligands as
antagonists. For these experiments, cells were treated with both
estradiol and a 10-fold excess of either TOT or ICI. For most of the
mutant receptors, 1 × 109 M estradiol
with 1 × 10
8 M antagonist was used.
However, for G521A, H524A, and L525A, where 1 × 10
9
M estradiol does not induce high levels of transactivation
(23), 1 × 10
8 M (for G521A and H524A)
and 1 × 10
7 M (for L525A) estradiol
were used with a corresponding 10-fold higher concentration of the
antagonist. In all cases, both TOT and ICI were effective antagonists,
suppressing estradiol-induced transactivation by greater than 85%
(data not shown).
We have probed the interaction that various ligands have with a portion of the ligand binding domain of the human estrogen receptor (ER) by alanine scanning mutagenesis, studying in addition to estradiol the behavior of three high affinity ligands: two nonsteroidal agonists, the simple, symmetrical synthetic ligand meso-hexestrol, and P1496, a derivative of the fungal produced estrogen zearalenone, as well as the partial antagonist, trans-hydroxytamoxifen, the potent metabolite of tamoxifen. As in our earlier study with estradiol, we found that the substitution of certain residues in this ER region to alanine caused a significant to marked reduction in the potency of these ligands in inducing transcriptional activity. In addition, the principal finding from this study is that each of the ligands displayed a distinct footprint of interacting residues in the alanine scanning mutagenesis, indicating that the structural differences in these ligands result in different patterns of interaction with amino acids of the ER (Table I).
We have confirmed that the alterations in ligand-induced transcriptional activity of the mutant receptors correspond to a dose shift (i.e. change in potency) in the transactivation response. Previously, with estradiol, we established that the decrease in transcription activation potency correlated well with a decreased binding affinity of estradiol to the mutant receptors (23). Thus, these interacting residues are considered to be ones that are important in determining the binding affinity of these ligands.
The effects of mutational change at certain sites were of particular interest. First, the L525A mutation was the only mutation that strongly affected the transcriptional potency of all four ligands (Table I). However, P1496, the bulkiest of the ligands, was least affected, possibly because it may best be able to compensate for the reduced bulk in ER that results from the Leu to Ala mutation. Second, the H524A mutation had a very strong effect on hexestrol and P1496-induced ER activity and a major effect on estradiol but no effect on TOT. It is of note that TOT is the only ligand that lacks a second hydroxyl group with which this histidine may be interacting. Third, the G521A substitution strongly affected estradiol and P1496 transactivation yet had only a modest effect on TOT and no effect on hexestrol. As discussed later, the smaller size of certain portions of the hexestrol and TOT structures, compared with estradiol and P1496, might enable them to tolerate the increased residue size resulting from the G521A substitution. Fourth, the K520A, M522A, N532A, and P535A substitutions had an effect on TOT induced transactivation but did so by reducing the maximal transcriptional activity of the mutant ER-TOT complex rather than by shifting the dose response curve. This decreased transactivation potential suggests that these residues do not involve the binding interaction between TOT and ER but rather affect the manner in which the liganded complex interacts with other components important for activating transcription. It is interesting that certain specific contacts between TOT and ER appear to affect only its balance of agonist and antagonist activity (such as residues 520, 522, and 535), whereas others affect only its binding affinity (such as residues 515, 516, and 525).
From our previous study on the effect of alanine scanning mutagenesis
on the binding of estradiol and ligand-induced transactivation of the
ER (23), we showed that the residues most affected by alanine
substitution, Gly521, His524,
Leu525, and Met528, could be displayed on three
adjacent turns on one face of an -helix. By sequence comparison with
the human retinoic acid receptor-
(RAR) and the rat thyroid hormone
receptor-
1 (TR), these sites lie on the inner face of helix-11 and
correspond to residues in the RAR-retinoic acid (RA) and TR-thyroid
hormone (T3) structures that are in close contact with the
bound ligands (32, 33). In the case of RAR-RA, these residues are in
contact with the apolar end of the ligand, the
-ionone portion, and
in the case of TR-T3, these residues contact the distal
phenolic ring, with His381 in TR (corresponding to
Gly521 in ER), making a hydrogen bond to the distal
phenolic hydroxyl group.
In this study, we find that the pattern of residues in the 515-535
region of ER that affect ligand-induced transcription differs depending
on the structure of the activating ligand. A comparison of the
structures of these ligands (Fig. 1) together with their interacting
residues (Table I) allows one to formulate a model for the basic
orientation of these ligands within the binding cavity of the ER (Fig.
6). By analogy with the TR-T3 and RAR-RA structures, this model shows the 515-535 region of ER as a helical region (corresponding to a portion of helix-10 and all of helix-11) and
the beginning of the loop region between helix-11 and -12. The residues
that affect the transcriptional potency of each of the four ligands are
indicated schematically with circles. The helix and loop of ER and the
structures of the ligands are presented on the same dimensional scale
to illustrate the differential interaction that these four ligands have
with the ER.
The four activating ligands, estradiol, hexestrol, P1496, and TOT, all have one common structural feature, a phenol, yet they show different patterns of residues whose substitution with alanine affects their transcriptional potency. These differences suggest that the ligands are not making contact with the 515-535 portion of the ER through their structurally common A-ring like feature. The rather narrow footprint of residues that affect hexestrol recognition (principally two residues, His524 and Leu525, on one turn of the helix) suggests that contact is being made with a portion of the hexestrol structure that is narrower than the corresponding region in estradiol (whose potency is affected principally by three residues, Gly521, His524, and Leu525, on two helical turns). In contrast, the much wider footprint of residues that affect P1496 binding (four or more residues on at least three turns) suggests that their interaction is with a region of the ligand that is larger than the corresponding region in estradiol. Thus, if the phenol rings of these three ligands are oriented similarly and away from the 515-535 region, then it is the D-ring end of estradiol and the corresponding narrower region of hexestrol and the wider region of P1496 that are likely to be in contact with this region of the ER.
The pattern of residues that affect the transcriptional potency of TOT is rather unique. The interaction of TOT with the middle of the helical region is limited. In particular, its lack of interaction with His524 suggests that in lacking a second hydroxyl group, TOT is indifferent to the hydrogen bonding characteristics of the residue at this site. On the other hand, TOT is the only ligand whose transcriptional potency is affected by residues near the N terminus of the examined region (Arg515 and His516), suggesting that its side chain may extend to be in contact with this region of the receptor.
Altogether, the comparison of ligand structure with the pattern of residues where mutation affects their transcriptional potency suggests that selected residues in the 515-535 region of the receptor are in contact with a portion of these ligands that corresponds to the D-ring end of estradiol. Thus, a rough comparison can be made between the orientation of these estrogens in ER and the RA and T3 ligands in RAR and TR, respectively. It is the D-ring end of estradiol that corresponds to the apolar end of RA and the phenol of T3 that is in contact with the helix-11 region of these nuclear receptors. The A-ring or phenolic portion of the estrogen ligands, which corresponds to the polar end of the RA and T3 ligands, is directed away from helix-11.
It is well recognized now that the ER and related nuclear receptors show transcriptional activity that is modulated by the nature of the particular gene promoter and the cellular background, consistent with ligand-receptor interaction with cell- and promoter-specific factors and transcriptional coactivators (5, 34). The interplay between the shape of an ER ligand and the residues that support its binding to ER, as studied here, may potentially underlie the selective actions of different ER ligands in various target cells and promoter contexts.
We thank Donald Seielstad for helpful discussions.