We have been interested in understanding how the
estrogen receptor (ER) binds estrogens and discriminates between
different classes of steroids with closely related structures. Using
insights from our prior studies on ER and from sequence comparisons of steroid receptors, we identified three residues in the hormone-binding domain of the human ER, Leu345, Thr347,
and Glu353, that we considered were likely to be involved
in steroid A-ring recognition and therefore estrogen versus
androgen discrimination. We then tested the effect on ER activity of
mutating these ER residues to the corresponding androgen receptor
residues. Specifically, we examined the ability of the mutant receptors
to bind and be activated by 17
-estradiol and three different
androgens. No change in receptor activity was observed with the T347N
mutation, while the L345S mutation greatly reduced ER activity in
response to all ligands. Interestingly, the E353Q substitution behaved
as expected, causing a 9-fold reduction in the transactivation potency of estradiol and a concomitant 10-140-fold increase in the
transactivation potency of different androgens. These reciprocal
changes in the transcriptional effectiveness of estrogens and androgens
correlated with a decreased affinity of the E353Q ER for estradiol
binding and an increased affinity for androgen binding. Therefore,
amino acid Glu353 appears to be playing a significant role
in binding the A-ring phenolic group of estradiol and in receptor
discrimination between estrogens and the most closely structurally
related steroids, androgens. Based on this data and our earlier
observations, we propose a model for the orientation of ligand within
the binding pocket of ER in which the A-ring 3-phenol of estradiol is
hydrogen bonded to Glu353 in helix-3 and the 17
-hydroxyl
of estradiol is hydrogen bonded to His524 in helix-11. Our
findings with estrogen and androgen suggest that this orientation of
the steroid in the ligand-binding pocket, with the steroid A-ring in
contact with helix-3 and the D-ring in contact with helix-11 residues,
is likely to be general for all the steroid hormone receptors.
 |
INTRODUCTION |
The distinctive biological effects of the different classes of
steroid hormones were recognized early in this century, when the basic
physiology of the endocrine system was being elucidated. Despite their
overall structural similarity, each class of steroid hormones does have
characteristic structural features. In this regard, the estrogens
(C18 steroids) have a characteristic aromatic A-ring with a
phenolic hydroxyl at C-3, which uniquely distinguish them from the
other four steroid classes, androgens, progestins, glucocorticoids, and
mineralocorticoids, all of which are 3-keto steroids. Among the latter,
the structural differences are principally in their C- and D-rings. The
androgens (C19 steroids) are identical to the estrogens in
this region, whereas the progestins and corticosteroids (C21 steroids) all have distinctive hydroxylation and/or
oxidation patterns in their C- and D-rings.
The steroid hormones are now known to act through specific receptors
that belong to the nuclear hormone receptor superfamily, many of whose
members have been cloned within the last decade (1-5). These complex
proteins have separate DNA-binding and hormone-binding domains and act
as ligand-dependent transcription factors. Despite their
similar structures, each of the ligands acts specifically through its
own receptor in vivo. The specificity of the estrogen receptors for estrogens is particularly strong. Nearly all good ligands
are A-ring phenols, whereas those lacking this group, including
androgens, corticosteroids, and progestins, are extremely poor ligands
(6). Conversely, among receptors for the 3-ketosteroids, less
specificity in binding between the four classes of ketosteroids is
observed (7). Direct sequence comparison of the ligand-binding domains
of the steroid receptors now reveals that the 3-ketosteroid receptors
are more closely related to each other than they are to the estrogen
receptor (8).
We have been intrigued by the structural features of the estrogen
receptor, as well as the other steroid receptors, that underlie their
ability to discriminate among ligands from the different steroid
hormone classes. To gain a better understanding of the interactions
between receptor and hormone, we have taken a structure/sequence-guided mutagenesis approach to study how estrogen receptor-
(ER)1 discriminates between
estrogens and androgens. Because androgens differ from estrogens only
in the A-ring region (Fig. 1), we felt a comparison between these
classes of ligands would be most direct and informative. We focussed
our attention to defining the regions of ER most likely to discriminate
between estrogens and androgens, that is, A-ring binding.
In this study, we selected three residues in ER that we considered
likely to determine ligand A-ring specificity, Leu345,
Thr347, and Glu353, and used site-specific
mutagenesis to replace them with the corresponding residues in the
androgen receptor (AR). We then investigated the ability of the mutant
receptors to bind and be activated by estradiol and a set of three
androgens. The greatest shift in specificity occurred with the E353Q
substitution, which caused an 9-fold reduction in interaction with
estradiol and a 10-140-fold increase in interaction with different
androgens. Mutations at the other positions, as well as combinations of
these mutations, had less pronounced effects on ligand discrimination. We conclude that the interaction of the C-3 phenolic hydroxyl group of
estradiol directly with Glu353 in ER is a major determinant
of ER's specific recognition of estrogens. By analogy, we would expect
that the interaction of the 3-keto group in the other steroid hormones
with the corresponding glutamine residue is a major determinant of the
recognition of the 3-ketosteroids by their corresponding receptors.
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EXPERIMENTAL PROCEDURES |
Reagents--
The plasmids (ERE)2-pS2-CAT (9),
pCMV5-hER (10), pCMV
(CLONTECH, Palo Alto, CA),
pCH110 (Pharmacia Biotech, Piscataway, NJ), and pTZ19R (11) have been
described. Plasmid DNAs used for transfection were purified either by
CsCl gradient centrifugation (12) or by Qiagen plasmid preparation kit
according to the manufacturer's instructions (Qiagen, Chatsworth, CA.)
Ligands, 17
-estradiol, testosterone, 5
-dihydrotestosterone, and
19-nortestosterone were obtained from Sigma. Restriction enzymes and
VENT DNA polymerase were purchased from New England Biolabs (Beverly,
MA). Oligonucleotides were purchased from Life Technologies, Inc.
(Gaithersburg, MD). Cell culture media, calf serum, and other reagents
for cell culture were purchased from Life Technologies, Inc. and Sigma.
For Western blot analysis, nitrocellulose membrane was obtained from
Millipore (Marlborough, MA), anti-ER H226 antibody was kindly provided
by Dr. Geoffrey Greene (University of Chicago), and rabbit anti-rat IgG was purchased from Zymed (San Francisco, CA). Radioisotopes for
chloramphenicol acetyltransferase (CAT) assays, sequencing, hormone
binding assays, and Western blotting were purchased from NEN Life
Science Products (Boston, MA) and Amersham.
Plasmid Constructions--
Plasmids pCMV5-E353Q, pCMV5-L345S,
pCMV5-T347N, pCMV5-T347N,E353Q, and pCMV5-L345S,
pCMV5-L3455,T347N,E353Q were constructed using
oligonucleotide-directed single-stranded DNA mutagenesis as
described (13, 14). The oligonucleotides used were as follows: E353Q,
5
-GCAGACAGGCAGCTGGTTCAC-3
; L345S, 5
-CGATGATGGGATCTCTGACCAACCTG-3
; T347N, 5
-CGATGATGGGCCTTTTAAACAACCTGGCAGACAG-3
; T347N, E353Q, 5
-CGATGATGGGCCTTTTAAACAACCTGGCAGACAGGCAGCTGGTTCACATG-3
;
L345S,T347N,E353Q, 5
-CGATGATGGGATCTTTAAACAACCTGGCAGACAGGCAGCTGGTTCACATG-3
. All constructs were sequenced (Sequenase 2.0, U. S. Biochemical
Corp./Amersham) to confirm the presence of the mutation.
Plasmids pET15b-ER and pET15b-E353Q were generated by polymerase chain
reaction cloning of the wild type and E353Q hormone-binding domain
sequences into the vector pET15b (Novagen, Madison, WI). Polymerase
chain reaction primers were NdeF,
5
-GGGAATTCCATATGGAAAACAGCCTGGCCTTGTCCCTG-3
, and S554R,
5
-GAATGGATCCTCAGCTAGTGGGCGCATGTAGGCG-3
. Polymerase chain
reaction amplification was performed with VENT DNA polymerase (New England Biolabs) according to the manufacturer's recommendations, with 300 µM dNTPs, 0.5 µM primers, 100 ng
of template, 2 mM MgSO4, and 1 unit of
polymerase in 100 µl total volume. Reactions were heated to 95 °C
for 5 min followed by three rounds of 95 °C for 1 min, 45 °C for
1 min, and 72 °C for 4.5 min, and 10 rounds of 95 °C for 1 min,
55 °C for 1 min, and 72 °C for 4.5 min with a final extension
time of 10 min at 72 °C. The resulting fragment was then digested
with BamHI and NdeI and cloned into the
BamHI and NdeI sites of pET15b.
Cell Culture and Transfections--
COS and MDA-MB-231 breast
cancer cells were maintained and transfected as described previously
(14-16). All transfections were done using the calcium phosphate
precipitation method (17). For transactivation studies, cells (in 10-cm
plates) were transfected with 100 ng of ER expression plasmid, 0.8 µg
of pCMV
, 2.0 µg of (ERE)2-pS2-CAT reporter plasmid,
and pTZ19R carrier plasmid to 15 µg of total DNA. ER activity was
determined by CAT activity of whole cell extracts normalized to
-galactosidase activity from the co-transfected pCMV
plasmid
(18).
Western Immunoblot Analysis, Hormone Binding Assays, and Relative
Binding Affinity Assays--
Cell extracts for Western blots and
hormone binding studies were prepared from COS cells or from MDA-MB-231
cells transfected with 10 µg of ER expression plasmid, 0.6 µg of
pCH110, and 4.4 µg of pTZ19R/10-cm plate (14). At 24 h after
transfection, cells were harvested in TNE (40 mM Tris, pH
7.5, 140 mM NaCl, 1.5 mM EDTA), centrifuged at
200 × g for 5 min, and resuspended in 100 µl of cell
lysis buffer (20 mM Tris, pH 7.4, 0.5 M NaCl,
1.0 mM dithiothreitol, 10% glycerol, 50 µg/ml leupeptin,
50 µg/ml aprotinin, 2.5 µg/ml pepstatin A, and 0.2 mM
phenylmethylsulfonyl fluoride). Cells were then subjected to three
rounds of freezing on dry ice/ethanol and thawing on ice followed by
centrifugation at 15,000 × g for 5 min at 4 °C.
Western blot analyses were performed as described (19, 20).
Nitrocellulose blots were probed with anti-hER antibody H226 at 2.0 µg/ml. This antibody detects an epitope in the N-terminal A/B domain
of the ER. Blots were then incubated with rabbit anti-rat IgG (1 µg/ml) followed by 125I-conjugated protein A. The amount
of extract loaded onto the gel for each receptor was normalized for
transfection efficiency by
-galactosidase activity.
Estradiol hormone binding assays and Scatchard analyses were performed
as described previously (14, 18, 21). Relative binding affinity
measurements were determined, with slight modifications, as previously
reported (22). The tritiated tracer, [3H]estradiol (51 Ci/mmol), was used at 2 nM to give the assay greater sensitivity to measure the low affinity of the androgens to wild type
ER. COS cell extracts containing full-length wild type or mutant ER
were diluted with TG buffer (50 mM Tris-HCl, pH 7.5, 10%
glycerol) to ~1 nM receptor concentration. The protein
solution was incubated with buffer alone or with several concentrations of unlabeled competitor, together with 2 nM
[3H]E2 at 0 °C for 18-21 h. The protein
was stable under these conditions. Free ligand was removed using the
hydroxylapatite assay (23). The unlabeled competitors were diluted into
1:1 dimethylformamide:TG buffer to ensure solubility; the final
concentration of dimethylformamide in the assay was 7%. All data are
reported relative to E2 which is set at 100%.
Fluorescence Spectrophotometric Assays--
Fluorescence spectra
were acquired on a Spex Fluorolog 2 (model IIIC) instrument using Spec
DM3000 software. The data was collected at 4 °C, in a ratio mode,
using photomultiplier correction and 2.5-mm slits. Excitation was 379 nm. Unliganded ER fluorescence was subtracted as background. The ER
hormone-binding domains were expressed in Escherichia coli
in a pET15b vector, using standard methods (Novagen) (12) and purified
by batchwise adsorption onto a nickel resin, according to the
manufacturers suggestions (Qiagen). HBDs were diluted to 6 nM in TG buffer and incubated for 60 min at 0 °C with 2 nM tetrahydrochrysene (THC)-nitrile (24, 25), or no ligand
for background. The free THC-nitrile was not removed since under these
conditions nearly all of the ligand is bound. Spectra were measured at
several ratios of ER to THC-nitrile to establish that free ligand was
not affecting the position of the bound fluorescence peak.
 |
RESULTS |
Selection of Sites in the Estrogen Receptor Likely to Determine
Ligand A-Ring Specificity--
In our recent studies of
ligand-receptor contact sites (14, 26), we used alanine scanning
mutagenesis over a 21-residue region of ER to identify likely ER-ligand
contact points. This region of the ER hormone-binding domain (HBD) is
now thought, based on comparisons with the retinoic acid
receptor-retinoic acid (27) and the thyroid hormone
receptor-triiodothyronine (28) HBD crystal structures, to form a
portion of helix-11 and the loop between helix-11 and -12. When we
investigated the footprint of ligand contacts for four ER ligands that
had very similar A-ring structures but differed substantially in their
D-ring regions, we found that each ligand had a unique, characteristic
footprint (26). We therefore concluded that helix-11 in ER formed a
portion of the ligand-binding pocket that accommodated the D-ring
region of estradiol and related estrogens. Interestingly, sequence
similarity in the helix-11 region of different steroid receptors is
low, consistent with a requirement to accommodate hormones that have different and distinctive patterns of D-ring polar functions, suggesting that all the steroids may bind to their receptors in this
orientation.
If the steroids bind with their D-rings near helix-11 and then orient
the rest of their structure along the same axis as does the ligand in
the retinoic acid receptor-retinoic acid and thyroid hormone
receptor-triiodothyronine crystal structures (27, 28), then the A-rings
would extend toward and contact portions of helix-3 and -5. We searched
the sequences of the steroid receptors over these regions for potential
A-ring C-3 recognition/discrimination sites, amino acid differences
likely to reflect the characteristic functional difference between
binding a 3-phenolic versus a 3-keto steroid (Fig.
1). Because the steroid hormone receptor
sequences in helix-5 are highly conserved, we did not select any sites
in that helix.

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Fig. 1.
Structure and abbreviation of the ligands
used in this study. The two estrogen (estradiol and
tetrahydrochrysene nitrile) and three androgen (testosterone,
5 -dihydrotestosterone and 19-nortestosterone) ligands are
shown.
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We chose to investigate three sites in helix-3 which are highlighted in
the sequence alignments shown in Fig. 2.
These sites appear to represent positions where the ER residue is of a
different character (presumably selected to favor interaction with a
3-phenol) from that at the corresponding position in the sequence the
other four steroid receptors (presumably selected to favor interaction with a 3-keto group). Leucine 345 in ER is replaced by a smaller, more
polar residue (serine or threonine) in the other receptors. Similarly,
threonine 347 in ER is replaced by an asparagine, and glutamate 353 in
ER is replaced by a glutamine. At the other positions shown in Fig. 2,
the residues in ER versus the other four receptors are
either identical or highly homologous, or they show no systematic change between the receptor classes.

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Fig. 2.
Sequence alignment for a portion of helix-3
of the different classes of steroid receptors. The sequence of
ER is shown at the top and the relationship to the sequences of the
other four steroid hormone receptors (33) is indicated with the
symbols: identity or high homology (vertical
line), characteristic differences between 3-phenolic and 3-keto
classes ( ), no systematic difference (no symbol). This
portion of helix-3 is close to ligand contact sites noted in the
crystal structure of the retinoic acid receptor complex with retinoic
acid (shown with solid dots at the bottom).
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The glutamate versus glutamine substitution at position 353, in particular, represents the type of change expected for recognizing the functional group difference between estrogens and 3-ketosteroids at
the ligand C-3 position. The glutamate of ER, a strong hydrogen bond
acceptor, would pair well with the strong hydrogen bond donor phenol of
estrogens. Conversely, the good hydrogen bond donor glutamine is
matched with the good hydrogen bond acceptor 3-keto function in the
ligands for androgen, progesterone, glucocorticoid, and
mineralocorticoid receptors (AR, progesterone receptor, glucocorticoid receptor, mineralocorticoid receptor, Fig.
3).

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Fig. 3.
Matching of hydrogen bonding character
between ligand and receptor. In the estrogen/ER situation, the
phenolic function on the ligand is a strong hydrogen bond donor and the
carboxylate on Glu353 in ER, a strong acceptor. In the
androgen/androgen receptor situation, the roles of ligand and receptor
are reversed, with the 3-keto function on the ligand now a good
hydrogen bond acceptor, and the carboxamide from the corresponding
glutamine residue in the receptor a good donor.
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Transcriptional Response of Estrogen Receptor Mutants with
17
-Estradiol and Androgens--
Using site-directed mutagenesis of
the ER cDNA, we mutated Leu345, Thr347, and
Glu353 of the HBD to match the AR amino acid sequence;
serine, asparagine, and glutamine, respectively. We generated three
single amino acid substitutions, as well as a double and a triple
mutation. We then tested the ability of the mutant ERs to activate
transcription in response to E2, as well as three
androgens, 5
-dihydrotestosterone (DHT), testosterone (T), and
19-nortestosterone (norT) (Fig. 1). These results are shown in Figs.
4 and 5,
and in Table I. Transcriptional activity
was measured in ER-negative human breast cancer MDA-MB-231 cells
transfected with an ER expression vector and an estrogen response
element (ERE)-containing reporter gene construct,
(ERE)2-pS2-CAT. Activity was measured over a range of
ligand concentrations (1 × 10
12-1 × 10
5 M) to assess the potency and efficacy of
these different ligands to induce transcriptional activity of the wild
type and mutant ERs.
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Table I
Relative transactivation potency of estradiol and the three androgens
with wild type and mutant estrogen receptors
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Fig. 4 shows the ability of the wild type
ER and the different ER mutants to be activated by estradiol
(E2). For wild type ER, E2 was a 50,000-fold
more potent ligand than the androgens (Fig. 4, panel A).
However, all the ligands, at high concentrations, activated wild type
receptor to the same maximal level. The T347N mutation had no effect on
ER's ability to be activated either by estrogens or androgens,
and the transactivation curves closely paralleled those seen with wild
type ER (Fig. 4A).

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Fig. 4.
Transactivation profiles for wild type
(wt) estrogen receptor and mutant ERs in response to
estradiol and three androgens. MDA-MB-231 cells were cotransfected
with (ERE)2-pS2-CAT reporter, pCMV internal control
plasmid, and the indicated ER expression plasmid. Transfected cells
were then treated with the indicated ligand for 24 h before
preparation of extracts. CAT activities were normalized to
-galactosidase activity and are expressed relative to the wild type
ER activity with 10 8 M estradiol
(100-140-fold stimulation in different experiments), which is set at
100%. The values represent the mean and S.D. from two or more
experiments. For some values, error bars are too small to be
seen. In panel A, data for wild type ER is shown with
filled symbols and dashed lines, and data for
T347N ER with open symbols and solid lines.
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As our model predicted, the E353Q mutation proved most interesting
(Fig. 4B). E353Q was 9-fold less sensitive to E2
than was the wild type ER. More striking, this mutant was 13-, 25-, and 143-fold more sensitive than wild type ER to the androgens (see Table
I), and maximal transcriptional activity was again elicited by these
ligands. Conversely, the L345S mutation greatly diminished the
effectiveness of all the ligands (Fig. 4C). For
E2, the dose-response curve was shifted markedly to the
right, and the maximal transcriptional ability of the ER in response to
E2 was also diminished. Likewise, no activation by
androgens was obtained.
Despite the fact that the E353Q ER responded better to androgens than
did wild type ER and that T347N ER showed ligand response identical to
that of wild type ER, the combination of these two mutational changes
in the ER (Fig. 4D, denoted TE-NQ ER) resulted in
a receptor that was only activated by very high concentrations of
E2. This double mutant exhibited little to no change in
androgen responsiveness compared with wild type. The triple mutant,
with changes at amino acids 345, 347, and 353 (Fig. 4D,
denoted LTE-SNQ), also showed poor response to
E2 and the same response as wild type ER to androgens.
In Fig. 5, the data are arranged to show
how the receptors respond to each of the ligands tested. Response to
estradiol was best with the wild type and T347N ERs, followed by E353Q
ER, and then L345S, with the double and triple mutants showing a
105-fold reduced transactivation effectiveness of estradiol
(Fig. 5A). Response to the three androgens (Fig. 5,
panels B-D) was best with the E353Q ER. The androgens were
approximately 20-fold less effective with the two other single or the
double mutant ERs. Of note, the L345S receptor showed no stimulation by
any of the androgens, yet was moderately responsive to estradiol (Fig. 4C).

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Fig. 5.
Response to 17 -estradiol (panel
A), testosterone (panel B), 5 -dihydrotestosterone
(panel C), and 19-nortestosterone (panel D) of
wild type ER and ER mutants. MDA-MB-231 cells were transfected and
processed for analysis of reporter gene activity exactly as described
in the legend to Fig. 5. Values represent the mean and S.D. from two or
more experiments. For some values, error bars are too small to be
seen.
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Western Blot Analyses of Mutant ER Protein Levels--
To
determine whether protein stability and/or expression levels
contributed to any of the differences in observed transactivation, we
performed Western blot analyses. The ERs were expressed in COS cells,
and extracts were separated by SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose, and probed with antibody specific for
the ER. Each of the mutant receptors was expressed at levels very
similar to that of wild type ER (data not shown). Similar results were
obtained when the ERs were expressed in MDA-MB-231 cells or in the
presence of 10
6 M E2 or
10
5 M DHT.
Hormone Binding Assays--
Transactivation data showed that the
E353Q mutation caused a 9-fold decrease in potency of the natural
ligand 17
-estradiol, while the potencies of the androgens were
increased 10-140-fold. To establish whether these shifts were the
result of corresponding changes in ligand binding affinities, we
examined the effect of the E353Q mutation on ligand binding by both
direct and competitive hormone binding studies.
In the direct binding studies, extracts from cells transfected with
wild type or E353Q ER expression vector were incubated with varying
concentrations of [3H]E2 or
[3H]E2 plus a 100-fold excess of unlabeled
E2. The level of ER-bound E2 was determined,
and Scatchard analyses were performed. The Kd
determined for wild type receptor was 0.055 ± 0.027 nM (n = 4); E353Q ER had a
Kd of 0.16 ± 0.08 nM
(n = 4) (data not shown). In the four experiments,
E353Q ER had a 3.1 ± 1.5-fold lower affinity for E2
than wild type ER.
Relative binding affinities for the androgens were also determined
using a competitive radiometric binding assay. For these experiments,
cell extracts containing expressed wild type ER or E353Q ER were
incubated with tritiated E2 plus increasing concentrations of unlabeled competitor (10
10-10
3
M E2, DHT, norT, or T). The results, presented
in Table II, show very low relative
binding affinities (0.052, 0.0014, and 0.00035% for DHT, norT, and T,
respectively) for the wild type ER, but very significantly increased
affinities of the androgens for the mutant E353Q ER (19.6, 4.39, and
3.03% for DHT, norT, and T, respectively). Thus, the improved
transactivation ability of these androgens with the E353Q ER can be
explained to a large degree by the greatly improved affinity of these
androgen ligands for this E353Q estrogen receptor.
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Table II
Relative binding affinity and transactivation potency of ligands with
wild type and E353Q estrogen receptors
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Fluorescence Probing of Ligand Receptor Interaction--
We have
developed a series of non-steroidal ligands for the ER, the
tetrahydrochrysenes (THCs, see Fig. 1) (24), that have excellent
fluorescent properties and have been used to assay the binding of
ligands to ER and the distribution of the receptor in cells (25, 29).
An unusual characteristic of these fluorescent estrogens is that they
emit at longer wavelengths when bound to ER than in aqueous solution.
We ascribed this receptor-induced red shift to a specific interaction
between the THCs and residues in the ER ligand pocket that stabilize
the excited state of the fluorophore (25). This stabilization could be
achieved through a polar or charge interaction between the phenolic
function of the THCs and a nearby residue in ER, such as
Glu353.
We therefore investigated this possibility by comparing the
fluorescence emission spectra of the tetrahydrochrysene nitrile (THC-nitrile, see Fig. 1) when bound to wild type ER versus
E353Q ER. To perform these experiments, large quantities of the HBDs of
these two receptors were expressed as His-tagged proteins in E. coli and were purified over a nickel column.
As shown in Fig. 6, the THC-nitrile when
bound to wild type ER shows a fluorescence emission of 510 nm, close to
that reported by us with uterine ER preparations (25, 29). In contrast, the emission of the THC-nitrile when bound to E353Q shifted even further to the red, emitting at 567 nm. Thus, the mutational change of
glutamate to glutamine at position 353 in ER has a very significant effect on the fluorescence of the THC-nitrile, providing further support to the direct interaction of this site with the phenolic function of estrogens.

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Fig. 6.
Fluorescence emission spectra of the
tetrahydrochrysene-nitrile bound to wild type and E353Q ERs.
THC-nitrile was bound to the ligand-binding domain of the wild type ER
(dashed line) or E353Q ER (solid line) at 3:1
receptor ligand-binding domain:ligand. Spectra are photomultiplier
corrected, and the background fluorescence from the protein has been
subtracted.
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 |
DISCUSSION |
Glu353 in the Estrogen Receptor Plays an Important Role
in Binding the A-Ring Phenolic Hydroxyl Group of Estradiol and in
Discrimination between Estrogen and Androgen--
We have used
information from steroid receptor sequence analysis, comparison with
crystal structures of related proteins, and previous mutational studies
on ER done by us to select residues in the ligand-binding domain of the
ER that appeared likely to be in contact with the estradiol A-ring
phenolic hydroxyl group. By investigating amino acid substitutions made
at these positions, we identified amino acid Glu353 as
playing a significant role in discriminating between estrogens and the
most closely structurally related steroids, androgens. The recognition
of estradiol at the Glu353 site is likely to occur by the
more favorable interaction that the anionic carboxylate in the
glutamate residue has with the phenolic hydroxyl of estradiol, than
with the 3-keto group of other steroids from the other hormone classes
(cf. Fig. 3).
We demonstrated that mutation of the glutamate residue at position 353 in the ER to the glutamine found in AR, progesterone receptor,
glucocorticoid receptor, and mineralocorticoid receptor, reduced the
potency of estradiol in inducing ER transactivation, while at the same
time increasing the potency of several androgens. The dramatic effect
that this single mutation has both in terms of the "loss of
function" toward estrogens and the "gain of function" toward
androgens is striking, and indicates that the role of this site in
discriminating between these classes of hormonal steroids is
significant. By contrast, mutations at two other positions, Thr347 and Leu345, did not have similar
effects. The T347N change had no observable affect on ER activation by
either estradiol or by androgens and is therefore unlikely to play a
role in ligand discrimination. L345S, however, reduced ability of both
estradiol and androgens to stimulate ER activity. This mutation, while
not affecting overall stability of the protein as demonstrated by
Western blot analysis, may be altering the overall conformation of the
receptor, rendering ER unable to bind ligand well or to activate
transcription when ligand is bound. The placement of Leu345
and Thr347 relative to Glu353 on an
-helical
wheel makes it unlikely that all three residues would contact
ligand.
Despite the marked reciprocal shift in the relative potency and
affinity of estradiol versus androgens that results from the E353Q mutation, the residue at this site alone does not fully define
the estrogen versus androgen hormonal specificity of these receptors. E353Q ER still binds androgens less well than estrogens, although with much less of a difference than wild type ER. Thus, it is
clear that amino acids at other locations are important contributors to
estrogen/androgen ligand discrimination by these receptors.
Functional mapping approaches have also been used to define ligand
contact sites in other receptors. Using the fact that chick progesterone receptor fails to bind the antiprogestin RU486, whereas human progesterone receptor binds it well, Garcia et al.
(30) used a segmental chimera approach followed by single site
substitutions to identify a residue in helix-3 (at a position that
would correspond to Ala350 in ER) as the receptor site most
likely to be close to substituents at the 11
-position of a
progesterone ligand. More recently, Vivat et al. (31) used a
similar segmental chimera/mutational approach to identify some regions
in AR and progesterone receptor that are important in discrimination
between ligands from the androgen and progestin hormonal classes.
Comparison between the Transcription Activation Potency and the
Binding Affinity of Estrogens and Androgens with ER Mutants--
The
transcription assays show that the E353Q mutation affects the relative
potency of estradiol and the three androgens, but not their efficacy,
as all ligands were able to induce maximal levels of transcriptional
activity at sufficiently high ligand concentrations. However, the issue
of changes in ligand transactivation potency versus ligand
binding affinity is interesting and complex. The E353Q mutation in ER
results in a 9-fold decrease in the potency of E2 as a
transcriptional activator, but only a 3-fold decrease in E2
binding affinity is observed. In our earlier study of alanine mutants
in helix-11 of the ER-HBD (14), we found that there was a good
correlation between the shift in E2 transactivation potency
and binding affinity in the three mutants for which we could measure
E2 binding; again, however, the reduction in E2 potency was about 3-4-fold greater than the reduction in
E2 binding affinity. Also, in a recent study (32), a
related mutation in the same region of helix-11 was found to cause a
greater decrease in transactivation potency than of binding affinity
for one enantiomer of the non-steroidal estrogen, indenestrol B. This
quantitative difference between the binding affinity versus
transcriptional potency might potentially reflect perturbations in the
coupling between the ligand-receptor complex and co-activator proteins such as SRC-1 that mediate the transcriptional response (5).
In this paper, we also examined the transcriptional potencies and
binding affinities of several androgens with wild type and E353Q ERs
(Table II). In competitive binding assays, using
[3H]E2 as tracer, the androgens showed
greatly increased binding to E353Q ER; even when the 3-fold difference
in E2 binding to the E353Q ER is taken into consideration,
the increases are 130-3,000-fold.
It is of note that the relative affinity of the three androgens for the
E353Q ER (DHT > norT > T) is the same as their relative affinity for AR itself. Relative to the androgen methyltrienolone (R1881), their affinities for AR are: DHT, 61 ± 17%
(n = 3); norT, 31 ± 2% (n = 2);
and T, 6.7 ± 1.4% (n = 6).2 The second item of note
are differences in the relative binding affinity and transactivation
potencies of the three androgens. This comparison is made in the
columns marked "Index" in Table II, which shows the ratio of
binding affinity to transactivation potency. By this index, the potency
of DHT with both wild type ER and E353Q ER is much less than expected
from its affinity; the index values for T and norT are much smaller.
Although these differences could be due to altered coupling between
co-activators and the ligand-receptor complex (5), the fact that DHT is
most affected suggests another explanation.
The binding assays are done with cell extracts under dilute protein
concentrations in the absence of serum, whereas the transactivation assays are done with cells in 5% calf serum. Calf serum is known to
contain a protein related to sex hormone binding globulin (33). Although the binding specificity of the bovine protein has not been
characterized in detail, human sex hormone-binding globulin binds DHT
20-fold better than E2, T is bound only 4-fold better, and
norT 3-fold less well then E2.2 Thus, greater
binding of DHT by serum components in the transactivation assay could
account for its reduced transactivation potency relative to receptor
binding affinity.
Functional Mapping of Ligand-Receptor Contact Sites: A Model for
the Orientation of Estradiol and Other Steroid Hormones in the Ligand
Binding Pocket of Their Receptors--
By combining our identification
in this study of Glu353 as the likely A-ring phenol-binding
site of estrogens in ER, with our earlier conclusion that the D-ring
portion of the ligand is in contact with helix-11 (26), we have defined
the basic axis and orientation of the ligand within the binding pocket
of the hormone-binding domain of ER. The D-ring of E2 is in
contact with helix-11, with the 17
-hydroxyl group most likely
hydrogen bonded to His524 (26), and the A-ring is projected
toward helix-3, with the 3-phenolic hydroxyl hydrogen bonded to
Glu353. Our findings further suggest that the other steroid
hormones will adopt the same A-ring/helix-3-D-ring/helix-11
orientation. It is of note that this orientation of steroid ligands in
the binding pocket of steroid receptors is opposite of that proposed by
others on the basis of homology models built from the
nonsteroid-receptor retinoic acid receptor-retinoic acid crystal
structure (34). Final verification of the ligand orientation within the
ER and other steroid receptors will need to await reports of the
crystal structures of these hormone-receptor complexes.
We thank Kathy Carlson, Arvin Gee, and Donald
Seielstad for assistance and helpful discussions.