Isolation and Characterization of Androgen Receptor
Mutant, AR(M749L), with Hypersensitivity to 17-
Estradiol
Treatment*
Tin Htwe
Thin,
Liang
Wang,
Eungseok
Kim,
Loretta L.
Collins,
Ravi
Basavappa, and
Chawnshang
Chang
From the George Whipple Laboratory for Cancer Research Departments
of Pathology, Urology, and Radiation Oncology, and the Cancer Center,
University of Rochester, Rochester, New York 14642
Received for publication, June 20, 2002, and in revised form, December 23, 2002
 |
ABSTRACT |
Estrogens, primarily 17
-estradiol
(E2), may play important roles in male
physiology via the androgen receptor (AR). It has already been shown
that E2 modulates AR function in LNCaP prostate cancer cells and xenograft CWR22 prostate cancer tissues. Using a
molecular model of E2 bound-AR-ligand binding domain (LBD)
and employing site-directed mutagenesis strategies, we screened several AR mutants that were mutated at E2-AR contact sites. We
found a mutation at amino acid 749, AR(M749L), which confers AR
hypersensitivity to E2. The reporter assays demonstrate
that E2 can function, like androgen, to induce AR(M749L)
transactivation. This E2-induced AR mutant transactivation
is a direct effect of the AR(M749L), because the transactivation was
blocked by antiandrogens. The hypersensitivity of AR(M749L) to
E2 is not due to increased affinity of AR(M749L) for
E2, rather it may be due to the existence of the proper
conformation necessary to maintain E2 binding to the AR-LBD
long enough to result in E2-induced transactivation.
AR(M749L) transactivation can be further enhanced in the presence of AR coregulators, such as ARA70 and SRC-1. Therefore, amino acid 749 may
represent an important site within the AR-LBD that is involved in
interaction with E2 that, when mutated, allows
E2 induction of AR transactivation.
 |
INTRODUCTION |
Estrogens play an important role in the normal and abnormal
processes of male physiology (1). Male estrogen is synthesized by
aromatization of the principle androgen, testosterone, in many tissues,
including brain, liver, adipose tissue, and prostate. The physiological
level of circulating 17
-estradiol
(E2)1 in the
adult male is ~0.1 nM (~73-184 pM or
12-34 pg/ml) (2), however, local aromatase activity may cause tissue
levels to be higher than the serum level. Estrogens exert feedback
control at the level of the hypothalamus and pituitary by decreasing
luteinizing hormone-releasing hormone and luteinizing hormone
production. The necessity of estrogens for male fertility was
discovered in aromatase knockout (3) and estrogen receptor
(ER
)
knockout (4) mice. Both strains of mice develop infertility and exhibit defects of the reproductive system. Furthermore, estrogen imprinting on
male reproductive organs causes poor semen quality, cryptorchidism, and
testicular and prostatic hyperplasia in male offspring with prenatal
exposure to high levels of estrogen (5).
Estrogen (C18 steroid) and androgen (C19 steroid) are similar in
structure, however, estrogen possesses a hydroxyl group at C3 of the
phenolic A ring, whereas androgen does not (6). Although estrogen binds
to the androgen receptor (AR), it has little influence on AR
transactivation at the physiological concentration in males (7).
Traditionally, the estrogen receptor (ER) has been considered the sole
mediator of estrogen action. However, several findings show that
estrogen can directly activate AR function after loss of ligand
specificity due to mutations of the AR ligand binding pocket (7, 8).
Alternatively, estrogen-dependent wild-type AR (wtAR)
transactivation can be promoted by selective coregulators, such as
ARA70 (9-12) and SRC-1 (10). These findings support the idea that
estrogen may modulate AR-mediated functions during embryogenesis and in
the adult (12).
Estrogen, primarily E2, activation of AR function has been
observed in androgen-independent prostate cancer cells lines containing three major AR mutations. Systems in which these mutations occur include the LNCaP cell line (T877A) (7), androgen-independent prostate cancer (T877S) (13), the xenograft CWR22 (H874Y) (14), and the
Finnish prostate cancer germ line mutant (R726L) (15). Recent
crystallographic studies of the structures of dihydrotestosterone (DHT)-bound wtAR-ligand binding domain (LBD) and DHT-AR-LBD (T877A) (16) show that the replacement of Thr-877 by alanine creates space to accommodate a larger substituent. Therefore, E2 is
not only able to bind but also activate AR(T877A) transactivation (7).
In terms of estrogen sensitivity, the induction level of AR(H874Y) is
higher compared with that of the AR(T877A), AR(T877S), and AR(R726L) in
response to E2 (14).
We are interested in the effect of E2 signaling via the AR.
ER
and
knockout mice show prostate hyperplasia (5), and this
evidence suggests the possibility that estrogen may act through AR to
influence prostate hyperplasia. In addition, patients who have partial
androgen insensitivity syndrome show an intact androgen-AR-coregulator pathway but loss of the E2-AR-coregulator pathway (9, 12). This also indicates that E2 may influence AR function, in
the presence of coregulators, by modulating androgen action in male reproductive organ development. Here, we report the isolation of an AR
mutant that confers hypersensitivity to E2. A screening system was applied to a series of site-directed AR mutants that were
generated by replacing AR-LBD residues with ER-LBD residues at the
predicted sites of E2 contact in the AR ligand binding pocket. The prediction of E2 contact sites was based on the
molecular model of the crystal structure of the AR-LBD (17) bound to
E2. The estrogenic profiles of the AR mutants have been
determined using AR response element (ARE)-based reporter assays in
both prostate and non-prostate cell lines, and the estrogenic activity of AR(M749L) was found to be a direct effect via the mutant receptor. The estrogen induction of AR(M749L) is similar to the androgen stimulation of wtAR, and the site of the mutation is also a hot spot
for mutations in AR-associated diseases, such as androgen insensitivity
syndrome (AIS) and prostate cancer.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
E2 (Sigma),
[3H]E2 (3H-labeled
(2,4,6,7)-17
-estradiol, specific activity 118 Ci/mmol, PerkinElmer
Life Sciences, Boston, MA), and trypsin (Promega) were purchased. ICI
176,334 (Micronised, Adm:44008/90 C:41567, Imperial Chemical
Industries, PLC Pharmaceuticals Division, Great Britain) was kindly
provided by Dr. R. Harrison, and hydroxyflutamide (HF) was obtained
from Schering-Plough Corp.
Modeling--
The crystal structure of the hAR LBD bound to
R1881 was used as the starting model (17). R1881 was replaced by
E2 in the model using SPDBV. Mutations in the
structure were introduced using the SWISS-MODEL (18). The resulting
models were energy-minimized using the CNS suite of programs (19) with
the parameter and topology files for the ligands obtained from the
HIC-Up server (x-ray.bmc.uu.se/hicup) (20).
Site-directed PCR Mutagenesis--
The positions of AR mutations
are based on the presumed E2 contact sites determined via
the molecular model of AR bound with E2, considering
residues within 4.5 Å of E2 (Fig. 1, A and
B). The AR residues were changed to the homologous residue
of ER (Fig. 1B). The AR(M749I) prostate cancer and AR(M749V)
androgen insensitivity phenotype mutations (8) were also constructed
using site-directed mutagenesis. In each case, the presence of the
correct mutation was confirmed by sequencing.
Cell Culture, Transfections, and Reporter Gene Expression
Assays--
AR-negative DU145 human prostate cancer cells and both AR-
and ER-negative COS-1 monkey kidney cells were maintained in phenol red-free Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate with 5% CO2, at 37 °C. Transfections and
luciferase assays were performed as previously described (12).
pCMV-
-galactosidase was used as an internal control. Data are
presented as the mean ± S.D. of at least three independent experiments.
Steroid Binding Assay--
Steroid binding was performed on
intact COS-1 cells as described previously (12). COS-1 cells were
transfected with either the wtAR or the mutant (5 µg). Forty-eight
hours after transfection, the cells were treated with
[3H]E2 ranging from 0.01 to 1 nM
in the presence or absence of a 100-fold molar excess of unlabeled
E2, for 2 h, at 37 °C, in a 5% CO2
incubator. Specific binding was determined from the difference between
radioactivity in the presence or absence of unlabeled E2.
Scatchard plot analysis was used to determine binding affinity. Data
are presented as a mean ± S.D. of three independent experiments.
E2 Dissociation Assay--
The whole cell-binding
assay was used to determine the effect of mutation on dissociation of
E2 from the AR (12). COS-1 cells were transfected with 5 µg of pSG5wtAR or mutant AR. After 24 h, the transfected cells
were treated with 1 nM [3H]E2 in
the presence or absence of 1000-fold excess of unlabeled E2
at 37 °C, in a 5% CO2 incubator. The cells were
harvested at various time points. The radioactivity of crude cell
lysates, containing AR, was determined using a scintillation counter.
Data are presented as the mean ± S.D. of three independent experiments.
Limited Trypsinization Assay--
In vitro
transcription/translation reactions were performed using the
TNT-coupled reticulocyte lysate system (Promega) in the
presence of [35S]methionine. Two microliters of labeled
translation mixture was incubated for 30 min at room temperature, with
2 µl of ethanol, 10 nM DHT, or 100 nM
E2. Limited trypsinization was performed by addition of 1 µl of trypsin solution (50 µg/ml) (dissolved in 50 mM
Tris-HCl/pH 7.5, 1 mM CaCl2) for various
lengths of time, at 25 °C. Reactions were stopped by addition of 10 µl of SDS sample buffer, and samples were separated by 11% SDS-PAGE.
Autoradiography was performed overnight.
Data Analysis--
Pooled data are reported as the mean ± S.D., and statistical significance was determined using the Student's
unpaired t test. Probabilities < 5%
(p < 0.05) were considered significant.
 |
RESULTS |
Molecular Model of the AR Ligand Binding Domain Bound to
E2--
The molecular models of the AR-LBD bound to either
E2 (Fig. 1) or DHT (data not
shown) were constructed based on the crystal structure of the human
AR-LBD bound to R1881, a synthetic androgen (17). The modeling was
limited to identifying the residues that might affect ligand binding
specificity and ligand specific activation. Fig. 1A shows
the fourteen residues that have been identified as being involved in
E2 contact with the AR-LBD. Each residue is located within
4.5 Å of E2. Fig. 1B lists the AR-LBD contact residues (column 1); corresponding ER-LBD residues
(column 2); the ligand, either DHT or E2, that
contacts each AR-LBD residue (column 3); and the AR helix
containing each AR-LBD residue (column 4). Leu-704, Leu-707,
Gly-708, Gln-711, Met-742, Met-745, Met-749, Arg-752, Phe-764, Met-787,
Leu-873, and Thr-877 are not only the contact sites identified by the
molecular model of AR-LBD bound to E2 but also the contact
sites in the molecular model of the AR-LBD bound to DHT (data not
shown). With the exception of residue Leu-707, the same residues have
been identified as the contact sites of R1881 in the AR-LBD crystal
structure (16, 17, 21). Conserved amino acids in the LBDs of AR and ER,
Leu-704, Leu-707, Arg-752, and Phe-764 indicate the importance of these
residues for the receptor-LBD pocket structure. Val-746 and Leu-873
have been identified as the primary E2 contact sites in the
AR-LBD.

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Fig. 1.
E2 contact sites of the AR-LBD
pocket. A, the molecular model of E2
bound to the AR-LBD was constructed based on the crystal structure of
the human AR-LBD with R1881, a synthetic androgen, as the bound ligand
(17). The model shows the selected residues of AR that are predicted to
be within 4.5 Å of E2. B, list of residues
identified as E2 contact sites in the AR-LBD pocket.
Columns shows AR-LBD residues (column 1),
corresponding ER-LBD residues (column 2), either ligand, DHT
or E2, that contacts each AR-LBD residue (column
3), and the AR helix containing each AR-LBD residue (column
4). Among these residues, Leu-704, Gly-708, Gln-711, Met-742, Met-745,
Met-749, Arg-752, Phe-764, Met-787, Leu-876, and Thr-877 are identified
as the contact sites for R1881 in the AR crystal structure (16, 20).
D, DHT; E, E2.
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AR(M749L) Mutant Confers Hypersensitivity to
E2--
To isolate an E2-sensitive AR mutant,
we used site-directed mutagenesis to introduce several point mutations
over 14 residues in the wtAR-LBD. The positions of mutations are based
on the E2 contact sites in the E2·AR
molecular model (Fig. 1A). The AR contact site residues were
replaced with the corresponding ER residues (Fig. 1B), and
the desired mutations were confirmed by sequencing. A luciferase
reporter assay was used to screen the AR mutants for their
transactivation under 1 or 10 nM E2 treatment
using the (ARE)4-pG1-luciferase reporter construct in AR-negative DU145 prostate cancer cells. The AR(H874Y) mutant construct was used as a
control for estrogen stimulation. In the reporter assays, E2-fold induction was expressed in comparison to the
induction level of each AR, wt, or mutant in response to ethanol. Fig.
2A shows that, after screening
all mutants, the AR(M749L) mutant showed the most significant
hypersensitivity to E2.
E2-dependent transcriptional activity of
AR(M749L) was highly induced by 1 nM E2 and
induction further increased at 10 nM E2.

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Fig. 2.
AR(M749L) confers hypersensitivity to
E2. A, a series of AR mutants were screened
in the presence of E2. AR-negative DU145 cells were
transfected with 3.5 µg of (ARE)4-pG1-luciferase, 10 ng of
pCMV- -galactosidase internal control plasmid, and 1.5 µg of either
the wtAR or an AR mutant. Transfected cells were treated with either
ethanol (as a control) or 1 or 10 nM E2 for
24 h. The CWR22:AR(H874Y) mutant construct was used as a positive
control for estrogen stimulation (14). Luciferase activities were
normalized according to -galactosidase activities and are expressed
as -fold induction relative to that with ethanol treatment. The values
represent the mean ± S.D. from three individual assays.
B, transactivation profile of the wtAR, AR(M749L), and
AR(H874Y) in the presence of DHT. DU145 cells were transfected with 3.5 µg of (ARE)4-pG1-luciferase, 10 ng of pCMV- -galactosidase internal
control plasmid, and 1.5 µg of wtAR, AR(M749L), AR(H874Y), or various
other AR mutants. Transfected cells were treated with ethanol (as a
control) or 1 or 10 nM DHT for 24 h. AR
transactivation in response to DHT was used to confirm the functional
ability of the AR(H874Y) construct. Weak E2 stimulation of
AR(H874Y) in DU145 cell, in contrast with previous studies using CV-1
cells (14), may be due to cell-specific effects. Luciferase activities
were normalized according to -galactosidase activity and are
expressed as -fold induction relative to ethanol treatment. The values
represent the mean ± S.D. from three individual assays.
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Interestingly, we found that E2 stimulation of the
AR(H874Y) mutant in DU145 cells was far less compared with a previous
report where 10 nM E2 treatment activated the
mutant receptor severalfold in CV-1 cells (14). We therefore tested the
functional ability of the AR(H874Y) mutant construct in response to DHT
in our system, using DU145 cells. Fig. 2B demonstrates that
wtAR, the AR(M749L) mutant, and the AR(H874Y) mutant are induced to
approximately the same level in response to DHT. This result confirms
that the AR(H874Y) plasmid is functional and suggests that there are
cell-specific factors that mediate differences in E2
induction of AR mutant in CV-1 versus DU145 cells.
Nevertheless, based on Fig. 2 (A and B), we can
conclude that the AR(M749L) mutant is highly sensitive to
E2 using the (ARE)4-pG1-luciferase reporter in DU145 cells. The AR(G708A), like wtAR, can be activated in response to DHT, but not
to E2 (Fig. 2, A (lanes 5 and
6) versus B (lane 4)). The AR(Q711E), AR(M742L), AR(M745L), AR(M787L), AR(L873G), and AR(T877L) are very weakly activated by DHT. We also tested the expression level
of each mutant and found similar expression levels of the AR mutants
compared with wtAR (data not shown).
Met-749 Is a Hot Spot for Mutation in AR-associated
Diseases--
We next searched the AR gene mutation data base (8)
to understand the physiological importance of the amino acid residue Met-749. Fig. 3A shows that
mutations at Met-749 have been correlated with various pathological
phenotypes. Interestingly, in the case of AR(M749L), a direct
connection between a pathological phenotype and this mutation has not
yet been found. Fig. 3B shows the side-chain differences
among mutants. From the AR gene mutation data base (8), there were
four reports describing two different mutations of AR at
position 749. These mutants involved a change from Met (M) to either
Ile (I) or Val (V). Met-749 mutated to Ile, AR(M749I), has been
detected in androgen-independent prostate cancer as a somatic mutation
(22). AR(M749) mutated to Val, AR(M749V), was isolated from a germ line
mutation associated with AIS, either complete or partial, and found in
individuals from different families (23, 24). These reports reflect the
possible significance of Met-749 as a hot spot for mutations in
AR-associated diseases.

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Fig. 3.
AR(M749) is a mutation hot spot for
AR-associated diseases. A, methionine (Met)
at AR position 749 is mutated to isoleucine (Ile) in
androgen-independent prostate cancer (22) and to valine
(Val) in complete or partial androgen insensitivity syndrome
(AIS) (23, 24). An AR mutant in which amino acid 749 is changed from
Met to Leu has not yet been identified to have linkage with
AR-associated diseases. B, side-chain differences among
amino acid residues at position 749 in AR mutants.
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AR(M749L) Responds to 0.1 nM
E2--
We generated the AR(M749I) prostate cancer mutant
and the AR(M749V) AIS mutant, using site-directed mutagenesis and
tested the E2-dependent transactivation of each
mutant. Fig. 4 shows the luciferase
activities of three AR mutants compared with the activity of the wtAR
using three different ARE reporters: (ARE)4-pG1-Luciferase, (ARE)-PSA-Luciferase, and (ARE)2-MMTV-Luciferase in DU145 cells. Ethanol treatment was used as a control for the basal level of receptor
activity. Fig. 4A shows the transactivation profiles of the
wtAR, AR(M749L), AR(M749I), and AR(M749V) in response to various
concentrations of either DHT or E2, using the
(ARE)4-pG1-Luciferase reporter in DU145 cells. Both wtAR and AR(M749L)
were activated in response to DHT. This activation started at the very
low concentration of 0.001 nM DHT and reached the maximum
level at 10 nM DHT (lanes 2-6). However, there
is undetectable or very low wtAR activity in response to E2
even at the 10 nM concentration. In contrast, AR(M749L)
activation was observed in response to 0.1 nM
E2 (physiological concentration), and increased activity
was demonstrated at 10 nM E2 (lanes
10-12). This suggests that AR(M749L) is highly sensitive to
E2 for functional activation. Similar to AR(M749L),
AR(M749V) showed clear activation at 10 nM DHT. However, in
contrast to AR(M749L), the lower sensitivity of AR(M749V) at the
physiological concentration of 1 nM DHT matches its
association with the AIS phenotype. In terms of sensitivity to
estrogen, AR(M749V) also demonstrated an estrogenic profile, similar to
M749L, with lower intensity and shows higher sensitivity than wtAR in
response to 10 nM E2 (lane 12). The
AR(M749I) mutant shows very weak activation in response to 10 nM DHT (lane 6) and a negligible level of
activation in response to 10 nM E2
(lane 12).

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Fig. 4.
AR(M749L) responds to 0.1 nM
E2. A, DU145 cells were transfected with
3.5 µg of (ARE)4-pG1-luciferase, 10 ng of pCMV- -galactosidase
internal control plasmid, and 1.5 µg of either wtAR or an AR mutant.
Transfected cells were treated with either ethanol (as a control)
(lanes 1 and 7), various concentrations of DHT at
0.001, 0.01, 0.1, 1, or 10 nM (lanes 2-6), or
various concentrations of E2 at 0.001, 0.01, 0.1, 1, or 10 nM (lanes 8-12) for 24 h. Luciferase
activities were normalized according to the -galactosidase activity
and are expressed as -fold induction compared with ethanol treatment.
The values represent the mean ± S.D. from three individual
assays. B, DU145 cells were transfected with 3.5 µg of
either the (ARE)-PSA-luciferase or the (ARE)2-MMTV-luciferase reporter,
10 ng of pCMV- -galactosidase internal control plasmid, and 1.5 µg
of the wtAR, AR(M749L), AR(M749I), or AR(M749V). Transfected cells were
treated with either ethanol (as a control) (lanes 1 and
6), 1 nM DHT (lanes 2 and
7), 10 nM DHT (lanes 3 and 8), 1 nM E2
(lanes 4 and 9), or 10 nM
E2 (lanes 5 and 10) for 24 h.
Luciferase activities were normalized according to -galactosidase
activity and are expressed as -fold induction compared with ethanol
treatment. The values represent the mean ± S.D. from three
individual assays.
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We also made use of two AR target gene (PSA and
MMTV) promoter reporters, to confirm the responsiveness of AR(M749L),
AR(M749I), and AR(M749V) to estrogen. Fig. 4B shows not only
high stimulation of AR(M749L) (lanes 4 and 5, and
9 and 10) in response to E2, but also
the relative stimulation of AR(M749V) by E2 (lanes
4 and 5, and 9 and 10), which are
effects that can be demonstrated consistently with different reporters.
The effects of DHT are less consistent among the mutants using various
reporter genes. AR(M749V)-mediated induction in response to 1 nM DHT is higher using the PSA reporter construct. 10 nM DHT weakly induced AR(M749I) transactivation of both the
(ARE)PSA (lane 3) and (ARE)2-MMTV (lane 8)
promoters, however, E2 did not significantly induce
transactivation of AR(M749I) using either of the promoters (lanes
4, 5, 9, and 10). Together, the
data indicate that Met-749 of AR is a critical amino acid involved in
the control of ligand specificity and that particular mutants of
Met-749 intensify the responsiveness of AR to E2.
AR(M749L) Strongly Responds to E2 but Not to
Progesterone or Dexamethasone--
Fig.
5A shows the sequence
alignment and homology of the helix 5 region of the human AR (hAR),
human progesterone receptor (hPR), human glucocorticoid receptor (hGR),
and human ER
(hER
) (17, 21). Most of the residues in helix 5 of
the AR are either identical or conserved among the steroid receptors.
The AR and ER crystal structures show that Met-749 of AR and Leu-391 of
ER are not only aligned but also reside in the ligand contact sites of
AR and ER, respectively. The difference between Met and Leu is that the
side chain of Met contains sulfur atoms, whereas that of Leu is
aliphatic. It is reasonable to expect that the selectivity could be
offset by steric factors due to differences in the shape of Met and
Leu, although the substitution of Leu for Met is a favorable amino acid
change according to the studies of context-dependent protein stabilization (25).

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Fig. 5.
AR(M749L) responds strongly to E2
but not to progesterone or dexamethasone. A, sequence
alignment and homology of the helix 5 region of the human AR
(hAR), human PR (hPR), human GR (hGR),
and human ER (hER ) is shown (21). The sequence
position is given for the hAR (top) and hER
(bottom). Residues conserved among steroid receptors are in
italics. Identical residues among the aligned receptor
sequences are shaded. The residue of focus in this study,
Met-749, is highlighted in boldface for comparison with
amino acids at the same position in other steroid receptors. At
position 749, hAR contains a methionine (M) residue, whereas
the same residue in hPR, hGR, and hER , after alignment, is leucine
(L). B, the luciferase activity of a receptor
gene in response to the wtAR, AR(M749L), AR(M749I), and AR(M749V) upon
treatment with 10 nM DHT, E2, progesterone, or
dexamethasone. COS-1 cells were transfected with 3.5 µg of the
(ARE)4-pG1-luciferase reporter, 10 ng of pCMV- -galactosidase
internal control plasmid, and 1.5 µg of either the wtAR, AR(M749L),
AR(M749I), or AR(M749V). Transfected cells were treated with either
ethanol (as a control) (lanes 1, 6,
11, and 16), 10 nM DHT (lanes
2, 7, 12, and 17), 10 nM E2 (lanes 3, 8,
13, and 18), 10 nM progesterone
(lanes 4, 9, 14, and 19),
or 10 nM dexamethasone (lanes 5, 10,
15, and 20) for 24 h. Luciferase activities
were normalized according to -galactosidase activity and are
expressed as -fold induction relative to ethanol treatment. The values
represent the mean ± S.D. from three individual assays.
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The sequence alignment indicates that all steroid receptors contain Leu
at amino acid 749, except AR, which contains Met at that position. We
next tested whether the broadened specificity of AR(M749L) involves
only E2 or applies to other steroids as well. Fig.
5B shows reporter transactivation mediated by the wtAR, AR(M749L), AR(M749I), and AR(M749V) in the presence of 10 nM DHT, 10 nM E2, 10 nM
progesterone, or 10 nM dexamethasone. The wtAR is activated
by 10 nM DHT (lane 2) but not by other steroids
(lanes 3-5), compared with no activation upon ethanol
treatment (lane 1). AR(M749L) is significantly activated by
10 nM DHT (lane 7) and 10 nM
E2 (lane 8), is moderately induced by 10 nM progesterone (lane 9), but shows no induction
with 10 nM dexamethasone (lane 10), compared
with no activation upon ethanol treatment (lane 6). 10 nM E2 stimulates AR(M749L) activity (lane
8) to a higher level compared with 10 nM DHT treatment
(lane 7), in COS-1 cells. The reporter induction mediated by
progesterone (lane 9) is one-third of the induction mediated
by E2 (lane 8). Induction of reporter activity
stimulated by progesterone is not surprising, because hPR and hAR share
the greatest structural homology among the classic steroid receptors.
10 nM dexamethasone did not stimulate AR(M749L) transactivation (lane 10) indicating that the position of
the AR mutation, M749L, is particularly important for
E2 stimulation. AR(M749V) is clearly activated by 10 nM DHT (lane 17) and weakly activated by 10 nM E2 (lane 18), compared with
ethanol treatment (lane 16), similar to the induction of
AR(M749V) activity by 10 nM DHT and 10 nM
E2 in DU145 cells (Fig. 4). Neither 10 nM
progesterone nor 10 nM dexamethasone stimulated AR(M749V)
transactivation (lanes 19 and 20), and AR(M749I)
shows negligible activation in response to 10 nM
progesterone or dexamethasone (lanes 14 and
15).
E2-induced AR(M749L) Transactivation Occurs via the AR
Not the ER--
We next tested whether the transactivation of AR
mutants induced by E2 occurs via the AR. The AR- and
ER-negative COS-1 cells were used for transfection. Antiandrogens, such
as HF and ICI 176,334 (casodex or bicalutamide), were used to block
transactivation of the AR mutants in response to E2. If
E2 induction of wtAR is inhibited by addition of
antiandrogens, it indicates that E2 action occurs through
AR. We first tested any partial agonist activities of HF and ICI
176,334. The cells transfected with wtAR, AR(M749L), and AR(M749V) were
treated with either 5 µM HF (lane 2), 1 µM ICI 176,334 (lane 3), or 10 µM ICI 176,334 (lane 4). In Fig.
6, lanes 2-4 show no
transactivation of the wtAR or AR(M749V) in the presence of HF or ICI
176,334. However, we saw minor activation (~5-fold) of AR(M749L) in
the presence of 5 µM HF or 10 µM ICI 176,334. Strong transactivation of both wtAR and AR(M749L) was detected
in the presence of 1 nM DHT, whereas weak activation of
AR(M749V) was observed at the same concentration (lane 5). The weak activation is similar to that observed in DU145 cells (Fig.
4), again suggesting that the physiological level of 1 nM DHT is not high enough to activate the AR(M749V) mutant.

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Fig. 6.
E2-induced AR(M749L)
transactivation is via the AR not the ER. AR-negative, ER-negative
COS-1 cells were transfected with 3.5 µg of the (ARE)4-pG1-luciferase
reporter, 10 ng of pCMV- -galactosidase internal control plasmid, and
1.5 µg of either the wtAR or an AR mutant. Transfected cells were
treated with ethanol as a control (lane 1) or with the
various combinations of ligand and antiandrogen as indicated.
E2 stimulation of AR(M749L) and AR(M749V) in COS-1 cells
supports the data obtained in DU145 prostate cancer cells, shown in
Fig. 4, demonstrating the lack of cell-specific effects. No significant
wtAR activity was detected at the supra-physiological concentration of
100 nM E2 (lane 10). The high
concentration of 100 nM E2 was used to
demonstrate the clear blocking effects of antiandrogens. Luciferase
activities were normalized according to -galactosidase activity and
are expressed as -fold induction compared with ethanol treatment. The
values represent the mean ± S.D. from three individual
assays.
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|
To demonstrate the antiandrogenic activity of HF and ICI 176,334 on
AR-mediated transactivation in the presence of DHT or E2,
transfected cells were treated with different combinations of DHT or
E2 with HF or ICI 176,334. Induction of the wtAR and AR(M749V) mutant was negligible, whereas there was moderate induction of the AR(M749L) mutant in the presence of 1 nM DHT and 5 µM HF (Fig. 6, lane 6). This partial
antagonist effect of HF on AR(M749L) is reasonable in that HF itself
has been shown to stimulate AR(M749L) (lane 2). The
transactivation of all three receptors, wtAR, AR(M749L), and AR(M749V),
was also suppressed in a dose-dependent manner in the
presence of 1 µM (lane 7) or 10 µM (lane 8) ICI 176,334.
E2-induced AR(M749L) transactivation was detected at both
10 and 100 nM E2 (lanes 9 and
10), whereas AR(M749V) transactivation was strongly induced
only at 100 nM E2. E2 stimulation
of AR(M749L) and AR(M749V) in COS-1 cells supports the data obtained in
DU145 prostate cancer cells, shown in Fig. 4,
demonstrating the lack of cell-specific effects. No significant wtAR
activity was detected upon treatment with the supraphysiological
concentration of 100 nM E2 (lane
10). The high concentration of 100 nM E2
was used to demonstrate the clear blocking effects of antiandrogens.
Repressive effects of HF on E2-induced AR(M749L) and
AR(M749V) transactivation were detected (lane 11). Also,
dose-dependent responses of the AR mutants to ICI 176,334 were observed when the antiandrogen was combined with various
concentrations of E2 (lanes 12-15). Similar
profiles of antiandrogen blockage of DHT or E2-induced wtAR
or AR mutant transactivation were observed when using DU145 prostate
cancer cells (data not shown). HF and ICI 176,334 can antagonize
E2 induction of AR(M749L) and AR(M749V) and therefore indicate that E2 modulates AR function directly.
AR(M749L) Slows E2 Dissociation from AR--
To test
whether E2 stimulation of AR mutants could be due to the
improved ability of AR to bind E2, COS-1 cells were
transfected with either the wtAR or the mutant. The affinity of wtAR or
a mutant for E2 was measured by the binding assay, using
[3H]E2 in the presence or absence of a
100-fold excess unlabeled E2. After performing Scatchard
analysis, the equilibrium binding affinity for
[3H]E2 determined by the whole cell binding
assay was not found to be altered significantly in AR(M749L) compared
with wtAR (Fig. 7A).
Therefore, the binding ability of E2 does not significantly contribute to the E2-dependent AR(M749L)
transactivation.

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Fig. 7.
AR(M749L) retards E2
dissociation. A, Scatchard plot analysis of equilibrium
binding of [3H]E2 to wtAR and AR mutant,
AR(M749L), was determined in whole cell binding assays as described
(12). COS-1 cells were transfected with either the wtAR or the mutant
(5 µg) and treated with 0.1-1 nM
[3H]E2 in the presence or absence of a
100-fold excess unlabeled E2 for 2 h at 37 °C. The
values represent the means ± S.D. of three individual assays.
B, counts (cpm) of [3H]E2 bound in
the absence of unlabeled E2 competitor to the wtAR,
AR(M749L), AR(M749I), or AR(M749V). C, counts (cpm) of
[3H]E2 bound to the wtAR, or AR(M749L), in
the presence of excess unlabeled E2. The wtAR or AR mutant
(5 µg) were transiently expressed in COS-1 cells. Transfected cells
were treated with 1 nM of [3H]E2
for 2 h, at 37 °C. After 2 h, the dissociation was
initiated with the addition of a 1000-fold molar excess of unlabeled
E2 with continued incubation at 37 °C. The cells were
harvested at the indicated times, and the radioactivity was measured.
The values are the means ± S.D. of three individual assays. *, p < 0.05, significant decrease in
[3H]E2 dissociation from the AR(M749L)
compared with the wtAR. D, the combination of E2
and DHT increased AR(M749L) transactivation. COS-1 cells were transfected with 3.5 µg of the (ARE)4-pG1-luciferase reporter, 10 ng of
pCMV- -galactosidase internal control plasmid, and 1.5 µg of either
the wtAR or an AR mutant. Transfected cells were treated with ethanol
as a control (lanes 1, 7, and 13),
0.01 nM DHT (castration or very low level) (lanes
2, 8, and 14), 1 nM DHT
(lanes 3, 9, and 15), 10 nM E2 (lanes 4, 10, and
16), the combination of 0.01 nM DHT plus 10 nM E2 (lanes 5, 11, and
17), or 1 nM DHT plus 10 nM
E2 (lanes 6, 12, and 18).
Luciferase activities were normalized to -galactosidase activity and
are expressed as -fold induction compared with the basal activity upon
ethanol treatment. The values represent the mean ± S.D. from two
or more assays.
|
|
We further tested whether the AR mutations influence the dissociation
of E2 from AR. Fig. 7B represents the
[3H]E2 binding ability of various ARs labeled
with 1 nM [3H]E2 without
unlabeled E2 competitor, to be used as a control. Fig.
7C represents [3H]E2 counts of AR,
wt, or the AR(M749L) mutant, after 2 h of labeling with
[3H]E2, followed by initiation of
dissociation by changing the medium to that which contains a 1000-fold
excess of unlabeled E2. The reduction in bound counts after
addition of excess E2 demonstrates the
[3H]E2 dissociation. We have performed this
assay over extended periods, including 1, 2, 3, 4, 5, and 8 h in
three independent assays, because the later time points are more
relevant and a more prolonged effect on ligand dissociation will have a
greater influence on AR transactivation. Fig. 7C shows that
over time there was a pronounced retardation in
[3H]E2 dissociation from AR(M749L), compared
with the wtAR, in the presence of excess unlabeled E2.
There was no statistically significant difference in the
[3H]E2 dissociation between the wtAR and
AR(M749I) or AR(M749V) (data not shown).
Additive Induction of AR(M749L) with Combined E2 and
DHT Treatment--
We next tested the combined effect of DHT and
E2 on wtAR, AR(M749L), and AR(M749V) transactivation. Fig.
7D shows weak (lane 2) and strong (lane
3) activation of wtAR detected in the presence of 0.01 and 1 nM DHT, respectively. Reduced activation of wtAR was
detected in the presence of either 0.01 nM (castration or low level) or 1 nM DHT and 10 nM E2
(lanes 5 and 6). We repeated this experiment in
DU145 cells and confirmed the antagonistic effect of 10-fold excess
E2 on DHT-induced AR transactivation (data not shown).
AR(M749L) shows a very weak response to 0.01 nM DHT
(castration level) in COS-1 cells (lane 7 versus
8). In contrast, significant levels of AR(M794L) activation
were detected in the presence of both E2 and DHT
(lanes 11 and 12) compared with transactivation
induced by either DHT or E2 alone (lanes 8-10).
Similar profiles were detected with the AR(M749V) mutant (lanes
17 and 18 versus lanes 14 and
15), but at lower activity levels. The differential
induction of wtAR versus AR(M749L), after combining DHT and
E2 treatment, could be explained by the existence of
different ligand saturation thresholds of the wtAR compared with
AR(M749L). E2 at 10-fold excess may compete with DHT for binding to wtAR and result in repression of DHT-induced wtAR
transactivation. In the case of the AR mutants, DHT may not be able to
saturate the receptors, and the addition of E2 enhances
receptor transactivation. Therefore, the AR(M749L) mutation may allow
more efficient and/or productive ligand binding for both DHT and
E2.
AR(M749L)-bound E2 Resists Trypsin Digestion--
To
understand the different effects of E2 on wtAR and
AR(M749L) transactivation, in vitro translated wtAR or
AR(M749L) was incubated in the presence or absence of ligands. Limited
trypsinization was performed for various lengths of time and
proteolysis-resistant fragments were analyzed. Fig.
8A shows the expression levels
of wtAR (lane 1) and AR(M749L) (lane 2). Fig.
8B shows a 29-kDa proteolysis resistant fragment of both
wtAR and AR(M749L), which formed in the first 10 min of incubation with
trypsin after addition of 10 nM DHT. This is consistent
with previous studies (26). In the absence of ligand, the wtAR and
AR(M749L) were completely degraded (lanes 1 and
13). However, we can see the 29-kDa proteolysis-resistant fragment of AR(M749L) after addition of 100 nM
E2 (lane 15), but a resistant wtAR fragment is
not visible (lane 3). As incubation time is increased,
another small fragment (20-kDa) becomes apparent and the 29-kDa
fragment begins to disappear (lanes 18 and 21). This suggests that E2-AR(M749L) takes on a different
conformation than E2-wtAR, resulting in differences in
resistance to trypsin digestion, or alternatively,
E2-AR(M749L) takes on a conformation similar to DHT-wtAR
resulting in a trypsin digestion pattern resembling that of
DHT-wtAR.

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Fig. 8.
AR(M749L)-bound E2 resists
trypsin digestion. In vitro transcription/translation
was performed in TNT-coupled reticulocyte lysate systems
(Promega) in the presence of [35S]methionine. Two
microliters of labeled translation mixture was incubated for 30 min at
room temperature with 2 µl of ethanol, 10 nM DHT, or 100 nM E2. Limited trypsinization was performed by
addition of 1 µl of a 50 µg/ml trypsin solution (dissolved in 50 mM Tris-HCl (pH 7.5), 1 mM CaCl2)
for the indicated time, at 25 °C. Reactions were stopped by addition
of 10 µl of SDS sample buffer, samples were separated by 11%
SDS-PAGE, and autoradiography was performed overnight. A,
the expression levels of wtAR (lane 1) and AR(M749L)
(lane 2). B, shows a 29-kDa proteolysis-resistant
fragment of both wtAR and AR(M749L), which formed in the first 10 min
of incubation with trypsin, in the presence of 10 nM DHT
(lanes 2 and 14). A 29-kDa proteolysis-resistant
fragments of AR(M749L), in the presence of 100 nM
E2 (lanes 15 and 18), was observed,
whereas there was no visible resistant wtAR fragment with
E2 treatment (lane 3). A small fragment (20 kDa)
of AR(M749L) with E2 became clear after prolonged
incubation with trypsin (lanes 21 and 24).
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AR(M749L) Influences Coregulator Modulation of AR
Transactivation--
We next tested whether AR mutations affect
coregulator modulation of receptor transactivation in response to
either 10 nM DHT, 10 nM E2, or 100 nM ICI 176,334. In fact, AR(M794I) was identified as a
somatic mutation in androgen-independent prostate cancer, which often
results from combined treatment involving total androgen withdrawal and
treatment with the antiandrogen drug bicalutamide (27). Therefore, we
tested whether the AR(M749I) and AR(M749L) mutations enable the
antagonist ICI 176,334 to become an agonist in the presence of coregulators.
AR-negative DU145 cells were transfected with 2.5 µg of
(ARE)4-pG1-luciferase reporter and 1 µg of various forms of AR in the
presence or absence of 3 µg of ARA70 or SRC-1. The parent pSG5 vector
(3 µg) was used to substitute for pSG5-ARA70 or pSG5-SRC-1 in the AR
alone transfection. Luciferase activity was normalized according to
-galactosidase activity, and the -fold luciferase activity is
expressed as the -fold induction relative to ethanol treatment (set as
1-fold). Fig. 9 shows that coregulators
of the AR, ARA70 and SRC-1, significantly promoted DHT- or
E2-dependent transactivation of the AR (M749L)
(lanes 5, 6, 8, and 9).
SRC-1 promoted E2-dependent AR(M749L)
transactivation to a higher level than ARA70 and compared with that of
the wtAR (lanes 9 versus 8). However,
there is negligible activation of the wtAR, AR(M749L), or AR(M749I) in
response to 100 nM ICI 176,334, even with overexpression of
ARA70 or SRC-1 (lanes 11 and 12).

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Fig. 9.
Coactivators enhance
E2-dependent AR(M749L) transactivation.
AR-negative COS-1 cells were cotransfected with 2.5 µg of the
(ARE)4-pG1-luciferase reporter and 1 µg of pSG5 wtAR or an AR mutant,
in the presence or absence of 3 µg of pSG5ARA70 or SRC-1, under 10 nM DHT (lanes 4-6), 10 nM
E2 (lanes 7-9), or ICI 176,334 (lanes
10-12) treatment. Equal amounts of pSG5 vector (3 µg) were used
to substitute for pSG5ARA70 or SRC-1 in the wtAR or mutant AR alone
transfection. Luciferase activity was normalized according to
-galactosidase activity, and -fold luciferase activity is expressed
based as -fold induction relative to ethanol treatment (set as 1-fold).
Data represent the mean ± S.D. of three individual
experiments.
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Analysis of the Effect of Met-749 in the AR-LBD Pocket--
To
understand the effects of AR mutations, we examined the locations of
various mutated residues in the AR-LBD crystal structure. For our
examination, we replaced the R1881 model with the E2 model (Fig. 10) and minimized the energy of
the resulting structure. We also changed the identity of residue 749 from Met to Leu or Val and minimized the energy of the resulting
structures. In all cases, energy minimization yielded final energies
that are very similar and did not greatly perturb the AR structure.
Modeling is still at an insufficient stage of development for us to
reliably discern which small-scale shifts in the structure might be of actual significance. However, absence of an increase in the model energy indicates that the AR structure is easily able to accommodate these changes. The sole mutation that has an effect on
E2-induced transactivation occurs at position 749. As shown
in Fig. 10, this residue forms the wall of the binding pocket that is
close to the O3 atom of the ligand. The O3 atom is bonded to the
A ring of the ligand and happens to be at one of the regions where the difference between E2 and DHT is greatest. In
E2, the O3 is part of a hydroxyl group, and the A ring is
aromatic (planar) in structure. In DHT, the O3 atom is a keto oxygen,
and the A ring is not aromatic and adopts a chair conformation instead.
Thus, the location of a mutation that confers E2
hypersensitivity is near one of the areas of significant difference
between E2 and DHT. Such positioning provides compelling
corroborating evidence that this residue is critical for discriminating
between DHT and other ligands in the process of transactivation.

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Fig. 10.
Locations of the mutations and their effects
on E2 hypersensitivity. The model is based on the
R1881·AR complex crystal structure (17), but the R1881 has been
replaced with E2 and the resulting complex is energy
minimized. A, the chemical structure profiles of DHT and
E2. DHT and E2 are similar in structure,
however E2 possesses a hydroxyl group at C3 of the phenolic
A ring, whereas DHT does not. B, residues of AR, when
changed to the corresponding residues of ER, have no effect on
E2 hypersensitivity are shown in cyan. The only
residue, Met-749, that when mutated allows E2 stimulation
of AR is shown in red. The location of this mutant is very
close to the O3 atom and the A ring of E2. C,
the binding cavity formed by the residues surrounding the ligand. The
perspective is the same as in B. The color coding
of the surface is as follows: yellow, cavity surface formed
by residues not mutated in this study; cyan, cavity surface
formed by residues that, when mutated, have no effect on E2
hypersensitivity; and red, cavity surface formed by residue
749 which, when mutated, confers E2 hypersensitivity.
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 |
DISCUSSION |
The residues of the ligand contact site of AR are hypothesized to
be responsible for the proper interaction and packaging of the ligand
within the receptor LBD pocket (27). The ligand selectivity does not
depend on the number of hydrophobic contacts made by the ligand within
the large LBD pocket (27) but rather on how the ligand interacts with
specific key contact sites within the pocket. After a ligand binds to
the receptor, the conformational change induced allows the recruitment
of cellular coregulators and the initiation of AR transactivation.
Although E2 could bind to the wtAR (Fig. 7) (12), it was
unable to induce wtAR transactivation. There are several possible
reasons for the inability of E2 to stimulate AR activity:
1) E2 may not bind properly within the wtAR-LBD pocket, 2)
weakly bound E2 may easily escape from the wtAR-LBD pocket,
and 3) the E2·AR complex may not adopt the proper conformation necessary for AR transactivation.
We were interested in isolating an AR mutant that is hypersensitive to
E2 to study how estrogen may regulate androgen targets. The
E2-bound AR modeling approach was used primarily to
identify residues that might affect ligand binding specificity and
ligand-specific activation. As shown in Figs. 1, 2, 4, and 5, Met-749
is the most influential residue of the E2 contact site
within the AR-LBD pocket and is important for E2 regulation
of AR function. The functional data show that AR(M749L) has two
significant characteristics: 1) it does not significantly diminish DHT
activation, suggesting that position 749 is not critical for DHT
induction, and 2) upon E2 binding, the AR(M749L) mutant is
hypersensitive to E2 stimulation, suggesting that the
residue identity at position 749 is a major determinant in allowing
E2-mediated AR induction. As shown in Figs. 7-9,
E2-dependent AR(M749L) transactivation may be
dependent upon the length of time E2 is able to remain
locked into the AR-LBD, providing the preferred conformation necessary
for coregulator recruitment and achievement of the final steps of AR
transactivation. Fig. 7C demonstrates that E2
dissociates more slowly from the AR(M749L) LBD pocket than from the
wtAR, suggesting that length of ligand association is a crucial factor
in induction of transactivation. There is no clear correlation observed
between the ligand binding affinity and the functional activity of
AR(M749L), with strong induction of transactivation observed in
response to E2. Such an effect is similar to previous
studies reporting that the binding properties and relative biological
potencies of a number of steroids do not always correlate (12, 15).
Furthermore, Fig. 7D shows that combined E2 and
DHT treatment increases the activity of AR(M749L) compared with the
response to DHT alone. In contrast, the activation of the wtAR by DHT
is reduced in the presence of E2, suggesting that there is
competition between DHT and E2 for binding to the wtAR. The
additive effect of DHT and E2 in the activation of
AR(M749L) indicates the possibility of differential structural
conformation between AR(M749L) and wtAR, with that of AR(M749L)
allowing more efficient and/or productive binding of both DHT and
E2. Differences in the susceptibility of the wtAR and
AR(M749L) to proteolysis in the presence of E2 are
demonstrated in Fig. 8, indicating that the conformation of
E2-bound AR(M749L) is different from that of
E2-bound wtAR, as well as the existence of complex specific coregulator recruitment. In parallel, Fig. 9 shows that coregulators of
AR, such as ARA70 and SRC-1 (10, 12), significantly increase DHT- or E2-dependent AR(M749L) transactivation
compared with wtAR transactivation, and these data indicate that DHT-
or E2-bound AR(M749L) produces an active interface for
coregulator interaction, resulting in modulation of transactivation.
SRC-1 promoted E2-dependent AR(M749L)
transactivation to a higher level than did ARA70, suggesting a
preferential E2-dependent modulation effect by
SRC-1. It is also possible that SRC-1 is better able to relieve
repression by one or more AR-bound corepressors than is ARA70.
Therefore, AR(M749L) harbors changes in the LBD that not only permit
AR(M749L) to accept E2 but also allow the conformational
changes that allow the modulation by coregulators that is necessary for
AR(M749L) transactivation. Mutation of Met-749 to Leu leads to 1)
retardation of E2 dissociation from the mutant receptor
pocket, 2) adoption of an active conformation, and 3) recruitment of
the modulators necessary for the induction of E2-induced
AR(M749L) activity.
In terms of steroid structure, there is a significant difference
between E2 and DHT in the A ring of each molecule (Fig.
10A). The A ring is aromatic in E2 but not in
DHT. Residue 749 of AR abuts the A ring of a steroid when it is bound.
It is possible that the Met residue at this position allows DHT binding
and resultant receptor transactivation while limiting access to
E2. The mutation of Met-749 to Leu or Val reduces this
selectivity and allows both DHT and E2 to activate AR.
Although both Leu and Val have similar side-chain characteristics, the
differential effects of Leu versus Val in the intensity of
E2-dependent transactivation is not readily explained in structural terms by examining the crystal structure of the
AR·ligand complex or by simple modeling exercises. The other major
difference between DHT and E2 structure is the presence of
a methyl group in DHT (C19). When DHT is bound by AR, the only side
chain within the van der Waals distance of the C19 methyl group is that
of Gln-711. A mutation of the Gln-711 residue may affect the proper
accommodation of E2 in the AR-LBD pocket. In our
experiments, we screened a mutant wherein glutamine, Gln-711, had been
changed to glutamic acid, Glu, which does not result in responsiveness
to E2 (Fig. 2A) but instead mediates a loss of
responsiveness to DHT (Fig. 2B).
It is also interesting that, as shown in Fig. 3, different amino acid
substitutions at Met-749 are associated with various AR-related
diseases, including prostate cancer and complete or partial AIS.
AR(M749I) was identified as a somatic mutation in androgen-independent
prostate cancer, which often results from combined treatment involving
total androgen withdrawal and the antiandrogen drug bicalutamide (28).
The AR(M749I) mutation disrupted the responsiveness of AR to DHT and
E2, and, although E2 may play a role in
androgen-independent prostate cancer progression, there is no influence
of E2 or other steroids, such as progesterone or
dexamethasone, on AR(M749I) transactivation. Furthermore,
overexpression of coregulators did not increase AR(M749I)
transactivation. Therefore, androgen-independent prostate cancer
progression in patients with this AR mutation may be mediated via
alternate signaling pathways rather than resulting from reduced ligand
specificity of AR(M749I).
Another naturally occurring AR mutant, AR(M749V), has been linked to
the AIS phenotype, a condition characterized by loss of androgen action
in male development. AR(M749V) is sufficiently activated in response to
10 nM DHT but not by 1 nM DHT, the
physiological concentration of androgen. This weak response of
AR(M749V) to physiological androgen levels may be the cause of the
defective AR signaling that leads to the development of androgen
insensitivity. AR(M749V) is activated to a higher level by 10 nM E2 than is wtAR, but this stimulation does
not reach the level of wtAR induction by DHT. Therefore,
E2-AR(M749V) signaling could not compensate for the
insufficient DHT-AR(M749V) signaling in AIS patients.
In summary, AR(M749L), a mutant that responds to the agonistic effect
of E2 in a manner similar to the response of wtAR to DHT,
may be a useful tool with which to study the influence of estradiol on
AR function. Additional characterization of this hyperestrogenic
AR(M749L) mutant will also yield information regarding the influence of
estrogen on particular androgen targets.
 |
FOOTNOTES |
*
This work was supported by a George Whipple Professorship
Endowment and National Institutes of Health Grant DK60905.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology,
George Whipple Laboratory for Cancer Research, 601 Elmwood Ave., Box
626, University of Rochester, Rochester, NY 14642. Tel.: 585-273-4500;
Fax: 585-756-4133; E-mail: chang@URMC.rochester.edu.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M206172200
 |
ABBREVIATIONS |
The abbreviations used are:
E2, 17
-estradiol;
AR, androgen receptor;
wtAR, wild-type androgen
receptor;
PSA, prostate-specific antigen;
MMTV, mouse mammary tumor
virus;
LBD, ligand-binding domain;
DHT, 5
-dihydrotestosterone;
HF, hydroxyflutamide;
ARE, androgen receptor response element;
h, human;
CMV, cytomegalovirus;
AIS, androgen insensitivity syndrome;
PR, progesterone receptor;
GR, glucocorticoid receptor.
 |
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