From the Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
Received for publication, December 2, 2002, and in revised form, February 17, 2003
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ABSTRACT |
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The androgen receptor (AR) is a ligand-regulated
and sequence-specific transcription factor that activates or represses
expression of target genes. Here, we show that the N terminus of AR
contains an inhibitory domain located in an 81-amino acid segment lying upstream of the DNA-binding domain (DBD). The inhibitory domain interacted directly with DBD and repressed DBD binding to the androgen
response element. Mutations of the conserved amino acid residues (K520E
and R538E) within the inhibitory domain decreased its inhibiting
ability in vitro and increased AR trans-activation in
vivo. These data demonstrate the existence of a novel inhibitory domain in the N-terminal part of AR, which might play important roles
in the regulation of AR trans-activation.
The androgen receptor
(AR)1 mediates androgen
functions in the differentiation and maturation of the male
reproductive organs and in the development of male secondary sex
characteristics (1). Mutations in the AR gene are
associated with the androgen insensitivity syndrome (2, 3). Numerous
somatic mutations in the AR gene have been reported among
prostate cancer patients and as well as in prostate cancer cell lines
and xenografts (3, 4). Most of these mutations have been detected in
tumor tissues of late-stage prostate carcinoma, indicating that somatic
mutation of the AR gene might be involved in the progression
and aggressiveness of prostate cancer.
The AR is a member of the nuclear receptor superfamily (5).
These receptors are characterized by distinct functional domains: an
N-terminal part involved in ligand-independent transcription activation
(AF1), a DNA-binding domain (DBD), and a C-terminal ligand-binding
domain involved in ligand binding and
ligand-dependent transcription activation (AF2) (6). As for
other steroid receptors, ligand binding is generally believed to result
in a conformational charge in AR with consequent dissociation of heat
shock proteins/chaperones (7), dimerization, and binding to cognate
androgen response elements (AREs) in target genes and (through its AF1
and AF2 domains) interactions with various coactivators that facilitate
transcription by the general transcriptional machinery (8). The DBD
encompasses two zinc finger-like modules and binds as dimers to two
hexameric sequences orientated as direct or inverted repeats (9, 10). Although the DBD and the ligand-binding domain of steroid
hormone receptors are highly conserved, there is much less homology
among steroid hormone receptors in their N-terminal parts. The AR has a
long N-terminal part with a strong autonomous AF1 and interacts directly with AF2 in the C-terminal part (11, 12). The N- and
C-terminal interactions are important for androgen-induced gene
regulation, and disruption of these interactions may be linked to
androgen insensitivity syndrome (13, 14). The conserved FXXLF and WXXLF motifs within the N-terminal part
seem to be involved in pairwise interactions between AF1 and AF2 (15).
The N-terminal part contains stretches of glutamines (coded by CAG) and
glycine (coded by GGN) (16). Expansion of the CAG repeats is associated with X-linked spinal and bulbar muscular atrophy (17). A shorter CAG repeat is associated with an increased trans-activation of AR (18,
19), but the biological role of GGN repeats is less clear.
In this study, we demonstrated that AR contains a highly conserved
inhibitory domain within the N-terminal region. The inhibitory domain
interacted directly with DBD and inhibited the DBD-DNA interactions.
The mutations in the inhibitory domain resulted in decreased inhibitory
ability and increased AR trans-activation activity, indicating that
this domain might play important roles in the regulation of AR function.
Production and Purification of Recombinant Proteins--
The
human full-length AR was expressed in Sf9 cells via the
baculovirus expression vector pVL1393 (BD Biosciences), and the recombinant AR was purified as described previously (20). All of the AR
and glucocorticoid receptor (GR) cDNA fragments were amplified by
PCR with specific oligonucleotides, cut with NdeI and
BamHI, and subsequently cloned in the corresponding
restriction sites of the vectors pET15d (Novagen), pGEX-2TL (Amersham
Biosciences), and pcDNA3.1 (Invitrogen). The fragments were
expressed as His6-tagged (via pET15d) or GST fusion (via
pGEX-2TL) proteins in Escherichia coli BL21 and purified
through nitrilotriacetic acid Ni2+-agarose or
glutathione-Sepharose columns, respectively. Point mutations were
generated by using the QuikChange site-directed mutagenesis kit
(Stratagene) following the manufacturer's instructions and confirmed
by DNA sequencing analysis. The mutated proteins were expressed and
purified similarly.
Gel Shift Assay--
Two pairs of oligomers
(5'-AGCTTTTGCAGAACAGCAAGTGCTAGCTG-3' and
5'-AAATTCAGCTAGCACTTGCTGTTCTGCAA-3';
5'-AGCTTTTGCAGAATAGCAAATGCTAGCTG-3' and
5'-AAATTCAGCTAGCATTTGCTATTCTGCAA-3')
derived from the prostate-specific antigen gene ( Cell Culture and DNA Transfection--
PC3 cells were cultured
in RPMI 1640 medium containing 10% fetal bovine serum. Cells (5 × 105) were plated in each well of 24-well plates and
transfected with 100 ng of 4xARE-E4-Luc reporter plasmid, 2.5 ng of
control plasmid pRL-CMV, and various amounts of expression plasmids.
Cells were grown in the presence of 10 nM R1881 for 48 h after transfection and harvested for dual-luciferase activity assay
(Promega).
Protein-Protein Pull-down Assay--
GST and GST-DBD(AR537-644)
were expressed in bacteria and immobilized on glutathione-Sepharose
beads. Beads (10 µl) containing 100 ng of GST or GST-DBD proteins
were incubated with 5 µl of transcription and translation
coupled rabbit reticulocyte lysates containing
35S-labeled AR477-538 in BC150, 0.1% Nonidet P-40 for
2 h at 4 °C. After being washed with the incubation buffer,
beads were boiled with SDS sample buffer and subjected to SDS-PAGE
followed by autoradiography.
The Full-length AR Interacts with the Androgen Response Element
More Weakly than the DNA-binding Domain--
The
ligand-dependent interaction of AR with the ARE has been
demonstrated in vitro with crude AR-containing cell extracts (21). However, the AR-DNA interactions have not been studied with the
highly purified recombinant AR. To this end, the FLAG epitope-tagged
human AR was expressed in Sf9 cells and immunopurified under
high salt conditions (500 mM KCl) to strip off heat shock proteins associated with the unliganded AR. The recombinant AR preparation is near homogeneity (Fig.
1B, lanes 2 and
3) and contains two bands that migrated near the 110-kDa
position. The top band might be the phosphorylated form of
AR (22). Two minor polypeptides (70 and 55 kDa, indicated by
stars on the right) were recognized by the
anti-FLAG monoclonal antibody (data not shown), indicating that they
are degraded products of the full-length AR. A DNA probe containing the
ARE derived from the prostate-specific antigen promoter ( A Domain within the AR N Terminus Inhibits DBD-ARE
Interactions--
A C-terminal extension of the DBD of AR was found to
be required for specific and high affinity interactions of DBD with ARE (25). To investigate whether the sequences surrounding DBD would affect
DBD-DNA interactions, AR537-662 and AR477-644 (Fig. 1A) were expressed in and purified from bacteria (Fig. 1B,
lanes 5 and 6). AR537-662 strongly interacted
with the probe, similar to AR537-644 (Fig. 2A, lanes
2-4). However, AR477-644 completely lost the ability to interact
with the ARE probe even though much more protein (up to 1.6 pmol) was
used in the binding reaction (lanes 5-7). The N-terminal
extension of DBD (amino acid residues 477-558) was expressed and
purified (Fig. 2B, lane
1). Its molecular mass as determined by SDS-PAGE (16 kDa)
is much bigger than the calculated mass (10 kDa), and it was heavily
degraded (Fig. 2B, lane 1). This region contains
20% charged amino acids and 16% proline residues, which may be
responsible for this aberrant mobility of the protein. When AR477-558
was added to the binding reaction that contained the fixed amount (0.3 pmol) of AR537-644, the density of the DBD·ARE complex dramatically
decreased (Fig. 2C, lanes 3-8). These results
indicate that AR477-538 specifically inhibits the DBD-ARE interactions
in trans as well as in cis. We noticed that
different preparations of AR477-538 contained various amounts of the
full-length protein and that amounts of the full-length protein (Fig.
2B, lane 1, indicated by the top arrow
on the right) were correlated with the inhibition ability of
AR477-538. As negative controls, the recombinant prostate apoptosis
response-4 (26), 30-kDa Tat-interaction protein (27), and
39-kDa subunit of RNA polymerase C (28) expressed and purified
similarly did not significantly affect the DBD binding to the ARE
probe (Fig. 2D).
The Inhibitory Domain Interacts with DBD and Inhibits AR
Trans-activation--
The protein-protein pull-down assay was
performed to investigate whether the inhibitory domain (ID) interacts
directly with DBD. GST and GST-DBD(AR537-644) fusion protein were
expressed in bacteria and immobilized on glutathione-Sepharose beads
(Fig. 3A, lanes 2 and 3). The in vitro translated
35S-labeled AR477-558 (lane 4) bound to GST-DBD
(lane 6) and not to GST (lane 5). This result
indicates that the inhibitory domain interacts directly with DBD.
We then investigated the effect of the ID on AR trans-activation by
performing transient transfection assays. A luciferase reporter
containing four tandem copies of the same ARE used for the gel shift
assay upstream of the minimal adenovirus E4 promoter was cotransfected
with expression vectors for AR, AR477-558, or both into prostate
cancer PC3 cells in the presence of the synthetic androgen R1881. As
shown in Fig. 3B, AR activated the reporter gene ~25-fold,
and coexpressed AR477-558 showed a strong (62%) inhibition of this
activity. Coexpression of AR477-558 did not influence reporter gene
activity driven by p53, indicating that the inhibiting effect of
AR477-558 was specific for AR. Western blot analysis revealed that the
AR protein levels in the absence and presence of AR477-558 were
comparable (Fig. 3C, lane 3 versus lane 2). On the basis of in vitro studies (Fig.
2), the ID inhibited AR trans-activation most likely by
blocking the interaction of the AR with the ARE.
The Inhibitory Domain Is Specific for AR--
The DNA-binding
domains of AR, GR, progesterone receptor, and mineralocorticoid
receptor are highly conserved (29). Not surprisingly, they bind to the
same consensus DNA site (GGTACANNNTGTTCT) and can be considered
a subfamily of the nuclear receptor superfamily. However, the
inhibitory domain of AR is not conserved in the other receptors (Fig.
4A). GR418-525 and GR358-525
were expressed and purified (Fig. 4B, lanes 1 and
2). Gel shift assay demonstrated that GR358-525 and
GR418-525 bound the ARE probe similarly (Fig. 4C,
lane 3 versus lane 2). The lower band
(Fig. 4C, lane 3, indicated by a star
on the right) might contain a monomer of GR358-525. These
results indicated that the ID in AR is not conserved in GR; thus, the
inhibitory domain is specific for AR.
Mutations in the ID Enhance AR Trans-activation--
Sequence
alignment shows that the ID of AR is highly conserved through evolution
(Fig. 5A). To further
characterize the biological effects of this region, we mutated two
conserved residues (Lys-520 and Arg-538) in the ID and cDNAs
encoding the mutated AR (K520E and R538E) were transiently transfected
in PC3 cells with the luciferase reporter plasmid. The mutated AR had
elevated trans-activation activity compared with the wild-type AR (Fig.
5B), although the mutated and wild-type AR were expressed at
the same level in the transfected cells (Fig. 5C,
lanes 2-4). The ID (AR477-558) from the mutated AR (K520E
and R538E) were expressed and purified (Fig. 2B, lanes
2 and 3). The gel shift assay revealed that mutations of K520E and R538E decreased the inhibitory ability of ID (Fig. 5D, lanes 9-12 and 13-16 versus
lanes 5-8). Ten nanograms of the wild-type AR477-558
almost completely blocked AR537-644 binding to the ARE probe (Fig.
5D, lane 5). However, the same amount of the
mutated (K520E and R538E) AR477-558 inhibited the DBD-ARE interaction
only 65 and 35%, respectively (Fig. 5D, lanes 9 and 13). Thus, the enhancement of AR trans-activation by
mutations of K520E and R538E correlates with a decrease in the
inhibitory effect of ID on DBD-ARE interactions.
The N-terminal parts of nuclear receptors are the most divergent
among members of this superfamily of proteins, suggesting that each
receptor will take on a unique N-terminal conformation to determine its
specificity. This paper describes a highly conserved novel inhibitory
domain designated ID, which lies in N-terminal 81-amino acid residues
upstream of the DBD of AR. ID interacts directly with DBD and strongly
inhibits the DBD-ARE interactions in vitro and AR
trans-activation in vivo.
Much of the work devoted to understanding regulation of transcription
by the AR has focused on the N-terminal AF1 and the C-terminal AF2
(30). However, transcriptional inhibition may be equally important as a
way of preventing activation. Studies that deal with inhibition of
AR-dependent transcription have focused on silencing
mechanisms through recruitment of corepressors to the target promoters
and through receptor occupancy at one DNA site interfering with
transcription by an activator at an adjoining site (5, 31). We have now
demonstrated that negative function element exists in the AR molecule
itself and markedly suppresses the DNA binding activity of DBD. The ID
function is similar to that of the N-terminal region of TAF250 (the
250-kDa TATA box-binding protein-associated factor 1), which forms a
DNA-like structure, interacts with the DNA-binding surface, and
inhibits the DNA binding activity of TATA box-binding protein (32). In
contrast, the direct interactions between the ID and DBD suggest that
perhaps ID acts through intramolecular contacts. In this respect, ID is similar to p53, which exists in a latent DNA-binding form as a result
of the C-terminal tail-DNA-binding domain interactions (33, 34).
Phosphorylation of lysine residues in the C-terminal region leads to
the disruption of interactions between the C-terminal domain and the
core DBD, thus allowing the DBD of p53 to adopt an active conformation.
It is important to know whether modifications in the ID of AR or
interactions of this domain with the other proteins might regulate the
DNA binding activity of AR. A study on the rat AR indicated that the
unknown protein could enhance the DNA binding activity of the protein
fragment containing the DBD in a gel shift assay (35). Another study
has demonstrated that mutations on 668QPIF671
at the boundary of the hinge and ligand-binding domain of AR, resulting
in receptors that exhibit 2-4-fold increased activity compared with
the wild-type AR in response to dihydrotestosterone, and these
mutations have been detected in prostate cancer patients (36). However,
the molecular mechanism for this phenomenon is unclear.
Several mutations found in men with prostate cancer (37) and in men
with the androgen insensitivity syndrome (38, 39) localize in ID (Fig.
5A). These mutations might change the function of ID,
therefore affecting AR trans-activation. D528G mutation was detected in
a patient with prostate cancer (37), and we found that AR with D528G
mutation was more active (>3-fold) than the wild-type AR in transient
transfection assays (data not shown). Currently, we are investigating
whether the enhanced AR trans-activation is because of the decreased ID
function. Thus, ID may play an important regulatory role in AR
function, and dysfunction of ID may contribute to prostate cancer or
androgen insensitivity syndrome in some men.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
152 to
174)
were synthesized, annealed, and subcloned into HindIII and
EcoRI sites of the vector pBluescript II (Stratagene). The
underlined bases were mutated from their corresponding bases in the wild-type prostate-specific antigen gene sequence. The wild-type
and mutant ARE probes were made by cutting these constructs with
XhoI and XbaI and purification of fragments from
agarose gel. Probes were labeled with [
-32P]dCTP by a
fill-in reaction with the Klenow enzyme. In gel shift assays, 20-µl
reaction contains 20 mM HEPES, pH 7.9, 70 mM
KCl, 1 µg of poly(dI-dC), 1 mM dithiothreitol, 0.1%
Nonidet P-40, 100 µg/ml of bovine serum albumin, and various
proteins. The reaction mixture was incubated for 20 min at room
temperature, and the binding reaction was initiated by the addition of
the labeled probes (20,000 cpm) and then incubated for an additional 30 min at room temperature. The reaction mixture was loaded directly onto
a 4% (37.5:1, acrylamide:bisacrylamide) nondenaturing polyacrylamide gel with 0.25× Tris borate EDTA and run at 150 V for 2 h
at room temperature.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
152 to
174) (Fig. 1D) (23) was used for a gel shift assay. The
recombinant AR (0.9 pmol) shifted the probe (Fig. 1C, lane 2) while there was no ligand (androgen) dependence
(lane 3 versus lane 2). The band of
the AR·ARE complex (indicated by an arrow on the
left) is quite broad. However, mutations of the nucleotides
in the probe that are critical for AR-ARE interaction (24) (Fig.
1D) dramatically decreased the density of the AR-ARE band
(Fig. 1C, lanes 7 and 8 versus lanes 2 and 3), indicating that
the shifted band is specific. The DBD of AR (amino acid residues 537-644) (Fig. 1A) was expressed as a
His6-tagged fusion protein and purified through an
nitrilotriacetic acid Ni2+-agarose affinity column (Fig.
1B, lane 4). In the same assay, 0.3 pmol of
AR537-644 almost completely shifted the probe (Fig. 1C,
lane 4). The band of the AR537-644·ARE complex
(indicated by an arrow on the left) is much
sharper (Fig. 1C, lanes 4 and 5), and
mutations in the probe (Fig. 1D) completely diminished the
formation of the AR537-644·ARE complex (Fig. 1C,
lanes 9 and 10). The results indicate that the
binding affinity of DBD to the ARE is much stronger than that of the
purified full-length AR to the same ARE.
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Fig. 1.
The recombinant AR interacts with the ARE
weaker than the DBD. A, diagram of domains and
truncations of AR. B, SDS-PAGE analysis of the recombinant
AR and DBD. Proteins of 100 ng (lane 2) and 200 ng
(lane 3) of the purified recombinant AR expressed in
Sf9 cells and recombinant His6-tagged truncations of
AR expressed in bacteria (lanes 4-6) were subjected to
SDS-PAGE with Coomassie Blue R250 staining. Lane 1 is
standard protein markers (Bio-Rad). C, the gel shift assay
was performed using a DNA probe containing the wild-type ARE
(lanes 1-5) or the mutant ARE (lanes 6-10). 0.9 pmol of AR (lanes 2, 3, 7, and
8) and 0.3 pmol (lanes 4 and 9) or 0.6 pmol (lanes 5 and 10) of AR537-644 were used in
the binding reactions. The synthetic androgen R1881 (100 nM) was included in the reactions in lanes 3 and
8, and lanes 1 and 6 are probes only.
D, sequences of the wild-type (AREwt) and mutant
(AREmt) ARE and the ARE consensus. The mutated bases in
AREmt are underlined. PSA, prostate-specific
antigen. M, standard molecular markers. Qn and
Gn, stretches of glutamines and glycines, respectively.
LBD, ligand-binding domain. AR+L, the gel shift
assay performed in the presence of androgen R1881.
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Fig. 2.
AR477-558 blocks DBD binding to ARE.
A, AR477-644 did not interact with the ARE. 0.15 (lane 2), 0.3 (lane 3), or 0.6 pmol (lane
4) of AR537-662 and 0.4 pmol (lane 5), 0.8 pmol ng
(lane 6), or 1.6 pmol (lane 7) of AR477-644 were
used in the binding reactions. Lane 1 is the probe-only
control. B, SDS-PAGE of the purified recombinant wild-type
(lane 1), K580E (lane 2), or R538E (lane
3) AR477-558. The full-length proteins (top arrow) and
the main degraded products (bottom arrow) are indicated on
the right. Nonspecific background bands are marked with a
star on the right. C, AR477-558
inhibits AR537-643 binding to the ARE. The binding reactions contained
0.3 pmol of AR537-644 (lane 2) or 0.3 pmol of AR537-644
plus 0.0625 (lane 3), 0.125 (lane 4), 0.25 (lane 5), 0.5 (lane 6), 1 (lane 7), or
2 pmol (lane 8) of AR477-558. D, the purified
recombinant PAR-4, TIP30, and RPC39 proteins do not affect the
interaction of AR537-644 with ARE. The binding reactions contained 0.3 pmol of AR537-644 (lane 2) or 0.3 pmol of AR537-644 plus
0.225 (lane 3), 0.45 (lane 4), 0.9 (lane
5), or 1.8 pmol (lane 6) of prostate apoptosis
response-4 (Par-4) (lane 8) or 1.25 (lanes
7 and 10), 2.5 (lanes 8 and 11),
or 5 pmol (lanes 9 and 12) of 30-kDa
Tat-interaction protein (TIP30) (lanes 7-9) or
39-kDa subunit of RNA polymerase C (RPC39) (lanes
10-12), respectively. WT, wild type.
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Fig. 3.
AR477-558 interacted directly with DBD
in vitro and inhibited AR trans-activation in
vivo. A, AR477-558 interacted
directly with DBD(AR537-644) in vitro. SDS-PAGE analysis of
GST (lane 2) and GST-DBD (lane 3) expressed in
bacteria and immobilized on glutathione-Sepharose 4B beads is shown.
Bands corresponding to GST and GST-DBD fusion protein are indicated by
arrows on the right. The immobilized GST-DBD
pulled down 35S-labeled AR477-558 (lane 6).
Lane 4 is 10% 35S-labeled AR477-558 input
(IP) for the pull-down assay. B, AR477-558
inhibited AR trans-activation in vivo. PC3 cells were
transfected with 100 ng of the reporters PGL3-ARE-E4 or pGL3-GAL4-E4,
2.5 ng of the internal control reporter pRL-CMV, 20 ng of pcDNA-AR or
pcDNA-GAL4-p53-(1-53), and 18.5 ng of pcDNA-AR477-538 as indicated.
Cells were treated with 10 nM R1881 after transfection and
harvested 48 h later for the dual luciferase assay. C,
AR477-538 did not affect AR protein levels in the transfected cells.
Western blot analysis of cells transfected with pcDNA3.1 (lane
1), pcDNA-AR (lane 2), or pcDNA-AR plus
pcDNA-AR477-558 (lane 3) with the anti-AR antibody is
shown.
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Fig. 4.
The DBD-containing fragments of GR bind to
the ARE probe. A, sequence alignment of DBD and ID of
AR with the corresponding regions of rat GR. B, SDS-PAGE
analysis of the recombinant GR418-525 and GR358-525. 500 ng of
His6-tagged GR418-525 (lanes 1) and GR 357-525
(lane 2) expressed in bacteria were subjected to SDS-PAGE
with Coomassie Blue R250 staining. The bands corresponding to the
full-length protein fragments are indicated by arrows on the
right, and a nonspecific background band is marked by a
star on the left. The standard protein markers
(Bio-Rad) are indicated on the left. C, GR417-525 and
GR357-525 bind to the ARE. 0.3 pmol of GR417-525 (lane 2)
or GR357-525 (lane 3) was used in the binding reactions.
Lane 1 is the probe-only control.
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Fig. 5.
Mutations in ID enhanced AR trans-activation
and decreased ID inhibitory activity. A, sequence
alignment of ID and DBD of human (hAR), rabbit
(rAR), mouse (mAR), and Xenopus (xAR)
AR. Point mutations found in prostate cancer (PC) and in
complete (CAIS), mild (MIAS), or partial
(PAIS) androgen insensitivity syndrome patients are
indicated by arrows on the top. B,
mutations (K520E and R538R) enhanced AR trans-activation in
vivo. PC3 cells were transfected with 100 ng of the reporter
pGL3-ARE-E4, 2.5 ng of the internal control reporter pRL-CMV, or 10 ng
of pcDNA-wild-type AR or pcDNA-mutant (K520E or R538) AR as
indicated. Cells were treated with 10 nM R1881 after
transfection and harvested 48 h later for the dual luciferase
assay. Each value represents the mean ± S.D. of a representative
experiment performed in triplicate. C, Western blot analysis
of cells transfected with pcDNA3.1 (lane 1) or with
wild-type (lane 2), K520E (lane 3), and R538E
(lane 4) mutant AR. D, mutations of K520E and
R538E decreased ID inhibitory ability on DBD-ARE interactions. The
binding reactions contained 0.075 (lane 2), 0.15 (lane
3), or 0.3 pmol (lane 4) of DBD alone or 0.3 pmol of
DBD plus 0.625 (lanes 5, 9, and 13),
0.125 (lanes 6, 10, and 14), 0.025 (lanes 7, 11, and 15), or 0.005 pmol
(lanes 8, 12, and 16) of wild-type
(lanes 5-8), K520E mutant (lanes 9-12) or R538E
mutant AR477-558 (lanes 13-16). WT, wild
type.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Michael S. Worley for critical editorial review and Liliana DeGeus for expert assistance in the preparation of the paper.
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FOOTNOTES |
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* This work was supported in part by U. S. Department of the Army Grant DAMS17-01-1-0097, the Association for the Cure of Cancer of the Prostate, and Cancer Center Support Core Grant CA16672 from NCI, National Institutes of Health.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.
Present address: Dept. of Infectious Diseases, XiangYa Hospital,
Central-South University, Chang Sha 410008, People's Republic of China.
§ To whom correspondence should be addressed: Dept. of Cancer Biology, Unit 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030-4009. Tel.: 713-794-1035; Fax: 713-792-8747; E-mail: zhenwang@mdanderson.org.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M212229200
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ABBREVIATIONS |
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The abbreviations used are: AR, androgen receptor; AF, transcription activation; DBD, DNA-binding domain; ARE, androgen response element; GR, glucocorticoid receptor; GST, glutathione S-transferase; Luc, luciferase; ID, inhibitory domain.
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