From the Division of Biochemistry, Faculty of Medicine, Campus Gasthuisberg, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium
Received for publication, October 21, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Steroid receptors are transcription factors that,
upon binding to their response elements, regulate the expression of
several target genes via direct protein interactions with
transcriptional coactivators. For the androgen receptor, additional
interactions between the amino- and carboxyl-terminal regions have been
reported. The first amino acids of the amino-terminal domain are
necessary for this amino/carboxyl-terminal interaction. Deletion of a
FQNLF core sequence in this region blunts the interaction, as does a G21E mutation. We investigated the effect of the aforementioned mutations in the context of the full size androgen receptor on a series
of selective and nonselective androgen response elements. Strikingly,
the FQNLF deletion strongly reduced the androgen receptor capacity to
transactivate through nonselective motifs but did not affect its
activity on selective elements. Although the G21E mutation strongly
impairs the amino/carboxyl-terminal interaction, it does not
significantly influence androgen receptor activity on either selective
or nonselective elements. Surprisingly, this mutation leads to an
increased binding of the amino-terminal domain to the glutamine-rich
region of the steroid receptor coactivator-1 of the p160 family. Taken
together, these data suggest that the amino-terminal amino acids of the
androgen receptor play a key role in determining its transcriptional
activity by modulating the interaction with the ligand-binding domain
as well as interaction with p160 coactivators.
Androgens are involved in the differentiation of the male
reproductive organs in the embryo and the development and maintenance of secondary sexual characteristics. The biological actions of androgens are mediated by the androgen receptor
(AR).1 The AR is a
ligand-activated transcription factor and belongs to the class I
subgroup of the nuclear receptor superfamily. Two structural domains
are well conserved among the nuclear receptors (NR): the DNA-binding
domain (DBD), consisting of two zinc finger modules, and the
ligand-binding domain (LBD), containing a transcription activation
function (AF-2) (1, 2). The amino-terminal domain (NTD) and the hinge
region are more divergent. Because of their highly conserved DBD, it is
not surprising that the class I steroid receptors (AR, GR, progesterone
receptor, and mineralocorticoid receptor) recognize the same
palindromic (inverted) repeats of the 5'-TGTTCT-3' core sequence,
spaced by three nucleotides. The mechanisms that lead to the in
vivo steroid specificity of gene regulation are still not
completely understood (3-6). Selective DNA binding by the AR could be
one possible mechanism for hormone-specific gene regulation. It has
been reported that the AR-DBD does not only recognize response elements
organized as inverted repeats of 5'-TGTTCT-3' sequences with a
three-nucleotide spacer but can also bind to direct repeats of
5'-TGTTCT-3'-like sequences, which is proposed to contribute to the AR
specificity of transcriptional responses (7-14).
The NTD of the AR is indispensable for AR transactivation and contains
a strong transcription activation function (AF-1). Its activity is
affected by coregulators that influence a number of functional
properties of AR (15-18). Several putative coregulators (coactivators
and corepressors) have already been described (19). The best
characterized subgroup of receptor-interacting proteins is the family
of 160-kDa NR coactivators, also called p160 proteins, comprising
steroid receptor coactivator-1 (SRC-1) (20-23), the human and rat
transcription intermediary factor 2 (24-26) and its mouse
orthologue glucocorticoid receptor-interacting protein 1 (27), and
receptor-associated coactivator 3 (28, 29), also known as activator of
thyroid hormone receptor (ACTR) (30), thyroid
receptor-associated molecule 1 (TRAM-1) (31), amplified in breast
cancer (AIB) (32), p300/CBP co-integrator-associated protein (p/CIP)
(33), and SRC-3 (34). They interact with the LBD via a centrally
located NR-interacting region containing three highly conserved
The AR depends on a strong amino/carboxyl-terminal (N/C) interaction
for its full activity (18, 40-43). These N/C interactions have also
been demonstrated in the progesterone receptor (44) and the estrogen
receptor Plasmid Constructs--
The expression vectors
pSG5AR538-919 (encoding the hAR-DBD-LBD), pSG5AR
(expressing FLAG-tagged full-length hAR), and pSG5SRC-1e and the vector
Qr SRC-1 (expressing the Qr domain of SRC-1 fused to the Gal4-DBD) are
described elsewhere (14, 18, 39). The Gal4-DBD and the VP16 fusion
constructs hAR1-529, hAR1-529 Transfections--
All of the transfections were performed in
COS-7 African green monkey kidney cells, obtained from the American
Type Culture Collection (Manassas, VA). One day prior to transfection,
the cells were seeded in 96-well culture plates in Dulbecco's modified Eagle's medium (Invitrogen) containing 5% dextran-coated
charcoal-stripped fetal bovine serum at a density of 104
cells/well. They were transfected using FuGENE 6 transfection reagent
(Roche Molecular Biochemicals) as described by the manufacturer. The
amount of luciferase reporter construct was fixed on 100 ng/well, and
the amount of pCMV- Preparation of COS-7 Whole Cell Extracts Containing Full Size
AR--
COS-7 cells were plated in 10-cm Petri dishes and were
transiently transfected as described above with 1 µg of expression vectors. At 24 h after transfection, the cells were stimulated for
1 h with R1881 (10 Gel Shift Assays and Western Blots--
Synthetic complementary
oligonucleotides were hybridized, radioactively labeled, and used in
band shift assays as described previously (14). In brief, 5 µl of
total cell extract was preincubated with 1 µl of poly(dI-dC) (1 µg/µl), 10 µl of D100 (20 mM Hepes, 5 mM
MgCl2, 0.1 mM EDTA, 17% glycerol, 100 mM NaCl), 1 µl dithiothreitol (20 mM), 1 µl
Triton X-100 (1%), and 1 µl of water. Subsequently, the probe is
added and incubated for 20 min on ice. Bound probe was separated from
the free by an electrophoresis for 2 h at 120 V in a 5%
polyacrylamide gel. To obtain supershifts, a rabbit antiserum against
human AR was added prior to the probe (48). For Western blotting, equal
amounts of protein were separated by SDS-PAGE on an 8% gel and blotted
onto polyvinylidene difluoride membranes (Amersham Biosciences). The
membranes were probed with a monoclonal M2 anti-FLAG antibody
(Stratagene), and immunoreactive proteins were visualized with the
chemiluminescence reagent plus (PerkinElmer Life Sciences). For the
detection of VP16 fusion proteins, extracts were made in passive lysis
buffer. Western blot analysis was performed as described above.
Hormone-dependent Functional Interaction between the
LBD and the Amino-terminal Domain--
Previous studies have shown
that an FQNLF motif at position 23 in the amino-terminal domain of the
human AR plays a key role in the androgen-dependent
interaction between the NTD and the LBD. Another motif
(432WHTLF436) was also postulated as a possible
interaction site (40). We have studied the N/C interaction in a
mammalian two-hybrid system using a luciferase reporter construct
driven by the E1b promotor and containing a tandem repeat of the rat
tyrosine aminotransferase-GRE (2×TAT-GRE(E1b)-Luc). In these
experiments we tested and compared three AR-NTD (AR1-529)
mutants (Fig. 1A). The first
construct consists of a deletion of the first motif
(AR1-529 Role of the FQNLF Motif in the Transactivation through Different
Types of AREs--
The effect of the deletion of the FQNLF motif
(hAR
Two mutations were introduced within slp-HRE2
(slp-HRE2 mut-4T-A;+2A-T) and in sc-ARE1.2
(sc-ARE1.2 mut-4T-A; Role of the FQNLF Motif in Transactivation through Complex
Enhancers--
We tested four luciferase reporter constructs in
transient transfections of COS-7 cells (Fig.
4). One construct is driven by the
probasin proximal promotor (pPB-Luc). The three other constructs are
driven by the thymidine kinase minimal promotor and contain either the
C3 (1) intronic enhancer (pC3 (1)-TATA-Luc), the slp
enhancer (pSLP-TATA-Luc), or the sc enhancer (pSC-TATA-Luc). Three of them (pPB-Luc, pSLP-TATA-Luc, and pSC-TATA-Luc) are controlled by androgen-specific elements, whereas pC3 (1)-TATA-Luc is activated by
all class I steroid receptors (12). In contrast to the isolated
elements tested in Figs. 2 and 3, no difference in the effect of the
FQNLF deletion is seen between specific and nonspecific enhancers,
because for all reporter constructs, the FQNLF deletion reduces the
potency of the AR ~2-fold. The average induction factors are 15.0, 15.6, 35.0, and 49.8 for wtAR and 6.2, 4.6, 19.0, and 17.0 for
AR Analysis of Gly21 Flanking the FQNLF
Core--
The point mutation G21E shows a decreased AR N/C interaction
similar to the deletion of the FQNLF core in a mammalian two-hybrid assay (Fig. 1C). We tested whether the transcriptional
activity of the full size AR is affected by this mutation when specific or nonspecific AREs are used. Surprisingly, the transcriptional activation of the TAT-GRE construct is decreased only marginally when
the G21E mutation is introduced in the AR, and no clear effect is seen
when the androgen-selective slp-HRE2 was tested. When the
nonselective mutant of slp-HRE2 was used, the G21E mutation again had no effect on the functionality of the AR (Fig.
5).
Effects of Mutations within the NTD on DNA Binding--
To analyze
the influence of the deletion of the FQNLF motif and the G21E and the
L435P mutations on the in vitro interaction of the full size
receptor with nonspecific and specific AREs, band shifts assays using
wtAR, AR Analysis of the AF-1 Activity of the Mutated AR-NTDs and Binding
the Qr Region of SRC-1--
The AR-NTD contains a strong
ligand-independent activation function in the carboxyl-terminal region
of the AR-NTD (Tau-5). To analyze whether the aforementioned mutations
influence this constitutively active Tau-5 function, wild type and
mutant forms of the AR-NTD domains were fused to the Gal4-DBD and
transfected into COS-7 cells. As reporter construct, we used
(Gal4)5-TATA-Luc (Fig.
7A). Deletion of FQNLF and the
G21E mutation resulted in an activation comparable with that of
wtAR-NTD, whereas the L435P mutation resulted in a decreased
activation.
It has already been proposed that the efficient recruitment of
coactivators by the native AR occurs primarily through the NTD (18,
37-39). SRC-1 is a member of the p160 coactivator family and contains
a glutamine-rich region (amino acids 989-1240) called Qr, needed for
interaction with the amino-terminal domain of the AR in a
ligand-independent manner (18, 37-39). We tested whether the mutations
AR1-529 For the members of the steroid receptor superfamily such as
estrogen receptor The AR-NTD not only harbors a strong activation function (AF-1) (16)
but also displays a high affinity for the liganded LBD (18, 40-43).
LXXLL-related sequences in the AR-NTD have been investigated
for their interaction with the LBD. We already reported the involvement
of one LXXLL-like motif, 179LKDIL183
(18), the mutation of which (I182A/L183A) impairs the N/C interaction and the activity of the AR. Two other candidate motives have been described: 23FQNLF27 and
432WHTLF436 (40, 41). In this study, we
performed a functional analysis of these latter motifs. Deleting the
FQNLF motif destroys the ability of the AR-NTD to interact with the
AR-LBD. The mutation of Gly21 flanking the FQNLF core to
Glu strongly attenuates the N/C interaction. In contrast to another
report (40), LBD binding by the L435P mutated NTD is comparable with
that observed for the wtAR-NTD (Fig. 1C).
To investigate the role of the N/C interaction in AR functionality in
more detail, we analyzed the activity of the mutated receptors on
different responsive constructs. Next to the classical nonselective
class I elements, there is an additional group of AREs that are only
recognized by the AR. Examples of such androgen-specific elements are
the PB-ARE2 from the rat probasin promotor (7, 49, 51), the SC-ARE
present in the first exon of the human secretory component (SC) gene
(13, 52), the sc-ARE1.2 in the far upstream enhancer of the
human SC gene (8, 10), the slp-HRE2 from the upstream
enhancer of the mouse sex-limited protein (53), and the Pem ARE-1 and
ARE-2 in the proximal promotor of the murine pem gene (54).
Elsewhere, we hypothesized that an alternative dimerization mechanism
of the DNA-binding domain would be responsible for the androgen
specificity (11). We demonstrated indeed that the AR is the only
steroid receptor which binds direct repeats of 5'-TGTTCT-3'-like
sequences (9-11, 13), indicating that the AR could bind the specific
elements in a head-to-tail configuration and not in the conventional
head-to-head configuration. In this study, we compared the
transcriptional activity of the constructs AR We observed a decrease in the transcription activating capacity of
AR Nonspecific HREs seem to be more dependent on the N/C interaction
compared with the direct repeat AREs (Figs. 2 and 3). One exception is
the AR-specific PB-ARE2, although this might be explained by the fact
that PB-ARE2 (5'-GGTTCTnnnAGTACT-3') can be considered as a direct as
well as a palindromic repeat. It is clear from Fig. 6A that
there are no obvious differences between the wild type and the mutant
receptors in their binding characteristics for the different DNA elements.
The G21E mutation strongly reduces N/C interaction (Fig.
1C). It is therefore clear that not only the core FQNLF
motif but also some flanking residues contribute. This correlates with
the stronger conservation of this region among the AR of different species (Fig. 1B) (41, 55). Surprisingly, the differences seen on AR transcriptional activity at specific versus
nonspecific elements by the deletion of FQNLF were not observed with
this point mutation. This might be explained by the residual
AR-NTDG21E/AR-LBD interaction, observed in Fig.
1C, which could be sufficient for AR activity on the
nonspecific HREs.
The differences we observed for AR A second interesting feature for the G21E mutation is an almost 3-fold
increase in the affinity for the Qr of SRC-1 as measured in
double-hybrid assays (Fig. 7B). This increase in Qr binding is not seen for the AR-NTD Our data do not exclude the possibility of a stabilization by the NTD
of the conformation of the LBD, which would lead to an enhanced
recruitment of coactivators as postulated earlier (57). However, the
effects of the G21E mutation on Qr recruitment by the isolated NTD
(Fig. 7B) leads us to propose a more direct role in the p160 recruitment.
In summary, our findings demonstrate a dual role of the first amino
acids of the amino-terminal domain of the AR, which are essential for
the N/C interaction and which seem to have an additional function in
interaction with Qr of SRC-1. These results contribute to the
unraveling of the mechanisms involved in the AR-N/C interactions and
the mechanisms of SRC-1 recruitment. Recently, it has been described
that the relative contribution of the two interaction sites of SRC-1
with the AR, the LXXLL motifs and the Qr region, depend on
the nature of the enhancers (39). In addition, we provide the first
indication that transactivation by the AR through some selective AREs
(slp-HRE2 and sc-ARE1.2) is structurally
different from transactivation through other classical AREs.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical LXXLL motifs or NR boxes (20, 28, 29, 35, 36).
In addition, the glutamine-rich region of SRC-1 (Qr) can interact with
the NTD of the AR in a ligand-independent manner (18, 37-39).
(45, 46), but in these receptors the implication in
receptor activity is less pronounced. The AR N/C interaction involves
LKDIL and FQNLF core sequences, both present in predicted
-helical
structures within the NTD (18, 41). The FQNLF motif is located within
the thirty amino-terminal amino acids of the AR and has the ability to
bind AF-2 of the carboxyl-terminal domain in an
androgen-dependent manner. The LKDIL motif is located
between amino acids 178 and 183 and is important for the
transcriptional activation function AF-1. In this paper, we describe
the involvement of the FQNLF motif and a flanking conserved Gly in the
N/C interaction. In addition, we have analyzed the functional role of
this interaction on isolated direct and indirect repeat AREs and on
AR-specific versus nonspecific enhancers. Our data suggest
that the interaction between the AR-NTD and -LBD may be a prerequisite
for its transcriptional activity on nonspecific HREs, whereas it is
dispensable on specific HREs.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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FQNLF,
and hAR1-529L435P were made by a PCR-based method. The
construct hAR1-529 was made by using the following primers: 5'-GGCAGATCTCCATGGAAGTGCAGTTAGGGCTGGG-3' and
5'-GGATCCTCAACGCATGTCCCCGTAAGGTCCGGA-3'. As template, the expression
vector for the full-length human AR (pSV-AR), a kind gift of Dr.
A. O. Brinkmann (Erasmus University of Nijmegen, Nijmegen, the
Netherlands) was used. The
FQNLF deletion was generated with the
oligonucleotides 5'-CCTACCGAGGAGCTCAGAGCGTCCGCGAAGTG-3' and
5'-CGCGGACGCTCTGAGCTCCTCGGTAGGTCT-3'. To generate the L435P mutation, the following oligonucleotides were used:
5'-CCTGGCACACTCCCTTCACAGCCGAAG-3' and
5'-CTGGCCTTCTTCGGCTGTGAAGGGAGTGTGCCAGGATGAG-3', in
which the mutated base is underlined. These PCR-generated hAR-NTD
fragments were inserted in frame with the Gal4-DBD in the
BamHI restriction site of pABGal4 and in frame with the VP16
activating domain in the BglII site of pSNATCH-II (18).
Expression vectors for the hAR carrying the FQNLF deletion or the L435P
point mutation were made by insertion of a
Eco52I/Eco81I fragment of the PCR products in the
pSG5AR construct, which was cut with the same restriction enzymes. The
pSG5 construct expressing the G21E-mutated hAR was generated by
coincidence and was detected by sequencing. Restriction and modifying
enzymes were obtained from MBI Fermentas GmbH. The luciferase reporter
constructs containing the isolated elements TAT-GRE,
slp-HRE3, C3 (1)ARE, slp-HRE2,
sc-ARE1.2, slp-HRE2 mut-4T-A;+2A-T, and
sc-ARE1.2 mut-4T-A;
2A-T are driven by the thymidine kinase
minimal promotor and have been described elsewhere (Ref. 10 and
references therein). The reporter construct driven by the E1b promotor
and containing two copies of the rat tyrosine aminotransferase-GRE was
described previously (47). The thymidine kinase minimal promotor-driven
reporter construct containing the slp and sc
upstream enhancers and the C3 (1) intronic fragment as well as the
pb promotor-driven construct have also been described (10).
The reporter construct (Gal4)5TATA-Luc, a kind gift from Dr. M. G. Parker (Imperial Cancer Research Fund, London,
UK), was used for measuring the AF-1 AR activity. The
pCMV-
-galactosidase vector was obtained from Stratagene.
-galactosidase was fixed at 5 ng/well. Where
applicable, empty pGEM-T, empty pSG5, empty pSNATCH-II, or pABGal4
vector was added to keep the total amount of transfected DNA constant.
After transfection, the cells were incubated for 24 h with medium
containing 5% dextran-coated charcoal-stripped fetal
bovine serum and supplemented or not with hormones (10
8
M). The synthetic androgen R1881 (methyltrienolone) was
purchased from PerkinElmer Life Sciences. Finally, the cells were
washed twice with phosphate-buffered saline (Invitrogen) and lysed in 25 µl of passive lysis buffer (Promega). The luciferase and
-galactosidase activities were measured on 2.5 µl of the extracts
using the assay systems from Promega and Tropix (Westburg, the
Netherlands), respectively. The luciferase activity was corrected for
transfection efficiency by normalizing it according to its
-galactosidase activity. The values shown are the averages of at
least three independent experiments performed in triplicate. The error
bars are the S.E. values.
8 M). The medium was
removed, and the cells were washed twice with ice-cold
phosphate-buffered saline. The cells were collected in 1.5 ml of
ice-cold phosphate-buffered saline/dish and pelleted by centrifugation
(1 min). The phosphate-buffered saline was removed, and the cells were
resuspended in 100 µl of ice-cold extraction buffer containing 20 mM Hepes-KOH, pH 7.8, 450 mM NaCl, 0.4 mM EDTA, and 25% glycerol. Immediately before use,
dithiothreitol and phenylmethylsulfonyl fluoride are added to a final
concentration of 0.5 mM. The samples were freeze-thawed
three times. The pellet was collected, and the supernatant was removed
and stored at
80 °C. The protein concentrations of the extracts
were determined with the Coomassie protein assay kit (Pierce) and were
in each case ~4 µg/µl.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
FQNLF). The second construct has a point
mutation immediately amino-terminally of the FQNLF motif;
Gly21 is mutated to a Glu (AR1-529G21E). This
Gly is outside the FQNLF motif but inside a predicted
-helical
region and is highly conserved among different species (Fig.
1B). The third construct has a point mutation in the WHTLF
motif so that Leu435 is mutated into a Pro
(AR1-529L435P). Wild type and mutant forms of the AR-NTD
domain were fused to the VP16 transactivation domain and were
coexpressed in the mammalian two-hybrid assay with
pSG5AR538-919, which encodes a DBD- and LBD-containing fragment of the AR (Fig. 1C). Expression levels of the VP16
fusion constructs were similar (Fig. 1D). As expected,
the AR1-529 fragment interacts
hormone-dependently with AR538-919. The deletion of the FQNLF motif in AR1-529 completely
abolished the R1881-dependent interaction. These findings
are in agreement with previous reports (40, 41). Surprisingly, the G21E
mutation in the NTD, outside the FQNLF motif, also markedly
reduces N/C interaction. In contrast, no difference in the LBD
interaction is observed for the AR1-529L435P mutation.
Therefore, we suggest that the first amino-terminal amino acids of the
AR-NTD form the predominant interaction site for the AR-LBD.
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Fig. 1.
AR amino-terminal mutants and
their role in N/C interaction. A, schematical
representation of the amino-terminal mutated domains FQNLF, G21E,
and L435P fused to the VP16 transactivation domain. B,
alignment of the first amino acids of the AR. The alignment was
performed using MultAlin (55). The conserved residues are in bold
type, and Gly21 and FQNLF are underlined.
C, two-hybrid assay. PSG5AR-DBD-LBD (538-919) (50 ng/well)
was coexpressed in COS-7 cells with either the empty pSNATCH-II
expression vector or the same expression vector containing the wild
type or the indicated mutated AR-NTDs (50 ng/well). The assays were
performed using the 2×TAT-GRE(E1b)-Luc reporter (100 ng) and the
CMV-
-galactosidase reporter (5 ng/well) in the presence or absence
of 10
8 M R1881. The luciferase activity was
calculated as described under "Materials and Methods." The values
shown are the averages of at least three independent experiments
performed in triplicate. The error bars indicate the S.E.
values. D, Western blot analysis of indicated mutated
AR-NTDs fused to the VP16 activation domain depicted in
A.
FQNLF) was analyzed in the context of the full size AR on a
series of selective and nonselective AREs (Fig.
2). When using wild type AR, the
induction factors are 16.5, 19.9, and 8.9 for the nonspecific AREs
TAT-GRE, slp-HRE3, and C3 (1)-ARE, respectively, whereas
AR
FQNLF showed a markedly decreased induction of these reporter
constructs (6.0, 8.7, and 3.1, respectively). However, for
slp-HRE2 and sc-ARE1.2, which are
androgen-specific HREs, no difference is seen between the wtAR and the
AR
FQNLF. In contrast, the FQNLF deletion does affect the
transactivating abilities of the receptor when tested on the PB-ARE2,
which is another known androgen-specific ARE (7, 49).
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Fig. 2.
Transcriptional activation of the wtAR and
AR FQNLF through specific versus
nonspecific HREs. Thymidine kinase minimal promotor-driven
luciferase reporter constructs (100 ng) containing two copies of the
response elements, indicated on the right, were transiently
transfected into COS-7 cells and cotransfected with 20 ng of empty
vector, pSG5wtAR, or pSG5AR
FQNLF as indicated on the
left. The cells were incubated for 24 h without hormone
(open bars) or with hormone (R1881, 10
8
M, black bars (wtAR) or gray bars
(AR
FQNLF)). The bars represent the transcriptional
activities of the AR constructs, relative to the activity of the
construct containing the empty pSG5 vector in the presence of hormone,
which was set at 1. Luciferase activities were adjusted according to
the
-galactosidase activity in the same sample. The results are the
averages of at least three separate experiments performed in
triplicate, and the error bars indicate the S.E. values. The
induction factors represent the average luciferase/
-galactosidase
values of the stimulated cells divided by the values obtained without
hormone stimulation.
2A-T), changing these elements into
nonselective ones and leading to a functional loss of AR specificity
(10). As shown in Fig. 3, this loss of specificity correlates with a strong decrease in transactivation by the
AR
FQNLF in comparison with wtAR activity. Although the activity of
the
FQNLF mutant on the wild type slp-HRE2 and the sc-ARE1.2 does not significantly differ from that of wtAR,
androgen-mediated induction factors of the mutated sc and
slp response elements are two or three times lower for the
FQNLF construct compared with the wild type AR.
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Fig. 3.
Transcriptional activation by the wtAR and
AR FQNLF on slp-HRE2,
sc-ARE1.2, and their mutated forms. Luciferase
reporter constructs containing two copies of either the wild type or
mutated slp-HRE2 and sc-ARE1.2 motifs, indicated
on the left, were transiently transfected into COS-7 cells
and cotransfected with 20 ng of empty vector, pSG5wtAR, or
pSG5AR
FQNLF. The mutated nucleotides are underlined, and
the repeats of 5'-TGTTCT-3'-like sequences are indicated by black
arrows. The direction of each arrow indicates the
orientation of the half-site. The experimental values are presented as
in Fig. 2.
FQNLF on pC3 (1)-TATA-Luc, pPB-Luc, pSLP-TATA-Luc, and
pSC-TATA-Luc, respectively.
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Fig. 4.
Transcriptional activation of the wtAR and
AR FQNLF on specific and nonspecific
enhancers. COS-7 cells were transfected with 100 ng of the
reporter constructs indicated on the left and cotransfected
with 20 ng of empty vector, pSG5AR, or pSG5AR
FQNLF. The experimental
values are presented as in Fig. 2.
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Fig. 5.
Transcriptional activation of the wtAR and
ARG21E at specific versus nonspecific HREs. COS-7
cells were transfected with 100 ng of luciferase reporter constructs
containing two copies of the response elements indicated on the
left and cotransfected with 20 ng of empty vector, pSG5wtAR,
or pSG5ARG21E. The experiments are presented as in Fig. 2.
FQNLF, ARG21E, and ARL435P were performed (Fig.
6A). COS-7 cells were
transfected with expression vectors for wtAR, AR
FQNLF, ARG21E, and
ARL435P (1 µg). As a specific ARE, we used slp-HRE2, and
as nonspecific elements, we used slp-HRE2 mut-4T-A;+2A-T and
the TAT-GRE. The gel shift assays showed very similar binding of wtAR
and the mutant ARs to different response elements. Western blot
analysis of the extracts was performed to assess equal expression
levels of the different receptors (Fig. 6B).
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Fig. 6.
A, gel shift assays of the TAT-GRE,
slp-HRE2 and slp-HRE2 mut ( 4T-A;+2A-T) HREs
with wtAR, AR
FQNLF, ARG21E, and ARL435P. Labeled probes (indicated
at the top of the panels) were incubated with 5 µl of COS-7 extracts containing wtAR, AR
FQNLF, ARG21E, or ARL435P
(lanes 2 and 3, lanes 4 and
5, lanes 6 and 7, and lanes
8 and 9, respectively). For each probe, no protein was
added in lane 1 as a negative control. The supershifts were
performed in the presence of specific antibodies against AR (
-AR,
lanes 3, 5, 7, and 9). Free
probe, shifted, and supershifted complexes are indicated on the
right by an open arrow, an asterisk,
or a black arrow, respectively. B, immunoblot
analysis of the AR and AR mutants. Equal amounts of COS extracts
containing AR
FQNLF, ARG21E, and ARL435P as used in the supershift
assay and as indicated at the top were used as to perform
Western blot. The extracts were resolved on 8% SDS-polyacrylamide gel
and subjected to immunoblotting with monoclonal M2 anti-FLAG
antibody.
View larger version (20K):
[in a new window]
Fig. 7.
Tau-5 activation function of wtAR-NTD and
mutated NTDs, interaction with QrSRC-1 and coactivation of wtAR and AR
mutants by SRC-1e. A, study of the constitutive
activation domain Tau-5. COS-7 cells were transfected with 50 ng of the
AR-NTD and the mutated NTDs, fused to the Gal4-DBD, together with the
luciferase reporter construct p(Gal4)5-TATA-Luc. The
activities are depicted relative to the activity of the wtAR-NTD
construct, which was set to 100. B, Interaction of AR-NTD
and the mutated NTDs with QrSRC-1. pABGal4-DBDQrSRC-1e (989-1240)
(50 ng/well) was coexpressed in COS-7 cells with 50 ng of empty
pSNATCH-II, pSNATCH-IIAR1-529,
pSNATCH-IIAR1-529 FQNLF,
pSNATCH-IIAR1-529G21E, or
pSNATCH-IIAR1-529L435P. The assays were performed using
the (Gal4)5-TATA-luciferase reporter (100 ng). The
activities are depicted relative to the activity of the wtAR-NTD
construct, which was set to 100. C, coactivation of wtAR and
AR mutants by SRC-1e. COS-7 cells were transfected with 20 ng of empty
vector and pSG5 with wtAR, AR
FQNLF, ARG21E, or ARL435P and
cotransfected with 100 ng pGEM-T or pSG5SRC-1e. As reporter construct,
100 ng of 2×TAT-GRE (E1b)-Luc was used. The activities are depicted
relative to the activity of the pSG5wtAR construct in the presence of
androgens, which was set to 100. Luciferase activities were adjusted
according to the
-galactosidase activity in the same sample. The
results are the averages of at least three separate experiments
performed in triplicate; the error bars indicate the S.E.
values.
FQNLF, AR1-529G21E, and
AR1-529L435P, fused to the VP16 activation domain, are
still able to interact with the Qr of SRC-1, fused to the Gal4-DBD
(Fig. 7B). A striking characteristic for the point mutation
G21E is that, although the NTD shows a strongly reduced interaction
with the AR-LBD (Fig. 1C), there is a 2.5-fold better
interaction with Qr when compared with wtAR-NTD. This is not seen with
AR1-529
FQNLF or AR1-529L435P (Fig.
7B). To compare the interaction of Qr with AR-NTD and its mutants with the coactivation ability of SRC-1e, the constructs pSG5,
pSG5wtAR, pSG5
FQNLF, pSG5G21E, and pSG5L435P and the 2×TAT-GRE(E1b) luciferase reporter construct were transiently transfected into COS-7
cells and cotransfected with pGEM-T or pSG5SRC-1e (Fig. 7C).
Transcriptional activity of ARG21E and ARL435P increased to the same
level compared with wtAR in the presence of SRC-1e, whereas the net
coactivation of AR
FQNLF in the presence of SRC-1e is lower.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, GR, and progesterone receptor, a ligand-induced conformational change has been clearly demonstrated to result in a
hydrophobic recruitment surface (AF-2) for transcriptional coactivators. These coactivators interact with the LBD via three highly
conserved
-helical LXXLL signature motifs (35). The AR
has been reported to be different from the other steroid hormone receptors in that the AF-2 activity in the LBD is quite low when tested
in mammalian cells (2). However, the AR-LBD has the same overall
three-dimensional structure as the LBDs of the other nuclear hormone
receptors (50).
FQNLF, ARG21E, and
ARL435P on specific versus nonspecific response elements and
enhancers (Figs. 3-5). For the mutation L435P in the WHTLF motif and
the mutation I182A/L183A in the LKDIL motif, no difference on selective
versus nonselective elements was seen. In fact, the L435P
mutation did not affect the transcriptional activation by any of the
tested motifs (data not shown).
FQNLF compared with wtAR on nonspecific HREs, whereas no changes
were observed on the specific slp-HRE2 and
sc-ARE1.2 elements. However, when these AREs are mutated in
their left and/or right half-sites (slp-HRE2 mut-4T-A;+2A-T
and sc-ARE1.2 mut-4T-A;
2A-T) to become partial palindromic
repeats, AR transactivation was significantly affected by the FQNLF
deletion. A possible explanation for why no decrease in AR
transcriptional activity is observed on the two specific AREs is that
AR dimers, in which the DBD is dimerized in a head-to-tail
conformation, might be less dependent on the N/C interaction for
transactivation. Whether other factors or cofactors play a role in
these differences remains to be elucidated.
FQNLF transcriptional activity at
specific versus nonspecific elements are limited to isolated response elements because no effect is seen on specific and nonspecific enhancers. This could be due to the fact that hormone response elements
form part of more complex enhancers, whose activity is also tightly
controlled by additional transcription factors (12). The slp
enhancer, for example, contains three HREs: HRE-1, HRE-2, and HRE-3,
and other transcription factors binding to the slp-ARU were
identified as NF
B, octamer transcription factor, and AML3/CBF
1 (56).
FQNLF nor for the L435P mutated AR-NTD. It
should be noted that deletion of the FQNLF core and the G21E mutation
does not affect the intrinsic activity of the NTD, in contrast to L435P
mutation, which reduces the activity 2-fold (Fig. 1C).
Possibly, the lowered affinity of the NTDG21E for the LBD and its
effect on transactivation by the AR is compensated by the increased
affinity of the NTD for the Qr of SRC-1. SRC-1e coexpression seems to
rescue the ARG21E activity to the same level as wtAR and ARL435P (Fig.
7C).
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ACKNOWLEDGEMENTS |
---|
We thank R. Bollen, H. Debruyn, and K. Bosmans for excellent technical assistance. We are indebted to A. O. Brinkmann and M. G. Parker for providing several plasmids.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap and by grants from the Fonds voor Wetenschappelijk Onderzoek, Vlaanderen.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.
Recipient of a Postdoctoral Fellowship from the Fonds voor
Wetenschappelijk Onderzoek-Vlaanderen.
§ To whom correspondence should be addressed. Tel.: 32-16-345770; Fax: 32-16-345995; E-mail: frank.claessens@med.kuleuven.ac.be.
Published, JBC Papers in Press, December 31, 2002, DOI 10.1074/jbc.M210744200
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ABBREVIATIONS |
---|
The abbreviations used are: AR, androgen receptor; GR, glucocorticoid receptor; NR, nuclear receptor; ARE, androgen response element; HRE, hormone response element; GRE, glucocorticoid response element; NTD, amino-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain; wt, wild type; AF, activation function; Tau-5, transcription activation function 5; CMV, cytomegalovirus; Qr, glutamine-rich; SRC, steroid receptor coactivator; N/C, amino/carboxyl-terminal.
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