Ligand- and Coactivator-mediated Transactivation Function (AF2)
of the Androgen Receptor Ligand-binding Domain Is Inhibited by the
Cognate Hinge Region*
Qi
Wang
,
JinHua
Lu§, and
E. L.
Yong
¶
From the
Department of Obstetrics and Gynecology, and
§ National University Medical Institutes, National
University of Singapore, Republic of Singapore 119074
Received for publication, October 31, 2000
 |
ABSTRACT |
Transactivation functions (AF2) in the
ligand-binding domains (LBD) of many steroid receptors are well
characterized, but there is little evidence to support such a function
for the LBD of the androgen receptor (AR). We report a mutant AR, with
residues 628-646 in the hinge region deleted, which exhibited
transactivation activity that was more than double that of the wild
type (WT) AR. Although no androgen-dependent AF2 activity
could be observed for the WT ARLBD fused to a heterologous
DNA-binding domain, the mutant ARLBD(
628-646) was 30-40 times more
active than the WT ARLBD. In the presence of the p160 coactivator TIF2,
AR(
628-646) was significantly more active than similarly treated WT
AR. Deletion of residues 628-646 also enhanced TIF2-ARLBD activity
8-fold, an effect not present when the LBD-interacting
LXXLL motifs of TIF2 were mutated, suggesting that the
negative modulatory activity of residues 628-646 were exerted via
coactivator pathways. Although the AP-1 (c-Jun/c-Fos) system and NcoR
have been reported to interact with and repress the activity of some
steroid receptors, c-Jun, c-Fos, c-Jun/c-Fos, nor NcoR function was
consistently affected by the absence or presence of residues 628-646,
implying that the AR hinge region exerts its silencing effects in a
manner independent of these corepressors. Our data provide evidence for
the novel finding that strong androgen-dependent AF2 exists
in the ARLBD and is the first report of a negative regulatory domain in
the AR. Because mutations in this region are commonly associated with prostate cancer, it is important to characterize the mechanisms by
which the hinge region exerts its repressor effect on ligand-activated and coactivator-mediated AF2 activity of the ARLBD.
 |
INTRODUCTION |
The androgen receptor
(AR),1 a member of the
steroid-hormone superfamily of nuclear transcription factors, mediates
male sexual differentiation in utero, sperm production at
puberty, and prostate growth in the adult. Like other members of the
steroid/nuclear receptor superfamily, the AR has four major functional
regions: the N-terminal transactivation domain (TAD), a central
DNA-binding domain (DBD), a C-terminal ligand-binding domain (LBD), and
a hinge region connecting the LBD and the DBD (see Fig. 1
below). In the absence of hormone, nuclear receptors are
maintained in a repressed state by association with heat shock proteins
and/or corepressors (1). In the thyroid hormone (THR) and retinoic acid
receptors, ligand-independent repression of gene transcription occurs
and is mediated by nuclear receptor corepressor proteins such as NcoR
(2, 3) and SMRT (4). Upon exposure to hormone, steroid receptors are
released from their inactive states and the receptor-ligand complexes
translocate to the nucleus and bind to and activate hormone response
elements of target genes. Activation function 1 (AF1) and AF2 are
located in the TAD and the LBD, respectively, of the steroid receptors,
and activity of these is dependent on the recruitment of coactivator
molecules to form active preinitiation sites for gene transcription (5,
6). Strong AF2 function can readily be demonstrated in LBDs of retinoic
acid receptor (7), retinoic-X receptor (8), vitamin D receptor (9), glucocorticoid receptor (GR) (10), progesterone receptor (PR) (6),
peroxisome proliferator-activated receptor (PPAR
) (11), estrogen
receptor (ER) (12), and THR (13) but not in AR (5, 14-16). Although
p160 coactivators, like SRC1 and TIF2, can bind to the ARLBD in a
hormone-dependent manner (6, 17, 18), the ARLBD itself
demonstrates very little activation function when fused to heterologous
DNA-binding domains (5, 14-16). Deletion of the AR TAD results in an
LBD fragment that can bind ligand and androgen response elements (ARE)
but is relatively inactive in reporter gene assays in human cells. In
contrast, the AR TAD fragment alone has a hormone-independent AF1 that
is almost equal to the ligand-activated full-length AR. The very
existence of an AF2 in the AR has been questioned, and it has been
proposed that AR transactivation function is dependent on the strong
AF1 activity of the TAD, consequent to ligand-activated interactions between the TAD and the LBD (5, 16, 19, 20). Crystal structures of all
LBDs solved to date indicate that activation of the apo-receptor by
ligand involves a structural change, where C-terminal helix 12 of the
LBD is positioned over the ligand-binding pocket to complete the AF2
surface (21, 22). Helix 12 and helices 3, 4, and 5 form opposite ends
of a hydrophobic cleft for binding leucine-rich motifs of
nuclear-receptor-interacting domains of coactivator molecules (23-25).
Considering the high similarity in crystal structure of the AR LBD (21)
to that of the PPAR
(11), vitamin D receptor (26), ER (27), and PR (28), it is puzzling as to why the prominent AF2 activity present in
other steroid receptors cannot be elicited in the ARLBD.
Although the LBD and the DBD of the AR have been characterized in some
detail (29), the hinge region in between these major domains, defined
by residues 628-669, is less well understood (Fig.
1). Although this region is poorly
conserved among steroid receptors, several lines of evidence indicate
that key functional elements may reside in this AR domain. For example,
7 of 10 reported amino acid substitutions affecting residues 619-672
in the AR hinge region are associated with the
androgen-dependent tumor, prostate cancer (30).
Furthermore, a sequence located between amino acids 628 and 657 within
the hinge region contains a short stretch of basic amino acids that
resemble the nuclear targeting signals of the glucocorticoid receptor
and the SV-40 large T antigen (31) and has been described to form part
of a bipartite nuclear localization signal (NLS) (32). In addition,
transactivation activity of the ARLBD in yeast, but not in mammalian
cells, appears to be modulated when the hinge region is attached (33),
and coexpression of Ubc9, a ubiquitin-conjugating enzyme that interacts with the hinge region, can enhance AR transactivation activity (34). In
this study we explored the effects of deleting key residues in the
hinge region and observed that the ligand-activated and
coactivator-augmented transcriptional activity of the deletion mutant
was unexpectedly higher than the WT. Experiments were performed to
elucidate the mechanism whereby the deleted residues exert an
inhibitory effect on ARLBD function.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Structure of the AR and homology comparison
of the bipartite NLS and hinge regions of human AR, PR, GR,
ER , PPAR , and
THR . The vertical line
demarcates the well-conserved DBD from the poorly conserved hinge
region. The two basic domains forming the bipartite NLS in the AR are
underlined, and the 19 residues, which are deleted in
AR( 628-646), are boxed. Underlined residues
in PPAR (166) and THR- (206) have been described to
interact with the corepressor NcoR (2, 3). The black box in
the AR is the hinge region (residues 628-669), and the shaded
box is the DBD (residues 557-627).
|
|
 |
EXPERIMENTAL PROCEDURES |
Construction of Plasmids--
The full-length AR deletion
mutant, AR(
628-646), was created by site-directed mutagenesis using
two internal primers: Sense (dF),
5'-gaagcagggatgactctgggcagcaccaccagccccact-3' and the
antisense (dR), 5'-cagtggggctggtggtgctgcccagagtcatccctgcttc-3'. The
external primers were sense (DNA-B), 5'-gacttcaccgcacctgatgtg-3' and
antisense (QE3), 5'-cctggagttgacattggtgaa-3'. Initially, two primary
PCRs were performed using the AR expression plasmid pSV-AR
as the template. Primers DNA-B and dR were used to generate fragment 1, and primers dF and QE3 for fragment 2. These overlapping primary
amplification products were then denatured and allowed to anneal
together to produce a heteroduplex product with overlapping ends. The
recessed ends of the heteroduplexes were extended with Pfu DNA
polymerase to produce a fragment that is the sum of the two overlapping
products. A subsequent reamplification was performed for 30 cycles
using primers DNA-B and QE3 to generate the cDNA fragment with a
deletion of AR codons 628-646 (
628-646). The secondary
PCR product was double-digested with HindIII and
XhoI and ligated into pSV-AR, with the equivalent
fragment excised, to generate AR(
628-646). The pGAL4DBD-ARLBD chimeric vector comprised the
AR hinge and LBD regions (codons 628-919) fused
in-frame to the GAL4DBD residues 1-147 (23).
pGAL4DBD-ARLBD
628-646 was generated by using
AR(
628-646) as the template and amplifying
the cDNA fragment encoding the ARLBD(
628-646) with the forward primer
5'-cagcaccaccagccccactgaggagac-3' and the reverse primer
5'-gtttccaaagcttcactgggtgtggaa-3'. The PCR product, including the stop
codon in exon 8 was digested with HindIII and ligated
in-frame into the SmaI/HindIII site of
pM containing the Gal4DNA-binding domain. The
plasmid pSV-AR(N727K,M886V), containing the double mutations
M886V and N727K, was created by placing the
XhoI-EcoRI segment containing the N727K mutation
(17) into the M886V AR expression vector (18).
pGAL4DBD-ARLBD(N727K,M886V) was generated by using
pSV-AR(N727K,M886V) as the template and amplifying the
LBD with the forward primers 5'-ggcccggaagctgaagaaactt-3' and the reverse primer 5'-gtttccaaagcttcactgggtgtggaa-3'. The PCR
product, including the stop codon in exon 8, was digested with
HindIII and ligated in-frame into the
SmaI/HindIII site of pM containing the
GAL4DNA-binding domain
pGAL4DBD-ARLBD(
628-646), and
pGAL4DBD-ARLBD(
628-646,N727K,M886V) were
generated by amplifying the relevant fragments of pSV-AR or
pSV-AR(N727K,M886V) and fused in-frame into
GAL4DBD. pVP16-NcoRC' was generated by digesting the cDNA encoding NcoR (kind gift of Dr. G. Jenster,
Erasmus University, Rotterdam) with EcoRI and
BamHI to obtain a 1.9-kb fragment containing residues
1818-2453 of the C-terminal fragment of NcoR, and ligating it in-frame to the VP16 transactivation domain. This NcoRC'
fragment have both repression domains removed, but included amino acids 1859-2142 and 2239-2300 of steroid-receptor-interacting domains I (3)
and II (35), respectively. The pCMV-cJun encoding human c-Jun driven by the cytomegalovirus promoter (36), and the
pRSV-c-fos containing rat c-fos driven by
the RSV-LTR promoter (37) were kind gifts of Dr. R. Tjian (University
of California, Berkeley, CA). pSG5-TIF2 and
pSG5-mTIF2, encoding full-length TIF2 and mutant TIF2 (in
which all three LXXLL nuclear-receptor-interacting motifs were mutated to LXXAA), respectively, were provided by Dr.
H. Gronemeyer (Institut de Génetique et de Biologie
Moléculaire et Cellulaire, Strasbourg) (25). The reporter
vectors pARE-TATA-Luc and pGAL4-TATA-Luc,
contained five GAL4 DNA-binding sites and two tandem copies of the ARE
from the aminotransferase gene, respectively, driving the luciferase
reporter gene (17). All constructs were sequenced to confirm the
fidelity of enzymatic manipulations.
Transient Transfections--
HeLa cells were maintained in RPMI
1640 medium and COS cells in Dulbecco's modified Eagle's medium.
1.5-1.8 × 104 cells were seeded into each well of
24-well plates 20 h prior to transfections. For Western blotting,
COS-7 cells were seeded on P60 Petri dishes 29 h before
transfection. All transient transfections were performed using
LipofectAMINE (38), and appropriate amounts of empty parent vector were
added to the replicates, if indicated, to normalize the amount of total
DNA in each well to prevent general squelching.
Immunoblotting--
Immunoblotting was performed as previously
described (17). Transfected COS-7 cells were lysed, and 10 µg of
protein from the cell lysate was resolved on 8% SDS-polyacrylamide gel
electrophoresis. Proteins were transferred onto Hybond-C nitrocellulose
membranes, and AR protein was detected using the rabbit polyclonal
antibody, NH27, which recognizes amino acids 360-564 of the AR (gift
of Dr. A. Mizokami, Kitakyushu, Japan).
Confocal Immunofluorescence Microscopy--
COS-7 cells were
seeded on 15-mm diameter coverslips on 12-well pates and transfected
with LipofectAMINE. Five hours after transfection, the cells received
fresh medium with 10% charcoal-treated fetal bovine serum and were
cultured for an additional 20 h in the presence or absence of
increasing doses of DHT. Cells were fixed in 4% paraformaldehyde in
phosphate buffer saline and permeabilized with 1% Brij. AR protein was
detected with the antibody NH27 (1:50 dilution). Fluorescein
isothiocyanate-conjugated anti-rabbit secondary antibody was used for
visualization of the receptor protein under a confocal laser scanning
biological microscope (Olympus Fluoview IX70, Tokyo, Japan).
 |
RESULTS |
Effect of
628-646 on Full-length AR Activity--
To test the
consequences of deleting amino acids 628-646 from full-length AR, WT
AR or mutant AR(
628-646) was expressed in the HeLa cells and
transactivation activity measured with a multimeric ARE promoter linked
to a luciferase reporter gene (Fig. 2).
WT AR activity at 0.001 nM DHT was 2-fold higher than
replicates not exposed to hormone and reached a maximum of 1600-fold
increase in activity at 10 nM (Fig. 2A). Further
increases in androgen dose did not raise AR activity above this
maximum, indicating that saturating doses of hormone had been reached.
However, the transactivation response of mutant AR was biphasic and
differed from WT. Low doses of androgen (0.001-0.01 nM)
did not increase mutant AR activity significantly, resulting in WT AR
activity 10- to 44-fold higher than mutant AR. Surprisingly, this
pattern was reversed for doses of androgen of 0.1 nM. The
AR mutant, despite having its hinge region and the associated NLS
deleted, exhibited AR activity that was more than double that of the WT
AR for doses of DHT and mibolerone between 0.1 and 1000 nM
(Fig. 2). These differences in transactivation function were not due to
changes in protein expression, because both WT AR and deletion mutants were present in approximately equal amounts in the cells, as indicated by immunoblotting (Fig. 2C).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of hinge deletion on transactivation
function of full-length AR. WT and mutant AR( 628-646), were
expressed in HeLa cells and exposed to the indicated doses
(nM) of DHT (A) and mibolerone (B) at
low (left panels) and high doses (right panels)
for 48 h. AR activity was measured with the
ARE-TATA-Luc reporter gene and luciferase activity in
relative light units (RLU) as mean (±S.E.) of at least
three replicates. C, immunoblot of WT and hinge-deleted AR.
AR were expressed in COS-7 cells, exposed to the indicated amounts of
androgen, and 10 µg of lysate protein was separated on an
SDS-polyacrylamide gel electrophoresis gel. AR of ~110 kDa
(arrow) was specifically detected with the AR antibody,
NH27. Note that the deletion mutant ( AR)
migrated slightly faster than the WT AR. Control (Con) wells
were mock-transfected with the parent vector lacking the AR.
|
|
Effect of Hinge Deletion on Activation Function of the LBD--
To
measure activation function of the ARLBD, a chimeric construct,
comprising the GAL4DBD fused in-frame to the
ARLBD, was coexpressed with the GAL4-TATA-Luc
reporter gene (Fig. 3). WT ARLBD chimeric
protein did not demonstrate significant transactivation activity with,
or without androgen, consistent with previous studies indicating that
very little transactivation function resides in the AR LBD (5, 15, 19).
Unexpectedly, deletion of residues 628-646 from the ARLBD increased
androgen-dependent transactivation activity markedly. The
ARLBD(
628-646) fragment was about 30-40 times more active than the
intact WT LBD. Whereas no androgen-dependent increase in
AF2 activity was observed for the WT ARLBD, dose-dependent augmentation of ARLBD(
628-646) AF2 activity was observed for doses
of DHT and mibolerone from 0.01 to 1000 nM. This suggests that amino acids 628-646 may serve to inhibit the transactivation potential of AF2 of the ARLBD.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of hinge deletion on transactivation
function of the ARLBD. Fusion proteins consisting of GAL4DBD fused
to WT ARLBD or ARLBD( 628-646) deletion mutant were expressed in
HeLa cells and protein-protein interactions studied with the
GAL4-TATA-Luc reporter vector. Cells were exposed to DHT
(A) or mibolerone (B) at low (left
panels) or high doses (right panels) doses
(nM), and luciferase activity was measured.
|
|
Cellular Localization of AR Protein--
A short stretch of basic
amino acids located within amino acids 628-646 of the hinge region
forms part of a bipartite NLS, the other portion located 10 residues
upstream in the DBD (Fig. 1) (31). To determine whether nuclear
localization of the AR is affected by the hinge deletion,
immunofluorescence confocal microscopy was performed (Fig.
4). In the absence of androgen, transfected AR was located in both cytoplasm and the nucleus (Fig. 4,
top panels). With low doses of DHT (0.01 nM), WT
AR was observed increasingly in the nucleus. At doses of DHT >1
nM, WT AR protein was localized mainly in the nucleus, and
at 100 nM the WT AR signal was observed almost exclusively
in the nucleus (Fig. 4, bottom panels). The deletion mutant
surprisingly behaved in a similar manner, with most of the AR being
located in the nucleus in the presence of androgen doses >1
nM, except that the nuclear signals were marginally less
intense. Thus deletion of the hinge portion of the bipartite NLS in the
mutant AR did not prevent its localization to the nucleus, suggesting
that the intact portion located in the DBD was sufficient for this
purpose (32).

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 4.
Subcellular localization of AR. WT or
deletion mutant ( ) AR was expressed in COS-7 cells
grown on coverslips and exposed to the indicated doses of DHT for
20 h. Representative fields show cells that have been fixed with
formaldehyde and AR protein detected with the AR antibody, NH27.
Fluorescein isothiocyanate-conjugated anti-rabbit IgG was used for
visualization of the receptor protein. Fluorescence signals
(green dots) were superimposed on direct interference
contrast light transmission images of the same field, using a confocal
laser scanning microscope.
|
|
Effect of TIF2 on AF2 Function--
The effects of coactivator on
mutant AR were next investigated, because AF2 is dependent on their
efficient recruitment to the ARLBD. Of the three steroid coactivators
identified to date, TIF2 interacts the strongest with AR (39).
Consistent with this observation, TIF2 augmented full-length WT AR
activity by over 100% (Fig. 5).
Remarkably, the AR(
628-646) mutant, was observed to display even
greater TIF2-augmented activity, being 70% higher than similarly
treated WT AR. Thus the synergistic activity of TIF2 on full-length AR
function was present when the hinge region was deleted. To further
define this effect, a chimeric construct consisting of the GAL4DBD
linked in-frame to ARLBD was coexpressed with TIF2, and
transactivation activity was measured with a GAL4 reporter gene (Fig.
6A). In the absence of TIF2,
no AF2 function of the WT LBD was observed, whereas deletion of the
628-646 region resulted in significant ARLBD AF2 activity. The
presence of TIF2 augmented the AF2 function of the WT LBD by more than
80-fold. Strikingly, the activity of LBD(
628-646) fusion protein
with TIF2 was 8-fold higher than that of the corresponding
TIF2-stimulated WT LBD fragment, and 40-fold higher than that observed
with mutant LBD alone. In contrast, a TIF2 mutant, with three
LXXLL nuclear-receptor-interacting motifs mutated, was not
able to synergize with either WT LBD or LBD(
628-646)
transactivation function, indicating that residues 628-646 exert their
repressive actions via LXXLL motifs of steroid receptor
coactivators. To further delineate the sites of action of TIF2, LBD
mutants incorporating two substitutions, N727K and M886V, were
constructed. The N727K,M886V mutations have previously been
demonstrated to have partially defective interactions with TIF2 (17,
18), resulting in minimal androgen insensitivity and male infertility.
The WT ARLBD chimeric protein did not have any activity in the absence
of hormone, but displayed strong androgen-dependent activity in the presence of TIF2 (Fig. 6B). As expected, the
LBD(N727K,M886V) fusion protein was partially defective in the presence
of TIF2, having only half the activity of the WT. Nevertheless,
deletion of residues 628-646 resulted in TIF2-dependent
augmentation of mutant AR activity, such that triply mutated
LBD(
628-646,N727K,M886V) was more than 3-fold stronger than the
doubly mutated LBD(N727K,M886V) fragment. Thus mutations in AR residues
727 and 886, unlike TIF2 with mutated LXXLL motifs, did not
abrogate the stimulatory action of the coactivator on LBD(
628-646).
Overall, the data indicate that codons 628-646 harbor a functional
element that directly, or indirectly, represses the activity of TIF2,
and deletion of these codons enables maximal
androgen-dependent coactivator-induced AF2 function to be
expressed.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of coregulators on transactivation
function of full-length AR. WT AR or the deletion mutant was
coexpressed with c-Jun, c-Fos, or TIF2, as indicated, in the presence
or absence of 0.1 nM DHT. AR transactivation activity was
measured with ARE-TATA-Luc reporter gene. Luciferase
activity in relative light units (RLU) was mean (±S.E.) of
at least three replicates.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of TIF2 on LBD activation
function. A, fusion proteins consisting of GAL4DBD
fused to WT ARLBD or ARLBD( 628-646) were coexpressed with 100 ng of
full-length TIF2 or mTIF2 (wherein the last two leucines in all three
LXXLL motifs were mutated to alanine) with or without 0.1 nM DHT. B, chimeric proteins consisting of
GAL4DBD fused to WT LBD, or LBD mutants containing the double
substitutions N727K,M886V with, or without the 628-646 deletion
were coexpressed with TIF2 and exposed to 0.1 nM of MB.
Transactivation activity was measured with GAL4-TATA-Luc
reporter vector, and luciferase activity in relative light units
(RLU) was mean (±S.E.) of at least three replicates. The
horizontal bar in A denotes a change in scale,
and differences in scale between panels are due to adjustments in
luminometer sensitivity.
|
|
Effect of c-Jun and c-Fos on AF2 Function--
AP-1 is a
transcription factor whose components are the nuclear proteins encoded
by c-Fos and c-Jun proto-oncogenes (36). Expression of these
proto-oncoproteins can have a negative effect on steroid receptor
function (40). Because the sites of interaction of c-Jun with AR
include the DBD and the adjacent hinge region (41-43), the effects of
these proto-oncoproteins on AR activity were examined. The presence of
either c-Jun or c-Fos exerted a profound inhibitory effect on both WT
AR and AR(
628-646) activity (Fig. 5). The presence of c-Jun/c-Fos
together resulted in hormone-independent stimulation of reporter gene
activity but inhibition of DHT-stimulated AR(
628-646) activity. The
equal inhibitory effect on both WT and deletion AR indicates that
residues 628-646 are not likely to be the binding site for either
proto-oncoproteins. The effect of cotransfecting c-Jun and c-Fos on the
transactivation function of the chimeric ARLBD was examined
(Fig. 7A). Although c-Jun and c-Fos stimulated WT ARLBD activity slightly in a
hormone-independent manner, the presence of c-Jun reduced activity
of DHT-stimulated LBD(
628-646) action by 70%. Surprisingly c-Fos
had a strong hormone-dependent stimulatory effect on WT LBD
activity, increasing reporter gene activity by 100-fold compared with
LBD not exposed to c-Fos. Deletion of the hinge region enhanced this
stimulatory effect of c-Fos on LBD activation function. The presence of
both c-Jun/c-Fos resulted in hormone-independent activation of reporter
gene. It is important to note that, even in the absence of AR, the
activity of ARE and GAL4-driven reporter genes was increased 10-fold by
the Jun/Fos heterodimer (data not shown). In contrast, Jun or Fos alone
did not have this stimulatory effect on the reporter genes. Although the actions of c-Jun, c-Fos, and their heterodimers are complex and
dependent on the particular cellular systems examined (40), deleted
codons 628-646 did not appear to have a critical role in their
actions, because almost equivalent effects were observed for both
WT and deletion AR.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of the corepressors, c-Jun, c-Fos, and
NcoR on transactivation function of ARLBD fragment. A,
fusion proteins consisting of GAL4DBD fused to WT ARLBD or
ARLBD( 628-646) were coexpressed with 100 ng of c-Jun, c-Fos, or
c-Jun/c-Fos combination, with or without 0.1 nM of DHT, as
indicated. B, the mammalian two-hybrid assay was used to
measure interactions between WT ARLBD or ARLBD( 628-646)
fusion proteins, and a NcoR fragment, containing both
receptor-interacting regions, fused to the VP16AD. Transactivation
activity was measured with GAL4-TATA-Luc reporter vector,
and luciferase activity in relative light units (RLU) was
mean (±S.E.) of at least three replicates.
|
|
Binding of NcoR Fragment to ARLBD--
Because NcoR can interact
with the hinge regions of the THR (3) and PPAR
(2) (Fig. 1), we
investigated whether this corepressor mediates the effects of the AR
hinge region. To this end we made a VP16 construct fused in-frame to
the NcoRC' fragment, which incorporated both C-terminal steroid
receptor-binding domains (3, 35), but we excluded the repressor regions
of NCoR. This VP16-NcoRC' chimera was coexpressed with the LBD fragment
in the mammalian two-hybrid assay (Fig. 7B). Neither the
ARLBD nor the NcoRC' chimeras alone stimulated reporter gene
activity. Expression of WT ARLBD and NcoRC' together increased reporter
gene activity slightly, indicating that normal binding of NcoRC' to the
ARLBD was weak. As observed previously, the LBD(
628-646) chimera
exhibited marked AF2 activity, which was increased in an additive
manner in the presence of the NcoRC', indicating that NcoR was unlikely to bind to the deleted region.
 |
DISCUSSION |
The main finding in this report is that a strong AF2 domain in the
ARLBD can be demonstrated in mammalian cells and that this AF2 activity
is normally inhibited, directly or indirectly, by residues 628-646 in
the hinge region. Our deletion fragment was designed to be intermediate
between the previously reported deletions 629-633 (34) and 628-657
(31), both of which did not appear to affect AR transactivation
activity. For a wide range of androgen doses, deletion of residues
628-646 in the hinge region resulted in a mutant receptor with more
than double the activity of the WT AR. This repressive effect was even
more evident when the LBD was expressed as a fusion protein, because
the deletion mutant was capable of raising transactivation activity
30-fold higher than the WT. It is of interest to note that, although
GAL4DBD-ARLBD(
628-646) was transcriptionally more active than the
WT LBD, the overall activity of the chimeric LBD constructs was a
fraction of the full-length AR activity in terms of relative light
units, reflecting synergistic interactions between activation regions
of TAD and LBD in the intact receptor (14, 15). Our study provides a basis for understanding why AF2 function of the AR, unlike other steroid receptors, is weak and cannot be readily elicited.
Intriguingly, the activation function of full-length AR(
628-646)
was biphasic. At low doses of androgen (
0.01 nM), the
activity of full-length mutant AR was lower than the WT, but this was
reversed at higher doses. This biphasic pattern could reflect partially
defective AR nuclear transport at low androgen levels, due to deletion
of the hinge portion of the bipartite NLS (32). At higher androgen doses, the intact DBD portion of the NLS could permit sufficient nuclear transport for the stronger intrinsic transactivation activity of the deletion mutant to manifest. The observation that this biphasic
response was not observed with the chimeric mutant LBD protein supports
this hypothesis, because a strong NLS is present in the GAL4DBD moiety.
Mutagenesis studies indicate a functional link between AF2 activity in
the LBD and the binding of p160 coactivators, such as SRC1 and TIF2,
which interact specifically with the AF2 region of nuclear receptors
via distinct receptor-interacting domains containing LXXLL
motifs (11, 24, 27). Because AF2 function is thought to depend on the
recruitment of coactivators, we examined the effect of coexpressing
TIF2 on mutant AR activity. The transactivation activities of
full-length WT AR and the deletion mutant were both proportionately
enhanced by TIF2, such that TIF2-mediated mutant AR activity was
doubled that of the WT. This androgen-dependent effect was
also evident with the LBD chimera, wherein deletion of residues
628-646 enhanced TIF2-LBD activity 8-fold. In contrast, mutant TIF2,
which lacked active receptor-interacting motifs, was not able to
augment WT or mutant AR LBD activity. The enhancement of mutant AR
function by TIF2 suggests that the element demarcated by residues
628-646 normally inhibits the coactivator-AF2 complex and that its
removal enabled maximum coactivator function to be expressed. To
further define the mechanism of ternary interactions between TIF2, LBD,
and residues 628-646, we introduced naturally occurring substitutions
in the LBD that were known to reduce interactions with the coactivator.
Each of these substitutions, N727K (18) and M886V (17), causes minimal
androgen insensitivity and reduced spermatogenesis, partly by reducing
AR-TIF2 interactions. The presence of N727K and M886V substitutions
reduced, but did not abolish, the repressor effect of residues 628-646
on coactivator-AF2 function. Nonetheless, the above data suggest the
presence of a repressor element in the hinge region, which exerts an
inhibitory effect on coactivator-mediated AF2 function in the ARLBD.
Cross-talk occurs between the signaling pathways that convey signals to
the nucleus. AP-1, a transcription factor whose components are c-Fos
and c-Jun proto-oncoproteins, (37), is implicated in diverse aspects of
cell growth, differentiation, and development. First reports of
interactions between nuclear receptors and AP-1 came from the
observation that GR-induced transcription is strongly inhibited by
either c-Fos or c-Jun (44). In view of the association of hinge
mutations with prostate cancer, it is of interest that AP-1/c-Jun can
repress AR activity in the context of the prostate-specific antigen
promoter (43) and that c-Jun interacts with AR via the DBD and the
adjacent hinge region (41-43). Although the actions of c-Jun, c-Fos,
and c-Jun/c-Fos heterodimers on AR activity are complex and may be
either inhibitory or stimulatory depending on the receptor, cell, or
promoter contexts (40), the question arises whether the repressor
element in AR residues 628-646, exerts its effects through the AP-1
pathway. The presence of c-Jun, c-Fos, or c-Jun/c-Fos together
profoundly inhibited both WT and
628-646 AR activity. In contrast
to some studies suggesting that c-Jun is stimulatory (41), the
strongest inhibition was associated with c-Jun in our system. The
prominent AF2 activity of ARLBD(
628-646) fusion protein was reduced
by two-thirds in the presence of c-Jun. In sharp contradistinction,
c-Fos stimulated AF2 function in an androgen-dependent
manner. The AF2 activity of WT LBD increased by two orders of magnitude
in the presence of c-Fos, reminiscent of the activity of the
coactivator TIF2. The AF2 activity of ARLBD(
628-646) in the
presence of c-Fos was even higher than WT, indicating that c-Fos was
able to exert its effects even in the absence of residues 624-648.
Although we observed diverse effects of the AP-1 system on AR activity,
none of these appear to be affected by the absence or presence of
residues 624-648, suggesting that the repressive effects of the hinge
region were not predominantly mediated through these
proto-oncoproteins.
Nuclear receptor corepressors, like NcoR, are thought to silence
transcription by promoting a closed chromatin configuration through
histone deacetylation. In the THR, binding of NcoR to the hinge region
decreases transcription, and defective release of corepresssor by hinge
mutants are found in patients with resistance to thyroid hormone (45).
NcoR also preferentially associate with antagonist-occupied PR (46) and
may mediate the inhibitory effects of ER antagonists (4). A fusion of
NcoR repressor domain to the ER-LBD strongly inhibits
estrogen-dependent responses in breast cancer cells (47).
In our study, the C-terminal fragment of NcoR, NcoRC', containing two
independent receptor-interacting regions demonstrated very little
interaction with WT ARLBD or ARLBD(
628-646). Thus it is unlikely
that residues 628-646 are a critical binding site for NCoRC', because
interactions with WT and mutant LBD were equally weak.
Although the action of coactivators like those of the p160 and p300
family of proteins are well established (1), factors causing repression
of steroid receptor action, although less well understood, are
increasingly being reported. Thus NcoR and SMRT are found to be key
regulators of ligand-dependent transcriptional activity of
the human PR (46). Opposing effects of coactivators and corepressor
determine agonists and antagonists activity of glucocorticoid-regulated
gene expression (48-50). A 37-kDa REA protein potentiates the
activities of dominant negative ERs and anti-estrogen-liganded ER (51).
Binding of the orphan nuclear receptors TR4 to the AR (52) and SHP to
ER (53) can result in heterodimers that down-regulate target gene
expression. Although a negative modulation domain in the amino-terminal
region of the PR has been described (54), this is the first report of
an inhibitory element in the AR. Our data indicate that relatively
strong AF2 function exists in the AR LBD and that it is normally
inhibited by an element centered on residues 628-646 of the hinge
region. Despite deletion of a portion of NLS and slightly reduced
nuclear translocation, the deletion mutant exhibited significantly
stronger basal and TIF2-augmented activity than the WT AR. Our results predict that the mutations in the AR hinge region might result in a
release of a normal inhibitory function, leading to excessive AR
activity and possibly unrestricted growth of androgen-regulated tissues, making it imperative to determine the precise mechanisms whereby this region inhibits the AF2 domain.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. H. Gronemeyer, R. Tjian, and G. Jenster for plasmids and Dr. A. Mizokami for AR antibody, NH27.
 |
FOOTNOTES |
*
This study was supported by the National Medical Research
Council, Singapore.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
Obstetrics and Gynecology, National University Hospital, Level 2, Lower Kent Ridge Rd., Republic of Singapore 119074. Tel.: 65-772-4261; Fax:
65-779-4753; E-mail: obgyel@nus.edu.sg.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M009916200
 |
ABBREVIATIONS |
The abbreviations used are:
AR, androgen
receptor;
TAD, transactivation domain;
DBD, DNA-binding domain;
LBD, ligand-binding domain;
THR, thyroid hormone;
AF1, -2, activation
functions 1 and 2;
GR, glucocorticoid receptor;
PR, progesterone
receptor;
PPAR
, peroxisome proliferator-activated receptor;
ER, estrogen receptor;
ARE, androgen response element;
NLS, nuclear
localization signal;
WT, wild-type;
PCR, polymerase chain reaction;
DHT, dihydrotestosterone;
kb, kilobase(s);
RLU, relative light unit(s).
 |
REFERENCES |
1.
|
McKenna, N. J.,
Lanz, R. B.,
and O'Malley, B. W.
(1999)
Endocr. Rev.
20,
321-344[Abstract/Free Full Text]
|
2.
|
Dowell, P.,
Ishmael, J. E.,
Avram, D.,
Peterson, V. J.,
Nevrivy, D. J.,
and Leid, M.
(1999)
J. Biol. Chem.
274,
15901-15907[Abstract/Free Full Text]
|
3.
|
Horlein, A. J.,
Naar, A. M.,
Heinzel, T.,
Torchia, J.,
Gloss, B.,
Kurokawa, R.,
Ryan, A.,
Kamei, Y.,
Soderstrom, M.,
Glass, C. K.,
and Rosenfeld, M. G.
(1995)
Nature
377,
397-404[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Lavinsky, R. M.,
Jepsen, K.,
Heinzel, T.,
Torchia, J.,
Mullen, T. M.,
Schiff, R.,
Del-Rio, A. L.,
Ricote, M.,
Ngo, S.,
Gemsch, J.,
Hilsenbeck, S. G.,
Osborne, C. K.,
Glass, C. K.,
Rosenfeld, M. G.,
and Rose, D. W.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2920-2925[Abstract/Free Full Text]
|
5.
|
Bevan, C. L.,
Hoare, S.,
Claessens, F.,
Heery, D. M.,
and Parker, M. G.
(1999)
Mol. Cell. Biol.
19,
8383-8392[Abstract/Free Full Text]
|
6.
|
Onate, S. A.,
Boonyaratanakornkit, V.,
Spencer, T. E.,
Tsai, S. Y.,
Tsai, M. J.,
Edwards, D. P.,
and O'Malley, B. W.
(1998)
J. Biol. Chem.
273,
12101-12108[Abstract/Free Full Text]
|
7.
|
Durand, B.,
Saunders, M.,
Gaudon, C.,
Roy, B.,
Losson, R.,
and Chambon, P.
(1994)
EMBO, J.
13,
5370-5382[Abstract]
|
8.
|
vom Baur, E.,
Harbers, M.,
Um, S. J.,
Benecke, A.,
Chambon, P.,
and Losson, R.
(1998)
Genes Dev.
12,
1278-1289[Abstract/Free Full Text]
|
9.
|
Jiménez-Lara, A. M.,
and Aranda, A.
(1999)
J. Biol. Chem.
274,
13503-13510[Abstract/Free Full Text]
|
10.
|
Sheldon, L. A.,
Smith, C. L.,
Bodwell, J. E.,
Munck, A. U.,
and Hager, G. L.
(1999)
Mol. Cell. Biol.
19,
8146-8157[Abstract/Free Full Text]
|
11.
|
Nolte, R. T.,
Wisely, G. B.,
Westin, S.,
Cobb, J. E.,
Lambert, M. H.,
Kurokawa, R.,
Rosenfeld, M. G.,
Willson, T. M.,
Glass, C. K.,
and Milburn, M. V.
(1998)
Nature
395,
137-143[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Tora, L.,
White, J.,
Brou, C.,
Tasset, D.,
Webster, N.,
Scheer, E.,
and Chambon, P.
(1989)
Cell
59,
477-487[Medline]
[Order article via Infotrieve]
|
13.
|
Barettino, D.,
Vivanco Ruiz, M. M.,
and Stunnenberg, H. G.
(1994)
EMBO J.
13,
3039-3049[Abstract]
|
14.
|
Berrevoets, C. A.,
Doesburg, P.,
Steketee, K.,
Trapman, J.,
and Brinkmann, A. O.
(1998)
Mol. Endocrinol.
12,
1172-1183[Abstract/Free Full Text]
|
15.
|
He, B.,
Kemppainen, J. A.,
Voegel, J. J.,
Gronemeyer, H.,
and Wilson, E. M.
(1999)
J. Biol. Chem.
274,
37219-37225[Abstract/Free Full Text]
|
16.
|
He, B.,
Kemppainen, J. A.,
and Wilson, E. M.
(2000)
J. Biol. Chem
275,
22986-22994[Abstract/Free Full Text]
|
17.
|
Ghadessy, F. J.,
Lim, J.,
Abdullah, A. A.,
Panet-Raymond, V.,
Choo, C. K.,
Lumbroso, R.,
Tut, T. G.,
Gottlieb, B.,
Pinsky, L.,
Trifiro, M. A.,
and Yong, E. L.
(1999)
J. Clin. Invest.
103,
1517-1525[Abstract/Free Full Text]
|
18.
|
Lim, J.,
Ghadessy, F. J,
Abdullah, A. A.,
Pinsky, L.,
Trifiro, M.,
and Yong, E. L.
(2000)
Mol. Endocrinol.
14,
1187-1197[Abstract/Free Full Text]
|
19.
|
Alen, P.,
Claessens, F.,
Verhoeven, G.,
Rombauts, W.,
and Peeters, B.
(1999)
Mol. Cell. Biol.
19,
6085-6097[Abstract/Free Full Text]
|
20.
|
Ikonen, T.,
Palvimo, J. J.,
and Janne, O. A.
(1997)
J. Biol. Chem.
272,
29821-29828[Abstract/Free Full Text]
|
21.
|
Matias, P. M.,
Donner, P.,
Coelho, R.,
Thomaz, M.,
Peixoto, C.,
Macedo, S.,
Otto, N.,
Joschko, S.,
Scholz, P.,
Wegg, A.,
Basler, S.,
Schafer, M.,
Ruff, M.,
Egner, U.,
and Carrondo, M. A.
(2000)
J. Biol. Chem.
275,
26164-26171[Abstract/Free Full Text]
|
22.
|
Wurtz, J. M.,
Bourguet, W.,
Renaud, J. P.,
Vivat, V.,
Chambon, P.,
Moras, D.,
and Gronemeyer, H.
(1996)
Nat. Struct. Biol.
3,
87-94[Medline]
[Order article via Infotrieve]
|
23.
|
Feng, W.,
Ribeiro, R. C.,
Wagner, R. L.,
Nguyen, H.,
Apriletti, J. W.,
Fletterick, R. J.,
Baxter, J. D.,
Kushner, P. J.,
and West, B. L.
(1998)
Science
280,
1747-1749[Abstract/Free Full Text]
|
24.
|
McInerney, E. M.,
Rose, D. W.,
Flynn, S. E.,
Westin, S.,
Mullen, T. M.,
Krones, A.,
Inostroza, J.,
Torchia, J.,
Nolte, R. T.,
Assa-Munt, N.,
Milburn, M. V.,
Glass, C. K.,
and Rosenfeld, M. G.
(1998)
Genes Dev.
12,
3357-3368[Abstract/Free Full Text]
|
25.
|
Voegel, J. J.,
Heine, M. J.,
Tini, M.,
Vivat, V.,
Chambon, P.,
and Gronemeyer, H.
(1998)
EMBO, J.
17,
507-519[Abstract/Free Full Text]
|
26.
|
Rochel, N.,
Wurtz, J. M.,
Mitschler, A.,
Klaholz, B.,
and Moras, D.
(2000)
Mol. Cell.
5,
173-179[Medline]
[Order article via Infotrieve]
|
27.
|
Shiau, A. K.,
Barstad, D.,
Loria, P. M.,
Cheng, L.,
Kushner, P. J.,
Agard, D. A.,
and Greene, G. L.
(1998)
Cell
95,
927-937[Medline]
[Order article via Infotrieve]
|
28.
|
Tanenbaum, D. M.,
Wang, Y.,
Williams, S. P.,
and Sigler, P. B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5998-6003[Abstract/Free Full Text]
|
29.
|
Quigley, C. A.,
De Bellis, A.,
Marschke, K. B.,
el-Awady, M. K.,
Wilson, E. M.,
and French, F. S.
(1995)
Endocr. Rev.
16,
271-321[Medline]
[Order article via Infotrieve]
|
30.
|
Gottlieb, B.,
Lehvaslaiho, H.,
Beitel, L. K.,
Lumbroso, R.,
Pinsky, L.,
and Trifiro, M.
(1998)
Nucleic Acids Res.
26,
234-238[Abstract/Free Full Text]
|
31.
|
Simental, J. A.,
Sar, M.,
Lane, M. V.,
French, F. S.,
and Wilson, E. M.
(1991)
J. Biol. Chem.
266,
510-518[Abstract/Free Full Text]
|
32.
|
Zhou, Z. X.,
Sar, M.,
Simental, J. A.,
Lane, M. V.,
and Wilson, E. M.
(1994)
J. Biol. Chem.
1269,
13115-13123
|
33.
|
Moilanen, A.,
Rouleau, N.,
Ikonen, T.,
Palvimo, J. J.,
and Janne, O. A.
(1997)
FEBS Lett.
412,
355-358[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Poukka, H.,
Aarnisalo, P.,
Karvonen, U.,
Palvimo, J. J.,
and Janne, O. A.
(1999)
J. Biol. Chem.
274,
19441-19446[Abstract/Free Full Text]
|
35.
|
Seol, W.,
Mahon, M. J.,
Lee, Y. K.,
and Moore, D. D.
(1996)
Mol. Endocrinol.
10,
1646-1655[Abstract]
|
36.
|
Baichwal, V. R.,
and Tjian, R.
(1990)
Cell
63,
815-825[Medline]
[Order article via Infotrieve]
|
37.
|
Turner, R.,
and Tjian, R.
(1989)
Science
243,
1689-1694[Medline]
[Order article via Infotrieve]
|
38.
|
Wang, Q.,
Ghadessy, F. J.,
Trounson, A.,
Kretser, D.,
McLachlan, R.,
Ng, S. C.,
and Yong, E. L.
(1998)
J. Clin. Endocrinol. Metab.
83,
4303-4309[Abstract/Free Full Text]
|
39.
|
Ding, X. F.,
Anderson, C. M.,
Ma, H.,
Hong, H.,
Uht, R. M.,
Kushner, P. J.,
and Stallcup, M. R.
(1998)
Mol. Endocrinol.
12,
302-313[Abstract/Free Full Text]
|
40.
|
Shemshedini, L.,
Knauthe, R.,
Sassone-Corsi, P.,
Pornon, A.,
and Gronemeyer, H.
(1991)
EMBO J.
10,
3839-3849[Abstract]
|
41.
|
Bubulya, A.,
Wise, S. C.,
Shen, X. Q.,
Burmeister, L. A.,
and Shemshedini, L.
(1996)
J. Biol. Chem.
271,
24583-24589[Abstract/Free Full Text]
|
42.
|
Lobaccaro, J. M.,
Poujol, N.,
Terouanne, B.,
Georget, V.,
Fabre, S.,
Lumbroso, S.,
and Sultan, C.
(1999)
Endocrinology
140,
350-357[Abstract/Free Full Text]
|
43.
|
Sato, N.,
Sadar, M. D.,
Bruchovsky, N.,
Saatcioglu, F.,
Rennie, P. S.,
Sato, S.,
Lange, P. H.,
and Gleave, M. E.
(1997)
J. Biol. Chem.
272,
17485-17494[Abstract/Free Full Text]
|
44.
|
Yang-Yen, H. F.,
Chambard, J. C.,
Sun, Y. L.,
Smeal, T.,
Schmidt, T. J.,
Drouin, J.,
and Karin, M.
(1990)
Cell
62,
1205-1215[Medline]
[Order article via Infotrieve]
|
45.
|
Safer, J. D.,
Cohen, R. N.,
Hollenberg, A. N.,
and Wondisford, F. E.
(1998)
J. Biol. Chem.
273,
30175-30182[Abstract/Free Full Text]
|
46.
|
Wagner, B. L.,
Norris, J. D.,
Knotts, T. A.,
Weigel, N. L.,
and McDonnell, D. P.
(1998)
Mol. Cell. Biol.
18,
1369-1378[Abstract/Free Full Text]
|
47.
|
Chien, P. Y.,
Ito, M.,
Park, Y.,
Tagami, T.,
Gehm, B. D.,
and Jameson, J. L.
(1999)
Mol. Endocrinol.
13,
2122-2136[Abstract/Free Full Text]
|
48.
|
Schneikert, J.,
Hubner, S.,
Martin, E.,
and Cato, A. C.
(1999)
J. Cell Biol.
146,
929-940[Abstract/Free Full Text]
|
49.
|
Subramaniam, N.,
Treuter, E.,
and Okret, S.
(1999)
J. Biol. Chem.
274,
18121-18127[Abstract/Free Full Text]
|
50.
|
Szapary, D.,
Huang, Y.,
and Simons, S. S., Jr.
(1999)
Mol. Endocrinol.
13,
2108-2121[Abstract/Free Full Text]
|
51.
|
Montano, M. M.,
Ekena, K.,
Delage-Mourroux, R.,
Chang, W.,
Martini, P.,
and Katzenellenbogen, B. S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6947-6952[Abstract/Free Full Text]
|
52.
|
Lee, Y. F.,
Shyr, C. R.,
Thin, T. H.,
Lin, W. J.,
and Chang, C.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14724-14729[Abstract/Free Full Text]
|
53.
|
Johansson, L.,
Bavner, A.,
Thomsen, J. S.,
Farnegardh, M.,
Gustafsson, J. A.,
and Treuter, E.
(2000)
Mol. Cell. Biol.
20,
1124-1133[Abstract/Free Full Text]
|
54.
|
Huse, B.,
Verca, S. B.,
Matthey, P.,
and Rusconi, S.
(1998)
Mol. Endocrinol.
12,
1334-13342[Abstract/Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.