Specific Androgen Receptor Activation by an Artificial
Coactivator*
Xiaomei
Sui,
Kelli S.
Bramlett,
Michael C.
Jorge,
David A.
Swanson,
Andrew C.
von Eschenbach, and
Guido
Jenster
From the Department of Urology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
Transcription activation of steroid receptors,
such as the androgen receptor (AR), is mediated by coactivators, which
bridge the receptor to the preinitiation complex. To develop a tool for studying the role of the AR in normal development and disease, we
constructed artificial coactivators consisting of the transcription activation domains of VP16 or p65/RelA and the AR hinge and
ligand-binding domain (ARLBD), which has been shown
to interact with the AR N-terminal domain. The artificial
VP16-ARLBD and ARLBD-p65 coactivators
interacted with the AR N terminus and wild-type AR in an
androgen-dependent and androgen-specific manner.
VP16-ARLBD and ARLBD-p65 enhanced the AR
transactivity up to 4- and 13-fold, respectively, without affecting the
expression of the AR protein. The coactivators did not enhance the
transcription activity of the progesterone receptor (PR) or the
glucocorticoid receptor (GR), showing their specificity for the AR. In
addition, to construct PR- and GR-specific coactivators, the VP16
activation domain was fused to the PR and GR hinge/ligand-binding domain. Although VP16-PRLBD and VP16-GRLBD
interacted with the C-terminal portion of steroid receptor
coactivator-1, they did not enhance the transcription activity of their
receptor. The presented strategy of directing activation domains or
other protein activities into the DNA-bound AR complex provides a novel
means of manipulating AR function in vitro and in
vivo.
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INTRODUCTION |
Steroid receptors are hormone-dependent transcription
factors that regulate the expression of a large variety of genes
affecting cell growth, differentiation, development, and homeostasis.
How these nuclear receptors activate or repress transcription of target genes has been the focus of much research in the past years. A major
breakthrough in our understanding of these activities was the
identification of so-called coactivators that bridge the receptors to
the preinitiation complex (PIC)
(1-4).1 These coactivators
are recruited into the promoter-bound complex by the receptor and
facilitate assembly of basal transcription factors into a stable PIC,
likely via their activation domains. In addition to this bridging
function, some coactivators, including steroid receptor
coactivator-1 (SRC-1), cAMP response element-binding protein-binding
protein (CBP), p300, and ACTR, can also remodel chromatin by
acetylating histones (5-8). Moreover, the nuclear receptors, ACTR,
SRC-1, CBP, and p300 can recruit the p300/CBP-associated factor (9),
which also harbors an intrinsic histone acetyltransferase activity (6,
7, 10, 11). The model that emerges from these data takes the two
activities of coactivators into account; the liganded steroid receptor
binds as a homodimer to the hormone response element and recruits
coactivators and p300/CBP-associated factor. These cofactors loosen the
nucleosomal structure by targeted histone acetylation. The coactivators
can then initiate the stable assembly of the PIC by their bridging
function, which results in enhanced rates of transcription initiation
by RNA polymerase II (9).
To perform their bridging function, coactivators need to harbor at
least two domains: a receptor-interacting domain and a domain that
contacts and stabilizes assembly of the PIC. This latter domain will
almost certainly be a transcription activation domain that directly or
indirectly binds proteins of the PIC. The receptor-interacting domain
determines the binding specificity. Except for AR-associated protein 70 (12), none of the known coactivators is specific for a certain nuclear
receptor (1, 3). In addition, SRC-1 and CBP can mediate transactivity
of transcription factors other than nuclear receptors (13-15).
The objective of this study was to generate an AR-specific and very
potent coactivator to manipulate AR function in vitro and
in vivo. Such a coactivator could be used as a tool to study the role of the AR in normal development and disease. Moreover, this
coactivator could form the blueprint for the construction of artificial
corepressors that inhibit AR function in the presence of ligand, of
cofactors that direct any other enzyme activity into the AR DNA-bound
complex, and of coactivators that are specific for other nuclear receptors.
To form a coactivator that is AR-specific and more potent than the
known coactivators, a domain had to be found that interacts only with
the AR and not with other nuclear receptors. The first evidence for the
existence of such a domain came from the observation that the AR
ligand-binding domain (ARLBD) binds to the AR N terminus in
an androgen-dependent manner (16-18). However, it was
unknown whether the ARLBD could also interact with
the wild-type AR and whether this binding is AR-specific.
The activation domains are important determinants of coactivator
potency and are responsible for the direct interaction with proteins of
the PIC and/or recruitment of additional coactivators. There are a few
obvious choices for strong activation domains, including the activation
domain from viral protein 16 (VP16) (19) and p65/RelA (20). Both have
been shown to directly contact proteins of the PIC and associate with
coactivators (21-30).
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EXPERIMENTAL PROCEDURES |
Materials--
R1881 (17
-methyltrienolone) and R5020
(promegestone) were purchased from NEN Life Science Products.
Dexamethasone, estradiol, testosterone, hydroxyflutamide, cyproterone
acetate, and dihydrotestosterone were purchased from Sigma. Casodex
(bicalutamide, ICI-176, 334) was obtained from Zeneca
Pharmaceuticals (Newark, DE).
Plasmids--
The construction of pAR0 (encoding the wild-type
AR of 910 amino acids), pAR3, pAR5, pAR65, pAR106, pAR126, and pAR104
has been described previously (31, 32). The pcDNA-ARLBD
was constructed by inserting the partial EcoRI 1-kilobase
pair ARLBD fragment (encoding amino acids 605-910) from
pAR65 into the pcDNA3.1HisB vector (Invitrogen, Carlsbad, CA)
digested with EcoRI. A multiple cloning site (33) was
introduced before the AR stop codon by polymerase chain reaction
using the following primers: AR861A, 5'-GCGAGAGAGCTGCATCAGTTCAC-3', and
AR910mcsB, 5'-CTAGGGCCCGTTAACCTCGAGACCGCGGACTGGGTGTGGAAATAGATGGG-3'. The polymerase chain reaction fragment was digested with
BspEI and Bsp120I and inserted into
pcDNA3.1His- ARLBD digested with the same enzymes. The
resulting plasmid (pcDNA-ARLBDmcs) contains the unique
SacII, XhoI, HpaI, and
Bsp120I sites into which fragments can be inserted in frame
with the ARLBD. pcDNA-VP16 was constructed by inserting
the VP16 EcoRI fragment (encoding amino acids 411-490) from
pRSET-VP16 into pcDNA3.1HisC linearized with EcoRI.
pcDNA-ARLBDVP16 was constructed by in frame insertion
of VP16 from pcDNA-VP16 (BamHI filled in with Klenow and
Bsp120I-digested) into the pcDNA-ARLBD mcs
vector (XhoI filled in with Klenow and
Bsp120I-digested). pcDNA-VP16ARLBD was
generated by inserting the ARLBD (Asp718 filled in with
Klenow and XbaI-digested) into the pcDNA-VP16 vector
(NotI filled in with Klenow and XbaI-digested).
pcDNA-VP16ARLBDVP16 was constructed by inserting the
Bsp120I/BspEI fragment from
pcDNA-ARLBDVP16 into the
pcDNA-VP16ARLBD vector digested with the same enzymes. pcDNA-ARLBDp65 was constructed by inserting the
1.3-kilobase pair Asp718-Klenow/SalI fragment of p65/RelA
encoding amino acids 286-550 into the
XhoI/HpaI-digested pcDNA-ARLBDmcs
vector. pcDNA-VP16ARLBDp65 was constructed by inserting
the HindIII/BspEI fragment from
pcDNA-VP16ARLBD into the pcDNA-ARLBDp65
HindIII/BspEI-digested vector. pcDNA-p65 was
constructed by inserting the 1.3-kilobase pair
BamHI-Klenow/EcoRI p65 fragment into the
XbaI-Klenow/EcoRI-digested pcDNA3.1HisC. pcDNA-AR0mcs was generated by inserting the
MluI-Klenow/EcoRI AR sequence from pAR3 into
KpnI-T4 DNA polymerase-blunted/EcoRI pcDNA-ARLBDmcs vector. pcDNA-AR0 was constructed by
replacing the mcs sequence in pcDNA-AR0mcs
(EcoRI/XhoI-digested) with the wild-type sequence
from pAR0 (EcoRI/SalI fragment).
pcDNA-VP16PRLBD was constructed by inserting the
HincII-digested PRLBD fragment from
pABGAL-PRLBD into the XhoI-digested, filled-in
pcDNA-VP16 vector. pcDNA-p65GRLBD was generated by
inserting the filled-in ClaI/DraI
GRLBD fragment into the pcDNA-p65
XhoI-digested, filled-in vector.
pcDNA-p65PRLBD was constructed by inserting the
1-kilobase pair XhoI PRLBD fragment from
pcDNA-VP16PRLBD into the XhoI-digested pcDNA-p65 vector. pcDNA-PRLBD and
pcDNA-GRLBD were generated by releasing the p65
fragment from pcDNA-p65PRLBD and
pcDNA-p65GRLBD, respectively.
pcDNA-VP16GRLBD was constructed by inserting the HpaI/Bsp120I GRLBD fragment from
pcDNA-p65GRLBD into the
NotI/Klenow/Bsp120I-digested pcDNA-VP16
vector. pABGAL-SRC-1(0.9) was constructed by inserting the
HindIII-Klenow/BglII SRC-1(0.9) fragment from
pVLGST-SRC-1 (6) into PvuII/BamHI-digested
pABGAL1-147 (34). The reporter plasmid harboring two
androgen/progesterone/glucocorticoid response elements and a TATA-box
driving the luciferase gene ([ARE]2-E1b-luc) and the
(UAS)4TATA-luciferase reporter have been described
previously (9, 35). The pcDNA3.1His-LacZ plasmid (Invitrogen) was
used as an internal transfection efficiency control. Correct nucleotide sequence of all constructs was verified by DNA sequencing, and correct
protein expression in HeLa cells was determined by Western blotting analysis.
Cell Culture and Transient Transfections--
HeLa cells (human
epithelial cervix carcinoma (American Type Culture Collection)) were
maintained in minimal essential medium supplemented with 5% fetal
bovine serum and antibiotics. LNCaP cells (American Type Culture
Collection) were maintained in RPMI medium supplemented with 10% fetal
bovine serum and antibiotics. 24 h before transfection,
105 cells were plated in each well of 12-well dishes in
medium containing dextran-coated charcoal-stripped serum. Cells
were transfected with 0.3 µg of reporter plasmid, 0.1 µg of
pcDNA-LacZ, 30 ng of receptor construct, and 0.1 µg of
coactivator plasmid per well using Lipofectin (Life Technologies, Inc.)
according to the manufacturer's guidelines. 24 h later, cells
were washed and fed with medium containing stripped serum and the
indicated hormones. Cells were harvested 14 h later, and cell
extracts were assayed for luciferase activity using the luciferase
assay system (Promega). Luciferase values were corrected for the
-galactosidase internal control. Experiments were performed in
triplicate; data are presented as the means ± S.E. of at least
three independent experiments.
Immunoblots--
Transfected HeLa cells were lysed in 40 mM Tris-HCl pH 7.0, 1 nM EDTA, 4% glycerol, 10 mM dithiothreitol, 2% SDS, and protease inhibitors.
Protein concentration of the samples were determined by Bradford assay
using 10 µg of protein loaded in sample buffer on a 7.5%
SDS-polyacrylamide gel as described previously (36). Gels were blotted
onto nitrocellulose, and the protein of interest was visualized with
the AR-specific F39.4 antibody (37) or Xpress antibody (Invitrogen) and
a goat-anti-mouse horseradish peroxidase secondary antibody using the
ECL kit (Amersham Pharmacia Biotech). The pcDNA3.1His vectors that
were used for the construction of almost all plasmids described above
harbor the Xpress epitope tag to which the Xpress antibody is directed.
 |
RESULTS |
Construction of Artificial AR Coactivators--
To investigate
whether the ARLBD interacts with the wild-type AR, various
fusion proteins were constructed containing the ARLBD and
the activation domains from VP16 and p65. The various constructs that
were used in this study are depicted in Fig.
1. The wild-type AR (AR0) cotransfected
with the (ARE)2E1b-luciferase reporter in HeLa cells was
activated up to 50-fold by 1 nM synthetic androgen R1881
(Fig. 2A). Cotransfection with
empty pcDNA3.1His vector, ARLBD, VP16 activation
domain, or p65 activation domain did not significantly change the
capacity of AR to activate transcription. However, fusion proteins of
the ARLBD with VP16 or p65 greatly enhanced AR
transactivity. The location of the VP16 activation domain with respect
to the ARLBD did not influence the coactivation potential
of the VP16-ARLBD and ARLBD-VP16 fusion
proteins. We cotransfected all the different coactivators and their
components with the reporter plasmid in the absence and presence of
R1881 to test their potential effect on the transcription of the
reporter in the absence of the AR. As expected, none of the
coactivators or their components changed the basal transcription from
the (ARE)2E1b-luciferase reporter (data not shown). To show
that endogenous AR can also be superactivated by coactivators,
VP16-ARLBD and ARLBD-p65 were transiently
transfected with the reporter plasmid into the prostate cancer cell
line LNCaP. LNCaP cells express the AR and are sensitive to androgens
with respect to their growth. Transfection of the reporter alone showed
that R1881 can activate the endogenous AR (Fig. 2B).
Cotransfection of the ARLBD or p65 activation domain did
not significantly affect AR activity. However, as was observed in the
HeLa cell transfections, the ARLBD-p65 coactivator strongly enhanced the AR up to 9-fold.

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Fig. 1.
Schematic representation of constructs.
Various steroid receptors, including AR, GR, PR, and AR mutants, and
artificial coactivators consisting of the receptor hinge region with a
LBD and activation domain of VP16 or p65/RelA were used in this study.
In addition, the GAL4 DNA binding domain and a fusion with the
receptor-interacting part of the SRC-1 coactivator were used as bait in
the mammalian two-hybrid system. NT, N-terminal
domain.
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Fig. 2.
Superactivation of the AR by artificial
coactivators. A, AR transcription activity was examined
by cotransfection of the AR expression plasmid and the
(ARE)2E1b-luciferase reporter in HeLa cells. In addition,
empty vector, a coactivator fragment (ARLBD, VP16, and p65)
or a coactivator (ARLBD-VP16, VP16-ARLBD, and
ARLBD-p65) were cotransfected. B, LNCaP cells,
which express endogenous AR, were cotransfected with
(ARE)2E1b-luciferase reporter and empty vector, coactivator
fragment (ARLBD and p65) or ARLBD-p65
coactivator. Luciferase activity was determined from cell lysates of
transfected cells, which were cultured for 16 h in the absence
( ) or presence (+) of 1 nM synthetic androgen R1881.
Activities were corrected for a pcDNA3.1His-LacZ internal control
and are presented as the percentage of luciferase activity ± S.E.
relative to the AR activity in the presence of R1881 (second
lane from the left in both panels).
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Lack of Effect of Artificial Coactivators and Their Components on
AR Protein Expression--
To ensure that cotransfection of the AR
with the artificial coactivators did not increase AR expression and
consequently transcription activity from the reporter, HeLa cells were
cotransfected, and the AR protein expression was determined by Western
blot analysis. The AR expression was not affected by the coactivators
or their components (Fig. 3).

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Fig. 3.
Western blot analysis of AR protein
coexpressed with artificial coactivator or coactivator fragments.
HeLa cells were cotransfected with AR expression vector,
(ARE)2E1b-luciferase reporter, and empty vector,
coactivator fragment (ARLBD, VP16, and p65) or coactivator
(VP16-ARLBD, and ARLBD-p65). Protein extracts
of transfected cells were analyzed after culturing for 16 h in the
presence of 1 nM R1881. Proteins were separated by
SDS-polyacrylamide gel electrophoresis, blotted, and immunostained with
the F39.4 monoclonal antibody.
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The VP16-ARLBD and ARLBD-p65 Coactivators
Are Specifically Androgen-dependent--
Because the
artificial coactivators harbor the ARLBD, we tested whether
the VP16-ARLBD and ARLBD-p65 fusion proteins
enhanced the AR in a hormone-dependent manner. The AR5
mutant lacks the AR ligand-binding domain and is constitutively active
(32). As expected, the addition of R1881 to the HeLa cells
cotransfected with AR5 and the (ARE)2E1b-luciferase
reporter did not affect AR5 activity (Fig.
4). The artificial ARLBD-p65
coactivator did not enhance AR5 transactivity in the absence of ligand
but did superactivate transcription in the presence of 1 nM
R1881. This result was repeated with the VP16-ARLBD fusion
protein, showing that the coactivators containing the ARLBD
depend on ligand for function. To investigate to which part of the AR
N-terminal domain the ARLBD binds, we tested two different
AR mutants harboring different fragments of the N terminus for enhanced
transcription activation by ARLBD-p65. Both AR126 (amino
acids 1-370) and AR106 (amino acids 360-528) were only partially
enhanced by the artificial coactivator as compared with AR5,
indicating the necessity for both fragments for full
ARLBD-p65 interaction. In addition, the binding of
the artificial coactivator to the AR DNA-binding domain and
ligand-binding domain was analyzed using AR104, which completely lacks
the N-terminal domain. The transcription activity of this mutant is
very low (32), and in this study ARLBD-p65 did not enhance
its transcription (Fig. 4).

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Fig. 4.
Transcription enhancement of AR mutants by
artificial coactivators. HeLa cells were transfected with the AR
mutant, (ARE)2E1b-luciferase reporter, and empty vector or
ARLBD-p65 coactivator. Luciferase activity was determined
from cell lysates of transfected cells, which were cultured for 16 h in the absence ( ) or presence (+) of 1 nM R1881.
Activities were corrected for a pcDNA3.1His-LacZ internal control
and are shown as the percentage of luciferase activity ± S.E.
relative to AR5 (second column from the left),
AR126 (sixth column from the left), AR106
(tenth column from the left), or AR104
(fourteenth column from the left) in the presence
of hormone.
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We used this same experimental approach to determine whether the fusion
of the ARLBD to VP16 or p65 changed ligand specificity. Various agonists (R1881, testosterone, and dihydrotestosterone), antagonists (Casodex, hydroxyflutamide, and cyproterone acetate), and
other hormones (estradiol and the synthetic progestin R5020) were
tested for their ability to induce ARLBD-p65 binding to AR5 (Fig. 5). Only the androgens
testosterone, dihydrotestosterone, and R1881 significantly
induced AR5 superactivation, showing that the artificial coactivator
did not change ligand specificity and remained androgen-specific.

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Fig. 5.
Ligand specificity of the
ARLBD-p65 coactivator. HeLa cells were transfected
with AR5, (ARE)2E1b-luciferase reporter and empty vector or
ARLBD-p65 coactivator. Luciferase activity was determined
from cell lysates of transfected cells, which were cultured for 16 h in the absence ( ) or presence (+) of 1 nM R1881, 1 nM testosterone (T), 1 nM
dihydrotestosterone (DHT), 100 nM estradiol
(E2), 100 nM synthetic progestin R5020, 100 nM antiandrogen Casodex, 100 nM antiandrogen
hydroxyflutamide (OH-flu), or 100 nM
antiandrogen cyproterone acetate (CA). Activities were
corrected for a pcDNA3.1His-LacZ internal control and are presented
as the percentage of luciferase activity ± S.E. relative to the
AR5 activity in the absence of R1881 (first column from the
left).
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The VP16-ARLBD and ARLBD-p65 Coactivators
Are AR-specific--
To determine whether the ARLBD would
interact only with the AR and not with other steroid receptors, we
analyzed the effect of the artificial coactivators on two of the most
homologous nuclear receptors, the progesterone receptor (PR) and the
glucocorticoid receptor (GR). Cotransfection of the PR or GR with
ARLBD-p65 in the presence of R1881 and R5020 or
dexamethasone for activation of the PR and GR, respectively, showed no
superactivation (Fig. 6), indicating that
the ARLBD-p65 coactivator is AR-specific. Note that R1881
is a potent progestin and is able to activate the PR (Fig. 6).

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Fig. 6.
Superactivation of the AR, PR, and GR by
ARLBD-p65. HeLa cells were transfected with AR, PR, or
GR, (ARE)2E1b-luciferase reporter and empty vector, or
ARLBD-p65 coactivator. Luciferase activity was determined
from cell lysates of transfected cells, which were cultured for 16 h in the absence ( ) or presence (+) of 1 nM ligand or a
mix of ligands for AR (R1881), PR (R5020), and GR (dexamethasone).
Activities were corrected for a pcDNA3.1His-LacZ internal control
and are presented as the percentage of luciferase activity ± S.E.
relative to the activity of AR (second column from the
left), PR (sixth column from the
left), or GR (twelfth column from the
left) in the presence of ligand.
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Testing of Artificial PR and GR Coactivators--
To determine
whether the hinge/ligand-binding domains of the PR and GR can also
interact with their receptors, the VP16 activation domain was fused to
the PRLBD and GRLBD. Although their
construction was almost identical to that of VP16-ARLBD,
neither VP16-PRLBD nor VP16-GRLBD enhanced
transactivation of its full-length receptor (Fig.
7). For control purposes, we
cotransfected the activation domain of VP16 by itself with the
different receptors to analyze the effect of this potent activation
domain on receptor transactivity. The activity of all receptors was
reduced, probably because of sequestering of essential transcriptional
cofactors. Interestingly, the GR activity was the most severely reduced
(down to 25%). Cotransfection of the GR with VP16-ARLBD or
VP16-PRLBD in the presence of the various ligands again
showed the transcription-inhibiting effect of VP16 (Fig. 7). However,
in the absence of the ligand that interacts with the
VP16-ARLBD (R1881) or VP16-PRLBD (R5020), the
GR activity was again higher. One explanation for this phenomenon is
that in the unliganded VP16-ARLBD and
VP16-PRLBD, the VP16 is shielded by the heat shock proteins
that associate with the ligand-binding domain. In the presence of
ligand, the heat shock proteins dissociate, and VP16 is able to squelch
the GR transactivity.

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Fig. 7.
Functional analysis of VP16 and AR, PR, or GR
ligand-binding domain fusion proteins for superactivation of their
wild-type receptor. HeLa cells were transfected with AR, PR, or
GR, (ARE)2E1b-luciferase reporter and empty vector, or
VP16-ARLBD, VP16-PRLBD, or
VP16-GRLBD fusion proteins. Luciferase activity was
determined from cell lysates of transfected cells, which were cultured
for 16 h in the absence ( ) or presence (+) of 1 nM
ligand or a mix of ligands for AR (R1881), PR (R5020), and GR
(dexamethasone). Activities were corrected for a pcDNA3.1His-LacZ
internal control and are presented as the percentage of luciferase
activity ± S.E. relative to the activity of AR (second
column from the left), PR (fifteenth column
from the left), or GR (twenty-eighth column from
the left) in the presence of ligand.
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To check correct folding and the ability to bind hormone, we
cotransfected VP16-PRLBD and VP16-GRLBD with
the receptor interacting-part of the SRC-1 coactivator, which is fused
to the GAL DNA-binding domain (DBD) (GAL-SRC-1[1139-1441])
(38, 39). None of the VP16 fusion proteins interacted with the
GAL DBD (Fig. 8). However, VP16-ARLBD, ARLBD-VP16,
ARLBD-p65, VP16-PRLBD, and
VP16-GRLBD bound the GAL-SRC-1(1139-1441) in a
hormone-dependent manner, showing correct expression,
nuclear import, folding, and hormone binding.

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Fig. 8.
Mammalian two-hybrid analysis of binding of
AR, PR, and GR ligand-binding domain to the receptor interacting part
of SRC-1. HeLa cells were transfected with GAL DBD or
GAL-SRC-1(1139-1441), (UAS)4TATA-luciferase reporter and
empty vector, or VP16-ARLBD, ARLBD-VP16,
ARLBD-p65, VP16-PRLBD, or
VP16-GRLBD fusion proteins or fragments thereof. Luciferase
activity was determined from cell lysates of transfected cells, which
were cultured for 16 h in the absence ( ) or presence (+) of 1 nM ligand for AR (R1881), PR (R5020), and GR (dexamethasone
(dex)). Activities were corrected for a pcDNA3.1His-LacZ
internal control and are presented as the percentage of luciferase
activity ± S.E. relative to the activity of GAL-DBD or
GAL-DBD-SRC-1(1139-1441).
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 |
DISCUSSION |
Our results show for the first time that the ARLBD can
interact with the full-length AR but not with the PR or GR. The binding of the artificial coactivators occurred in the AR N-terminal domain and
was diminished when the first 360 amino acids or the last 158 residues
of the N terminus were deleted, confirming the previous observation
that two independent domains (the first 36 amino acids and residues
370-494) are necessary for full ARLBD binding (40).
Binding of the hinge/LBD to the N terminus or full-length receptor has
also been shown for the estrogen receptor
(41) and the PR (42).
However, VP16 fusion proteins with the PR and GR hinge/LBD did not
enhance the transcription activity of their receptor, indicating that
they did not bind the full-length receptor in our mammalian
protein-protein interaction system. The folding, ligand binding, and
nuclear import of VP16-PRLBD and VP16-GRLBD were correct because they interacted with the receptor-interacting domain of SRC-1 in a ligand-dependent manner (38, 39). The interaction of VP16-ARLBD and ARLBD-p65 with
SRC-1(1134-1441) was very weak compared with the binding of
VP16-PRLBD and VP16-GRLBD, confirming
previously published data showing that the AR binding to SRC-1 is the
weakest of all steroid receptors (39). The reason for the inability of
VP16-PRLBD and VP16-GRLBD to bind to their cognate receptor is not clear. We speculate that the three-dimensional structure of the AR DNA-bound homodimer is different as compared with
the PR and GR homodimers, allowing an additional ligand-binding domain
to be recruited into the AR complex.
Previously, Nyanguile et al. (43) constructed a completely
synthetic low-molecular-weight transcription activator by fusing FK506
to a short peptide with transactivating properties. FK506 interacts
with the FK-binding protein FKBP12, which was fused to the GAL
DNA-binding domain, thereby directing the transactivating peptide onto
the reporter gene. As a complementary strategy, we show that the
ARLBD, which binds the full-length AR also gives us the
opportunity to direct proteins into the DNA-bound AR complex. In
addition to the transcription activation domains described in this
manuscript, the effects of transcription repression domains, DNA-modifying enzymes, and chromatin-remodeling proteins on AR function
are currently being tested. These artificial cofactors provide a novel
means of manipulating AR activity and AR target gene expression to
investigate the role of AR function in normal and disease states.
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ACKNOWLEDGEMENTS |
We thank Dr. J. Trapman for helpful
discussions and careful reading of the manuscript, Drs. A. O. Brinkmann and J. Trapman for the F39.4 antibody, and Dr. B. W. O'Malley for the GAL-SRC-1 construct.
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FOOTNOTES |
*
This work was supported by the Physicians' Referral Service
and Prostate Cancer Research Program of The University of Texas M. D. Anderson Cancer Center.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: University of Texas
M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 026, Houston, TX 77030. Tel.: 713-792-8917; Fax: 713-792-4456; E-mail: gjenster{at}mdacc.tmc.edu.
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ABBREVIATIONS |
The abbreviations used are:
PIC, preinitiation
complex;
AR, androgen receptor;
LBD, ligand-binding domain;
PR, progesterone receptor;
GR, glucocorticoid receptor;
SRC-1, steroid
receptor coactivator-1;
CBP, cAMP response element-binding
protein-binding protein;
DBD, DNA-binding domain;
VP16, viral protein
16;
LNCaP, lymph node carcinoma of the prostate.
 |
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