Domain Interactions between Coregulator ARA70 and the Androgen Receptor (AR)
Zhong-xun Zhou,
Bin He,
Susan H. Hall,
Elizabeth M. Wilson and
Frank S. French
Departments of Pediatrics (Z-X.Z., S.H.H., E.M.W., F.S.F.) and
Biochemistry and Biophysics (B.H., E.M.W.), and The Laboratories for
Reproductive Biology, University of North Carolina School of Medicine,
Chapel Hill, North Carolina 27599-7500
Address all correspondence and requests for reprints to: Dr. Frank S. French, Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7500. E-mail:
fsfrench{at}med.unc.edu
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ABSTRACT
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The coregulator function of AR-associated protein 70
(ARA70) was investigated to further characterize its
interaction with the AR. Using a yeast two-hybrid assay,
androgen-dependent binding of ARA70 deletion mutants to the
AR ligand-binding domain (LBD) was strongest with ARA70
amino acids 321441 of the 614 amino acid ARA70 protein.
Mutations adjacent to or within an FxxLF motif in this 120-amino acid
region abolished androgen-dependent binding to the AR-LBD both in yeast
and in glutathione-S-transferase affinity matrix
assays. Yeast one-hybrid assays revealed an intrinsic
ARA70 transcriptional activation domain within amino acids
296441. In yeast assays the ARA70 domains for
transcriptional activation and for binding to the AR-LBD were inhibited
by the C-terminal region of ARA70. Full-length
ARA70 increased androgen-dependent AR
transactivation in transient cotransfection assays using a mouse
mammary tumor virus-luciferase reporter in CV1 cells. ARA70
also increased constitutive transcriptional activity of an AR
NH2-terminal-DNA binding domain fragment and bound
this region in glutathione-S-transferase affinity
matrix assays. Binding was independent of the ARA70 FxxLF
motif. The results identify an ARA70 motif required for
androgen-dependent interaction with the AR-LBD and
demonstrate that ARA70 can interact with the
NH2-terminal and carboxyl-terminal regions of
AR.
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INTRODUCTION
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THE AR IS a hormone-activated transcription
factor that mediates the differentiation, development, and
maintenance of male reproductive function (1, 2). AR
mutations identified in the androgen insensitivity syndrome exemplify
the essential role of AR in these processes (3, 4, 5, 6). AR
belongs to the nuclear receptor superfamily that comprises receptors
for steroid and thyroid hormones, vitamin D, retinoids, peroxisome
proliferator, and orphan receptors (7, 8, 9, 10, 11, 12). Nuclear
receptors have a conserved structural arrangement of the DNA and ligand
binding domains (13, 14, 15, 16, 17, 18, 19, 20), whereas the
NH2-terminal domains differ markedly in length
and sequence (7, 21).
Hormone-bound nuclear receptors bind DNA-responsive elements and
trigger a cascade of transcriptional events. To initiate transcription,
nuclear receptors interact in a ligand-dependent fashion with
coactivators and components of the transcriptional machinery such as
transactivation factor IIB (TFIIB), TATA-binding protein, TATA-binding
protein-associated factor, or TFIIH (22, 23, 24, 25, 26, 27). A
family of proteins, termed the p160 coactivators, includes steroid
receptor coactivator 1 (SRC-1), nuclear receptor coactivator 1
(28, 29, 30, 31); SRC-2, transcriptional intermediary factor 2
(TIF2), GR-interacting protein 1 (GRIP1), nuclear receptor
coactivator 2 (32, 33); and SRC-3, p300/CBP
cointegrator associate protein, receptor-associated coactivator 3,
activator of the thyroid and RAR, amplified in breast cancer
1, thyroid receptor activator molecule-1 (34, 35, 36, 37, 38, 39).
These p160 coactivators interact with nuclear receptors in a
ligand-dependent manner (16, 40, 41) through LxxLL motifs
that make up the nuclear receptor box (42, 43, 44). X-ray
crystallographic studies revealed that nuclear receptor box
peptides bind the hydrophobic cleft formed by nuclear receptor
ligand-binding domain (LBD) helices 3, 4, 5, and 12
(45, 46, 47). Multiprotein complexes of nuclear receptor
coactivators and chromatin remodeling factors gain access to target DNA
and activate transcription (26, 31, 48, 49, 50, 51, 52).
AR-associated protein 70 (ARA70) was isolated
from human brain (53) and prostate
(54) cDNA libraries by its binding to the human AR-LBD and
enhanced the transcriptional activity of AR in cotransfection assays.
Alen et al. (55) reported that
ARA70 bound TFIIB and p300/CBP-associated factor
in vitro and suggested that ARA70 acts
as a bridging factor between steroid receptors and components of the
transcription initiation complex. Herein we further characterize
ARA70 as an AR coactivator by analysis of its
interactions and functional domains. A sequence motif required for
androgen-dependent binding to the AR-LBD is identified. In addition,
regions containing intrinsic transcriptional activation and inhibitory
domains are delineated. We demonstrate also that
ARA70 can interact with the
NH2-terminal region of AR.
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RESULTS
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Isolation of a Partial ARA70 cDNA and Hormone
Requirements for Interaction with the AR-LBD
Yeast two-hybrid screening was used to identify human AR-LBD
interacting proteins expressed by a random primed cDNA library from an
androgen-stimulated LNCaP (lymph node-derived human prostate carcinoma)
cell line. The AR-LBD (amino acids 624919) included the hinge region
and LBD. Approximately 8 x 107 yeast
transformants of the VP16 library were screened. Among 18 positive
yeast clones, four clones with identical restriction maps were
sequenced and compared with data in GenBank.
ARA70 coding sequence for amino acids 321499
was identified and contained Gly 364 as in ARA70
(53) rather than Ala 364 as reported in RET-fused gene
(RFG) (57). In yeast two-hybrid assays, a dose-dependent
interaction of the AR-LBD with ARA70 321499 was
observed with dihydrotestosterone (DHT), T, or the synthetic androgen
R1881 (methyltrienolone) (Fig. 1A
).

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Figure 1. ARA70 Interaction with AR
A, Effects of DHT, T, and methyltrienolone (R1881) on the interaction
of ARA70 amino acids 321499 with AR-LBD in a yeast
two-hybrid assay. Yeast L40 was cotransformed with expression plasmids
pLexA-AR-LBD and VP ARA70 321499 and grown on Ura-, Trp-,
Leu- plates for 48 h. Colonies were picked and grown in liquid
culture in the presence of increasing concentrations of steroid.
ß-Gal activity was measured in yeast cell extracts of liquid
cultures. Liquid assay results are plotted in units as defined in
Materials and Methods. Shown are the mean and
SD of assays on three independent transformants. B, Effect
of ARA70 on AR transactivation. pSG5 expression vectors
coding for ARA70, and full-length AR (0.1 µg) were
cotransfected into 6-cm dishes of CV1 cells with MMTV-LUC (2.5 µg)
and incubated in the absence and presence of 0.1 nM DHT.
Control incubations included AR and MMTV-LUC alone or together with the
indicated amounts of pSG5 empty vector DNA (molar equivalents of the
pSG5ARA70) and pBR322 to make the same total weight of DNA.
Incubations and analysis of luciferase were as described in
Materials and Methods.
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ARA70 Enhances AR Transactivation
To investigate coregulator function of
ARA70, CV-1 cells were cotransfected with AR and
ARA70 expression vectors and the mouse mammary
tumor virus (MMTV)-luciferase reporter plasmid (Fig. 1B
). The
full-length ARA70 cDNA construct used in these
experiments had sequence identity to that of Yeh and Chang
(53). Transfection of pSG5ARA70 DNA
resulted in increases in DHT-dependent transcription above the level
with AR and reporter gene alone or in the presence of an equimolar
amount of pSG5 vector DNA. ARA70 had little
effect on background activity in the absence of DHT and no effect on
the transcriptional activity of a constitutive reporter vector, psLuc2
(data not shown). The level of endogenous ARA70
mRNA detected by northern hybridization in CV1 cells was similar to
that in COS cells and HeLa cells and 23 times higher than in the
prostate cell lines DU145, PC3, and LNCaP (data not shown).
ARA70 Sequence That Binds the AR-LBD
To determine the region of ARA70 that
interacts with the AR-LBD, a yeast two-hybrid assay was used. Yeast
strain L40 was transformed with LexA-AR-LBD and
VP16-ARA70 deletion mutants, and binding was
measured by lift and liquid ß-galactosidase (ß-gal) assays in the
presence of 10 nM DHT. Yeast transformed with LexA-AR-LBD and VP16
served as a negative control.
LexA-AR-LBD interacted with VP-ARA70 1614, and
binding increased 4-fold with deletion of the carboxyl-terminal region
(VPARA70 1499 and 1441) (Fig. 2A
). VPARA70
321499 was 6-fold higher than VPARA70 291614
or 320614 mutants that contained carboxyl-terminal sequence.
VPARA70 321441 had the highest AR-LBD binding
activity. NH2- and carboxyl-terminal deletions of
VPARA70 321441 resulted in a gradual decline of
binding activity. ARA70
NH2-terminal fragments in
VPARA70 1321, 1295, or 1118 did not bind
the AR-LBD despite the presence of a LxxLL sequence
(42, 43, 44) at residues 9296. Thus, 321441 contained the
amino acid sequence with highest binding activity for the AR-LBD. The
increase in binding activity with deletion of the
ARA70 carboxyl-terminal region suggested the
presence of an inhibitory sequence within residues 499614. This
apparent repression domain within ARA70 may
influence protein folding or interaction with a repressor protein.

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Figure 2. ARA70 Sequences That
Interact with the AR-LBD
A, Expression plasmids coding for fusion proteins of LexA-AR-LBD (AR
amino acids 624919) and pVP16AD-ARA70 were analyzed in
the yeast two-hybrid interaction assay. Numbers on the
bars indicate amino acid residues of ARA70 in the
fusion protein. Yeast containing an integrated ß-gal reporter gene
controlled by LexA binding sites was transformed with the different
expression plasmids. After transformation, yeast was grown on Ura-,
Trp-, and Leu- plates for 48 h, replica plated to similar plates
containing 10 nM DHT for 16 h for lift assays, or
grown in liquid medium for liquid assays. Liquid assay results are
expressed in units as defined in Materials and Methods.
Results are representative of three independent experiments. B,
Immunoblot analysis. Equal amounts of cell extract (1 x
108 cell equivalents) from transformed yeast L40 were
immunoblotted with a monoclonal antibody against VP16 amino acids 121
to measure relative expression levels of the VP16 ARA70
fusion proteins. C, In vitro interaction of
ARA70 deletion mutants with the carboxyl-terminal region of
AR. Equal amounts of whole Sf9 cell extracts expressing AR 507919 in
the presence or absence of 10 nM DHT were incubated with
glutathione-Sepharose beads containing bound GST-ARA70
1614, GST-ARA70 321499, GST-ARA70 321441,
(lanes 38), or GST alone (lanes 1 and 2). Beads were washed and bound
protein was eluted and analyzed by Western blot using AR52 rabbit
polyclonal anti-AR peptide antiserum (21 93 ). D,
In vitro interaction of full-length ARA70
and ARA70(2KA) mutant with AR-LBD.
[35S]ARA70 and
[35S]ARA70(2KA) were synthesized in
vitro and incubated in the presence or absence of DHT, 1
µM, with glutathione-Sepharose beads bound with
GST-AR-LBD (AR 624919). Control beads contained GST alone. Beads were
washed and bound [35S]-labeled proteins were eluted and
analyzed by SDS-PAGE and autoradiography. The input lane contained 15%
of the total radioactivity added.
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Immunoblot analysis using an antibody against the VP16 transcriptional
activation domain indicated that the VP16-ARA70
fusion proteins were expressed at similar levels (Fig. 2B
).
The ARA70 region that binds AR-LBD was further
investigated using glutathione-S-transferase
(GST)-fusion proteins. GST-ARA70 1614,
321499, or 321441 bound to baculovirus expressed AR DNA-binding
domain-LBD fragment, AR 507919 (Fig. 2C
). These fragments did not
bind to GST protein alone, but bound AR 507919 in the presence of
DHT, confirming that ARA70 residues 321441
contain a site for androgen-dependent interaction with the AR-LBD.
ARA70 K327A/K329A Abolished Androgen-Dependent Binding
to the AR-LBD
ARA70 residues 321441 contain basic amino
acids, R322, K327, and K329, and five evenly spaced cysteines at
residues 398, 404, 410, 416, and 422, each separated by five amino
acids. No similar sequence was identified in the GenBank database.
Missense mutations were created to test individual amino acid
requirements for ARA70 321441 interaction with
the AR-LBD. R322A decreased activity by 20% whereas the double
mutation K327A/K329A [ARA70(2KA)] reduced
binding to an undetectable level in the yeast two-hybrid assay (Fig. 2A
). C398A/C404A or C410A/C416A/C422A decreased binding by about 30%.
The results suggested that a sequence associated with the two basic
residues (K327, K329) has a key role in ARA70
binding to the AR-LBD.
Binding of full-length ARA70(2KA) to AR-LBD was
measured in GST affinity matrix assays (Fig. 2D
).
[35S]-labeled full-length
ARA70 and ARA70(2KA) were
synthesized in vitro and incubated with GST-AR-LBD in the
presence or absence of DHT. ARA70 binding to
GST-AR-LBD was increased about 2-fold in the presence of DHT while
there was no increase in ARA70(2KA) binding in
the presence of DHT.
To test the effect of the 2KA double mutation K327A/K329A on
coactivator activity, full-length ARA70(2KA) or
wild-type ARA70 was cotransfected into CV-1 cells
with AR and MMTV-luciferase (MMTV-LUC) reporter plasmids (Fig. 3A
). Transfection of 2.9 µg of
pSG5ARA70 DNA resulted in an increase in
luciferase activity relative to AR and reporter gene alone or an
equimolar amount of pSG5 vector DNA balanced with pBR322. AR
coactivation with the mutant ARA70(2KA) was
reduced compared with wild-type ARA70.

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Figure 3. Interaction of ARA70 and Mutant
ARA70(2KA) with the AR NH2-Terminal Region
A, pSG5 expression vectors coding for full-length AR, 0.1 µg, and
either ARA70 or ARA70(2KA), 2.9 µg, were
cotransfected into CV1 cells with MMTV-LUC, 2.5 µg, and incubated in
the absence and presence of 0.1 nM DHT. Control incubations
contained AR and MMTV-LUC alone or together with 2.0 µg pSG5 empty
vector DNA (the molar equivalent of 2.9 µg ARA70) and 0.9
µg pBR322 to make the same total weight of DNA. B, Expression
plasmids encoding an AR NH2-terminal-DNA binding domain
fragment, AR 1660, 15 ng, and ARA70 or
ARA70(2KA) mutant, 7.25 µg, were cotransfected into CV1
cells with MMTV-LUC, 2.5 µg. Control incubations contained AR 1660
and MMTV-LUC alone or together with 5 µg pSG5 empty vector DNA [the
molar equivalent of 7.25 µg ARA70 or
ARA70(2KA)] and 2.25 µg pBR322 to make the same total
weight of DNA. C, In vitro interaction of
ARA70 or ARA70(2KA) mutant with AR
NH2-terminal-DNA binding domain fragment, GST-AR
1660. [35S]ARA70 and
[35S]ARA70(2KA) mutant were synthesized
in vitro and incubated with glutathione-Sepharose beads
bound with GST-AR 1660. Control beads contained GST alone. Beads were
washed, and bound [35S]-labeled proteins were eluted and
analyzed by SDS-PAGE and autoradiography. The input lane contained 15%
of the total radioactivity added.
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ARA70 Interacts with the AR
NH2- Terminal Domain
The finding that ARA70(2KA) lacked
DHT-dependent binding to the AR-LBD, yet retained partial function as a
coactivator with full-length AR, raised the possibility that
ARA70 interacts with the AR
NH2-terminal region. The AR
NH2-terminal-DNA binding domain fragment (AR
1660) contains the AF1 region and has constitutive transcriptional
activity. We tested the effects of ARA70 and
ARA70(2KA) on transactivation by AR 1660 (Fig. 3B
). Expression plasmids encoding AR 1660 and
ARA70 or ARA70(2KA) were
cotransfected into CV-1 cells with the MMTV-LUC reporter.
ARA70 increased the transcriptional activity of
AR 1660 relative to control levels while the increase with
ARA70(2KA) was less than that of the wild-type
ARA70. In affinity matrix assays, both
[35S]-labeled wild-type
ARA70 and ARA70(2KA) bound
to GST-AR 1660 (Fig. 3C
), indicating that ARA70
interacts with the AR NH2-terminal-DNA binding
domain fragment.
FxxLF Is Required for Androgen-Dependent Binding of
ARA70 to the AR-LBD but Not for Interaction with the AR
NH2-Terminal Domain
After the discovery that the FxxLF motif is required for
androgen-dependent binding of the AR NH2-terminal
region to the AR-LBD (58), it was recognized that
ARA70 321441 contains a FxxLF sequence (328
FKLLF 332). To determine whether the FxxLF motif is required for
ARA70 binding to the AR-LBD, the sequence FKLLF
in full-length ARA70 was mutated to AKLAA(AxxAA),
and binding of
[35S]ARA70(AxxAA) to
GST-AR-LBD was analyzed in the affinity matrix assay (Fig. 4A
). ARA70(AxxAA)
lacked-DHT dependent binding to AR-LBD while the binding of wild-type
ARA70 in the presence of DHT was about 2 times
higher than the no steroid control. A second affinity matrix assay was
performed using [35S]AR-LBD (AR 624919) to
compare the binding of GST-ARA70 321407 with
binding to the same ARA70 protein fragment
containing the AxxAA mutation (Fig. 4A
). Binding in the absence of DHT
was similar to that of the GST control. DHT had no effect on
[35S]AR-LBD binding of the
ARA70 321407(AxxAA) mutant while it increased
binding to the ARA70 321407 fragment with
wild-type sequence several fold.

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Figure 4. Role of FxxLF in ARA70 Interactions
with the AR-LBD and the AR NH2-Terminal Domain
A, Effect of the ARA70 AxxAA mutation on
androgen-dependent ARA70 binding to the AR-LBD.
[35S]ARA70 and mutant
[35S]ARA70(AxxAA) were synthesized in
vitro and incubated with glutathione-Sepharose beads bound to
GST alone or GST-AR-LBD (AR 624919) either in the presence or absence
of DHT. Beads were washed, and the bound [35S]-labeled
proteins were eluted and analyzed by SDS-PAGE and autoradiography. The
input lane contained 15% of the total radioactivity added. In the
lower gel, [35S]AR-LBD was synthesized and incubated in
the presence or absence of DHT with glutathione-Sepharose bound to GST
alone, GST-ARA70 321407(AxxAA), or GST-ARA70
321407 containing wild-type sequence, and the bound
[35S]-labeled protein was analyzed as above. The input
lane contained 10% of the total radioactivity added. B, Effect of the
ARA70 AxxAA mutation on ARA70 coactivation with
full-length AR. pCMVhAR, 25 ng, and either pSG5ARA70 or
ARA70(AxxAA), in the amounts indicated, were cotransfected
into CV1 cells with MMTV-LUC, 2.5 µg, and incubated in the absence
and presence of 0.1 nM DHT. Control incubations contained
either AR and MMTV-LUC alone or together with 1, 2, or 5 µg pSG5
empty vector DNA (the molar equivalents of 1.45, 2.9, and 7.5 µg
ARA70) and 0.45, 0.9, or 2.25 µg pBR322 to make the same
total weight of DNA. C, Effect of the ARA70 AxxAA mutation
on ARA70 coactivation with the AR
NH2-terminal-DNA binding domain. Expression plasmids
pCMVhAR 1660, 10 ng, and pSG5ARA70 or
ARA70(AxxAA), 7.25 µg, were cotransfected into CV1 cells
with MMTV-LUC, 2.5 µg. Control incubations contained AR and MMTV-LUC
alone or together with 5 µg pSG5 empty vector DNA (the molar
equivalent of 7.25 µg ARA70 expression vector) and 2.25
µg pBR322 to make the same total weight of DNA. D, Binding of the
ARA70 AxxAA mutant to the AR NH2-terminal-DNA
binding domain. [35S]ARA70(AxxAA) and
[35S]ARA70 were synthesized in
vitro and incubated with glutathione-Sepharose beads bound to
GST alone or to GST-AR 1660. Beads were washed and the bound
[35S]-labeled proteins were eluted and analyzed by
SDS-PAGE and autoradiography. The input lane contained 15% of the
total radioactivity added.
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Since the ARA70(2KA) double mutation, K327A,
K329A, flanks F328 in the FxxLF motif, it likely altered the binding
function of the motif even though the consensus sequence FxxLF was
unchanged. The results indicate that the ARA70
FxxLF motif mediates DHT-dependent binding to the AR-LBD.
In cotransfection assays (Fig. 4B
), transcriptional activity of
full-length AR with full-length ARA70(AxxAA) was
less than that induced by wild-type ARA70. With
ARA70(AxxAA), there was no change in luciferase
activity with increasing amounts of transfected DNA, while wild-type
ARA70 caused a dose-dependent increase in
luciferase activity.
Cotransfection of ARA70(AxxAA) with AR 1660
increased the constitutive transcriptional activity of this AR
NH2-terminal fragment-DNA binding domain fragment
(Fig. 4C
), although it was less effective than wild-type
ARA70.
[35S]ARA70(AxxAA) binding
to GST-AR 1660 in the affinity matrix assay was similar to that of
wild-type [35S]ARA70
(Fig. 4D
), indicating that FxxLF is not essential for
ARA70 binding to the AR
NH2-terminal region. The reduced coactivation of
ARA70(AxxAA) with AR 1660 relative to that of
wild-type ARA70 may have resulted from disruption
of the ARA70 activation domain as indicated
below.
Transcriptional Activation Domain in ARA70
Intrinsic activation was investigated by fusing regions of
ARA70 to the pLexA DNA binding domain and
measuring transcriptional activity of a ß-gal reporter gene.
pLexA-lamin served as a negative control. Full-length
ARA70 1614 lacked activity; however, the
carboxyl-terminal deletion mutant 1499 induced ß-gal activity (Fig. 5A
), and LexA-ARA70
1441 activity was 2-fold higher, suggesting that residues carboxyl
terminal to amino acid 441 suppress the transcriptional activation
region. LexA-ARA70 1382 activity was lower
while 1321 was similar to 1441 as measured in both plaque lift and
liquid ß-gal assays. LexA-ARA70 1296 was
inactive. NH2-terminal deletion mutants
containing amino acids 291614 had low activity whereas 321499 was
5-fold higher, consistent with a repressor function in the
carboxyl-terminal region. LexA-ARA70 366499 and
384499 had only 20% of the activity of 321499. Western blots
probed with monoclonal antibodies to the LexA DNA binding domain
demonstrated that the LexA ARA70 fusion proteins
were expressed at similar levels (Fig. 5B
). Results of the yeast assays
indicated the presence of an ARA70
transcriptional activation domain within the region of
ARA70 amino acids 296441.

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Figure 5. Transcriptional Activation Domain in
ARA70
A, Analysis in yeast cells. L40 yeast cells containing an integrated
ß-gal reporter gene controlled by LexA binding sites were transformed
with expression plasmids coding for LexA DNA binding domain fused with
full-length ARA70 or with deletion mutants of
ARA70. Numbers on the bars indicate amino
acid residues of ARA70. Transformed yeast were grown on
Ura-, Trp- plates for 48 h and used for lift and liquid assays. On
the right is summarized ß-gal activity of the liquid
assays. Results are expressed as in Fig. 2 and are representative of at least three independent experiments. B, Immunoblot analysis of
expression in yeast. Equal amounts of cell extract (1 x 108 cell equivalents) from transformed L40 yeast were immunoblotted with
monoclonal antibody against LexA DNA binding domain to measure relative
expression levels of the various LexA-ARA70 fusion
proteins. C, Effects of ARA70 and ARA70( AD)
mutant on AR transcriptional activity. pSG5 expression vectors coding
for full-length AR, 0.1 µg, and ARA70, 2.9 and 5.8 µg,
or ARA70( AD), 2.7 and 5.4 µg, were cotransfected into
CV1 cells with MMTV-LUC, 2.5 µg, and incubated in the absence and
presence of 0.1 nM DHT. Control incubations contained AR
and MMTV-LUC alone or with equimolar weights of pSG5 empty vector DNA,
2 or 4 µg, and 0.7 or 1.4 µg pBR322 to make the same total weight
of DNA. D, Relative effects of TIF2 and ARA70 on
transcriptional activation of the AR DNA-binding domain-LBD fragment.
The expression vector pCMVhAR 507919 coding for the AR DNA-binding
domain-LBD (50 ng) and MMTV-LUC (2.5 µg) were cotransfected into CV1
cells with pSG5TIF2 or pSG5ARA70 and incubated in the
absence and presence of 0.1 µM DHT. Weights of the two
transfected coactivator expression vectors were kept equimolar. Control
incubations contained an equimolar weight of pSG5 empty vector DNA with
pBR322 to make the same total weight of DNA.
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Deletion of amino acids 296441 from ARA70
created ARA70(
AD) for testing with AR in
transient cotransfection assays using CV1 cells (Fig. 5C
). Transfection
of 2.9 or 5.8 µg wild-type pSG5-ARA70 DNA
resulted in increases above that with AR and reporter gene alone or
balanced with equimolar pSG5 empty vector and pBR322. Under the same
conditions, ARA70(
AD) induced substantially
less transcriptional activity than did wild-type
ARA70 and had an effect similar to that of the
ARA70(AxxAA) mutant.
To assess the strength of the
ARA70 activation domain, we compared its effect
on the transcriptional activity of the AR DNA binding domain-LBD
fragment AR 507919 with that of the p160 coactivator TIF2 (Fig. 5D
).
The expression vector pCMVhAR 507919 containing the AF2 domain was
cotransfected with pSG5-TIF2 or pSG5-ARA70 and
MMTV luciferase reporter. Androgen-dependent activity of AR 507919
was minimal in the absence of cotransfected coactivator but greatly
enhanced by TIF2, as reported previously (58, 59).
However, transcriptional activity was increased less than 2-fold by
ARA70, suggesting that
ARA70 and TIF2 have different mechanisms of
coactivation.
ARA70 Activation Domain Is Not Required for
ARA70 Binding to the AR NH2-Terminal Domain but
Is Necessary for Full Transactivation
[35S]ARA70(
AD)
was synthesized and binding to GST-AR 1660 tested in an affinity
matrix assay (Fig. 6A
).
ARA70(
AD) bound GST-AR 1660 but not
GST-glutathione-Sepharose alone. The amount of bound
ARA70(
AD) relative to the input fraction was
similar to that of wild-type
[35S]ARA70.

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Figure 6. Mutant ARA70( AD) Interaction with AR
NH2-Terminal Domain
A, Binding to the AR NH2-terminal-DNA binding domain
fragment. [35S]ARA70 and
ARA70( AD) were synthesized in vitro and
incubated with Sepharose beads containing bound GST-AR 1660. Beads
were washed, and bound protein was eluted and analyzed by SDS-PAGE and
autoradiography. The input lane contained 15% of the total
radioactivity added. B, Coactivation with the AR
NH2-terminal domain. Expression plasmids encoding the AR
NH2-terminal-DNA-binding domain fragment pCMVAR 1660, 15
ng, and pSGARA70, 7.25 µg, or ARA70( AD),
6.75 µg, were cotransfected into CV1 cells together with MMTV-LUC,
2.5 µg. Control incubations contained 5 µg pSG5 empty vector DNA
[the molar equivalent of 7.25 µg pSGARA70 or 6.75 µg
pSGARA70 ( AD)] and 2.25 µg pBR322 to make the same
amount of total DNA.
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Investigation of the involvement of this activation
domain in ARA70 coactivation with AR is
complicated by the presence of the FxxLF motif within the activation
domain. Deletion of the activation domain also deleted the FxxLF motif
and abolished androgen-dependent binding to the AR-LBD. However,
since FxxLF was not required for ARA70 binding to
NH2-terminal-DNA binding domain fragment AR
1660, the ARA70 transcriptional activation
domain within amino acids 296441 could be tested for its effect on
the constitutive transcriptional activity of AR 1660. Cotransfection
assays in CV-1 cells were performed with ARA70 or
ARA70(
AD) and MMTV-LUC reporter (Fig. 6B
).
ARA70 increased luciferase activity relative to
AR 1660 alone or balanced with equimolar pSG5 empty vector and
pBR322. Under the same conditions, ARA70(
AD)
stimulation was about 50% of wild-type ARA70.
Since the binding of ARA70(
AD) to AR 1660
was similar to that of wild-type AR, the reduced coactivation with
ARA70(
AD) is consistent with the presence of
an activation domain in the region between amino acids 296441.
Similar reduced coactivation by ARA70(2KA) and
(AxxAA) mutants with AR 1660 suggested that these mutations
interfered with the function of the ARA70
activation domain.
ARA70 Coactivation with AR Is Dependent on Androgen
Activation of AR at Physiological Concentrations of Hormone in the
Human Male
Ligand specificity of ARA70 coactivation was
tested in CV-1 cells cotransfected with AR and the MMTV-LUC reporter in
the presence of DHT, E2, or progesterone (P) (Fig. 7
). At a concentration of 1
nM DHT, ARA70 increased luciferase
activity several fold above that of AR balanced with pSG5 vector and
pBR322. E2 or P at a concentration of 1 nM showed little or
no increase in luciferase activity either in the presence or absence of
ARA70 (Fig. 7A
). When steroid concentrations were
increased to 10 nM E2 or P, a concentration well above the
blood levels found in human males, ARA70
increased AR transcriptional activity (Fig. 7B
). The results indicate
that within the physiological range of circulating free steroid
concentrations in the human male, ARA70
coactivation of AR is androgen specific. Similar findings were observed
with the AR coregulator, protein inhibitor of activated STAT1
(60), and the p160 coactivator thyroid receptor activator
molecule-1/SRC3 (39), suggesting that
steroid-induced AR activation is a prerequisite for coactivation. In
the human male, total E2 levels are highest in testicular venous blood
and in caput epididymis (see Ref. 61 for review) but much
lower than T and would have relatively little influence on AR
activation. Cotransfection results with 10 nM E2 in CV1
cells are in agreement with those of Yeh et al.
(62), who performed CAT assays in DU145 cells, but
contrast with those of Gao et al. (54), who did
not observe substantial ARA70 coactivation with
AR in the presence of E2 using MMTV-LUC assays either in CV1 or DU145
cells.

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|
Figure 7. Comparison of DHT, E2, and P Inducement of
ARA70 Coactivation
pSG5 expression vectors coding for ARA70, 2.9 µg, and
full-length AR, 0.1 µg, were cotransfected into CV1 cells with
MMTV-LUC, 2.5 µg, and incubated in the absence and presence of 1
nM (panel A) and 10 nM (panel B) concentrations
of the indicated steroids. Control incubations contained 2 µg pSG5
empty vector DNA (the molar equivalent of 2.9 µg
pSGARA70) and 0.9 µg pBR322 to make the same amount of
total DNA.
|
|
ARA70 Coactivation with PR and GR
Receptor specificity of ARA70 coactivator
function was investigated in CV-1 cells cotransfected with human PR or
GR with the MMTV-LUC reporter (Fig. 8
).
Steroid concentrations were 10 nM to optimize for
activation of PR and GR. Transfection of 5.8 µg of
pSG5-ARA70 increased transcriptional activity of
both PR and GR. These results indicate ARA70 is
not a specific coactivator for AR and are in agreement with results of
others (54, 55, 62). ARA70 is also
reported to be a coactivator with the PPAR
(63).

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|
Figure 8. Effects of ARA70 on Human AR, PR, and
GR Transcriptional Activity
pSG5 expression vectors coding for ARA70, 5.8 µg, and the
different steroid receptors, 0.1 µg, were cotransfected into CV1
cells with MMTV-LUC, 2.5 µg, and incubated in the absence and
presence of 1 nM DHT or 10 nM P or
dexamethasone. Control incubations contained 4 µg pSG5 empty vector
DNA (the molar equivalent of 5.8 µg pSGARA70) and 1.8
µg pBR322 to make the same amount of total DNA.
|
|
 |
DISCUSSION
|
---|
We mapped the region of ARA70 required for
androgen-dependent interaction with the AR-LBD, identified an intrinsic
transcriptional activation domain (ARA70
296441), and found that the carboxyl-terminal region of
ARA70 has an inhibitory effect on these
ARA70 functional domains in yeast. The sequence
exhibiting maximum AR-LBD binding was ARA70 amino
acids 321441. These amino acids were contained within the sequence
isolated by two-hybrid screening of a cDNA library using the AR-LBD as
bait. Androgen-dependent binding of ARA70
321441 to the AR-LBD was demonstrated also in cell-free assays
in vitro. A double mutation, K327A, K329A, which changed
KFKLLF to AFALLF in an FxxLF motif within this
ARA70 region, eliminated androgen-dependent
ARA70 binding to the AR-LBD in yeast and in the
GST affinity matrix assays. Mutating FxxLF to AxxAA also eliminated
androgen-dependent binding to the AR-LBD, indicating this motif is
required for the interaction. Since coactivation by
ARA70(2KA) or ARA70(AxxAA)
mutants with full-length AR was not abolished, the possibility remained
for another site of ARA70 interaction with AR
outside the AR-LBD. We observed that ARA70 binds
the AR NH2-terminal-DNA binding domain fragment
and enhances its constitutive transcriptional activity. Binding to the
AR NH2-terminal region was not dependent on an
intact FxxLF motif or the presence of an activation domain
(ARA70 296441). However, deletion of the
activation domain diminished ARA70 coactivation
with the AR NH2-terminal-DNA binding domain
fragment. This result in a mammalian cell line supported the presence
of an ARA70 activation domain as defined in
yeast. Our results are in agreement with existing evidence
(53, 54, 55) that ARA70 is an AR
transcriptional coactivator and indicate further that
ARA70 coactivation results from interactions with
both the NH2-terminal and LBDs of AR.
In pull-down assays, Alen et al. (55) observed
that both GST-ARA70/ELE1
and
GST-ARA70/ELE1ß bound full-length
[35S]AR in an androgen-independent manner.
ELE1
is identical to full-length ARA70 while
ELE1ß contains a deletion of amino acids 238566. Our results
indicate that ELE1ß would not be expected to bind the AR-LBD in an
androgen-dependent manner since the ELE1ß lacks the FxxLF motif. The
results of Alen et al. (55) could be explained
by binding to the AR NH2-terminal domain;
however, binding to this region was not detected in their assay.
Like other nuclear receptors AR NH2-terminal and
LBDs contain transcriptional activation subdomains designated
activation function 1 (AF1) and AF2, respectively (1, 23, 64, 65, 66, 67). When AR is activated by androgen binding, it dimerizes
on androgen response element DNA through an interaction of the
NH2- and carboxyl-terminal regions referred to as
the N/C interaction (67, 68, 69, 70, 71). As an isolated domain, the
AR NH2-terminal activation function, AF1, is
stronger than AF2 in the carboxyl-terminal region (56, 64, 70). Recent studies indicated the AR
NH2-terminal domain recruits p160 coactivators
while AF2 is the major contact site for the N/C interaction
(59). The N/C interaction is dependent on the presence of
an FxxLF motif in the AR NH2-terminal domain, and
it is this motif that interacts with the AR AF2 (58).
Because ARA70 contains an FxxLF motif, it likely
also interacts with AF2 in the AR-LBD. The
NH2-terminal region of
ARA70 contains an LxxLL motif. This sequence
motif is known to be important in the binding of p160 coactivators to
AF2 of other nuclear receptors (42, 43, 44, 45, 46, 47). However,
ARA70 sequences containing this motif (amino
acids 1118, 1295, and 1321) did not bind the AR-LBD in the yeast
assay. These results are in agreement with recent studies showing that
the AR AF2 region has higher affinity for the FxxLF motif than it does
for the LxxLL motif (71A ).
Studies with TIF2 (72), GRIP1 (73), and SRC1a
(74) point out the importance of the AR
NH2-terminal domain in p160
coactivator-stimulated gene transcription. p160 coactivator mutants
with deleted LxxLL motifs bound the AR
NH2-terminal region and retained coactivator
function despite the lack of binding to AF2 (72, 74). AF1
of the ER bound to sequences near the p160 coactivator carboxyl
terminus (75). SRC-1 interacted with both
NH2- and carboxyl-terminal regions of the PR
(29), and GRIP1 stimulated the transcriptional activation
of both AF1 and AF2 in the ER (75). Tremblay et
al. (76) demonstrated that ligand-independent
phosphorylation of MAPK sites in AF1 of ERß stimulated its
interaction with SRC-1. In the AR NH2-terminal
region, phosphorylation sites that include serines 81, 94
(77), and 515 (78) may be modulators of the
ARA70 interaction.
Molecular interactions of the ARA70
transcriptional activation domain remain to be determined, and the
precise relationship between the FxxLF motif and the activation domain
is not yet known. The partial coactivation maintained by the
ARA70 activation domain deletion mutant (deletion
of amino acids 296441) with the full-length AR and its reduced
activity with the AR NH2-terminal domain suggest
the possibility of a second activation domain.
ARA70/ELE
and
ARA70/ELEß binding to p300 CBP-associated
factor and TFIIB reported by Alen et al. (55)
may involve a second activation domain since the domain within amino
acids 296441 is deleted in ARA70/ELEß.
The p160 coactivator TIF2 contains two distinct activation domains AD1
and AD2. Transcriptional activity of AD1 results from direct
interaction with cAMP response element binding protein while the
mechanism mediating AD2 appears independent of cAMP response element
binding protein but remains undefined (32).
Deletion of AD1 from GRIP1 had little effect on its enhancement of AR
transactivation, indicating it has a minor role in AR coactivation
(73). With regard to inhibition of the
ARA70 intrinsic activation domain by its own
carboxyl-terminal region, it may be relevant that AD2 activity of the
SRC-1a isoform was suppressed by the 56 amino acids that are unique to
its carboxyl-terminal region, thereby reducing its ability to enhance
ER-mediated transcription (79). Whether this region
suppressed AD2 by masking it directly or through interaction with
another protein remains to be established. In
ARA70, carboxyl-terminal inhibition of
androgen-dependent binding to the AR-LBD in addition to inhibition of
intrinsic transactivation might be an indication of the close proximity
of these functional domains within amino acids 296441.
Spermatogenesis is an androgen- and AR-dependent process
(3). Expression of ARA70 in testis
could modulate AR regulation of spermatogenesis. Since
ARA70 is not AR specific, it may also be a
coactivator with other nuclear receptors in testis both in Sertoli
cells and spermatogenic cells. In Sertoli cells
ARA70 is one of several potential AR
coregulators. Others include the p160 family (39), protein
inhibitor of activated STAT-1 (PIAS-1) (60), and related
members of the PIAS family (80, 81). SNURF, a ring finger
protein (82), and ANPK, serine/threonine kinase
(83), add to the growing number of potential AR
coregulators that may be important in spermatogenesis.
 |
MATERIALS AND METHODS
|
---|
Materials
The following cells and reagents were used: monkey kidney CV1
cells from the American Type Culture Collection (Manassas,
VA); DMEM with high glucose with or without phenol red from JRH Biosciences (Lenexa, KS); bovine calf serum from HyClone Laboratories, Inc. (Logan, UT); Enhanced Chemiluminescence
Western Blotting Detection Kit from Amersham Pharmacia Biotech (Piscataway, NJ); unlabeled deoxynucleotide
triphosphates, glutathione Sepharose 4B, and pGEX (GST gene fusion)
vector; deep-Vent polymerase, T4 DNA ligase, and restriction
endonuclease from New England Biolabs, Inc. (Beverly,
MA); T4 polynucleotide kinase, MMLV RNA reverse transcriptase, RNase H,
and Escherichia coli DNA polymerase I from Promega Corp. (Madison, WI); oligo(dT) cellulose columns and XL2-Blue
MRF' supercompetent cells from Stratagene (La Jolla, CA);
X-gal and
o-nitrophenyl-ß-D-galactopyranoside
from Sigma (St. Louis, MO); prestained protein mol wt
standards from Life Technologies, Inc. (Gaithersburg, MD);
X-OMAT-AR diagnostic x-ray film from Kodak (Rochester,
NY); D-luciferin from Analytical Luminescence
Laboratory (San Diego, CA) ; cell lysis buffer from Ligand Pharmaceuticals, Inc. (San Diego, CA); Immobilon from
Millipore Corp. (Bedford MA); buffers and chemicals from
Fisher (Suwanee, GA), EM Science (Ft. Washington, PA), and
Sigma. psLuc2 was from S. K. Nordeen, University of
Colorado Health Sciences Center (Denver, CO). Full-length cDNA of RFG
containing a stop codon within the coding region was from M. Santoro,
Universita Adegli studi di Napoli (Naples, Italy) (57).
Yeast vectors and strains from S. Hollenberg, Oregon Health Sciences
University (Eugene, OR) (84) included pLexA, the LexA DNA
binding domain fusion vector; pVP16, the library vector for expressing
fusion protein with the transcriptional activation domain of the herpes
simplex virus VP16; and controls pLex-lamin, pLex-da, and pVP16MyoD.
Yeast strains were L40 [MaTa (trp1901 his3D 200 leu23, 112 ade2
LYS2:: (lexAop)4 -HIS3 URA3::
(lexAop)8 -lacZ GAL4] and AMR70 [MaTa his3 lys2
trp1 leu2 URA3:: (lexAop)8 -lacZ
GAL4].
Construction of Plasmids
Plasmids expressing the indicated amino acids of
ARA70 as fusion proteins were constructed using a
cDNA with sequence identical to that of ARA70
(53). VP16-ARA70 and pGEX
(GST-ARA70) fusion plasmids were constructed by
PCR of the modified RFG using 5'-BamHI and
3'-EcoRI-stop primers or by site-directed mutagenesis
(85, 86). Products were digested (BamHI
/EcoRI) and cloned into pVP16 or pGEX vectors.
pLexA-ARA70 fusion plasmids were created by PCR
of the modified RFG. Products were blunt-ended with T4 DNA polymerase,
digested (BamHI) and cloned into pLexA digested with
PstI, blunt-ended, and digested with BamHI.
pSG5-ARA70 and mutants
pSG5-ARA70(2KA), (amino acids
327KFK329 changed to
327AFA329,
pSG5-ARA70(AxxAA) (amino acids
328FKLLF332 changed to
328AKLAA332, or
pSG5-ARA70(
AD) (amino acids 296441 deleted)
were constructed by site-directed mutagenesis (85, 87).
TIF2 construct was provided by H. Gronemeyer (32). AR
1660 was as described previously (64). pLexA-AR-LBD,
GST/AR-LBD (both contain AR amino acids 624919), and GST-AR 1660
were derived by PCR of pCMV5hAR. Deep-Vent
polymerase was used to minimize PCR errors. PCR conditions for 500-bp
regions were: 1 cycle 94 C for 5 min, 55 C for 2 min, 72 C for 3.5 min;
11 cycles of 95 C for 1.5 min, 55 C for 2 min, 72 C for 3 min; and 1
cycle of 95 C for 1.5 min, 55 C for 2 min, 72 C for 8
min. PCR-amplified constructs were verified by sequencing.
Library Screening and Analysis of ARA70
Functional Activity
Random primed LNCaP cell cDNA library and two-hybrid screening
were performed as described by Hollenberg and associates
(84) with modifications. LNCaP cells were treated for
4 d with 5 nM T. Total RNA was extracted using
guanidinium isothiocyanate and cesium chloride. Poly(A)RNA was purified
on oligo(dT) cellulose. First-strand cDNA was synthesized using random
hexamers and reverse transcriptase at 42 C and second strand with
E. coli DNA polymerase I and RNase H (Promega Corp.). DNA ends were blunted with T4 DNA polymerase and ligated
with 500-fold molar excess of NotI adaptors. cDNAs of
350800 nucleotides in length were purified on agarose gels and PCR
amplified. cDNAs were digested with NotI, repurified, and
ligated into pVP16 in NotI. E. coli were
transformed with the ligation mixture to produce the cDNA fusion
library. For the two-hybrid screen, coding sequence for the human AR
hinge region and LBD residues 624919 was cloned into pLexA to create
LexA-AR-LBD.
Before screening the library, we tested the extent of nonspecific
activation of the reporter gene by bait protein. Yeast strain L40
containing an integrated ß-gal reporter gene controlled by eight LexA
binding sites was transformed with pLexA-AR-LBD and pVP16 and plated on
Ura-, Trp-, Leu-, and His- yeast complete medium containing 1
nM dihydrotestosterone (DHT) and the histidine
antimetabolite 3-aminotriazol (20 mM) and incubated for
three d. The transformants grew and had detectable ß-gal activity in
this yeast strain as reported previously (56). To minimize
transcriptional activity of the AR-LBD, transformants were plated on
Ura-, Trp-, Leu- plates without DHT for 2 d and replica plated to
the same plates containing 10 nM DHT. ß-gal Activity was
determined after 16 h using the lift assay. Under these
conditions, yeast transformed with lexA-AR-LBD and pVP16 did not turn
blue within 3 h. Yeast strain L40 was cotransformed with
LexA-AR-LBD and the above random-primed cDNA fusion library using
lithium acetate (84) and 10% dimethylsulfoxide.
Transformants were plated in Ura-, Trp-, Leu- medium at 30 C for 2
d and replica plated to Ura-, Trp-, Leu- medium containing 10
nM DHT and incubated for 16 h. About 8 x
107 transformants were assayed by the filter lift
method for ß-gal activity (84). Results were considered
positive if the blue color developed within 3 h at 25 C.
pLexA-lamin with pVP16 MyoD or pLexA-AR-LBD with pVP16 were negative
controls. pLexA-da with pVP16 MyoD or pLexA-AR-LBD with AR
NH2-terminal residues 1583 in pVP16 were
positive controls. Positive colonies were further analyzed after
eliminating LexA-AR-LBD. Bait plasmids were introduced by mating with
AMR70 containing pLexA-AR-LBD (84).
Sequence requirements of ARA70 interaction with
the AR-LBD (amino acids 624919) were determined using pLexA-AR-LBD
and pVP16-ARA70 with wild-type or mutant
ARA70 sequence. The ARA70
transcriptional activation domain was determined using
ARA70 fragments cloned into pLexA. Yeast strain
L40 containing an integrated ß-gal reporter gene controlled by eight
LexA binding sites was transformed with the different fusion protein
expression vectors. ß-gal Activity was measured by lift and liquid
assays.
Liquid ß-Gal Assays
L40 yeast were transformed and plated in Ura-, Trp-, and Leu-
media and incubated for 2 d at 30 C. Triplicate colonies were
grown overnight in Leu- and Trp- liquid medium at 30 C. Cultures were
diluted with fresh medium to OD600 0.02 in the
presence or absence of 10 nM DHT and grown for 16 h to
late log phase (OD600 0.60.8). Cells were
permeabilized with three cycles of freeze/thaw and incubated at 30 C
with
o-nitrophenyl-ß-D-galactopyranoside.
Activity was calculated as ß-gal = 1000 x
[OD420/(t) (v) (OD600)]
where OD420 is the absorbance of hydrolyzed
o-nitrophenol; t, time of reaction in min; v, volume of
culture in milliliters; and OD600, cell density
at the start of assay (88).
Immunoblots
Yeast extracts were prepared by a urea-SDS method
(89). Extracted proteins were fractionated on a
SDS-polyacrylamide gel and transferred to nitrocellulose. Blots were
probed either with monoclonal antibody Vp16 121 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) against the Vp16
transcriptional activation domain of the different Vp16
ARA70 fusion proteins or with monoclonal antibody
LexA 212 (Santa Cruz Biotechnology, Inc.), against the
LexA-DNA binding domain of the LexA-ARA70 fusion
proteins. Primary antibodies were detected with goat-antimouse IgG
coupled to horseradish peroxidase and detected with the enhanced
chemiluminescence system.
ARA70-AR Interaction in Vitro
Glutathione S-transferase fusion protein expression,
purification, and binding were described previously (86).
In brief, overnight E. coli cultures transformed with pGEX
expressing different regions of ARA70, AR-LBD (AR
624- 919), or the AR NH2-terminal-DNA binding
domain fragment (AR 1660) were diluted in LB medium and incubated at
37 C for 1.5 h. GST fusion protein expression was induced with 0.1
mM isopropylthiogalactoside (Sigma)
at 30 C for 4 h. Bacterial cultures were pelleted at 5,000 x
g for 5 min at 4 C and resuspended in 0.1 volume NENT (20
mM Tris, pH 8.0, 0.1 M
NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing
the proteinase inhibitors 0.5 mM
phenylmethylsulfonyl fluoride, 1 µM leupeptin,
and 0.1 µM aprotinin. Bacteria were sonicated
and centrifuged at 12,000 x g for 5 min at 4 C.
Supernatants were incubated for 30 min at 4 C with 30 µl
glutathione-Sepharose and washed three times with NENT. Sepharose with
bound GST-ARA70 1614,
GST-ARA70 321499, or
GST-ARA70 321441 was incubated for 45 min at 4
C with equal amounts of the AR DNA binding-LBD fragment (AR 507919)
from Sf9 cell extracts (68) as determined by immunoblot
analysis, washed five times with NENT, and eluted with SDS. Bound
protein was separated on SDS-acrylamide gels and immunoblotted using
AR52 antibody (21, 90).
In an affinity matrix assay using radiolabeled proteins,
glutathione-Sepharose bound to GST alone, GST-AR 624919, or AR 1660
was incubated for 90 min at 4 C in the absence or presence of 1
µM DHT with 35 µl
[35S]methionine-labeled full-length wild-type
ARA70 or mutant ARA70(2KA),
(AxxAA), or (
AD) synthesized using the TNT Coupled Reticulocyte
Lysate System (Promega Corp.). Sepharose was washed
repeatedly, the bound protein was eluted, and then fractionated by
SDS-PAGE, and the radioactive protein was visualized by
autoradiography. The input lane contained 15% of the total
radioactivity added.
In a similar assay [35S]AR-LBD (AR 624919)
was incubated in the presence and absence of 1 µM DHT
with glutathione-Sepharose bound to GST alone,
GST-ARA70 321407 wild-type sequence, or
GST-ARA70 321407 (AxxAA) mutant, and bound
radioactive protein was measured as above. In this assay the input lane
contained 10% of the total radioactivity added.
Cell Culture and Transfections
CV1 cells were maintained in 10% FCS in DMEM containing high
glucose and antibiotics. Cells were plated at 4.5 x
105 cells per 6-cm dish. Expression vectors
pSG5hAR (0.1 µg), pCMVhAR (0.025 µg), or pCMVAR 1660 (0.015 µg)
and MMTV-LUC (2.5 µg) were cotransfected with
pSG5ARA70 or mutant
pSG5ARA70 using calcium phosphate. Transfections
with pSG5AR and MMTV-LUC alone were included as a control. As an
additional control, equimolar amounts of the pSG5 empty vector were
used to balance pSG5ARA70, and pBR322 was added
to equalize the total transfected DNA. After 40 h with or without
0.1 nM or 10 nM DHT, cells were harvested and
luciferase activity was assayed (91). Results are
expressed as mean ± SD light units of three
replicates and are representative of three or more experiments.
It was reported that cotransfection of pSG5 expression vectors in COS
cells can inhibit the expression of AR from pSG5AR, presumably due to
depletion of transcription factors (92), and we confirmed
this observation by AR immunoblotting (our unpublished results).
In contrast to COS cells that make multiple copies of pSG5 vector DNA,
pSG5 is not amplified in CV1 cells. Nevertheless, we transfected
equimolar amounts of pSG5 empty vector DNA in an attempt to maintain a
balance of pSG5 in control and treated cells.
 |
ACKNOWLEDGMENTS
|
---|
We thank De-Ying Zang, Michelle Cobb, and Raymond T. Johnson,
Jr., for excellent technical assistance, and S. Hollenberg for yeast
strains and plasmids. Cell culture and cotransfections were performed
in the Cell Separation and Tissue Culture Core of the Laboratories for
Reproductive Biology.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grants HD-04466 (F.S.F.) and HD-16910
(E.M.W.), Fogarty International Center D43TW/HD00627 Training and
Health in Population and Research, by The Andrew W. Mellon Foundation,
and by NICHD/NIH through cooperative agreement U54 HD-35041 as part of
the Specialized Cooperative Centers Program in Reproduction
Research.
Abbreviations: AF1 and AF2, Activation function
1 and 2; ARA70, AR-associated protein 70; DHT,
dihydrotestosterone; ß-gal, ß-galactosidase; GRIP, GR-interacting
protein; GST, glutathione-S-transferase; LBD,
ligand-binding domain; LNCaP, lymph node-derived human prostate
carcinoma cell line; MMTV-LUC, mouse mammary tumor virus-luciferase; P,
progesterone; RFG, RET-fused gene; SRC, steroid receptor-coactivator;
TF, transactivation factor.
Received for publication July 19, 2000.
Accepted for publication October 8, 2001.
 |
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