RFG (ARA70, ELE1) Interacts with the Human Androgen Receptor in a Ligand-Dependent Fashion, but Functions Only Weakly as a Coactivator in Cotransfection Assays
Tianshu Gao,
Ken Brantley,
Erol Bolu and
Michael J. McPhaul
Department of Internal Medicine The University of Texas
Southwestern Medical Center Dallas, Texas 75235-8857
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ABSTRACT
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Abnormalities of the human androgen receptor (hAR)
cause a range of clinical defects in male development. A large
proportion of these mutations are single amino acid substitutions in
the hormone-binding domain (HBD) that alter AR function by interfering
with the capacity of the AR to bind androgen or to form stable
hormone-receptor complexes. Prior studies have suggested that the
formation of such stable, active hormone-receptor complexes is a
crucial step in the modulation of genes by the AR. It is presumed that
these hormone-receptor complexes interact with other proteins to
participate in the formation of active transcription complexes at the
initiation sites of androgen-responsive genes.
Using a yeast two-hybrid screening method, we isolated a partial cDNA
encoding the carboxy terminus of a protein that interacts with the
hAR-HBD (amino acid residues 623917) in a ligand-dependent fashion in
a yeast two-hybrid assay. Sequence analysis of this clone revealed that
it encoded a portion of a protein that had been previously
characterized as RFG (RET Fused Gene). Using
glutathione-S-transferase (GST) fusions of the hAR HBD and
immunoprecipitation of the in vitro translated proteins, we
have demonstrated that this interaction can be reproduced in
vitro. To determine the capacity of this protein to modulate the
activity of the AR in transfection assays, we expressed full-length RFG
in the CV1 and DU145 cell lines, in combination with an AR expression
vector and model androgen-responsive genes [mouse mammary tumor virus
(MMTV) and PRE2-tk luciferase]. Our results
demonstrate that RFG alters the induction of these reporter genes very
weakly (no greater than 2-fold compared with transfections without the
RFG expression plasmid). Thus, while our findings are in agreement with
published reports which indicate that RFG interacts with AR-HBD in a
ligand-dependent fashion, in our assays RFG does not exert major
effects on the activity of the hAR in response to androgen or to other
steroid hormones.
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INTRODUCTION
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The human androgen receptor (hAR) is a ligand-dependent
transcription factor that modulates the actions of androgen. Mutations
that impair the function of the AR lead to abnormalities of male sexual
development (1, 2). Definition of the mutations in the AR that cause
androgen resistance have demonstrated that a large proportion of these
are caused by amino acid substitutions in AR protein, and the majority
of these amino acid replacements are localized to the hormone-binding
domain (HBD). Detailed studies of such mutant ARs have revealed that an
impairment of ligand binding or an instability of ligand binding is a
common characteristic of substitution mutations in the AR HBD (3).
Furthermore, studies of the ARs expressed in individuals with complete
testicular feminization caused by single amino acid substitutions in
the HBD of the AR indicated that increased doses of hormone or
increased frequency of hormone addition can restore near-normal
receptor function (3).
Such studies have focused attention on understanding the conformational
changes that the AR undergoes after the binding of androgen and the
protein-protein interactions that such changes facilitate. To this end,
in parallel with studies of many members of the nuclear receptor
family, attempts have been made using a variety of techniques to define
the mechanisms by which androgens activate the AR. Studies by two
groups explored the conformational changes of the AR in response to
androgen agonists and antagonists. Using a limited proteolysis
technique, these researchers demonstrated that a 29- to 30-kDa fragment
of the AR agonist complex showed an increased resistance to trypsin
digestion. By contrast, proteolysis of AR complexed to antagonists
identified a distinct 35-kDa fragment of hAR that was resistant to
proteolysis (4, 5), indicating that the conformation change induced by
androgen was distinct from that stimulated by antiandrogens. Such
inferences are in agreement with similar studies using limited
proteolysis conducted in the study of other members of the nuclear
receptor family (6, 7, 8, 9) and consistent with the more detailed
information derived from crystallographic studies (10, 11, 12, 13, 14, 15).
While providing information regarding changes in the conformation of
the AR protein, such studies do not identify the components of the
transcriptional apparatus that are contacted by the AR when bound to
agonists or antagonists. Experiments employing both biochemical and
genetic approaches have identified a number of proteins that appear to
serve as bridges between members of a nuclear receptor family and the
transcription apparatus (16, 17).
Recently, cDNAs encoding a 70-kDa human protein, termed ARA70, were
reported by Yeh and Chang (18). Sequence analysis indicated that this
protein shares 94% homology with a previously characterized protein,
RFG (RET-fused gene) (19). In their studies, these investigators found
that RFG interacted with AR in an androgen-dependent manner and
increased the activation of a reporter gene 10-fold in a cotransfection
assay. In the studies detailed here, we isolated cDNAs encoding
proteins that interacted with the HBD of the hAR (amino acid residues
623917) in a ligand-dependent fashion. We found that one of these
encoded a fragment of the RFG protein. In the present report, we have
analyzed the capacity of the full-length RFG protein to interact with
the AR. Our experiments demonstrate that in cotransfection assays, RFG
behaves only weakly as a coactivator for the AR in the CV1 and DU145
cell lines and does not alter the ligand responsiveness of the AR.
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RESULTS
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The RFG Protein Interacts with the hAR in a Ligand-Dependent
Fashion
To understand the mechanisms regulating the function of the hAR,
we used a two-hybrid screening method to identify proteins that
interacted with the HBD of the hAR (20). One of the clones () that
we isolated corresponded to a protein previously identified by Santoro
et al. (19) as a fusion to the RET protooncogene in a human
papillary thyroid cancer (Fig. 1
). This
protein, which they termed RFG (RET-fused gene), at the time was not
known to perform any function (19).

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Figure 1. Schematic Structures of the Plasmids
Using a yeast two-hybrid screening method, we isolated a partial cDNA
encoding a protein fragment that interacted with the hAR-HBD in a
ligand-dependent fashion (clone 235). Sequence analysis of this
partial cDNA revealed a 99% homology with a cDNA isolated by Santoro
and co-workers (19 ), which they termed RFG (RET-fused gene). Three
nucleotide sequence differences were noted compared with the originally
reported sequence. These differences predict three amino acid
differences at residues 316, 317, and 364, as indicated (FL RFG). Yeh
and Chang (18 ) also identified the same differences in their isolate,
compared with the sequence published for RFG (19 ). As noted in
Materials and Methods, complete sequence of the cDNA
provided to us by Santoro et al. revealed these same
alterations, suggesting that the differences from the sequence reported
by these investigators represent inadvertent inaccuracies in the
nucleotide sequence that was originally reported (Genbank accession
number X77548).
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Initial experiments focused on examining the interaction with the AR
HBD that was inferred from the conditions in which the clone 235 had
been isolated. To address the specificity of this interaction, we
transformed yeast with plasmids encoding analogous fusion proteins of
the glucocorticoid receptor (GR) and progesterone receptor (PR) HBDs in
the pAS vector and incubated the cells in the presence of their
respective cognate ligands. Measurements of ß-galactosidase activity
indicate that RFG displays increased interaction with the AR HBD in the
presence of androgen. Parallel incubations of yeast cells harboring
pACT plasmids encoding the PR or GR HBD indicated a ligand-dependent
interaction of the RFG fusion protein with the PR, but not with the GR.
Similar experiments examining the interaction of steroid receptor
coactivator 1 (SRC-1) with these ligand-binding domains (LBDs)
were performed to contrast with the RFG results and demonstrated a
strong hormone-dependent interaction with the liganded PR and a weaker
association with the liganded AR (Fig. 2
).

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Figure 2. Interactions of RFG and the Human Androgen,
Progesterone, and Glucocorticoid Receptors Using the Yeast Two-Hybrid
Assay
Partial cDNAs encoding analogous segments of the hAR (amino acid
residues 623917), human GR (amino acid residues 483777), and human
PR (amino acid residues 639933) were generated by PCR and subcloned
into pAS vector. These plasmids were transformed with the 235/pACT
plasmid (encoding the segment of the RFG protein depicted in Fig. 1 )
into yeast strain Y190 to examine the strength and specificity of
interaction occurring between the receptors and RFG. As shown, while
RFG exhibits a strong interaction with AR HBD in the presence of
androgen and the PR HBD in the presence of progesterone, no such
ligand-independent interaction could be detected when the pAS plasmid
encoding the HBD of the GR was employed. In parallel experiments, a
ligand-dependent interaction was observed in experiments assessing the
interaction of the PR HBD with the carboxy terminus of SRC-1. No such
interaction was observed in experiments using the AR or GR HBDs,
although a ligand-dependent interaction of fusion proteins containing
the GR LBD and the carboxyl terminus of GRIP-1 could be detected (data
not shown).
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As the RFG protein bears no significant similarity to other
transcriptional coactivators or corepressors that have been identified,
we performed experiments to determine whether the two proteins interact
directly. Our first experiments employed a GST pull-down assay to
examine the interactions of these two proteins. As shown in Fig. 3A
, beads to which the HBD of the AR has
been bound displayed increased retention of the labeled RFG protein
synthesized using an in vitro transcription/translation
system. While the results of these experiments were suggestive of a
direct interaction, this conclusion was tempered by concerns regarding
the poor efficiency of synthesis of AR protein in bacteria that is
capable of binding hormone (21). For this reason, we employed a second
technique to examine whether a direct interaction was occurring. In
these experiments, we in vitro transcribed and translated
both the hAR and the RFG protein. As shown in Fig. 3B
, antibodies that
sequester the in vitro transcribed/translated hAR
precipitate the RFG protein in parallel. By contrast, little or no RFG
protein is precipitated by resin to which no anti-AR antibody has been
linked.

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Figure 3. Physical Interactions of the RFG Protein and the
hAR
A, Radiolabeled RFG was tested for its ability to bind to GST or to
GST-AR-HBD fusion protein immobilized on glutathione-Sepharose beads. A
sample of the input-labeled RFG protein is shown in lane 2. Aliquots of
the supernatants after incubation with GST-AR or GST-alone beads are
shown in lanes 3 and 4, respectively. After incubation, the amount of
radiolabeled RFG released after incubation with the GST-AR-HBD fusion
protein beads and four successive washes are shown (lanes 58). The
proteins released by solubilization in loading buffer of the GST
control beads (lane 9) and the GST-AR-HBD beads (lane 10) after the
four washes are shown. The level of nonspecific binding of RFG to the
glutathione beads is evident in lane 9. B, Samples of the labeled RFG
and hAR proteins were prepared in vitro as described in
Materials and Methods. Samples of the input-labeled AR
and input-labeled AR/RFG mixture are shown in lanes 2 and 3,
respectively. Supernatants resulting from the incubation of the labeled
mixture with beads to which no antibody (control), antibody directed
the AR amino terminus (U402), or at an internal epitope (U407) are
shown in lanes 46, respectively. The radiolabeled proteins
that remained absorbed to the beads to which the different anti-AR
antibodies had been affixed are shown in lanes 8 and 9, respectively.
Molecular weight markers are shown in lane 1, and the positions of the
in vitro translated AR and RFG proteins are shown to the
right.
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Effects of RFG Expression on AR Function
To examine the effects of RFG expression on AR function, CV
1 cells were transfected with varying ratios of the expression plasmids
encoding RFG and hAR. In these experiments, the quantities of DNA
employed, particularly the cytomegalovirus (CMV) expression plasmid,
were held constant. As detailed in Fig. 4
, only small alterations of AR function
could be observed and were similar whether expressed as fold induction
or in terms of the level of reporter gene activity that was measured.
These small effects were not limited to experiments in which the mouse
mammary tumor virus (MMTV) promoter was used to drive the reporter
plasmid (panel A). Experiments in which the PRE2-tk
luciferase reporter plasmid was employed also demonstrated only small
alterations of AR function (panel B).

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Figure 4. Effect of RFG on AR Function Assayed in the CV1
Cell Line Using the MMTV and PRE2-tk Luciferase Reporter
Genes
To assess the effect of RFG expression on the functional responsiveness
of an androgen-responsive reporter gene, expression plasmids encoding
the normal hAR, RFG, and either the MMTV luciferase (column A) or
PRE2-tk luciferase (column B) reporter plasmid were
introduced into CV1 cells. In each instance the data are expressed as
either the percent of fold induction (open bars) or the
percent of stimulated activity (stippled bars), with the
degree of responsiveness observed after transfection with the AR
expression plasmid alone after stimulation with 2 nM
5 -dihydrotestosterone (DHT) (included in each experiment) set
at 100%. Each of the data sets within columns A and B is derived from
six separate transfected cell samples (three basal and three stimulated
with hormone). The corrected basal and DHT-stimulated activities of the
cells transfected with the AR expression plasmid (included in each
experiment) are shown for each group in Table 1 .
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A prior report had suggested that RFG exerts potent effects on AR
function in assays conducted in cells that do not express the RFG
protein, such as has been reported for the DU 145 cell line (18). To
examine this possibility, we performed an additional series of
experiments using the DU145 cell line. Figure 5
presents the results of experiments in
which the recipient DU145 cell line was transfected with expression
vectors encoding RFG and hAR. These experiments included conditions
representing transfection with both small and large quantities of both
expression vectors. Examination of this figure reveals similar levels
of reporter gene activity in cells transfected with 50, 100, or 200 ng
of AR expression plasmid. As expected, transfection with larger amounts
of AR cDNA led to decreased levels of reporter gene activity. In
general, when cells were transfected with a single level of the AR
cDNA, transfections with increasing amounts of the RFG expression
plasmid led to a gradual decline in the level of reporter gene activity
that was observed. These patterns are similar whether expressed in
terms of the absolute level of stimulated reporter gene activity or as
the fold induction of reporter gene activity, as summarized in Fig. 6
for the MMTV-luciferase reporter
plasmid. Cotransfection assays using cDNAs encoding SRC-1 and GRIP-1
(GR interacting protein 1) yielded modest effects on AR function (data
not shown).

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Figure 5. Effect of RFG on AR Function Assayed in the DU145
Cell Line Using the MMTV and PRE2-tk Luciferase Reporter
Genes
To assess the effect of RFG expression on the functional responsiveness
of an androgen-responsive reporter gene in the DU145 cell line, these
cells were transiently transfected with an expression plasmid encoding
the normal hAR, RFG, and either the MMTV luciferase (column A) or
PRE2-tk luciferase (column B) reporter plasmids. In each
panel, the data are expressed as either the percent of fold induction
(open bars) or the percent of stimulated activity
(stippled bars) after stimulation with 2 nM
DHT, and the level of response observed is expressed relative to the
level observed in cells transfected with the AR expression plasmid
alone (as 100%). Each of the data sets is derived from six separate
transfected cell samples (three unstimulated and three stimulated with
hormone). The corrected basal and DHT-stimulated activities of the
cells transfected with the AR expression plasmid (included in each
experiment) are shown for each group in Table 1 .
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Figure 6. Summary of Experiments Using the MMTV Luciferase
Reporter Plasmid in the DU145 Cell Line
Because of the variability of the individual experiments depicted in
Figs. 4 and 5 , the results from three separate experiments are
summarized by relating the data to the activity of the reporter gene in
cells transfected with 0.1 µg of AR expression plasmid after
stimulation with DHT (included in each experiment). The data are
presented as percentage of the stimulated value (panel A) or the fold
induction (panel B) observed. Similar results were obtained using the
PRE2-tk-luciferase plasmid as the reporter plasmid (data
not shown).
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Effect of RFG Expression on the Responsiveness of hAR
Yeh and Chang (18) have reported that the expression of the RFG
protein in cells causes an alteration of the hormone responsiveness of
the AR in functional assays. In their experiments, incubations of cells
transfected with expression plasmids encoding RFG (ARA 70) and hAR
displayed responsiveness to estrogen, which was not displayed when the
RFG plasmid was omitted from the transfection mixture (22). To examine
this issue, we conducted such experiments in both the DU145 and CV1
cell lines. As shown in Fig. 7A
, while
small increments of reporter gene activation were observed after
treatment with estrogen when such experiments were conducted in CV1
cells, these increases were evident only when extremely high
concentrations of estradiol were employed. In addition, the stimulation
that occurred at these supraphysiological estradiol concentrations did
not require the expression of the RFG plasmid, and no such increases
were observed when the experiments were performed using lower, more
physiological concentrations of hormone. Diethylstilbestrol
(DES) was unable to stimulate this effect, even when used at high
concentrations (1 µM; data not shown). When such
experiments were repeated using the DU145 cell line (Fig. 7B
), estrogen
at low concentrations had a limited capacity to induce a model
androgen-responsive gene, and the small changes of expression were not
altered by the expression of the hAR or RFG expression plasmids. At
supraphysiological estrogen concentrations, an approximately 2-fold
increase of reporter gene activity was observed. This effect was not
seen when DES (1 µM) was employed or when lower, more
physiological concentrations of estradiol were employed.

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Figure 7. RFG Expression Does Not Alter the Ligand
Responsiveness of the hAR
A, CV1 cells were transfected with expression plasmids encoding
the hAR and the RFG, alone and in combination, and the MMTV luciferase
reporter plasmid. After transfection, the cells were incubated with no
hormone, DHT, or estradiol at the indicated concentrations. Reporter
gene activity was measured 48 h later. The data are plotted as
percent of fold induction (open bars) or percent of
stimulated reporter gene activity (stippled bars),
relative to the activation observed using saturating doses of DHT. In
this experiment, the basal and DHT-stimulated activities were 3142 and
162,011 relative light units (RLU) (52-fold induction). No
stimulation was observed when DES was used in place of estradiol (data
not shown). B, The results of similar experiments conducted in the
DU145 cell line using the MMTV luciferase reporter plasmid are shown.
In this experiment, the basal and DHT-stimulated activities were 137
and 929 RLU (7-fold induction). As in panel A, the data are plotted
relative to the reporter gene activity measured after stimulation with
saturating doses of DHT. No stimulation was observed when DES was used
in place of estradiol (data not shown).
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Detection of the RFG Protein
The interpretation of the experiments described above requires
that the RFG expression plasmid employed be capable of directing the
expression of the RFG protein in the transfected cells. To examine this
in the systems that we have employed, we performed immunoblots using an
antiserum raised to segments of the RFG protein to detect the
expression of the RFG protein in transfected cells. Figure 8
presents the results from one such
experiment. While no immunoreactivity is observed in untransfected
cells (data not shown), a single band is observed in extracts of cells
transfected with the RFG expression plasmid that migrates in
SDS-polyacrylamide gels with an apparent molecular mass of 70 kDa. The
detection of this protein can be blocked if the anti-RFG antibody is
preincubated with the RFG fusion protein.

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Figure 8. Immunoreactive RFG Is Detected in Cells Transfected
with the RFG Expression Plasmid
To examine whether the intact RFG protein could be detected in cells
transfected with the RFG expressions, COS cells were transfected with
either the RFG expression plasmid or the empty expression plasmid. Cell
extracts were prepared after which immunoblots were prepared
using antibodies raised against the RFG protein. The RFG protein is
detected in the extracts of the cells transfected with the RFG
expression plasmid and migrates with an apparent molecular mass of
about 70 kDa as shown in panel A. This species is efficiently competed
when the anti-RFG antiserum is preincubated with purified RFG protein
before use in immunoblotting experiments (panel B). This immunoreactive
band is also absent from extracts of COS cells that were not
transfected with the RFG expression plasmid (data not shown).
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DISCUSSION
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Our studies of AR containing a variety of amino acid substitutions
within the HBD demonstrated that the inability to form a stable
hormone-receptor complex was a common attribute of such mutant ARs.
This observation emphasized the need to identify the proteins that
interact with liganded androgen receptor to mediate its effects on the
activities of responsive genes (3).
In our attempts to approach this problem, we employed a yeast
two-hybrid screening method to identify proteins interacting with the
liganded hAR HBD. In this screen, we isolated a partial cDNA encoding
of protein previously identified by Santoro and co-workers (19) and
termed RFG by these investigators. A cDNA encoding a part of the same
protein was identified by Yeh and Chang (18) and termed ARA 70; it was
shown to interact with the hAR HBD in a ligand-dependent fashion when
expressed as a fusion protein in yeast. These latter investigators
reported that RFG (ARA70) was capable of acting as a
specific coactivator for AR function and capable of modifying the
hormonal responsiveness of the AR (22).
We have conducted experiments in yeast in vitro and in
mammalian cells to examine the capacity of this protein to interact
with the AR and to modulate its function. In yeast, our experiments
showed that the interaction of the AR HBD with the carboxy terminus of
RFG was maximal in the presence of agonist, and that this interaction
was greatly diminished when ligand was not bound to the AR HBD. This
interaction was not specific for the hAR, as the RFG fusion protein was
capable of interacting with the PR in a similar fashion in response to
progesterone in the yeast two-hybrid assay. By contrast, while a strong
interaction could be demonstrated between the LBD of the human PR and
the carboxy terminus of the SRC-1 coactivator, our studies indicated
that the ligand-dependent interaction of the LBD of the hAR with SRC-1
was substantially weaker.
To examine whether these findings were the result of a direct
interaction between the AR and RFG proteins, we performed GST pull-down
and immunoprecipitation experiments. Although GST pull-down experiments
suggested that an interaction was occurring between the full-length RFG
and the GST AR fusion protein, our interpretation of this result was
tempered by the results of our previous studies in which we
demonstrated that much of the AR fusion protein synthesized in this
fashion is not capable of binding ligand. To address this potential
shortcoming, we also performed immunoprecipitation experiments using
labeled full-length RFG and AR proteins derived from in
vitro translation results. The results of these experiments also
suggested that the RFG and AR proteins interact directly.
Despite these indications that a ligand-dependent interaction occurs
between these two proteins, our experiments testing the functional
significance of this interaction did not demonstrate an important role
of this protein in modulating gene activation by the hAR. In the
cotransfection assays reported here, RFG exhibited only subtle effects
on AR function, results that were similar whether performed in the DU
145 or CV1 cell lines. The results that we obtained were not dependent
on the promoter plasmids employed and yielded similar results using
both the MMTV and PRE2-tk luciferase reporter plasmids.
We were unable to detect an effect of RFG expression on the ligand
responsiveness of the hAR. In our experiments, only small increases of
luciferase activity could be observed in cells transfected with the hAR
and stimulated with estradiol. These increases were observed only at
high estradiol concentrations and did not require transfection with the
RFG expression plasmid. Such observations are reminiscent of the
activation of the AR that is effected by estradiol seen in cells that
do not oxidize steroids at the 17-hydroxyl group (23). Since estradiol
is capable of displacing androgen in binding assays when added at high
concentrations (24), such a finding would be most consistent with
activation occurring as the result of the binding of estradiol to the
AR in a nonphysiological fashion.
In summary, we have demonstrated that the interaction of the AR with
the RFG (ARA70) protein can be demonstrated in the presence of an AR
agonist ligand in a variety of systems. This interaction appears to be
direct, but is not specific for the AR, as a similar
ligand-dependent interaction can also be demonstrated for the PR. Most
importantly, although our studies confirm the predicted sequence
difference detected by Yeh et al. (22) and the
synthesis of the predicted protein product after transfection of the
cDNA into mammalian cells, we are unable to demonstrate an important
effect of this protein in modulating responsiveness of model
androgen-responsive genes. At present, we are unable to reconcile our
findings with those previously published on this subject.
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MATERIALS AND METHODS
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Yeast Two-Hybrid Screens
The Gal4 DNA-binding domain fusion plasmid encoding the AR HBD
was prepared by cloning a PCR-amplified fragment encoding amino acid
residues 623917 of hAR into the NdeI and BamHI
restriction endonuclease cleavage sites of the pAS vector (25).
Transformation of the pACT cDNA library into Y190 yeast cells (prepared
from human prostate RNA by Dr. S. Elledge) was performed using the
method of Rose et al. (26), except that 40 µg of plasmid
DNA and 4.0 mg of carrier DNA were added to 800 µl of LISORB (100
mM lithium acetate, 10 mM Tris-Cl, pH 7.5, 1
mM EDTA, 1.0 M sorbitol). This mixture was used
to transform 10- to 100-µl aliquots of competent cells. After
transformation, the cells were plated on YNB Trp (-) Leu (-) His (-)
plates containing 3-amino-1,2,4-triazole and mibolerone.
Expression of the AR Fusion Protein in Bacteria
The GST fusion protein encoding the AR HBD has been described
previously (27). After induction with isopropylthiogalactoside,
the bacteria were lysed and the soluble fraction was incubated with
[3H]mibolerone at 4 C for 20 h. After labeling, the
extract was passed over a glutathione-Sepharose column and washed
extensively. The bound fraction was eluted with reduced glutathione,
pooled, and dialyzed before use in the experiments examining the
interaction with labeled RFG.
Plasmids
The RFG expression plasmid was constructed by subcloning an RFG
cDNA (obtained from M. Santoro, Universita degli Studi di Napoli,
Naples, Italy) into the eukaryotic expression plasmid CMV5. In
vitro transcription/translation experiments employed the same cDNA
inserted into the Bluescript plasmid (pBSKS M13+). An expression
plasmid of full-length SRC was provided by B. OMalley (Baylor College
of Medicine, Houston, TX) and was inserted into the expression plasmid
CMV5. The plasmids GR HBD/pAS and PR HBD/pAS were constructed by
subcloning analogous fragments of GR (amino acid residues 483777)
(28) and PR (amino acid residues 639933) (29), which were generated
by PCR and cloned into the pAS vector (25).
GST Pull-Down Assay
RFG labeled with [35S]methionine was prepared
using the full-length RFG contained in an expression vector (inserted
in the expression vector pBSKS M13+) in a coupled in vitro
transcription/translation system (Promega Corp., Madison,
WI). Partially purified GST-hAR-HBD fusion protein (prebound to
mibolerone, see above), was incubated overnight with a 20-µl aliquot
of glutathione-Sepharose beads at 4 C. After the overnight incubation,
unbound fusion protein was washed away and the beads were incubated
with an aliquot of the in vitro transcribed translated
[35S]methionine-labeled RFG protein at 4 C. Beads were
washed four times with the binding buffer, resuspended in 50 µl of
2x SDS loading buffer, and analyzed by SDS-PAGE and
autoradiography.
In Vitro Transcription/Translation and
Immunoprecipitation Assays
The assay was performed as described previously (30) with
modification: in vitro-translated and
35S-radiolabeled protein was obtained using a transcription
and translation system (TNT-coupled reticulocyte lysate system,
Promega Corp.). Crude lysate (40 µl) diluted at
ratio 1:10 with modified CSK (mCSK) buffer (10 mM
1,4-piperazinediethane sulfonic acid, pH 6.8, 100 mM
NaCl, 300 mM sucrose, 1 mM MgCL2, 1
mM EGTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) containing 0.1
Triton X-100. Four 100-µl aliquots of mixed lysates were
incubated overnight with 20 µl beads conjugated with no hormone,
U402, U407, and R489 anti-AR antibodies, respectively, at 4 C (31).
After the overnight incubation, the lysate was centrifuged for 1 min at
10,000 x g, and the beads were washed three
times (1 ml mCSK buffer with 0.3 M NaCl). Both supernatant
and washed beads were solubilized in 2x SDS loading buffer and
analyzed by SDS-PAGE and autoradiography.
Transient Transfection Assay
The day before transfection, CV1 and DU145 cells were
plated into six-well plates at a density of 2 x 105
cells per well. Twenty hours later, each plate was transiently
transfected by the addition of a calcium phosphate precipitate
containing the indicated amount of expression plasmid DNAs, the
reporter plasmid MMTV or PRE2-tk luciferase (10 µg), and
a control plasmid, CMV-ß-galactosidase (1 µg), in culture
medium for 1418 h. Twenty hours later, the medium was replaced with
fresh medium containing 5% charcoal-stripped serum and either no
hormone or a hormone at the indicated concentrations. Cells were
harvested 48 h after hormone addition, and the levels of
luciferase and ß-galactosidase activity were measured. In
transfections in which the concentrations of plasmids (e.g.
CMV RFG or CMV hAR) were varied, the quantity of CMV5 vector was
adjusted so that the total amount of CMV5 expression plasmid remained
constant.
Sequence Analysis
Nucleotide sequence analysis was performed using a PE Applied Biosystems (Norwalk, CT) automated sequencer that
utilized the Sanger dideoxy method of DNA sequencing with a
fluorescent. The sequence of the RFG provided by Dr. Santoro was
determined on both strands over the complete cDNA fragment, as detailed
in the figures and text. The nucleotide sequence of the partial RFG
cDNA isolated in the pACT plasmid in the two-hybrid assay was also
sequenced on both strands.
Anti-RFG Antiserum
Antibodies directed at RFG were generated by immunizing rabbits
with fragments of the recombinant RFG expressed in bacteria (32). The
resulting antiserum was purified using an affinity column to which the
purified RFG peptides were covalently attached (K. Brantley, T. Gao,
and M. J. McPhaul, unpublished data).
Note Added in Proof
The studies of Alen et al. (33) also demonstrated
only minor effects of the RFG/ELE1/ARA70 protein on the transcriptional
activity of the human AR as assayed in cotransfection experiments.
 |
ACKNOWLEDGMENTS
|
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We wish to thank Judy Gruber and Diane Allman for superb
technical assistance.
 |
FOOTNOTES
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Address requests for reprints to: Dr. Michael McPhaul, Internal Medicine, Room J6.110, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8857.
This work was supported by NIH Grant DK-03892 and by Welch Foundation
Grant I-1090.
Portions of this work were reported in abstract form at the 75th Annual
Meeting of The Endocrine Society (Minneapolis, MN, 1997).
Received for publication September 22, 1998.
Revision received May 20, 1999.
Accepted for publication June 25, 1999.
 |
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