Interaction of the Putative Androgen Receptor-Specific Coactivator ARA70/ELE1
with Multiple Steroid Receptors and Identification of an Internally Deleted ELE1ß Isoform
Philippe Alen,
Frank Claessens,
Erik Schoenmakers,
Johannes V. Swinnen,
Guido Verhoeven,
Wilfried Rombauts and
Ben Peeters
Division of Biochemistry (P.A., F.C., E.S., W.R., B.P.) and
Laboratory for Experimental Medicine and Endocrinology (J.V.S.,
G.V.) Faculty of Medicine, Campus Gasthuisberg University of
Leuven B-3000 Leuven, Belgium
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ABSTRACT
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Steroid-regulated gene transcription requires the
coordinate physical and functional interaction of hormone receptors,
basal transcription factors, and transcriptional coactivators. In this
context ARA70, previously called RFG and ELE1,
has been described as a putative coactivator that specifically enhances
the activity of the androgen receptor (AR) but not that of the
glucocorticoid receptor (GR), the progesterone receptor, or the
estrogen receptor (ER). Here we describe the cloning of the cDNA for
ELE1/ARA70 by RT-PCR from RNA derived from
different cell lines (HeLa, DU-145, and LNCaP). In accordance with the
previously described sequence, we obtained a 1845-bp PCR product for
the HeLa and the LNCaP RNA. Starting from T-47D RNA, however, an 860-bp
PCR product was obtained. This shorter variant results from an internal
985-bp deletion and is called ELE1ß; accordingly, the longer isoform
is referred to as ELE1
. The deduced amino acid sequence of ELE1
,
but not that of ELE1ß, differs at specific positions from the one
previously published by others, suggesting that these two proteins are
encoded by different nonallelic genes. ELE1
is expressed in the
three prostate-derived cell lines examined (PC-3, DU-145, and LNCaP),
and this expression is not altered by androgen treatment. Of all rat
tissues examined, ELE1
expression is highest in the testis. This is
also the only tissue in which we could demonstrate ELE1ß expression.
Both ELE1
and ELE1ß interact in vitro with the AR, but
also with the GR and the ER, in a ligand-independent way.
Overexpression of either ELE1 isoform in DU-145, HeLa, or COS cells had
only minor effects on the transcriptional activity of the human AR.
ELE1
has no intrinsic transcription activation domain or histone
acetyltransferase activity, but it does interact with another
histone acetyltransferase, p/CAF, and the basal transcription factor
TFIIB. The interaction with the AR occurs through the ligand-binding
domain and involves the region corresponding to the predicted helix 3.
Mutation in this domain of leucine 712 to arginine greatly reduces the
affinity of the AR for ELE1
but has only moderate effects on its
transcriptional activity. Taken together, we have identified two
isoforms of the putative coactivator ARA70/ELE1
that may act as a bridging factor between steroid receptors and
components of the transcription initiation complex but which lack some
fundamental properties of a classic nuclear receptor coactivator.
Further experiments will be required to highlight the in
vivo role of ELE1 in nuclear receptor functioning.
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INTRODUCTION
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The androgen receptor (AR) is a member of the superfamily of
nuclear receptors (NR), which includes the receptors for the other
steroid hormones, for thyroid hormones, retinoids, and vitamin D
(1, 2, 3, 4). The NRs constitute a large group of evolutionarily related
proteins with a common structural organization, yet diverse
physiological functions. Apart from their role in embryonic
development, metabolic homeostasis, sex determination and
differentiation, and fertility, they are also implicated in a variety
of pathologies, including cancer.
NRs are ligand-inducible transcription factors with a modular
structure, encompassing an N-terminal transactivating domain, a
centrally located DNA-binding domain (DBD), and a C-terminal
ligand-binding domain (LBD). They have two transcription activation
functions, AF1 and AF2 (3, 4). AF1 is located in the N-terminal domain
and can activate transcription constitutively (5, 6), whereas the AF2
function colocalizes with the LBD and requires the addition of ligand
(7). The crystal structures of the LBDs of unliganded human retinoid X
receptor-
(hRXR
) (8), of ligand-bound human retinoic acid
receptor-
(hRAR
) (9), thyroid hormone receptor (TR) (10), and
progesterone receptor (PR) (11) and of agonist and antagonist-bound
estrogen receptor (ER) (12) revealed a common overall layered structure
consisting of 12
-helices. The most remarkable difference between
free and ligand-bound receptors is the position of the most
C-terminally located helix 12 (H12), which contains the core
AF2-domain. In the absence of ligand, H12 is projected away from the
hydrophobic ligand-binding pocket. Binding of ligand repositions this
helix, thereby closing the hydrophobic core as a lid (13).
Although direct interactions between NRs and components of the basal
transcription machinery have been described (14, 15, 16, 17), they cannot fully
explain the ligand-dependent transactivation of AF2. Therefore, another
class of proteins called coactivators must be taken into account (18, 19). A number of candidate NR coactivators have been described,
including RIP140 (20, 21), TIF1 (22), TRIP-1/SUG1 (23, 24), and a
family of related 160-kDa proteins comprising SRC-1 (25, 26, 27),
GRIP1/TIF2 (28, 29, 30), and RAC3/AIB1/ACTR/TRAM-1/p/CIP (31, 32, 33, 34, 35). While
the functions of RIP140, TIF1, and TRIP-1/SUG1 remain to be
established, the p160 proteins exhibit all the characteristics expected
for NR coactivators: 1) they interact with the LBD of NRs in a
ligand-dependent and AF2 integrity-dependent way both in
vivo and in vitro through a conserved
-helical
LXXLL-motif (27, 36, 37, 38, 39, 40); 2) upon cotransfection in mammalian cells
they potentiate the ligand-dependent transcriptional activity of
several NRs; 3) they harbor autonomous transcription activation
functions; and 4) they have intrinsic histone acetyltransferase (HAT)
activity and/or interact with yet other HATs such as CREB-binding
protein (CBP) and p300/CBP-associated factor (p/CAF). Thus, apart from
the recruitment of basal transcription factors to the promoter and the
formation of a stable preinitiation complex, steroid receptor
transactivation of target genes in vivo also involves
chromatin remodeling, probably through targeted histone acetylation by
the recruited coactivators (41).
Yeh and Chang (42) reported the first receptor-specific coactivator,
ARA70 (70 kDa AR-activator), which stimulates the
transcriptional activity of the AR but not that of the glucocorticoid
receptor (GR), the PR, or the ER. This protein had originally been
identified by others in thyroid cancer cells and named RFG (43) and
ELE1 (44, 45). In many cases of thyroid papillary carcinoma, the RET
protooncogene, a transmembrane receptor of the tyrosine kinase family,
is found activated, due to chromosomal rearrangements resulting in
recombinant genes. One such recombination, caused by a paracentric
inversion within band q11.2 of chromosome 10 (45), leads to the fusion
of the genomic region encoding the RET tyrosine kinase (TK) domain with
the 5'-terminal region of the ele1 gene (43, 44). The
resulting protein, called RET/PTC3, is expressed under the control of
the ele1 gene promoter and consists of the first 238 amino
acids (aa) of ELE1 fused to the RET TK-domain.
The goal of our study was to characterize the putative AR-specific
coactivator properties of ARA70/RFG/ELE1. Since our results
indicate that it lacks some of the fundamental properties of a
bona fide NR coactivator, we prefer the nomenclature ELE1 to
ARA70.
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RESULTS
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cDNA Cloning
To obtain the ELE1 cDNA, we performed RT-PCR on total RNA from
HeLa cells, LNCaP cells, and T-47D cells using primers based on the
sequence published by Yeh and Chang (42). For the HeLa and LNCaP RNA, a
cDNA of the expected 1845 bp was obtained, but surprisingly, the T-47D
RNA generates a 860-bp cDNA (Fig. 1A
).
The full-length cDNA will further be referred to as ELE1
;
accordingly, the shorter variant will be called ELE1ß. Sequence
analysis revealed that ELE1ß results from an internal 985-bp deletion
from nucleotides 790-1674 (the A of the ATG translation startcodon
was arbitrarily given the number +1). This deletion does not cause a
frame shift but results in a cDNA encoding a protein of approximately
35 kDa, and the 238 aa before the deletion correspond to the
ELE1-derived fragment in the RET/PTC3 fusion protein (43). Moreover,
Q238 is the last residue encoded by exon 5 of the ELE1 gene (46) (Fig. 1B
). Remarkably, the ELE1
cDNA clones derived from HeLa RNA, as well
as those from LNCaP RNA, contain five base substitutions (see Table 1
) as compared with the sequence
published by Yeh and Chang (42) and by Santoro and co-workers (43). We
have analyzed several ELE1
clones from different PCR reactions and
consistently found the same mutations, irrespective of the RNA or the
polymerase used (Taq or Pfu). Furthermore, in
some clones amplified with Taq polymerase but not in those
obtained with the Pfu polymerase, which has a lower error
rate, we found additional random mutations that only occurred in single
clones and are therefore likely due to misreading by the polymerase. In
the ELE1ß cDNA, however, we did not find the mutations listed in
Table 1
.
Expression of ELE1
Because of its putative role in androgen physiology and the
possible implications for the development of prostate cancer, we
examined the expression of ELE1 at the RNA level in three different
human prostate cancer-derived cell lines (DU-145, LNCaP, and PC-3),
grown in the absence or the presence of the synthetic androgen R1881.
As shown in Fig. 2A
, all cell lines
express ELE1 and only contain the ELE1
mRNA as can be concluded from
the size of the radioactive band. Of the three cell types tested,
ELE1
expression is highest in DU-145 cells, and the expression is
not substantially affected by androgen treatment.

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Figure 2. ELE1 Expression in Human Prostate Cancer-Derived
Cell Lines and Rat Tissues
Northern blot analysis of total RNA from three human prostate
cancer-derived cell lines, LNCaP, PC-3, and DU 145, grown in either the
absence or the presence of 1 nM R1881(panel A), and of
total RNA from different rat tissues (panel B). The blots were
hybridized with a radiolabeled probe encompassing the first 638 bp of
the ELE1 and the ELE1ß cDNA. RNA integrity and equal sample
loading were verified with a 18S probe. The positions of the 28S and
the 18S RNA bands are indicated as size markers. The
asterisk in panel B marks a band that possibly
corresponds to the ELE1ß mRNA.
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Since the main targets for androgens are the organs and tissues of the
male reproductive tract, we also analyzed the expression of ELE1 in rat
prostate and testis and made a comparison with other tissues. The blot
was probed with a radiolabeled cDNA fragment encompassing the first 638
bp of the ELE1 cDNA, which can hybridize to both the ELE1
and the
ELE1ß mRNAs. As shown in Fig. 2B
, ELE1
is expressed in all tissues
examined at comparable and moderate levels. The lowest expression is
found in the brain and the prostate, whereas the testis clearly
contains much higher levels of ELE1
-mRNA. Moreover, in the testis a
second and faster migrating mRNA band appears, possibly corresponding
to the shorter ELE1ß variant. We could not detect it in any of the
other tissues examined, even after prolonged exposure. It should be
noted that a human cDNA fragment was used to probe the rat multiple
tissue RNA blot that therefore had to be exposed approximately 10 times
longer to obtain a clear signal.
In Vitro Interaction of ELE1 with NRs
For the characterization of the interaction between ELE1 and NRs,
we performed in vitro glutathione S-transferase
(GST) pull-down experiments. To ensure that the in vitro
produced human AR (hAR) binds hormone correctly and that it undergoes
the associated conformational changes, we measured the binding affinity
of the AR for [3H]mibolerone and analyzed the tryptic
digest patterns of the ligand-free receptor and its complexes with
agonists and antagonists. We measured a Kd for mibolerone
of 0.46 nM, which is consistent with the values reported by
others (47) (Fig. 3A
, left
panel). Furthermore, in the absence of ligand the receptor is
completely digested by limited amounts of trypsin. Addition of the
agonists dihydrotestosterone (DHT) and R1881 or the antagonist
hydroxyflutamide (HO-F), however, results in the protection of small
but different cores against trypsinization, indicating that the
specific conformational changes that are associated to ligand binding
do occur (Fig. 3A
, right panel) (48).

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Figure 3. Analysis of the in Vitro Interaction
of ELE1 with NRs
A (left panel), In vitro produced hAR was
incubated with different concentrations of
[3H]mibolerone, ranging from 0.1 to 10 nM, in
the absence or the presence of a 100-fold excess of unlabeled hormone.
Free (F) and bound (B) mibolerone were separated with dextran-coated
charcoal. The Kd (0.46 nM) was calculated from
the slope of the Scatchard plot. A (right panel),
35S-labeled hAR was incubated without hormone, with 10
nM of DHT or R1881 or with 10
µM of hydroxyflutamide (HO-F) and subjected to a
limited proteolytic digest as described (47 ). The reaction products
were separated by SDS-PAGE and visualized by fluorography. B,
35S-labeled full-length hAR was produced by in
vitro coupled transcription-translation in the absence of
ligand or in the presence of 10 nM R1881 or 10
µM HO-F and incubated with GST, GST-ELE1 , or
GST-ELE1ß immobilized on Glutathione Sepharose resin. After stringent
washings, the resin-bound proteins were eluted with SDS-loading buffer,
analyzed by SDS-PAGE, and visualized by fluorography. The input lane
represents 10% of the reticulocyte lysate that was used in each
experiment. C, 35S-labeled hAR, mER, and rGR were produced
in vitro and tested for the interaction with GST,
GST-ELE1 , and GST-ELE1ß in the absence of ligand as described for
panel B and in Materials and Methods. D (upper
panel), Linear diagram of the hAR and the deletion mutants used
in this study. The numbers refer to amino acid
positions. The thin line represents the N-terminal
domain, the shaded box represents the DBD, and the
bold line represents the LBD. D, (lower
panel), The hAR and the deletion mutants depicted in the
upper panel were produced and radiolabeled by in
vitro transcription translation and subsequently analyzed for
interaction with GST or GST-ELE1 by the GST-pull down assay. Bound
proteins were eluted with SDS-loading buffer, separated by SDS-PAGE,
and visualized by fluorography.
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To analyze the ELE1-AR interaction, 35S-labeled full-length
hAR was added to a glutathione Sepharose matrix to which either GST
alone or a fusion protein of GST with ELE1
or ELE1ß was coupled.
The proteins were allowed to interact in the absence of ligand or in
the presence of an agonist (R1881) or an antagonist (HO-F). As shown in
Fig. 3B
, the AR interacts with both ELE1
and ELE1ß, although this
interaction is not ligand dependent and is not antagonized by HO-F. No
differences were observed when either R1881 or DHT were used to
activate the receptor (data not shown). Furthermore, the interaction is
not specific for the AR, at least in vitro, since the rat GR
(rGR) and the mouse ER (mER) also bind to ELE1
and ß with
affinities comparable to that of the hAR (Fig. 3C
).
To further characterize the region of the AR that is required for this
interaction, we performed GST pull-downs with full-length ELE1
and
C-terminally truncated receptors. In a first series of mutants, we
deleted the C-terminal half of the LBD and the complete LBD and found
that the region between aa 563 and 772 is sufficient for interaction
with ELE1
(data not shown). Next, we further
deleted the receptor to P722 (end of exon 4, mutant
1), P670 (mutant
4), and G626 (end of exon 3, mutant
5). As shown in Fig. 3D
, mutant
1 still interacts with ELE1
, whereas
4 does not. The
N-terminal domain is not required for the interaction, since deletion
of aa 1537 had no effect on the binding (data not shown). Based on
the alignment made by Wurtz et al. (49), the region between
P670 and P772 corresponds to helices 13 at the N terminus of the LBD.
We therefore generated mutants ending after helix 2 (mutant
2, aa
1696) and after helix 1 (mutant
3, aa 1683). These truncated
receptors do not interact with ELE1
(Fig. 3D
).
Thus, the domain that is sufficient for the in vitro
interaction of the hAR with ELE1
is located between E696 and P722
and corresponds to the predicted helix 3 (H3).
Cotransfection of the hAR with ELE1 and TIF2
DU-145 prostate cancer cells were cotransfected with the
mouse mammary tumor virus (MMTV)-luciferase reporter vector and
expression plasmids for the hAR and either ELE1
or ELE1ß, but we
did not observe strong coactivator properties (data not shown). Since
we have demonstrated that DU-145 cells express ELE1
, they offer no
obvious advantage over HeLa cells for use as a model system to analyze
the properties of ELE1. Therefore, these cells were also cotransfected
with the hAR and ELE1
or ELE1ß. As shown in Fig. 4
(upper panel), coexpression
of either ELE1 isoform results in a maximal 2-fold increase of AR
activity (compare lanes 2 and 3 to lane 1). As a control, we included
the p160 coactivator TIF2 (lane 4) which, under the same conditions,
stimulated the hAR 4-fold. Since TIF2 has been shown to function better
in COS cells than in HeLa cells (30), we performed the same experiments
in COS-7 cells. As shown in Fig. 4
(lower panel), also in
these cells coexpression of ELE1
or ELE1ß has only moderate
effects on the activity of the hAR (2- to 3-fold increase in receptor
activity), whereas coexpression of TIF2 results in an almost 20-fold
increase.

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Figure 4. Analysis of the Coactivator Activity of ELE1
HeLa cells (upper panel) or COS7 cells (lower
panel) were transfected with 250 ng of pCMV-ßGAL, 300 ng of
MMTV-luc reporter vector, 100 ng of pSG5-hAR, and 600 ng of either
empty pSG5, pSG5-ELE1 , pSG5-ELE1ß, or pSG5-TIF2. The cells were
treated for 24 h without (open bars) or with
(solid bars) 1 nM of R1881 before the
luciferase and ß-galactosidase activities were measured. The results
shown are the mean ± SEM of three measurements. The
luciferase activity measured in the absence of coactivator and in the
presence of R1881 was arbitrarily set to 100.
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In Vitro Interaction of ELE1 with p/CAF and TFIIB
Many transcriptional coactivators have intrinsic HAT activity or
act as a platform for the recruitment of other HATs and the basal
preinitiation complex. Using the liquid HAT assay, we could clearly
demonstrate HAT activity for bacterially produced p/CAF but not for a
GST-ELE1
fusion protein. Single hybrid experiments in HeLa cells
also did not reveal an intrinsic transcription activation function for
a GAL4-ELE1
fusion protein (data not shown).
We next tested the ability of ELE1
and ELE1ß to interact in
vitro with p/CAF, TFIIB, the cointegrator CBP, and the p160
coactivators SRC-1a, SRC-1e, and TIF2 (Fig. 5
). We observed in vitro
binding to p/CAF and to TFIIB. The in vivo relevance of
these interactions needs to be further analyzed. Coactivators of the
p160 family and CBP did not bind to the GST-ELE1
fusion protein,
whereas mutual interactions between the two first components were
detected in control experiments.

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Figure 5. ELE1 and ELE1ß Interact in
Vitro with TFIIB and with p/CAF
p/CAF, the general transcription factor TFIIB, CBP, and the p160
coactivators TIF2, SRC1a, and SRC1e were synthesized and radiolabeled
in vitro in rabbit reticulocyte lysate. They were
allowed to interact with purified GST, GST-ELE1 , or GST-ELE1ß (for
TFIIB and p/CAF), GST-CBP[20582163] (for TIF2, SRC1a, and SRC1e),
or GST-SRC1[781988] (for CBP) prebound to a Glutathione Sepharose
matrix, under the conditions described in Materials and
Methods. After extensive washings the bound proteins were
eluted, gel electrophoresed, and visualized by fluorography. The
arrows indicate the positions of the respective
35S-labeled proteins.
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Analysis of H3-Mutants of the hAR
We have shown that the region in the hAR required for the in
vitro interaction with ELE1
corresponds to the predicted H3 of
the LBD (Fig. 3D
). To identify single residues that are involved in
this interaction, we generated point mutations in this domain. Since
in vitro the interaction is not AR specific, we chose to
mutate the following moderately or highly conserved amino acids in the
C-terminal part of H3: leucine 712 to arginine (L712R), valines 715 and
716 to alanine (V715A/V716A), tryptophan 718 to alanine (W718A),
alanine 719 to lysine (A719K), and lysine 720 to alanine (K720A) (Fig. 6A
). The ability of these mutants to bind
hormone was addressed in a single point ligand-binding assay (1
nM mibolerone) of transiently transfected COS 7 cells (Fig. 6B
). The mutants V715A/V716A and K720A can bind hormone equally well as
the wild-type receptor, whereas L712R has a lower affinity. W718A and
A719K, on the other hand, retained less than 10% of the amount of
ligand bound by the wild-type receptor. A Western blot with extracts
from transfected COS cells was performed to ensure adequate expression
of all receptor mutants.

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Figure 6. In Vitro and in Vivo
Analysis of hAR H3 Mutants
A, Sequence alignment of the LBD region of SRs corresponding to the
predicted H3, according to Wurtz et al. (49 ). The
numbers refer to amino acid positions. The mutations are
indicated by asterisks and given below
the sequence of the hAR. B, COS 7 cells were transiently transfected
with an expression vector for either the wild-type AR or for one of the
mutants described in panel A. For the single-point ligand-binding
assay, the cells were incubated with 1 nM
3H-labeled mibolerone with or without 100 nM
cold mibolerone. After a 2-h incubation at 37 C, they were washed and
the radioactivity that was specifically retained within the cells
was measured. The shown values are the mean of five
measurements ± SEM. The activity measured for the wt
AR was arbitrarily set to 100. The lower panel shows a
Western blot of extracts of COS 7 cells, probed with an anti-AR
antibody, to control the expression of the respective mutants. C,
In vitro interaction assays of GST or GST-ELE1 and
35S-labeled wt or mutated hAR were performed as in Fig. 3B
and as described in Materials and Methods. The mutations
are described in panel A. D, HeLa cells were transiently transfected
with the MMTV-luc (left panel) or the
(GRE)2-oct-luc (right panel)
reporter vectors (1 µg) together with expression vectors for either
the wt hAR or one of the H3 mutants (100 ng) and pCMV-ßGAL (250 ng)
as internal control. The cells were treated with (solid
bars) or without (open bars) 10-9
M R1881 for 24 h before harvesting and measuring the
luciferase and ß-galactosidase activities. The activity of the wt hAR
in the presence of hormone was taken as 100, and the relative values of
test measurements were calculated. At least three independent duplicate
experiments were performed, and the results are shown as the mean
value ± SEM. E, HeLa cells were transfected with
MMTV-luc and expression vectors for the wild-type AR
(triangles) or the L712R mutant (squares)
as in Fig. 6D and treated for 24 h without hormone or with 0.001,
0.01, 0.1 or 1 nM of R1881 before the
ß-galactosidase and the luciferase activity were measured. The
activity of the wt hAR in the presence of 1 nM R1881 was
taken as 100.
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The ability of these mutants to interact with ELE1
was tested in a
GST pull-down assay. As shown in Fig. 6C
, the mutant L712R has a
reduced affinity for ELE1
compared with the wild-type receptor,
since less protein is retained on the GST-ELE1
beads. The mutants
V715A/V716A, W718A, and A719K have affinities comparable to that of the
unmodified receptor, whereas that of K720A is slightly higher. We
examined the transcription-activating properties of these mutant
receptors in transient transfection experiments. HeLa cells were
cotransfected with MMTV-luc or (GRE)2-oct-luc
reporter constructs and expression vectors for either the wild-type
receptor or one of the mutants. As shown in Fig. 6D
, all mutants retain
some ability to activate the transcription of the MMTV-driven reporter
in response to 1 nM R1881. Comparable results are obtained
with the (GRE)2-oct-luc reporter. Mutation of
leucine 712 to arginine caused only a slight (
35%) decrease in the
ligand-dependent transcriptional activity of the AR, despite the clear
effects of this mutation on the AR-ELE1
interaction. Surprisingly,
mutation of the highly conserved lysine at position 720 to alanine,
which severely impaired the transcription activation function of the
mER (50), had no effect on the hAR. The double mutant V715A/V716A
showed a slightly higher activity on the
(GRE)2-oct-luc reporter, but a slightly reduced
activity on the MMTV-luc reporter.
Next, we compared the dose-response curve for the wild-type receptor
and the L712R mutant in HeLa cells, using MMTV-luc as reporter and
increasing concentrations of R1881 (0.001 to 1 nM). As
shown in Fig. 6E
, the response of the L712R mutant to a wide range of
R1881 concentrations is similar to that of the unmutated receptor,
except that the maximal response to androgens is allways
35%
lower, which is consistent with its reduced affinity for the
ligand.
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DISCUSSION
|
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The goal of our study was to characterize the interaction of ELE1
with the hAR and to evaluate its role as an AR-specific transcriptional
coactivator. Therefore we cloned the ELE1 cDNA by RT-PCR. In addition
to the expected full-length cDNA (ELE1
), we obtained an internally
deleted variant (ELE1ß). This deletion is likely caused by the
removal of one or more exons, since the last residue before the
deletion (Q238) is the last amino acid encoded by exon 5 of the
ele1 gene (46). Moreover, the NH2-terminal
region of 238 aa from ELE1 (
and ß) corresponds to the
ELE1-derived fragment in the RET/PTC 3 fusion-protein (43).
Specific base changes in the ELE1
but not in the ELE1ß cDNA
indicate, furthermore, that both proteins are encoded by two nonallelic
genes with different expression patterns and RNA processing
characteristics.
In view of its proposed role as an AR-specific coactivator, we analyzed
the expression of ELE1 RNA in different rat tissues and in three human
prostate cancer-derived cell lines. All cells, including DU-145 cells,
were shown to express ELE1
. A possible explanation for the lack of
ELE1
expression in these cells reported by Yeh and Chang (42) could
be that these authors used a high-passage cell line (personal
communication) with altered characteristics compared with our cells, or
that they used a different DU-145 subclone. The high level of ELE1
expression in the rat testis could be indicative of its role in AR
functioning. The prostate, however, another important androgen target
tissue, contains much less ELE1
.
We tested the coactivator properties of ELE1
and ELE1ß in
cotransfection experiments, and for both isoforms we found a very weak
(
2-fold) increase in AR transcripional activity, irrespective of the
cell line used (DU-145, HeLa, or COS7). The very poor effects of
cotransfection of the AR with ELE1
or ELE1ß could be due to
sufficient levels of endogenous protein in all cells. It should be
noted, however, that under the same conditions the p160 coactivator
TIF2 effeciently enhances the AR activity, most notably in COS cells,
despite the fact that the p160 family members are also ubiquitously
expressed.
The interaction between the hAR and ELE1 was reported to be ligand
dependent in yeast and specific for the AR (42). In vitro,
we observed a strong interaction of both ELE1
and ELE1ß with the
hAR, although this interaction does not require the addition of ligand.
The affinity of the AR is comparable for both ELE1 isoforms, indicating
that the 328 amino acid region deleted in ELE1ß is not involved in
this interaction. This apparent discrepancy between the in
vitro ligand-independent interaction described here and the ligand
dependency observed in yeast (42, 51) might be explained by the fact
that in yeast cells, additional proteins may interact with the AR-LBD
in the absence of ligand, thereby preventing interaction with ELE1.
Addition of hormone could then release this block and allow the LBD to
bind to ELE1. Apart from the hAR, other steroid receptors (mER and rGR)
also interact in vitro with ELE1. Thus, the interaction is
not receptor specific. Furthermore, Treuter et al. (51)
recently reported the cloning of the ELE1 cDNA by a yeast double-hybrid
screen using the LBD of the peroxisome proliferator-activated
receptor as bait. Thus, ELE1 also interacts with NRs that do not
belong to the subclass of the SRs. This is not exceptional, however,
since none of the coactivators that have been described so far display
receptor specificity. The ER, as well as the PR, TR, RAR, and RXR,
interacts with all of the p160 family members (25, 28, 29, 30, 31, 33, 35).
Likewise, Trip1 interacts with both the TR and RXR (28), and TIF1
interacts with RXR
, RAR
, vitamin D receptor, PR, and ER (23, 24).
We mapped the domain of the AR that is required for the interaction
with ELE1
to the region between aa 696 and 722, which corresponds to
the predicted H3 of the LBD (49). This region has been shown to be
important for transcription activation by the ER (50). Moreover,
crystallographic studies on the LBDs of the ER, TR, PR, RXR, and RAR
have indicated that binding of the appropriate ligand repositions helix
12, comprising the core AF2, which subsequently makes direct contacts
with amino acids in H3 and H4, thereby providing a new surface for
interaction with coactivators (13). Feng et al. (52)
systematically mutated specific residues in the LBD of the TR and found
that the AF2 is formed by residues from the helices 3, 5, 6, and 12.
The AF2 core is dispensable for the hAR-ELE1
interaction, which
again demonstrates that the interaction between the AR and ELE1 is
clearly different from the interaction between the p160 coactivators
and NRs. Furthermore, both ELE1
and ELE1ß contain a LXXLL motif
(LYSLL) between aa 92 and 96. It has been shown that the integrity of
the AF2 core domain is essential for the interaction of NRs with this
motif in p160 coactivators (37). Since a truncated receptor lacking H12
can still interact with ELE1
, it is very unlikely that this binding
occurs through the LYSLL motif.
ELE1 has no intrinsic transactivating properties and does not interact
with CBP, but it can bind to TFIIB and to p/CAF. Although the in
vivo relevance of these protein-protein interactions needs to be
examined in more detail, it is tempting to speculate that ELE1 might
function as a linker protein between the NR and the basal transcription
machinery, thereby recruiting or stabilizing the PIC on the promoter.
Furthermore, through the interaction with p/CAF it might contribute to
the recruitment of HATs and the subsequent chromatin remodeling. Such
an activity, however, would not become apparent under the conditions of
transient transfection as used in this work.
We tried to identify single residues that are critical for the
interaction of ELE1 with the hAR. Sequence comparison of the H3 regions
of steroid receptors reveals that this domain is best conserved at the
C terminus of the helix. Mutation of a series of moderately or highly
conserved residues in H3 revealed that the leucine at position 712 is
essential for ELE1 binding. In the hGR the corresponding residue is a
valine, and in the human mineralocorticoid receptor it is a
methionine. All these residues have hydrophobic side chains, indicating
that the binding between ELE1
and the AR could occur through
hydrophobic interactions. Despite the dramatic effects of this mutation
on the ELE1-hAR interaction, the mutated receptor is still
transcriptionally active on both the MMTV-luc and the
(GRE)2-oct-luc reporters, although the maximal
activity is
35% less than that of the wild-type receptor. This
correlates well with the reduced ligand-binding affinity observed in
COS 7 cells. Thus, the slight decrease in transcriptional activity most
probably reflects a decrease in ligand binding, rather than resulting
from an impaired interaction with ELE1. A striking observation in the
analysis of the H3 mutants is the fact that mutation of the highly
conserved lysine residue at position 720 to an alanine (K720A), which
severely impairs the function of the ER (50), had no effect on the
transcriptional activity of the AR. This indicates that the mechanism
of activation by the AR may be different from the ER. Similar
conclusions can de drawn from other observations: whereas the ER-LBD
harbors an autonomous transcription activation function (AF2) when
fused to a heterologous GAL4-DBD (7, 50), the AR-LBD requires the
NH2-terminal AF1 function to gain ligand-dependent
transcription activation properties (53).
In conclusion, our results reveal the existence of two nonallelic
variants of ELE1. They confirm that ELE1 interacts with the LBD of the
hAR but also demonstrate that, at least in vitro, this
interaction is not ligand dependent and that ELE1 can also interact
with other members of the NR superfamily. Although we demonstrate that
ELE1 may act as a bridging factor between steroid receptors and the
basal transcription machinery or chromatin-remodeling enzymes, it does
not act as a classic coactivator in mammalian cells in our experimental
conditions. In fact, mutation of specific residues in the LBD of the
hAR that severely impair the AR-ELE1 interaction have only moderate
effects on the AR transcriptional activity. Thus, the in
vivo function of ELE1 should be further investigated.
 |
MATERIALS AND METHODS
|
---|
Materials
All restriction and modifying enzymes were obtained from either
Pharmacia (Uppsala, Sweden), Life Technologies (Grand Island, NY), or
Boehringer (Mannheim, Germany). Cell culture reagents were obtained
from Life Technologies. The TNT rabbit reticulocyte lysate in
vitro coupled transcription translation kit was purchased from
Promega (Madison, WI). L-[35S]methionine
(>1000 Ci/mmol) and [3H]mibolerone (82.3 Ci/mmol) were
purchased from Amersham (Buckinghamshire, UK). The hormones
5
-dihydrotestosterone, R1881 (methyltrienolone), and mibolerone were
from Dupont-New England Nuclear (Boston, MA). Hydroxyflutamide was a
kind gift from Schering (Kenilworth, NJ).
Plasmids
For the construction of pSG5-hAR, the hAR cDNA was PCR amplified
from pSV-ARO (54) with Expand High Fidelity polymerase (Boehringer) in
the presence of 4% dimethylsulfoxide and using the primers
5'-GGTGGATCCATGGAAGTGCAG-TTAGGGCTGGGAAGGTCTAC-3' and
5'-TACGTGGATCCTCACTGGGTGTGGAAATAGATGGGCTTGACT, and the re-sulting
PCR product was cloned in the BamHI restriction site of
pSG5. Deletion mutants were made by PCR using the Pfu
thermostable polymerase (Stratagene, La Jolla, CA) and pSG5-hAR as
template and the following primers: 5'-TAGGATCCATGTTGGAGACTGCCAGGGAC-3'
(forward primer; FP), 5'-TAAGATCTGGATCCTCAAGGCAAGGCCTTGGCCC-3'
(reverse primer for
1), 5'-TAAGATCTGGATCCTCAAAAGGAGTCGGGCTGGTT-3'
(reverse primer for
2),
5'-TAA-GATCTGGATCCTCATACACCTGGCTCAATGGC-3' (reverse primer for
3), 5'-TAAGATCTGGATCCTCAGGGCTGACA-TTCATAGCC-3' (reverse primer for
4), 5'-TAAGATCTGGATCCTCATCCCAGAGTCATCCCTGC-3' (reverse primer for
5).The resulting PCR products were digested with
HindIII and BglII and exchanged for the
corresponding fragment in pSG5-hAR.
Site-directed mutagenesis was performed using a standard PCR-based
method. The plasmid pCR(538918;wt) was constructed by inserting a
fragment encoding the entire DBD and LBD, PCR amplified from pSG5-hAR
with the primers FP and 5'-GCTGCAATAAACAAGTTCTGC-3' (reverse primer;
RP), and subsequently cloned in the pCR-SCRIPT vector (Stratagene).
Next, two sets of PCR reactions were performed. In the first set, with
pSG5-hAR as template, oligonucleotide FP was used as forward primer and
the following oligonucleotides as reverse primers:
5'-CACGTGTACACG-CTGTCTCTC-3' (PCR a),
5'-CTTGTACACGCCGCCAAGTGGGCCAAG-3' (PCR c),
5'-GTGGTCAAGGCGGCCAAGGCC-3' (PCR e),
5'-GTCAAGTGGAAGAAGGCCTTG-3' (PCR g), and
5'-CAAGTGGGCCGCGGCCTTGCC-3' (PCR i). Similarly, the
oligonucleotide RP was used as reverse primer and the following
oligonucleotides as forward primers: CACGTGTACACGCTGTCTCTC-3' (PCR
b), 5'-CTTGGCCCACTTGGCGGCGTGTACAAG-3' (PCR d),
5'-GGCCTTGGCCGCCTTGACCAC-3' (PCR f),
5'-CAAGGCCTTCTTCCACTTGAC-3' (PCR h), and
5'-GGCAACGCCGCGGCCCACTTG-3' (PCR j). The templates for
the second set of reactions, using the oligonucleotides FP and RP as
primers, were a combination of the reaction products from PCRs
a and b, c and d,
e and f, g and h, and
finally i and j. The PCR products from this
second set of reactions were digested with HindIII and
BglII and exchanged for the corresponding fragment in
pCR-(538918;wt), giving rise to pCR-(538918;ab),
pCR-(538918;cd), pCR-(538918;ef),
pCR-(538918;gh), and pCR-(538918;ij).
Finally, Tth111I-NcoI fragments from these
constructs were exchanged for the corresponding fragment in pSG5-hAR,
resulting in AR expression plasmids carrying the following mutations:
L712R, V715A/V716A, W718A, A719K, and K720A. For the construction of
pSG5-TFIIB, the TFIIB cDNA was isolated from pGEX-TFIIB (a gift from
Dr. B. OMalley) and subcloned in pSG5. Expression vectors for TIF2
and SRC-1 were kindly provided by Dr. H. Gronemeyer and Dr. M. G.
Parker. The reporter gene construct
(GRE)2-oct-luc, which contains two GREs in front
of the minimal TATA box derived from the oct6 gene promoter
and the luciferase gene, was the kind gift of Dr. A. O.
Brinkmann.
Molecular Cloning of the ELE1 cDNA
The ELE1 cDNA was cloned by RT-PCR from RNA derived from HeLa
cells, LNCaP cells, and T-47D cells. Briefly, 3 µg of total RNA were
reverse transcribed with the primer
5'-CTAGGAATTCTCACATCTGTAGAGGAGTTC-3' (30 min at 42 C), which generates
an EcoRI restriction site immediately downstream of the ELE1
stop codon. The remaining RNA was digested with RNase H for 10 min at
55 C, and the first-strand cDNA was PCR amplified using oligonucleotide
5'-GATCGAATTCATATGAATACCTTCCAAGAC-3' as forward primer and the above
mentioned oligonucleotide as reverse primer. PCR was performed with
either Taq or Pfu (Stratagene) thermostable
polymerases. The reaction products were gel purified and subsequently
cloned in the pGEM-T (Promega) or pCR-SCRIPT (Stratagene) vectors,
respectively, and analyzed by sequencing. For expression as a
GST-fusion protein, the cDNA was cloned in the EcoRI site of
the pGEX-2TK vector (Pharmacia); for expression in mammalian cells or
in vitro transcription translation, it was cloned in the
EcoRI site of pSG5.
Northern Blotting
RNA from LNCaP, PC-3, and DU-145 cells, grown in either
the absence or the presence of 1 nM R1881, was prepared,
electrophoresed, and blotted as described (55, 56). The probe for
Northern hybridization was prepared by PCR with the primers
5'-GATCGAATTCATATGAATACCTTCCAAGACC-3' and
5'-C-CACTGGCAGGTTTGCTTCC-3', which generates a fragment comprising
the first 638 bp of the ELE1 cDNA. One microliter of this reaction was
used in a 100-µl labeling reaction as previously described (55). The
18S RNA probe was labeled by random priming. The membrane was
prehybridized for 2 h at 42 C in 5 x SSC (20 x SSC: 3
M NaCl and 3 M sodium citrate), 2 x
Denhardts (100 x Denhardts: 2% Ficoll 400, 2%
polyvinylpyrolidone) containing 50% (vol/vol) formamide, 0.1% SDS,
and 100 µg/ml sonicated salmon sperm DNA and subsequently hybridized
with probe (106 cpm/ml) for 18 h at 42 C in the same
buffer. The blot was washed once at room temperature with 2 x SSC
containing 0.5% SDS and twice for 20 min at 65 C with 0.2 x SSC
containing 0.1% SDS and finally autoradiographed at -80 C using
intensifying screens.
In Vitro Ligand Binding and Limited Trypsinization
Assay
The in vitro ligand-binding and limited
trypsinization assays were performed as described previously (47, 48).
GST Pull-Down Assay
Expression and purification of GST or the GST fusion proteins
using the pGEX-system (Pharmacia) and coupled in vitro
transcription-translation with the TNT system (Promega) were performed
according to the manufacturers instructions. 35S-labeled
proteins were added to the glutathione Sepharose containing either GST
or the GST fusion protein in NENT/B [NENT buffer (100 mM
NaCl, 1 mM EDTA, 0.02% NP-40, 20 mM Tris, pH
8) containing 1 mg/ml BSA] in a total volume of 200 µl and incubated
for 30 min at room temperature and for 30 min at 4 C. The glutathione
Sepharose was washed five times with 1 ml of NENT/B. Bound proteins
were eluted by boiling the resin in 30 µl of 2 x SDS loading
buffer (20% glycerol, 2% SDS, 0.0025% bromophenol blue, 10%
ß-mercaptoethanol), separated by SDS-PAGE and visualized by
fluorography. For the interactions of ELE1 with TFIIB, SRC-1, TIF2,
p/CAF, and CBP, NENT buffer without BSA but containing 0.1% NP-40 was
used.
Cell Culture
HeLa, T-47D, LNCaP, DU-145, and COS 7 cells were obtained from
the Amercian Tissue Type Collection (ATTC, Manassas, VA) and were
routinely maintained in DMEM (Life Technologies) containing 1 g/liter
glucose and supplemented with antibiotics (penicillin, streptomycin;
Life Technologies) and 10% heat-inactivated FBS. For DU-145 cells,
L-glutamine was added to a concentration of 2
mM.
Transfections
Cells were transfected using the standard calcium phosphate
coprecipitation procedure (57). Twenty four hours before transfection,
HeLa cells, COS 7 cells, or DU-145 cells were trypsinized and seeded in
24-well culture plates at a density of 7.5 x 104
cells per well in medium containing 5% dextran-coated
charcoal-stripped FBS (DCC-FBS). For cotransfection studies, the
following amounts of plasmids were used (per well): 300 ng MMTV-luc,
100 ng pSG5-hAR, and 600 ng of either empty pSG5, pSG5-ELE1
,
pSG5-ELE1ß, or pSG5-TIF2. For the analysis of the different receptor
mutants, HeLa cells were transfected with 1 µg of reporter vector
(MMTV-luc or (GRE)2-oct-luc) and 100 ng of
expression vector for the wild-type or mutated AR per well.
p-CMV-ß-GAL (250 ng) was always included as internal control. The
cells were incubated for 24 h in the absence or the presence of 1
nM R1881 before the luciferase, ß-galactosidase, and
protein concentrations were determined.
For the single-point ligand-binding assay and for Western blotting, COS
7 cells were transfected with pSG5-hAR or expression vectors for the
respective receptor mutants. Forty eight hours after transfection, the
medium was replaced with DCC-FBS containing 1 nM
[3H]mibolerone, either with or without a 100-fold excess
of cold mibolerone. After 2 h incubation at 37 C, the cells were
placed on melting ice, washed twice with ice-cold PBS, and lysed in 1%
Triton X-100, 0.1 N NaOH, and their radioactivity was counted. For
Western blot analysis, the cells were scraped in 1 ml PBS and pelleted
by centrifugation. The pellet was dissolved in 50 µl of high-salt
extraction buffer (20 mM Tris, pH 7.8, 420 mM
NaCl, 1 mM EDTA), and the cells were lysed by two cycles of
freezing in liquid nitrogen and thawing on ice. Insoluble material was
pelleted by centrifugation, and 10 µl of the soluble extract were
separated by SDS-PAGE in a 9% gel and electroblotted onto a
polyvinylidene fluoride membrane. The blot was probed with a
polyclonal antiserum against the N terminus of the hAR, and the
proteins were visualized with the enhanced chemiluminescence system
(Amersham).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. A. O. Brinkmann (Erasmus University,
Rotterdam, The Netherlands), Dr. B. W. OMalley (Baylor
University, Houston, TX), Dr. M. G. Parker (Imperial Cancer
Research Fund, London, UK) and Dr. H. Gronemeyer (Institut de Genetique
et de Biologie Moleculaire et Cellulaire, Illkirch-Cedex,
France) for the kind gift of plasmids; Dr. Neri (Schering Plough,
Kenilworth, NJ) for the gift of hydroxyflutamide; R. Bollen and H. De
Bruyn for excellent technical assistance; and V. Feytons for the expert
synthesis of many oligo-nucleotides.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. B. Peeters, Department of Biochemistry, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. e-mail: ben.peeters{at}med.kuleuven.ac.be
This work was supported in part by a grant Geconcerteerde
Onderzoeksactie van de Vlaamse Gemeenschap, by grants from the
Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek and by a
grant Interuniversity Poles of Attraction Programme, Belgian State,
Prime Ministers Office, Federal Office for Scientific, Technical and
Cultural Affairs. P.A. was holder of a scholarship Vlaams Instituut
voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in
de Industrie. F.C. and J.V.S. are Senior Research Assistants of the
Fund for Scientific Research Flanders (Belgium).
Received for publication June 4, 1998.
Revision received August 31, 1998.
Accepted for publication September 21, 1998.
 |
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