Biotechnology Centre of Oslo (T.S., I.K., T.B., F.S.), and
Department of Biochemistry (T.S.), Department of Biology (I.K., T.B.),
and Institute for Clinical Medicine (F.S.) University of Oslo
0349 Oslo, Norway
Department of Physiology (J.P.)
Institute of Biomedicine University of Helsinki FIN-00014
Helsinki, Finland
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ABSTRACT |
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INTRODUCTION |
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The precise sequences and mechanisms that contribute to the AF-1 activity have not been conclusively established. One reason for this is that the N terminus of nuclear receptors is very divergent, both in sequence and length (3, 4, 5). It is also possible that some nuclear receptors do not possess AF-1 function due to their relatively short N terminus. In AR, AF-1 maps to two distinct regions and displays promoter and cell line specificity (8, 10, 11).
On the other hand, a short region that mediates AF-2 activity in
the C terminus of nuclear receptors is highly conserved and has been
studied in detail through mutational analyses (12, 13, 14, 15, 16, 17, 18). Recently,
determination of the three-dimensional structures of the various apo-
and holo-LBDs has greatly facilitated the study of AF-2
structure-function relationship (for a review, see Ref. 19). According
to the crystal structures, the nuclear receptor LBDs display a common
fold with 12 -helices (H1H12) and one ß-turn, together arranged
as a three-layer
- helical sandwich (19). The core AF-2 is
formed by H12 that is localized close to, but at variable distances
from, the C terminus of the LBD in different nuclear receptors (for a
review, see Ref. 19 and references therein).
The H12 is of varying length and flanked by different sequences in the nuclear receptors studied so far. Comparison of this structure in agonist- vs. antagonist-bound receptors, or an apo-receptor with a holo-receptor of different type, suggests that upon ligand binding and activation, H12 is substantially reorganized, contributing to the formation of a coactivator binding surface (19, 20, 21, 22, 23). The loss of transcriptional activation upon mutagenesis of H12 was interpreted as a loss of interaction with coactivators, which is supported by biochemical data (18, 20, 24, 25, 26, 27). These findings were recently confirmed by cocrystallization of liganded nuclear receptors and nuclear receptor interaction domains of coactivators (21, 22, 23).
The activity of nuclear receptors is modulated by interactions with other proteins. These could be mediated through heterodimeric interactions within the nuclear receptor superfamily, such as those between retinoid X receptors (RXRs) and thyroid hormone, retinoic acid, and vitamin D receptors (T3Rs, RARs, and VDRs) in which the heterodimer has an increased ability to activate transcription (for a review, see Ref. 28). On the other hand, AP-1 complexes, composed of either Jun homodimers or Jun-Fos heterodimers (for a review, see Ref. 29), interfere with ligand-dependent transactivation by some nuclear receptors including AR (for a review, see Ref. 30). Reciprocally, liganded nuclear receptors, as first described for the glucocorticoid receptor (GR), interfere with AP-1 activity (for a review, see Ref. 30). The molecular mechanisms of this cross-talk have not been definitively established but may involve competition for a common cofactor, such as CREB-binding protein (CBP) (30, 31, 32, 33). Nuclear receptors can also cross-talk with other transcription factors, but these have not been studied in as much detail (e.g. Refs. 34, 35).
Most recently, proteins that act as putative coactivators or corepressors and which physically interact with nuclear receptors have been identified (for reviews, see Refs. 36, 37, 38). Most of these cofactors are expressed ubiquitously and can interact with more than one type of nuclear receptor. Furthermore, it appears that multiple cofactors may regulate nuclear receptor function at any one time. Therefore, the exact contribution of these cofactors to the activities of different receptors in vivo is still not well understood.
Given the variations in the context, sequence, and length of the
AF-2 core domains in various nuclear receptors and to gain a deeper
insight into its functioning, we carried out a detailed mutational
analysis of the AF-2 core in AR. This was based on information provided
by the crystal structures of other nuclear receptors (19) and previous
mutational studies on the AF-2 of T3R, T3Rß, ER, and GR (12, 13, 14, 15, 16, 18). We aimed to target surface exposed residues that are likely to
form recognition surfaces for AR regulatory proteins. Through this
analysis, we identified various single amino acid substitutions that
significantly compromised the transactivation potential of AR. There
were both similarities and distinct differences regarding the
importance of specific residues and the mechanisms involved in their
loss of function when compared with identical or similar mutations of
these conserved residues in other nuclear receptors, as well as a
recent mutational analysis of AR H12 (50). In addition, none of the
mutations that blocked transcriptional activation affected interference
with AP-1 activity, indicating that transactivation and transrepression
are mediated by different interaction surfaces in AR. These results
indicate the presence of receptor-specific differences in the function
of the AF-2 core that may contribute to differential activities of
nuclear receptors.
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RESULTS |
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Since all known activities of AR are dependent on activation by
androgens, we then tested the mutant proteins for proper ligand binding
properties. COS-1 cells were transfected with expression vectors
specifying the production of the wild-type or the mutant receptors, and
the cells were incubated with increasing amounts of
[3H]R1881. After the excess hormone was washed
away, specifically bound [3H]R1881 was
measured, and dissociation constants (Kd) were
calculated. As shown in Fig. 1A, M2-M6 had Kd
values that were comparable to those of wild-type AR, whereas M1 was
significantly compromised in its Kd, being 6% of
that of wild-type AR. M1 is anticipated to be saturated with hormone in
the experiments that are described below since approximately 12-fold
excess of hormone over the Kd was used in these
experiments. Since mutagenesis of the corresponding residue in other
nuclear receptors did not appreciably affect ligand binding
[e.g. in T3R
, T3Rß, and estrogen receptor (ER) (12, 16, 18)], these results indicate that the contribution of specific
residues in the conserved C-terminal region of AR to ligand binding may
be different than the corresponding residues in other nuclear
receptors.
As the transcriptional activity of AR depends on its ability to bind
DNA, we next performed mobility shift assays to compare wild-type AR
with the mutants for their DNA binding activities. Proteins were
expressed by in vitro transcription/translation and then
used in the mobility shift assay with the androgen response element
(ARE) in the first intron of the rat C3 gene as probe (46). As shown in
Fig. 1C, unprogrammed lysate resulted in a nonspecific band, whereas
lysates containing wild-type AR resulted in a slower migrating band
(depicted by an arrow). This slower migrating band is
specific for AR as shown by a supershift experiment with an AR-specific
antiserum and by competition studies using an excess of unlabeled ARE
(T. Slagsvold, unpublished data, and Ref. 46). All the mutants
displayed similar DNA binding activity compared with wild-type AR,
except M1, which was severely impaired in its ability to bind to the
ARE. Competition studies indicated that M2-M6 had similar DNA binding
affinity, except for M3, for which binding affinity was lower
than the other mutants (data not shown). In addition, M1 was defective
in ligand-induced change in mobility compared with wild-type AR and the
other mutants in the mobility shift assay (data not shown). These
results indicate that in addition to a substantial decrease in ligand
binding, M1 is also impaired in its DNA binding activity, whereas the
other mutants are comparable to wild-type AR in these respects.
Divergent Effects of C-Terminal Mutations on Transcriptional
Activities of AR
To assess the possible effects of the AF-2 core mutations to
biological activities of AR, we first tested the wild-type and mutant
receptors for their ability to stimulate androgen-dependent expression
of -285PB-LUC (8) in which a deletion derivative of the rat probasin
promoter drives expression of the luciferase (LUC) reporter gene. CV-1
cells were cotransfected with -285PB-LUC and either an empty
expression vector or expression vectors encoding the wild-type or
mutant ARs. After transfection, the cells were either left untreated or
treated with R1881 for 18 h and LUC activities were determined. As
shown in Fig. 2A, in the presence of
R1881, wild-type AR activated transcription of -285PB-LUC by 5-fold.
The mutants M2, M3, and M4 also stimulated transcription, but their
transcriptional activities were reduced by 20%, 30%, and 10%
compared with wild-type AR, respectively. In contrast, M1, M5, and M6
were inactive, as their activities did not significantly differ from
that of the empty expression vector pSG5. In a similar experiment for
the transcriptionally compromised mutants, M1, M5 and M6, qualitatively
comparable results were obtained in HeLa cells, except that the mutants
had approximately 20% activity compared with wild-type AR (Fig. 2B
),
indicating that the transcriptional defects of these mutants are not
cell type specific.
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Another activity of AR is its ability to interfere with AP-1
transcriptional activity (for a review, see Ref. 30). We therefore
tested the mutant proteins for their ability to interfere with AP-1. An
AP-1-dependent reporter in which a deletion derivative of the
collagenase promoter is fused to the chloramphenicol acetyltransferase
(CAT) reporter gene, -73Col-CAT (39), was cotransfected into HeLa
cells with expression vectors specifying the wild-type AR or the
mutants M1-M6. After transfection, the cells were treated with
12-O-tetradecanoylphorbol 13-acetate (TPA) to maximize AP-1
activity and either left untreated or treated with R1881 for 18 h
and CAT activities were determined. As was previously shown (32, 47),
the liganded wild-type AR efficiently decreased -73Col-CAT expression
(Fig. 2D). Interestingly, all mutants were similar to wild-type AR in
their ability to inhibit -73Col-CAT expression, suggesting that the
ability to activate transcription in response to R1881 is not required
for AR to interfere with AP-1 activity, and that these two activities
can be dissociated.
Interaction of AR and Its AF-2 Core Mutants with GRIP1
(GR-Interacting Protein 1) and CBP
Our earlier work with T3R showed that mutations of the
conserved Glu residue in the AF-2 domain inhibit interaction of T3R
with GRIP1 (42), a putative coactivator protein for a number of nuclear
receptors, thus decreasing the ability of T3R
to activate
transcription (18). To test whether this also applies to the AR
mutants, we considered the possibility that there were alterations in
the ability of the mutants to interact with GRIP1.
We first used the transient transfection assay to test the ability of
GRIP1 to increase transcription by wild-type AR and the
transcriptionally deficient mutants M5 and M6. The other
transcriptionally compromised mutant M1 was not included in further
analyses due to its severely impaired DNA and hormone binding
properties. -285PB-LUC was transfected into HeLa cells with expression
vectors encoding wild-type AR, M5, or M6 in the presence or absence of
an expression vector for GRIP1. In the absence of GRIP1, the liganded
wild-type AR activated -285PB-LUC expression by 3-fold, and this
activity was further increased by 4-fold when GRIP1 was coexpressed
(Fig. 3A). Interestingly, M5 and M6,
which are severely defective in their R1881-dependent transactivation
function (see Fig. 2
, AC), were significantly stimulated and thus
were in part rescued by GRIP1 coexpression. In the presence of GRIP1,
M5 had 125% and M6 had 150% of the activity elicited by the wild-type
AR in the absence of GRIP1.
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CBP was recently identified as a putative coactivator for AR (32, 33). We therefore tested whether there were any differences in the
ability of CBP to increase stimulation of transcription by wild-type
AR, M5, or M6. To that end, -285PB-LUC was transfected into HeLa cells
with expression vectors encoding wild-type AR, M5, or M6 in the
presence or absence of an expression vector for CBP. After
transfection, the cells were either left untreated or treated with
R1881 for 18 h and LUC activities were determined. In the absence
of CBP, the wild-type AR activated -285PB-LUC expression approximately
4-fold that was increased a further 3.5-fold in the presence of R1881
and CBP (Fig. 3C). Interestingly, M5 and M6 were also stimulated by CBP
expression: M5 had nearly 65% and M6 had 40% of the activity elicited
by the wild-type AR in the absence of CBP. Thus, similar to the results
obtained with GRIP1, M5 and M6 activity was partially rescued by
ectopic expression of CBP.
We then tested the wild-type and mutant ARs for their ability to
bind CBP in vitro. An N-terminal region of CBP (amino acid
residues 1452), which we have previously shown to bind AR (32), was
expressed in E. coli as a GST fusion protein and used in the
GST pull-down assay with cell-free translated
35S-labeled wild-type and mutant ARs in the
presence or absence of R1881. As shown in Fig. 3D, AR binding to GST
alone was weak and not enhanced by the addition of R1881. In contrast,
wild-type AR efficiently bound to GST-CBP in the absence or presence of
R1881. M5 and M6 also bound GST-CBP, although the binding was somewhat
decreased compared with that of wild-type AR. These results suggest
that the decreased transcriptional activity of M5 and M6 compared with
wild-type AR was not due to their inability to physically interact with
CBP.
AR Shows Significantly Diminished Ability to Bind GRIP1 in
Vitro Compared with T3R
In the GST pull-down experiments presented above, AR displayed
weak binding to GRIP1, which was only modestly enhanced in the presence
of hormone. In contrast, previous studies have indicated that the
in vitro interactions between GRIP1 and some other nuclear
receptors, such as T3R, are significantly stronger than shown in
this study for AR (e.g. Refs. 18, 27). We therefore
compared the ability of AR and T3R
to interact with GRIP1 under the
same experimental conditions. To that end, cell-free translated
35S- labeled wild-type AR or T3R
was used
in the GST pull-down assay with GST-GRIP(415812) in the presence or
absence of R1881 or T3, respectively. As shown in
Fig. 4A
, T3R
did not significantly
bind to GST either in the absence or presence of
T3, but it displayed weak binding to
GST-GRIP(415812) in the absence of T3, which
was further increased by 4-fold in the presence of
T3. In contrast, AR bound to GST-GRIP(415812)
weakly, and the addition of hormone resulted in only approximately
2-fold increase in binding. The ability of T3R
to bind
GST-GRIP(415812) in the presence of hormone was approximately 5-fold
stronger than that of AR. Addition of increasing amounts of
GST-GRIP(415812) in the binding reaction increased T3R
binding
further (3- to 4-fold), but only modestly affected AR binding (data not
shown). These data suggest that the AR-GRIP1 interaction in
vitro is substantially weaker compared with interaction of GRIP1
with other nuclear receptors, such as T3R
.
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GRIP1 Can Activate the AR AF-2
We next tested whether the C terminus of AR is also sufficient for
interacting with GRIP1 and studied the possible effects of AF-2 core
mutations on this interaction by a different approach. To that end, we
fused the LBD of wild-type AR, or M5 and M6, to the DBD of the yeast
transcriptional activator GAL4 (43) (GAL4-AR-LBD-WT) and performed
transfection assays using 5XGAL4-LUC (40) as reporter in which five
copies of the GAL4 response element drive expression of the LUC
gene. GAL4 DBD alone (data not shown), GAL4-AR-LBD-WT,
GAL4-AR-LBD-M5, or GAL4-AR-LBD-M6 did not activate 5XGAL4-LUC in
the presence or absence of R1881 (Fig. 5A). However, when GRIP1 was coexpressed
with GAL4-AR-LBD in the presence of R1881, approximately 10-fold
activation of 5XGAL4-LUC was observed, suggesting that the LBD of AR
directly interacts with and is activated by GRIP1. Even though
GAL4-AR-LBD-M5 and -M6 were inactive in the absence of GRIP1, they were
activated by GRIP1 coexpression 36% and 63%, respectively, compared
with the wild-type AR construct (Fig. 5A
). These data suggest that the
LBD of AR requires GRIP1, or a comparable cofactor, to activate
transcription. In addition, these results indicate that Glu897 in the
AF-2 core domain is responsible, at least in part, in mediating
interactions between AR and GRIP1.
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Mutations in the AR AF-2 Core Disrupt Interactions between the LBD
and the NTD
The LBD of AR has recently been shown to interact with the NTD in
a strictly hormone-dependent fashion (6, 8, 9). To assess the possible
role of the AF-2 core region in this interaction, HeLa cells were
transfected with the 5XGAL4-LUC reporter and GAL4-AR-LBD-WT, or the
GAL4 fusions of the mutants M5 and M6, in the presence or absence of an
expression vector specifying the NTD of AR [AR(1566)]. As shown in
Fig. 6, GAL4-AR-LBD-WT did not affect
activity of the reporter construct in the presence or absence of R1881;
similar results were obtained for the mutants M5 and M6 (data not
shown). Coexpression of AR(1566), specifying expression of the NTD,
with GAL4-AR-LBD-WT did not activate 5XGAL4-LUC in the absence of
R1881; however, there was 16-fold activation of the reporter in the
presence of R1881. In contrast to GAL4-AR-LBD-WT, no significant
reporter gene activation was observed in comparable experiments using
GAL4-AR-LBD-M5 or GAL4-AR-LBD-M6. These results indicate that AF-2 core
region in AR has an important role in mediating the intramolecular
interactions between the LBD and NTD.
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DISCUSSION |
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Substitution of Met894 with Asp (M1) significantly impaired the ability
of AR to activate from AREs (Fig. 2, AC). This is in line with
previous work that has shown that the same mutation in the
corresponding residue in T3R
(L398D) prevented transcriptional
activation (18). However, the mechanisms for the loss of
transactivation in these receptors appear to be quite different: AR-M1
is significantly reduced in its ability to bind either ligand or to DNA
(Fig. 1
, A and C), in contrast to the corresponding L398D mutation in
T3R
, which did not significantly affect either of these functions
(18). In addition, the characteristic shift in mobility of AR-ARE
complexes in a band shift experiment in response to ligand binding
(e.g. Ref. 46) is lost in M1 (data not shown). These data
suggests that Met894, although conserved as a hydrophobic residue in
most nuclear receptors, has a different role in the context of AR
compared with in T3R
and other nuclear receptors. This hypothesis is
supported by the finding that substitution of Met894 with Ala (M2) did
not significantly affect AR activity, which is in stark contrast with
the same mutation in the context of T3R
(18) and T3Rß (16) in
which all transcriptional activity was lost. Furthermore, double
mutants in ER (L543A/L544A) and GR (M758A/L759A) that include the
corresponding mutation were incapable of transcriptional activation in
response to ligand (12). None of these mutants, in the context of T3Rs,
ER or GR, were significantly impaired in either DNA or hormone binding
(12, 16, 18).
While this work was being prepared for publication, a mutational analysis of AR LBD was reported (50). In this study, mutagenesis of the hydrophobic pair Met894 and Met895 to alanine, resulted in the loss of all hormone-dependent transcription. Since mutagenesis of single amino acid residues was not performed, it is not possible to determine which of the methionine residues that were altered was responsible for the loss of activity. In our analysis, however, mutagenesis of Met894 into Ala (M2) did not significantly affect AR activity, suggesting that Met895 is the more crucial amino acid residue contributing to AR function. However, when Met894 was substituted to Asp (M1), transactivation ability of AR was lost due to substantially diminished DNA and hormone binding activities. Collectively, these results suggest that both Met894 and Met895 have important contributions to AR function, which may be mediated through different mechanisms.
In contrast to Met894, mutants of Ala896 and Glu897 (M3-M6) were
similar to wild-type AR with respect to their hormone and DNA binding
activities (Fig. 1, A and C). Substitution of Ala896 with either Val or
Leu (M3 and M4) did not significantly affect AR transcriptional
activity, which is consistent with the activities of similar mutations
in T3R
and T3Rß (16, 18). Since Ala896 is less conserved in
nuclear receptors and is substituted frequently with more distant amino
acids, these data support the notion that Ala896 does not directly
contribute to hormone-dependent transcriptional activity of AR.
Substitution of Glu897, which is completely conserved in
different nuclear receptors, either with Val or Ala (M5 and M6),
significantly diminished hormone-dependent activation by AR (Fig. 2, AC). In contrast to our results, it was recently reported that the
transcriptional activity of an AR double mutant, in which Glu893 is
substituted along with Glu897, was not significantly altered (50). One
possible explanation for this apparent discrepancy is that the
substitution that was carried out at Glu897 in the double mutant was a
glutamine compared with valine or alanine in our study. Thus, the
differences in size and properties of the substituted residues could
differentially affect the final structure of the AF-2 core region.
Alternatively, the effect induced by substitution of Glu897 is
neutralized by additional substitution at Glu893. More detailed
mutagenesis will be required to assess these possibilities.
In the context of other nuclear receptors, the impaired transcriptional
activity of the M5 and M6 mutants is consistent with the findings on
similar mutations generated at this residue in T3R and T3Rß (14, 16, 18), but different from that of ER
and GR in which mutants
analogous to M5 were as active as the wild-type receptors (12). This
suggests that the conformation, and perhaps function, of Glu897 in AR
is more like that of the corresponding residue in T3Rs than in other
steroid receptors, such as ER
and GR, to which AR is more closely
related overall. Assessment of this hypothesis must await the
delineation of the AR holo LBD crystal structure and its comparison
with those of other nuclear receptors.
It was previously demonstrated that the loss in transactivation
potential of many nuclear receptor AF-2 core mutants may be due to
disruption of fruitful interactions with putative coactivator
protein(s) (e.g. see Refs. 18, 25, 27). For example, the
loss of ligand-dependent transactivation by the inactive AF-2 core
mutants in T3R could largely be reversed when GRIP1 was ectopically
expressed (18). For the transcriptionally defective mutants of M5 and
M6 of AR, we have made similar observations in vivo: GRIP1
significantly stimulated, and thereby rescued, M5 and M6
transactivation potential, yielding 125150% of wild-type AR activity
in the absence of GRIP1 (Fig. 3A
). Similarly, CBP coexpression resulted
in partial rescue of M5 and M6 activity, which was smaller (4065%)
than that obtained with GRIP1 (Fig. 3C
).
The simplest explanation for these results is that the substitutions in
M5 and M6 decrease the affinity of the AR for GRIP1, or another
coactivator such as CBP, which in turn results in a significant loss of
AR transactivation potential. Thus, when an excess of coactivator is
available, this defect is in part overcome in that the ability of M5
and M6 to activate transcription significantly increases. Consistent
with this model, we have earlier found an excellent correlation between
T3-dependent in vitro association of
GRIP1 with T3R AF-2 mutants and their ability to support
T3-dependent transcriptional activation (18).
Similar observations were made with ER AF-2 mutants and GRIP1 binding
in vitro (27). However, in contrast to these findings, AR-M5
and AR-M6 displayed in vitro GRIP1 and CBP binding
characteristics that were comparable to those of wild-type AR (Fig. 3
, B and D).
Recent findings demonstrated that interaction of AR with SRC-1, another
putative coactivator, is mediated by the N terminus of AR and that the
AF-2 core does not play an important role in this process (50). In
addition, the transcriptional activity of AR N terminus is stimulated
by GRIP1 and SRC-1 (54). These results suggested that the major
interaction site of coactivators with AR is in the N terminus. This is
consistent with our findings since for both wild-type and the mutant
ARs, binding to GRIP1 in vitro was only modestly increased
in response to ligand binding and did not change for CBP (Figs. 3B and 3D
). This is in contrast to a substantial hormone-induced increase in
interactions of other nuclear receptors, such as ER and T3R
, with
GRIP1 (Fig. 4A
, and Refs. 18, 27) and CBP (data not shown) studied
under similar conditions. It is possible that additional factors are
involved in the interaction between AR and coactivators in
vivo, such as GRIP1, which give rise to these divergent
results.
AR LBD did not activate transcription when fused to a heterologous DBD
(Fig. 5A), indicating that the AF-2 function of AR is weak compared
with other nuclear receptors (e.g. ER and GR), which is
consistent with recent reports (50, 51). Ectopic expression of GRIP1
along with AR LBD fused to the GAL4 DBD resulted in hormone-dependent
transactivation (Fig. 5A
), indicating that an excess of a coactivator
can evoke the activity of AR AF-2 in isolation, which is consistent
with the findings summarized above and other recent findings (54).
Our mammalian two-hybrid data show that the wild-type AR LBD and GRIP1
[including the additional regions that were previously suggested to be
required for AR interaction (52)] strongly interact in intact cells
(Fig. 5B). In the same assay, M5 was significantly reduced in its
ability to interact with GRIP1, whereas M6 was comparable to wild-type
AR. Thus, although the decrease in the ability of M5 to interact with
GRIP1 in vivo may explain, at least in part, its loss of
transcriptional activation, this correlation does not hold for M6.
Therefore, the latter results, coupled to the in vitro
interaction data, suggest that additional mechanism(s) that could
account for the loss of transactivation potential of these mutants
should be at work in the cell.
It was recently shown that the interactions between N and C termini of
AR are involved in receptor stabilization, reduction in ligand
dissociation, and increase in DNA binding affinity (6, 8, 9, 53). The
data we present in Fig. 6 suggest that the disruption of fruitful
intramolecular contacts between the LBD and NTD is responsible for the
significant loss in transactivation potential of the M5 and M6 mutants.
While this manuscript was being prepared for publication, similar
findings on N/C interaction were reported using two mutants in the AR
AF-2 (51, 54). Collectively, these data suggest that the AF-2 core
domain mediates ligand-dependent intramolecular interactions between
the LBD and the NTD, which are required for full transcriptional
activation by AR.
Similar to many other nuclear receptors, an additional transcriptional
property of AR is its ability to interfere with AP-1-mediated
transactivation (32, 33, 47). Recent studies suggested that amino acid
residues in or close to the AF-2 core domain may be involved in the
cross-talk between AR and AP-1 (for a review, see Ref. 30). We found
that all the mutants, regardless of their hormone-dependent
transcriptional activation function, were similar to wild-type AR in
efficiently inhibiting AP-1 activity (Fig. 2D). It is of note that the
M1 mutant, which has substantially diminished ability to bind either
hormone or DNA, can still interfere with AP-1 in a fashion that is
comparable to wild-type AR. This suggests that the DNA binding activity
is not critical for the ability of AR to interfere with AP-1,
consistent with previous studies on the cross-talk between other
nuclear receptors and AP-1 (for a review, see Ref. 30). These data are
also consistent with the hypothesis that a cofactor, such as CBP,
mediates the cross-talk between AR and AP-1 (32, 33), since all AR AF-2
mutants bound CBP as efficiently as the wild-type AR in
vitro (Fig. 3D
, and data not shown). These results support our
earlier findings and suggestion (18) that transactivation and
transrepression by nuclear receptors are mediated by two different
interaction surfaces.
In summary, we identified important differences between the contribution of specific, conserved residues in the AF-2 core domain to AR activity compared with other nuclear receptors. These differences may reflect the fact that the structure of the AR AF-2 core domain, its function, its role in contacting the NTD, and the AR coregulatory proteins that it interacts with are different compared with other nuclear receptors. Such variations may be responsible for selective activation of different nuclear receptors by ubiquitous cofactors and thereby help determine specificity of transcriptional activation. Decisive assessment of this hypothesis must await the crystal structures of AR apo- and holo-LBD and AR NTD, as well as their functional characterization.
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MATERIALS AND METHODS |
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Cell Culture, Transient Transfection, LUC, and CAT
Assays
CV-1, HeLa, COS-1, and COS-7 cells were maintained in DMEM
supplemented with 5% newborn calf serum (NCS) or 5% or 10% FBS,
respectively. The calcium phosphate coprecipitation method was used to
transfect both the CV-1 and HeLa cells. CV-1 cells were transfected
with 0.25 µg of the reporter plasmid and the indicated plasmids and
pUC18 to a total of 0.67 µg DNA per well on 12-well plates. HeLa
cells were transfected with 0.67 µg of reporter plasmid, indicated
levels of expression vectors, and pUC18 to a total of 2 µg of DNA per
well on six-well plates and similar to CV-1 cells when 12-well plates
were used. After 56 h of incubation with the precipitates, CV-1 cells
were washed once with PBS and then maintained in DMEM supplemented with
0.5% charcoal-treated NCS in the presence or absence of R1881
(10-7 M, NEN Life Science Products, Boston, MA). After transfection, HeLa cells were
treated with 15% glycerol in PBS for 2 min, washed with PBS, and then
maintained in DMEM supplemented with 0.5% charcoal-treated NCS in the
presence or absence of R1881. In the experiments with -73Col-CAT,
cells were treated with TPA (10-7 M)
after transfection to maximize AP-1 activity. Sixteen hours after
transfection, cells transfected with the luciferase reporter were
washed once with cold PBS and harvested in Tris-MES solution (1
mM dithiothreitol, 0.5% Triton X-100, and 50
mM Tris-MES, pH 7.8), and the LUC activities were
determined. The cells that were transfected with the CAT reporter were
washed once with cold PBS, incubated with hypotonic buffer (25
mM Tris-HCl, pH 7.5, and 2 mM
MgCl2) for 5 min, and then lysed in Triton lysis
buffer (0.25 M Tris-HCl, pH 7.8, and 0.5% Triton X-100) to
make the extracts. CAT activities in these extracts were determined as
previously described (13).
R1881-Binding Assay
The calcium phosphate coprecipitation method was used to
transfect COS-1 cells with 0.5 µg of expression vector per well in
12-well plates. After 46 h incubation, the cells were shocked with
15% glycerol for 2 min, washed once with PBS, and then maintained in
DMEM supplemented with 0.5% charcoal-treated FBS. After 16 h, the
cells were washed twice with PBS, and increasing amounts of
[3H]R1881 (NEN Life Science Products) were added in serum-free DMEM. After incubation for
2 h, the cells were washed twice with cold PBS, harvested, and
directly counted in scintillation fluid to determine the
[3H]R1881 that remained bound in the cells. The
dissociation constants were determined using GraphPad software
(GraphPad Software, Inc., San Diego, CA).
Western Analysis
For Western analysis, the polyethylenimine (PEI) method (44) was
used to transfect COS-7 cells with 15 µg of expression vectors
specifying the production of wild-type or mutant ARs in 10-cm dishes.
After incubation for 46 h, the cells were washed with PBS and
maintained in DMEM supplemented with 10% FBS. Thirty six hours later,
whole cell extracts were prepared. Briefly, cells were washed twice in
cold PBS, resuspended in Dignam C solution (20 mM HEPES, pH
7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, and 0.5
mM dithiothreitol) with protease inhibitors, and lysed by
three cycles of freeze/thaw. The extracts were run on SDS-PAGE, blotted
onto a nylon membrane, and probed with a polyclonal antiserum raised
against the LBD of AR (45).
GST Pull-Down Assay
In vitro interactions between AR and GRIP1 or CBP
were examined by the GST pull-down assay as described previously (18, 32). Briefly, GST and GST-GRIP1 (amino acids 415812) (42), or
GST-CBP(1452) fusion proteins were expressed in E. coli
and purified on glutathione-Sepharose beads (Amersham Pharmacia Biotech, Arlington Heights, IL). AR and its mutants and T3R
were translated in vitro using the TNT-coupled
transcription/translation system (Promega Corp., Madison,
WI) in the presence of [35S]methionine,
1.3 x 10-6 M
ZnCl2, and in the presence or absence of R1881
(10-6 M) or
T3 (10-7
M), for AR and T3R
, respectively. In the GST
pull-down reactions, the translated proteins were incubated on ice for
20 min in the presence or absence of R1881 (10-6
M) or T3
(10-7 M) before the
addition of fusion proteins. The reactions were incubated for 12 h on
ice in NETN buffer (40 mM NaCl, 4
mM Tris-HCl, pH 8.0, 0.1% Nonidet P-40, and 0.2
mM EDTA) containing protease inhibitors and the
same amounts of ligand as above with occasional mixing. The beads were
then washed three times with NETN buffer, resuspended in sample buffer,
and size fractionated on SDS-polyacrylamide gels. Labeled proteins
visualized by fluorography were analyzed by using a PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA).
Mobility Shift Assay
Wild-type AR and mutant proteins were prepared by in
vitro transcription/translation as described above and tested for
binding to a 32P-labeled androgen response
element (ARE) of the first intron of rat C3 gene in the presence
of R1881 (10-6 M) (46).
Briefly, the translated proteins were incubated at room temperature for
15 min in binding buffer (20 mM HEPES, pH 7.9, 50
mM KCl, 1 mM
MgCl2, 0.4 mM EDTA, 10%
glycerol, 0.05% Nonidet P-40, and 20 mg/ml BSA) in 10 µl volume.
After hormone binding, 32P-labeled ARE was added
and binding reactions were supplemented with dithiothreitol (2
mM), and 200 ng
poly(dI-dC)2 were added, yielding a total volume
of 20 µl, and the reaction mixtures were incubated for 25 min at room
temperature. The binding reactions were then run on a 5%
nondenaturating polyacrylamide gel (29:1) in 0.25x
Tris-borate-EDTA buffer, dried, and visualized by
autoradiography.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by grants from the Norwegian Cancer Society, Norwegian Research Council, University of Oslo, The Odd Fellow and Jahre Foundations, the Medical Research Council of the Academy of Finland, the Finnish Foundation for Cancer Research, the Sigrid Jusélius Foundation, and Biocentrum Helsinki.
Received for publication February 3, 2000. Revision received July 13, 2000. Accepted for publication July 20, 2000.
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REFERENCES |
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