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Departments of Endocrinology and Reproduction (C.A.B., A.O.B.) and Pathology (P.D., K.S., J.T.) Erasmus University 3000 DR Rotterdam, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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For the hAR, two separate transcription activation units (TAUs) in the NH2-terminal domain were defined (8). One of these transactivation units [TAU-1; amino acids (aa) 100–370] is only active in the full-length ligand-activated AR. In a truncated hAR, which lacks the LBD, another region (TAU-5, aa 360–485) functions as a constitutively active transactivation domain. The role of TAU-5 in the full-length AR is not yet clear.
The LBDs of various nuclear receptors contain a ligand-dependent
transactivation function, AF-2 (9, 10, 11). An autonomous activating domain
(AD) in this AF-2 region, AF-2 AD, is conserved among many nuclear
receptors and is located in the C-terminal part of the LBD. A core
region in the AF-2 AD, 
-helix 12, appeared to be important for
transcriptional activity (9, 10, 12, 13) and the hormone-dependent
interaction with TIFs. These TIFs or coactivators can modulate the
transcriptional activity of a broad range of nuclear receptors
(14, 15, 16, 17). Mutations in the AF-2 AD core abolish the in vitro
association of the receptor with these coactivators. The recent finding
that a coactivator displays histone acetyltransferase activity
has provided further insights into the molecular events occurring at
the chromatin level during transcription activation (18). Wurtz
et al. (19) proposed a general mechanism for nuclear
receptor activation, in which the AF-2 AD core, present in helix 12,
plays a central role in the generation of an interaction surface,
allowing binding of TIFs to the LBD. The functional role of the AF-2 AD
core in the AR is not yet well understood.
Recently, for the estrogen receptor (ER) a functional, ligand-dependent, in vivo association between the NH2-terminal domain and the LBD was described (20). Also for the AR a functional in vivo interaction between the NH2-terminal domain and the LBD was demonstrated (21, 22). In the study presented here, we determined in more detail AR subdomains involved in the functional interaction of the LBD with the NH2-terminal domain. The data indicated that two NH2-terminal regions, together with the AF-2 AD core region in the LBD, are important for a functional in vivo interaction. Induction of AR AF-2 activity by the transcriptional coactivator TIF2 also required an intact AF-2 AD core. This enhancement of AR AF-2 activity by TIF2 appeared to be promoter dependent.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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To determine regions in the NH2-terminal domain involved in
the functional interaction with the AR-LBD, different deletion mutants
of the NH2-terminal domain were constructed, as shown in
Fig. 1A
(AR.N1-AR.N7). The deletion
mutants were transiently transfected into CHO cells, and the
appropriate expression of the proteins was assessed by immunoaffinity
purification and Western blot analysis of cytosolic fractions (Fig. 1B, lanes 1–7). Constructs (AR.N1- AR.N6) were expressed as proteins of
expected molecular mass and at comparable expression levels. The
expression level of AR.N7 could not be determined because this protein
lacks an epitope for the available antibodies.
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; AR.C), using mouse mammary tumor
virus (MMTV)-LUC as a reporter gene, is shown in Fig. 2. As shown previously (22), neither
AR.N1 nor AR.C could, when expressed separately in CHO cells, activate
transcription from the reporter in response to androgens. The lack of
activity of AR.C is not due to the experimental setup, because a
similar glucocorticoid receptor (GR) fragment, GR.C (aa 369–777),
highly induced luciferase activity in CHO cells (24-fold) upon addition
of 10 nM dexamethasone (data not shown). Coexpression of
AR.N1 and AR.C resulted in an androgen-dependent induction (18-fold) of
luciferase expression (Fig. 2; AR.N1), which is comparable to
full-length AR activity. Deletion mutants AR.N2 and AR.N3 showed
similar responses as seen with the intact NH2-terminal
domain. However, deletion of amino acid residues 371–494 markedly
reduced the androgen-induced LUC-activity (AR.N4). Deletions in the
first part of the NH2-terminal domain (AR.N5, AR.N6, AR.N7)
almost completely abolished the R1881-mediated response. These data
indicate that, for a functional interaction of the
NH2-terminal domain with the LBD of the AR in CHO cells,
amino acid residues 1–51 and 371–494 are important. This suggests
that the TAU-5 domain, located in the latter region, is involved in
this functional in vivo interaction. The region between
residues 51 and 360, harboring TAU-1, appears not to be involved.
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Truncated forms of the AR NH2-terminal domain were fused to
the transactivation domain of GAL4 (Gal4-TAD), in the high-expression
vector pACT2 (Fig. 3A). AR-LBD was fused
to the DBD of GAL4 (Gal4-DBD). Figure 3B shows the protein expression
in yeast Y190 cells, as assessed by immunoblot analysis using specific
antibodies directed against either the AR (lanes 1–4) or Gal4-TAD
(lanes 5–10). The immunoblot shows expression of most fusion proteins
of the appropriate length. However, the expression of GalAD-AR.N9 (lane
2) appeared to be somewhat lower. GalAD-AR.N8 became visible only at an
extended exposure time (lane 1a), indicating a very low protein
expression level.
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). The observed
reporter gene activity for GalAD-AR.N8 was lower as compared with
GalAD-AR.N9, GalAD-AR.N10, and GalAD-AR.N12, most likely due to the
very low protein expression level of GalAD-AR.N8. The constructs
showing a high ß-GAL activity (GalAD-AR.N8, -N9, -N10, and -N12) all
share one common region, spanning residues 3–36. Deletion of this
region from constructs GalAD-AR.N12 and GalAD-AR.N15, resulting in
constructs GalAD-AR.N13 and GalAD-AR.N16, respectively, caused an
intense drop in ß-GAL activity. Therefore, in accordance with the CHO
results, the data imply an important role of NH2-terminal
amino acid residues 3–36 in the functional interaction with the
AR-LBD.
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As reported previously (22), the measured ß-GAL activities are partly
due to Gal4-TAD transactivity, but could also be influenced by the
intrinsic transactivating activity of the AR NH2-terminal
domain. The contribution of the intrinsic AR activity to the total
measured ß-GAL activity could vary between different constructs and
thereby affect the mapping of interacting regions. To discriminate
between AR transactivity and AR interaction potential, the Gal4-TAD
part was deleted from these GalAD-AR.N constructs that showed a
considerable interaction with GalDBD-AR.C (as depicted in Fig. 4A).
This resulted in constructs AR.N8-AR.N12 and AR.N15. Protein expression
of these constructs, as determined by immunoblot analysis, is shown in
Fig. 3B (lanes 11–16).
The activities of the AR.N constructs, in the two-hybrid assay with
GalDBD-AR.C, are shown in Fig. 4B. AR.N11 and AR.N15 did not induce a
functional interaction, although the corresponding Gal4-TAD chimeric
constructs, as shown in Fig. 4A, were active. This indicates that
AR.N11 and AR.N15 only contain interaction potential and not
transactivity. These constructs have the 3–36 fragment in common.
Deletion of the region between residues 244 and 360 (AR.N9) did not
reduce ß-GAL activity; AR.N12 (missing residues 37–243) also showed
a clear response. This indicates that both these proteins still have
transactivating activity and interaction potential. However, by
deleting residues 371–494 (AR.N10), ß-GAL activity dropped by
approximately 80%, a decrease comparable to the data obtained in CHO
cells (Fig. 2; AR.N4). These results narrow the second interacting
region (aa 244–494) to involvement of residues 371–494 as a
major transactivation domain in the functional interaction with the
LBD.
Taken together, the yeast two-hybrid data imply a prominent role of amino acid residues 3–36 of the AR NH2-terminal domain in the interaction with the LBD. A second region seems to be mainly constrained to residues 371–494 and further supports the involvement of TAU-5, as a transactivating region, in the functional in vivo interaction with the LBD.
A Mutation in the LBD Affects the Functional Interaction with the
NH2-Terminal Domain, but not LBD
Dimerization
To determine regions in the AR-LBD involved in the functional
interaction with the NH2-terminal domain, mutational
analysis was constrained to the conserved amphipathic helix 12 in the
LBD. This helix constitutes the AF-2 activation domain (AD) core
region, of which the charged residues might generate a protein-protein
interacting surface (19). A single amino acid substitution in the AF-2
AD core was introduced: glutamic acid at position 888, which is highly
conserved among nuclear receptors, was replaced by glutamine
(E888Q).
Both the wild-type and the mutated LBD construct (Fig. 1A; AR.C and
AR.C(EQ), respectively) were transfected into CHO cells. The
appropriate expression of both proteins is shown in Fig. 1B (lanes 8
and 9). To determine whether the AF-2 AD mutation had an effect on
hormone binding, an in vivo androgen binding assay in
transfected CHO cells was performed. Scatchard analysis of the data
revealed that both proteins had similar Kd values for
R1881: AR.C, 0.37 nM, and AR.C(EQ), 0.33 nM,
respectively, indicating that the E888Q mutation did not affect hormone
binding.
The effect of the E888Q mutation on the functional in vivo
interaction of the LBD with the NH2-terminal domain was
investigated by cotransfecting AR.N1 with increasing amounts of either
AR.C or AR.C(EQ) and the MMTV-LUC reporter into CHO cells (Fig. 5A). As expected, no response to 1
nM R1881 was observed in the absence of the LBD constructs.
Coexpression of AR.C and AR.N1 resulted in a high induction (to
24-fold) of luciferase activity. The E888Q mutation strongly reduced
the response to R1881 (to maximally a 7-fold induction) as compared
with the wild-type LBD.
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; NH2/LBD). The effect of the E888Q mutation
appeared to be even more pronounced than in CHO cells, as ß-GAL
activity measured for the interaction of both AR domains (set at 100%
for the intact LBD) dropped more than 90% upon introduction of the
E888Q mutation. The data from CHO cells and from yeast indicate that
the AF-2 AD core region in the AR-LBD is important for a functional
interaction with the NH2-terminal domain.
Previously, homodimerization of the AR-LBD was demonstrated in yeast
(22, 23). To determine whether the E888Q mutation had an effect on LBD
dimerization, both LBDs (wild-type and mutant) were coupled to Gal4-TAD
and transformed together with the corresponding LBD domains coupled to
Gal4-DBD (Fig. 5C). In this LBD/LBD interaction assay, the wild-type
AR.C and the mutant AR.C(EQ) showed similar ß-GAL levels, indicating
that the E888Q mutation had no effect on LBD dimerization.
TIF2-Induced Activity of AR AF-2 in CHO Cells Is Affected by the
E888Q Mutation
The functional role of the AR AF-2 AD core region was further
characterized by comparing the functional in vivo
interaction of the AR-LBD and the AR NH2-terminal domain
with the interaction of the AR-LBD and the coactivator TIF2. The
coactivator TIF2 is able to enhance the AF-2 activity of several
steroid receptors including the AR (17). The involvement of the AR AF-2
AD core region in the functional in vivo interaction of TIF2
and the AR-LBD was investigated by examining the effect of the E888Q
mutation.
TIF2 was cotransfected with the MMTV-LUC reporter and increasing
amounts of the wild-type (AR.C) or the mutant LBD (AR.C(EQ)) into CHO
cells (Fig. 6). In the absence of the LBD
constructs, no response to 1 nM R1881 was observed. As
expected, coexpression of TIF2 and AR.C highly induced luciferase
activity (up to 26-fold). The E888Q mutation reduced luciferase
activity by approximately 70%, compared with AR.C. This decrease in
functional interaction of AR-LBD with TIF2, caused by the E888Q
mutation, was comparable to the negative effect of the mutation on the
interaction with the NH2-terminal domain (as shown in Fig. 5A).
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). The luciferase activities obtained
were compared with the transactivating activities measured after
separate transfection of the different plasmids. Individual expression
of TIF2 and the NH2-terminal domain induced the
transcriptional activity of AR.C on the MMTV promoter up to 24-fold
(panels a and d, respectively). When coexpressed with 50 ng/well AR.N1
(panel b), TIF2 slightly enhanced the luciferase activity induced by
the functional interaction of AR.N1 and AR.C (up to 34-fold). When
higher amounts of AR.N1 were transfected, no additional effects of TIF2
could be observed (panels c and d). These data most likely exclude
synergy between TIF2 and the NH2-terminal domain for the
interaction with the AR-LBD, indicating that TIF2 is not necessary for
the functional interaction between the AR domains.
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). AR.C was
cotransfected into CHO cells with increasing amounts of either AR.N1
(Fig. 8, A and C) or TIF2 (Fig. 8, B and D), together with either
MMTV-LUC (Fig. 8, A and B) or GRE-TATA-LUC (Fig. 8, C and D) as
reporter constructs. In the absence of either AR.N1 or TIF2, no
significant response to 1 nM R1881 could be detected. AR.N1
as well as TIF2 induced AF-2 activity on the MMTV-LUC promoter
(maximally 39- and 44-fold, respectively). Luciferase activity was also
highly induced by AR N.1 on the minimal, GRE-TATA, promoter (Fig. 8C).
In contrast, the TIF2-induced luciferase activity appeared to be much
more affected on this promoter (Fig. 8D). This indicates that the level
of transcriptional response induced by TIF2 depends on the promoter
context.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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It was described recently, for the ER and AR, that functional in vivo interactions between the separate NH2-terminal domain and the LBD can occur (20, 21, 22). Whether this association is direct or requires additional factors is unknown. Neither is it clear which regions in these AR domains are involved in this functional interaction. For a better understanding of AR action, we determined the subdomains in the AR involved in the functional interaction between the NH2-terminal domain and the LBD.
Cotransfection experiments of the AR-LBD with NH2-terminal
deletion mutants in CHO cells and in yeast imply a major role for amino
acid residues 3–36 of the NH2-terminal domain in the
functional in vivo interaction with the LBD. A previous
report by Langley et al. (21) indicated residues 14–150 to
be involved, suggesting that the interaction domain
might be constrained to residues 14–36. In this respect it is
interesting that region 16–36 is potentially capable of forming an
amphipathic -helix, if projected on a helical wheel diagram.
Experiments in CHO cells revealed a second region, amino acid residues 371–494, important for a functional in vivo interaction with the AR-LBD. The involvement of this NH2-terminal region was confirmed by data obtained with the two-hybrid system in yeast. This region harbors TAU-5 (8), postulating a role for this transcription activation unit in the functional interaction with the LBD. A recent report by McEwan and Gustafsson (24) described the interaction of the AR NH2-terminal domain (aa 142–485) with the general transcription factor TFIIF. It remained unclear which of the transactivation units (TAU-1 or TAU-5) in the AR NH2-terminal domain is involved in the interaction with TFIIF, as both TAUs are present in the AR domain used in this study. Therefore, the role of TAU-5 in the interaction with the AR-LBD might be, at least in part, by recruiting the transcriptional machinery to the target promoter.
The in vivo experiments described here imply an important role for the AR AF-2 AD core in the functional interaction with the NH2-terminal domain. The E888Q mutation markedly decreased the interaction between the two AR domains, without altering hormone binding. As the E888Q mutation did not completely reduce the interaction, it is likely that also other residues or regions in the LBD are involved in the formation of a proper interacting surface. Wurtz et al. (19) proposed a general mechanism for nuclear receptor activation, in which hormone-induced conformational changes within the LBD result in a close contact of helix 12 and helix 4, thereby creating an interaction surface that allows binding of coactivators to the AF-2 activation domain. As the formation of this interaction surface might also be important for the functional interaction of the LBD with the NH2-terminal domain, this would suggest that helix 4 is involved. Two-hybrid experiments in yeast showed that the E888Q mutation affected the functional interaction with the NH2-terminal domain but did not influence LBD homodimerization. This implies that different regions in the AR-LBD are involved in the interaction with the NH2-terminal domain and in LBD dimerization.
The E888Q mutation also decreased the stimulatory effect of the
coactivator TIF2 on AR AF-2 activity. This suggests that the AF-2 AD
core region of the hAR is involved in interacting with both the
NH2-terminal domain and the coactivator TIF2. A possible
mechanism of TIF2 action could be as a bridging factor in the process
of transcription activation by the AR. McInerney et al. (25)
demonstrated that the steroid receptor coactivator SRC-1 enhanced the
functional interaction between the NH2-terminal and
C-terminal domain of the ER. From these data it was suggested that
SRC-1 may act as an adaptor protein that promotes the integration of
NH2-terminal and C-terminal ER domains. However,
cotransfection experiments with TIF2 and the AR
NH2-terminal domain in CHO cells did not show synergy
between TIF2 and the AR NH2-terminal domain in the
induction of AR AF-2 activity (Fig. 7). Therefore, it is unlikely that
TIF2 functions as a bridging factor for the functional interaction of
the NH2-terminal domain and the LBD of the AR.
The AR appears to be a unique member in the nuclear receptor superfamily, because the AR C-terminal domain, containing the AF-2 AD core region, is inactive in the absence of the NH2-terminal domain or TIF2. This lack of activity is not due to the experimental setup or the cell type used (CHO), because a similar C-terminal construct of the GR is active. The most likely explanation of this difference is that interactions of the AR-AF2 AD with endogenous coactivators are very weak and therefore undetectable. Overexpression of coactivators (e.g. TIF2) might enhance the interaction and consequently transcriptional activity. Another explanation for the absence of AR AF-2 activity in CHO cells is that endogenous levels of TIF2 are not sufficient for activation of the MMTV promoter in the absence of AR.N1.
A signature motif in transcriptional coactivators that mediated binding to liganded nuclear receptors was described recently (26, 27, 28). Most coactivators, including TIF2, harbor several of these leucine-rich motifs (LXXLL). Two-hybrid experiments in yeast revealed that the interaction of the ER-LBD with LXXLL motifs was abolished by mutating the AF-2 AD core region in the LBD (26). Despite the demonstration that the NH2-terminal domain of the AR also interacts with the AF-2 AD, it does not harbor such a motif, suggesting a different type of cooperation between the two AR domains.
The induction of AF-2 activity by TIF2 appeared to be promoter dependent. TIF2 strongly stimulated AR AF-2 activity on a MMTV-LUC reporter but much less on a minimal GRE-TATA promoter. The AR NH2-terminal domain could, in contrast to TIF2, induce AF-2 activity on both promoters. Possibly, TIF2 not only affects the AR but also interacts with other transcription factors that bind to the complex MMTV promoter and not to the minimal promoter.
From the data presented here, together with previously obtained results (22), the following model for functional interactions between AR domains is proposed. Androgen binding induces a conformational change in the receptor that facilitates the AF-2 AD core in the LBD to interact with the NH2-terminal domain. This interaction might be either direct or indirect, requiring additional factors, and results in AR transcription activation. Enhancement of AR activity by the coactivator TIF2 also involves the AF-2 AD core. In addition to a functional NH2-terminal domain/LBD interaction, a LBD/LBD interaction is proposed that requires a region different from the AF-2 AD core.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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51–211 and AR 244–360 (30),
AR124, AR127, and AR106 (8) respectively : plasmids were digested with
KpnI and EcoRI and religated by a
KpnI-EcoRI linker. AR.N4 was obtained by
insertion of a RsrII-EcoRI linker into AR.N1.
pSVAR(EQ) was constructed by site-directed mutagenesis of pSVAR (31),
using the following oligonucleotides (the modified codon is shown
underlined): AR/EQ1: 5'-ACAGCCAGTGTGTCCGAATG-3';
AR/EQ2: 5'-CTTGCACAGAGATGATTTGTGCCATC-3';
AR/EQ3: 5'-GATGGCACAAATCATCTCTGTGCAAG-3';
AR/EQ4: 5'-CAAGGGGCTTCATGATGTCC-3'.
AR.C(EQ) was prepared by digestion of pSVAR(EQ) with
EcoRI and ligating the 465-bp fragment into AR.C. MMTV-LUC
reporter plasmid was kindly provided by Dr. Dijkema (Organon, Oss, The
Netherlands) and was described previously (32). GRE-TATA-LUC,
containing the TATA-box and a Sp1-site derived from the Oct-6 gene
promoter, has been described previously as
pJH4-(ARE)2-OCT-LUC (33). GR.C (aa 369–777) was
constructed by digestion of RshGR (34) with BstXI and
religation with a compatible DNA fragment obtained by annealing the
following oligos: 5'-AGCCCGGGACCATGGGAT-3' and
5'-CATGGTCCCGGGCTATCC-3'. The obtained plasmid was digested with
SmaI and DraI, and the 1074-bp fragment,
containing the GR DBD and LBD, was ligated into
SmaI-digested pSV328A (35).
The GAL4(DBD1-147) two-hybrid cloning vector pGBT9 and the parental GAL4(TAD768-881) cloning vector pGAD424 (low expression), or the high-level expression derivative pACT2 (all from Clontech, Palo Alto, CA), were used to generate all yeast fusion protein constructs. GalAD-AR.N8 (low) was described previously as pGAL4(TAD)AR(TAD) (22). GalAD-AR.N11 (low) was generated by digesting GalAD-AR.N8 (low) with NcoI and SalI, followed by religation of the blunted vector. The previously described pTZ19NAR (22), containing an additional BamHI site by addition of a BamHI linker to the blunt-ended EcoRI site in the polylinker, was digested with SmaI and NcoI, and the resulting vector was blunt ended and religated, yielding pTZ19AR.N12. The AR fragment was excised with BamHI and cloned in frame into the corresponding site in pGAD424, yielding GalAD-AR.N12 (low). The 0.4-kb EcoRI-Acc651 (blunt ended) fragment from the previously described pSVAR-123 (8), containing an additional EcoRI site by addition of an EcoRI linker to the blunt-ended XbaI site, was cloned in frame into pGAD424 via EcoRI and SmaI compatible ends, yielding GalAD-AR.N14 (low). GalDBD-AR.N8 (low) [previously described as pGAL4(DBD)-AR(TAD) (22)] was digested with RsrII and Acc651, and the blunted vector was religated, yielding GalDBD-AR.N10 (low). The AR fragment was excised with BamHI and cloned in frame into the corresponding site in pGAD424, yielding GalAD-AR.N10 (low). GalAD-AR.N15 (low) was generated by deletion of the internal PstI fragment from GalAD-AR.N12 (low). GalAD-AR.N16 (low) was constructed by digesting GalAD-AR.N15 (low) with EcoRI and NcoI, followed by religation of the blunted vector. GalAD-AR.N13 (low) was prepared by integration of the 0.5-kb GalAD-AR.N12 (low) PstI fragment into the homologous GalAD-AR.N16 (low) site. AR.N8, containing the AR fragment cloned in frame to the SV40 large T antigen nuclear localization signal in pGAD424, was described previously as pAR(TAD3-494) (22). AR.N12 (low) was generated by exchanging the Acc651 (blunt ended)-EcoRI fragment of pGAD424 with the 0.9-kb BamHI (blunt ended)-EcoRI fragment of pTZ19AR.N12. AR.N15 (low) was prepared by digesting GalAD-AR.N15 (low) with Acc651 and BamHI, followed by religation of the blunt-ended vector. All high expression derivatives were constructed by exchanging the internal HindIII fragments with the internal pACT2 HindIII fragment. AR.N9 (high) was generated by exchanging the internal 0.9-kb StuI-EcoRI fragment of AR.N8 (high) with the 0.5-kb StuI-EcoRI fragment of AR.N3 (described above). AR.N11 (high) was generated by digesting AR.N8 (high) with NcoI and EcoRI, followed by religation of the blunt-ended vector. Similarly, the high expression level AR.N10 was generated by digesting AR.N8 (high) with RsrII and EcoRI, followed by religation of the blunt-ended vector. Only the high expression AR.N constructs were used in the two-hybrid assays. The expression vectors GalDBD-AR.C (low expression) and GalAD-AR.C (high expression) were previously described as pGAL4(DBD)AR(LBD) and pGAL4(TAD)AR(LBD), respectively (22). GalDBD-AR.C(EQ) was prepared by integration of the 0.7-kb StuI-SalI fragment of pSVAR(EQ) (as described above) into the homologous GalDBD-AR.C sites. Similarly, the high-expression GalAD-AR.C(EQ) derivative was generated by integration of the 0.45-kb EcoRI fragment of pSVAR(EQ) into the homologous GalAD-AR.C site.
CHO Cell Culture, Transfection, and LUC Assay
Chinese hamster ovary (CHO) cells were maintained in DMEM/F12
culture medium, supplemented with 5% dextran-coated charcoal-treated
FCS (Life Technologies, Gaithersburg, MD). For transcription activation
experiments, CHO cells were plated in 12-well plates at a density of
0.6 x 105 cells per well (7 cm2) and
grown overnight. Cells were transfected using the calcium phosphate
precipitation method as described previously (36), with AR expression
plasmids and where indicated with TIF2, reporter plasmids (200
ng/well), and pTZ19 carrier plasmid to a total DNA concentration of 2
µg/well. After an overnight incubation, the cells were washed
and R1881 (methyltrienolone; New England Nuclear, Boston, MA) or
vehicle (0.1% ethanol) was added. After overnight incubation the cells
were harvested for the luciferase (LUC) assay, as described previously
(37). For the GR experiments, GR.C (50 ng/well) was cotransfected with
MMTV-LUC and pTZ19 carrier plasmid as described above. After overnight
incubation, dexamethasone (10 nM) was added, and luciferase
activity was measured the next day. For the coexpression studies of
TIF2 and AR.N1, the total amount of vector, added to each well, was
equalized by the addition of empty vector, to a total concentration of
800 ng/well. In addition, pTZ19 carrier plasmid was added to a total
concentration of 2 µg DNA/well.
Yeast Growth and Methods
All yeast studies were performed in strain Y190 (MATa,
ura3–52, his3-200, ade2–101, trp1–901, leu2–3, leu2–112,
GAL4, GAL80, URA3::GAL-lacZ,
cyhr, LYS2::GAL-HIS3), which was
purchased from CLONTECH. Yeast cells were grown in the appropriate
selective minimal medium [0.67% (wt/vol) yeast nitrogen base without
amino acids and 2% (wt/vol) dextrose, pH 5.8] supplemented to the
nutritional requirements of the yeast transformants. Yeast
transformations were carried out according to the lithium acetate
method (38).
Quantitative ß-GAL activity assays, indicative of AR domain interactions, were performed as described previously (22).
Immunoprecipitation and Immunoblotting
CHO cells were plated in 80-cm2 culture flasks
(1 x 106 cells per flask), grown overnight, and
transfected with 4 µg AR-plasmid and 16 µg pTZ19 as carrier
plasmid. The next day, medium was refreshed and the cells were grown
for another day. Cytosol of the transfected cells was prepared as
described before (22). Immunoprecipitation was performed as described
previously (39), using monoclonal antibodies F112.1.1 [directed
against synthetic peptide SP197; AR aa 1–20 (40)], F39.4.1 [directed
against SP061; aa 301–320; (41)], or F52.24.4 [directed against
SP063; aa 593–612; (39)]. Subsequent to SDS-PAGE and Western
blotting, the membrane was blocked with 5% nonfat dry milk and
incubated with polyclonal antisera against SP197, SP061, or SP066
[directed against AR aa 892–910 (40)]. The proteins were visualized
by chemiluminescence detection.
AR proteins in yeast were isolated as described previously (22). Yeast-lysate samples (2.5 µl) were run on SDS-polyacrylamide gels, after which the gels were electroblotted under semidry conditions. The blots were blocked overnight with 5% nonfat dry milk and incubated with GAL4AD monoclonal antibody (CLONTECH) or the polyclonal AR-antibody SP197. Proteins were visualized by chemiluminescence detection.
In Vivo Hormone Binding Assay
CHO cells were plated in six-well plates at a density of 1
x 105 cells per well, grown overnight, and transfected as
described above with AR-C or AR-C(EQ) (0.75 µg/well). The whole-cell
binding assay was performed 48 h later. Cells were washed once
with PBS and incubated with various [3H]R1881 (New
England Nuclear) concentrations (0.01–30 nM) in the
presence or absence of a 200-fold molar excess of unlabeled R1881 in
DMEM/F12 for 1 h at 37 C. The cells were washed four times with
PBS, collected in PBS, and transferred to a centrifuge tube. After
centrifugation (10 min, 800 x g) the pellet was lysed
in 0.5 M NaOH (15 min at 56 C), and radioactivity was
determined by liquid scintillation counting.
|
ACKNOWLEDGMENTS |
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plasmid, and Dr. Kuil for making the GR.C construct. |
FOOTNOTES |
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1 These authors contributed equally to this study. 
Received for publication November 6, 1997. Revision received April 13, 1998. Accepted for publication May 1, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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D. Szapary, Y. Huang, and S. S. Simons Jr. Opposing Effects of Corepressor and Coactivators in Determining the Dose-Response Curve of Agonists, and Residual Agonist Activity of Antagonists, for Glucocorticoid Receptor-Regulated Gene Expression Mol. Endocrinol., December 1, 1999; 13(12): 2108 - 2121. [Abstract] [Full Text]
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P. Alen, F. Claessens, G. Verhoeven, W. Rombauts, and B. Peeters The Androgen Receptor Amino-Terminal Domain Plays a Key Role in p160 Coactivator-Stimulated Gene Transcription Mol. Cell. Biol., September 1, 1999; 19(9): 6085 - 6097. [Abstract] [Full Text] [PDF]
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M. J. Tetel, P. H. Giangrande, S. A. Leonhardt, D. P. McDonnell, and D. P. Edwards Hormone-Dependent Interaction between the Amino- and Carboxyl-Terminal Domains of Progesterone Receptor in Vitro and in Vivo Mol. Endocrinol., June 1, 1999; 13(6): 910 - 924. [Abstract] [Full Text]
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F. Schaufele Regulation of Estrogen Receptor Activation of the Prolactin Enhancer/Promoter by Antagonistic Activation Function-2-Interacting Proteins Mol. Endocrinol., June 1, 1999; 13(6): 935 - 945. [Abstract] [Full Text]
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X. Sui, K. S. Bramlett, M. C. Jorge, D. A. Swanson, A. C. von Eschenbach, and G. Jenster Specific Androgen Receptor Activation by an Artificial Coactivator J. Biol. Chem., April 2, 1999; 274(14): 9449 - 9454. [Abstract] [Full Text] [PDF]
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B. He, J. A. Kemppainen, and E. M. Wilson FXXLF and WXXLF Sequences Mediate the NH2-terminal Interaction with the Ligand Binding Domain of the Androgen Receptor J. Biol. Chem., July 21, 2000; 275(30): 22986 - 22994. [Abstract] [Full Text] [PDF]
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M. I. Gonzalez and D. M. Robins Oct-1 Preferentially Interacts with Androgen Receptor in a DNA-dependent Manner That Facilitates Recruitment of SRC-1 J. Biol. Chem., February 23, 2001; 276(9): 6420 - 6428. [Abstract] [Full Text] [PDF]
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Q. Wang, J. Lu, and E. L. Yong Ligand- and Coactivator-mediated Transactivation Function (AF2) of the Androgen Receptor Ligand-binding Domain Is Inhibited by the Cognate Hinge Region J. Biol. Chem., March 2, 2001; 276(10): 7493 - 7499. [Abstract] [Full Text] [PDF]
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C. E. Petre, Y. B. Wetherill, M. Danielsen, and K. E. Knudsen Cyclin D1: Mechanism and Consequence of Androgen Receptor Co-repressor Activity J. Biol. Chem., January 11, 2002; 277(3): 2207 - 2215. [Abstract] [Full Text] [PDF]
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A. Bubulya, S.-Y. Chen, C. J. Fisher, Z. Zheng, X.-Q. Shen, and L. Shemshedini c-Jun Potentiates the Functional Interaction between the Amino and Carboxyl Termini of the Androgen Receptor J. Biol. Chem., November 21, 2001; 276(48): 44704 - 44711. [Abstract] [Full Text] [PDF]
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