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Originally published In Press as doi:10.1074/jbc.M209322200 on October 4, 2002

J. Biol. Chem., Vol. 277, Issue 51, 49230-49237, December 20, 2002
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Characterization of the Two Coactivator-interacting Surfaces of the Androgen Receptor and Their Relative Role in Transcriptional Control*

Valerie ChristiaensDagger §, Charlotte L. Bevan§||, Leen CallewaertDagger , Anna HaelensDagger **, Guy VerrijdtDagger **, Wilfried RombautsDagger , and Frank ClaessensDagger **DaggerDagger

From the Dagger  Division of Biochemistry, Faculty of Medicine, Campus Gasthuisberg, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium and  Prostate Cancer Research Group, Department of Cancer Medicine, Imperial College London, London W12 ONN, United Kingdom

Received for publication, September 11, 2002, and in revised form, October 3, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The androgen receptor interacts with the p160 coactivators via two surfaces, one in the ligand binding domain and one in the amino-terminal domain. The ligand binding domain interacts with the nuclear receptor signature motifs, whereas the amino-terminal domain has a high affinity for a specific glutamine-rich region in the p160s. We here describe the implication of two conserved motifs in the latter interaction. The amino-terminal domain of the androgen receptor is a very strong activation domain constituent of Tau5, which is mainly active in the absence of the ligand binding domain, and Tau1, which is only active in the presence of the ligand binding domain. Both domains are, however, implicated in the recruitment of the p160s. Mutation analysis of the p160s has shown that the relative contribution of the two recruitment mechanisms via the signature motifs or via the glutamine-rich region depend on the nature of the enhancers tested. We propose, therefore, that the androgen receptor-coactivator complex has several alternative conformations, depending partially on the context of the enhancer.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The androgen receptor (AR)1 is a member of the steroid receptor family of transcription factors. Steroid receptors are ligand-inducible sequence-specific transcription factors with highly conserved DNA binding domains (DBDs), moderately conserved ligand binding domains (LBDs), and divergent amino-terminal domains (NTD) (1-4). Two transactivating functions (AFs) have been characterized, AF1 in the NTD and AF2 in the LBD. For the AR, AF1 has strong constitutive activity, since deletion of the LBD results in a molecule that can activate a reporter gene to the same extent as the full-length receptor in the presence of ligand, whereas AF2 appears to be weak (5-8). This is in contrast to what occurs in most other nuclear receptors, for example for the estrogen receptor (ER), in which AF2 is the major activation domain (9). The precise residues and mechanisms that contribute to the AF1 activity of the AR have not been conclusively established. Almost the entire NTD is required for full transcriptional activity of the full-length receptor, whereas a core region located between residues 101 and 360 (Tau1) contributes 50% of activity (10). When a constitutively active AR mutant lacking an LBD is studied, the region necessary for transcriptional activation shifts to the region 370-494 (Tau5) (10).

Binding of the appropriate hormones to the steroid hormone receptors causes a translocation of the receptors to enhancer elements in the promoters of target genes. Transcriptional coactivators are recruited to the promotor through protein-protein interaction with the receptor (11-14). Most known coactivators are complex proteins that harbor multiple activation domains and receptor-interacting domains (15-16). The best studied group of coactivators is the p160 family of 160-kDa proteins. Three family members have been identified. The first p160 coactivators cloned were the human steroid receptor coactivator 1 (SRC1) and the transcription intermediary factor 2 (the human orthologue of glucocorticoid receptor-interacting protein 1, or GRIP1). The third member was reported simultaneously by several groups as receptor-associated coactivator 3, p300/CREB-binding protein-interacting protein, activator of thyroid hormone receptor, and thyroid receptor associated molecule 1 (17-20). Upon recruitment to the promoters by nuclear receptors, the coactivators affect transcription by modifying the chromatin structure through the recruitment of cofactors that possess histone acetyltransferase and histone methyltransferase activity. More specifically, p160 proteins appear to recruit both CREB-binding protein, which in turn interacts with the p300/CREB-binding protein-associated factor (21-25), and coactivator-associated arginine methyltransferase 1 (CARM1) (26-27). There is also evidence that coactivators are directly contacting and modifying the activity of the transcription initiation complex (12-13).

The interaction of p160 coactivators with the most nuclear receptors is strictly ligand-dependent and requires an intact AF2 domain. The nuclear receptor interacting domain of the p160s is located in the central region of the protein and contains three conserved alpha -helical motifs with a consensus amino acid sequence LXXLL (where L stands for leucine, and X stands for any amino acid) (28-32). The mutation of these motifs to LXXAA (where A stands for alanine) abolishes the interaction with the AF2 of nuclear receptors. However, it has been shown that a SRC1 mutant containing no functional LXXLL motif is still able to potentiate the transcriptional activity of the AR (33). This led to the discovery of a second, ligand-independent mode of recruitment of p160 coactivators to the amino terminus of the AR. In this study, we map the interaction surfaces between the androgen receptor and the p160 coactivators in detail and integrate them in a new working hypothesis on the mechanism of action of the androgen receptor as a transcription factor.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Plasmid Construction-- Restriction and modifying enzymes were obtained from either Fermentas or Invitrogen. The oligonucleotides were purchased from Eurogentec. All constructs created by PCR amplification were verified by sequencing.

The pCMV-beta Gal vector was supplied by Stratagene. The reporter plasmid pMMTV-luc was obtained from Dr. P. Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch Cedex, France). The expression vectors for the full-length AR (pSV-AR) and for the NTD/DBD were a kind gift from Dr. A. O. Brinkmann (Erasmus University of Nijmegen, Nijmegen, The Netherlands). The DBD/LBD fragment was PCR-amplified from pSV-AR with primers incorporating BamHI restriction sites and inserted in the BamHI restriction site of the pSG5 vector. The expressing vector for wild type SRC1e (pSG5SRC1e), for SRC1e with mutation of the three LXXLL-motifs (pSG5SRC1eM123), the reporter plasmid containing five Gal4-binding sites (pGal4-luc), the vectors containing the SRC1 fragments fused to the Gal4DBD, and the expression vector for the ER LBD fused to the VP16 activation domain were kind gifts from Dr. M. G. Parker (Imperial Cancer Research Fund, London, UK). To generate VP16 fusion proteins, the plasmid pSNATCH-II was used (34). The pSNATCH-II vector was modified to create an extra SmaI restriction site in the multiple cloning site (7). The AR NTD, the AR LBD, and the ER NTD, fused to the VP16 activation domain, were made by PCR amplification from pSV-AR or from pSG5-ER with primers incorporating BamHI restriction sites and insertion in the BglII restriction site of the modified pSNATCH-II vector. The deletion and mutation fragments of the glutamine-rich (Qr) domain of SRC1e were PCR-amplified from pSG5SRC1e with primers incorporating BglII or BamHI restriction sites. For the expression of SRC1 fragments fused to the DBD of the yeast transcription factor Gal4 (Gal4DBD), the PCR-generated fragments were inserted in the BamHI restriction site of pAB-Gal4 (35). To generate the SRC1-VP16 fusion proteins, the PCR-generated fragments were cloned in-frame with the VP16 activation domain in the BglII restriction site of pSNATCH-II. The yeast vectors encoding the LexA DNA-binding site fused to different fragments of SRC1e were created by PCR cloning into the BamHI restriction site of the BTM116 vector. The reporter plasmid TAT-GRE-E1BTATA-luc was obtained from Dr. G. Jenster (Erasmus University of Rotterdam, Rotterdam, The Netherlands). The Gal4-AR AF1 fusion vector has been described previously (33). GST fusion vectors were created by insertion of the relevant fragment (SRC1-(1050-1185) or SRC1-(1050-1185 mutA/B)) into the BamH1 restriction site of the pGEX-5x-3 vector. The expression vectors for deletion constructs of the NTD in the full-size AR (AR Delta 37-494, AR Delta 370-494, AR Delta 1-360) were a kind gift from Dr. A. O. Brinkmann (Erasmus University of Nijmegen, Nijmegen, The Netherlands). The expression vectors for the isolated Tau1 or Tau5 region of the AR fused to the VP16 activation domain were created by PCR cloning into the BglII restriction site of the pSNATCH-II vector.

Cell Culture, Transfection, Luciferase, and beta -Galactosidase Assays-- The COS7 cells and CV1 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 1000 mg/liter glucose supplemented with 100 units/ml penicillin, 0.1 mg/ml streptomycin, 4 µg/ml insulin, and 10% heat-inactivated fetal calf serum (Invitrogen) at 37 °C and 5% CO2. Twenty-four hours before transfection, cells were plated in 96-well plates (Nunc) at a density of 10,000 cells/well (COS7 cells) and 2,000 cells/well (CV1 cells) in the same culture medium but with 5% fetal calf serum; endogenous steroids were removed by an incubation with dextran-coated charcoal. The COS7 cells and CV1 cells were transfected with the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) in accordance to the manufacturer's instructions. To evaluate the transfection efficiency, 20 ng of an expression plasmid containing the beta -galactosidase reporter gene under control of the cytomegalovirus promotor (pCMV-beta Gal) was cotransfected as an internal control. The first day after transfection, the medium was refreshed, and 1 nM synthetic androgen R1881 (PerkinElmer Life Sciences) or 1 nM estradiol (PerkinElmer Life Sciences) was added when appropriate. After a further 24 h, cells were harvested in 25 µl of passive lysis buffer (Promega). Luciferase activity of 2.5 µl of lysate was measured in a Lumiskan Ascent luminometer (Labsystems) using the luciferase assay kit (Promega) according to the protocol provided by the company. beta -Galactosidase activity of 2.5 µl of lysate was determined with the chemiluminescent reporter assay kit from Tropix (Westburg, The Netherlands). All transfection experiments were performed at least 3 times independently (n = 3) in triplicate. Luciferase activity is corrected for transcription efficiency by normalizing against beta -galactosidase activity. Error bars represent S.E.

Method for COS1 Transfection Reporter Assay-- Twenty-four hours before transfection, cells were plated in 24-well plates (Falcon) in phenol red-free medium supplemented with 5% dextran charcoal-stripped fetal calf serum. Transfection was performed using a modified calcium phosphate method (36), each well receiving 50 ng of AR expression vector, 1 µg of androgen-responsive reporter plasmid, and 100 ng of pCMV beta -galactosidase plasmid together with varying amounts of coactivator expression plasmid plus empty vector to standardize the amounts of DNA. After incubation for 16 h, the cells were washed, and fresh medium was added containing 10-8 M mibolerone (a synthetic androgen, PerkinElmer Life Sciences) or vehicle (ethanol). After a further 24 h, cells were washed twice with phosphate-buffered saline and lysed in 0.5 M Hepes, pH 7.8, 2% Triton N101 (Sigma), 1 mM CaCl2, 1 mM MgCl2. Extracts were analyzed for luciferase activity using the Luclite kit (PerkinElmer Life Sciences), and values were corrected for beta -galactosidase activity, measured using the Galacto-Light chemiluminescent assay (Tropix).

Yeast Culture and Transfection-- The yeast strain L40 (MATalpha , trp1, his3, leu2, ade2, LYS2::(LexAop)4-HIS3, URA3::(LexAop)8-LacZ)), containing a LexA-responsive lacZ reporter, was transformed by electroporation with vectors encoding fusion proteins and transformants selected for the appropriate plasmid markers. To perform two-hybrid assays, transformants were grown to late log phase in 10 ml of selective medium (yeast nitrogen base containing 1% glucose and appropriate supplements) where appropriate in the presence of 10-7 M mibolerone. Cells were then harvested, washed, suspended in 0.1 M Tris-HCl, pH 7.5, 0.5% Triton X-100, snap-frozen in a dry ice-ethanol bath, and thawed. An aliquot of this extract was assayed for beta -galactosidase activity as described previously (37), and the protein content was measured by reading the optical density at 600 nm. Activity was calculated as (1000 × A420)/(A600 × reaction time in min) and expressed as beta -galactosidase units. Each assay was repeated on at least three independent transformants, and the data represent the mean ± S.D.

Preparation of COS7 Cellular Extracts-- COS7 cells were plated in six-well plates (Nunc) in Dulbecco's modified Eagle's medium at a density of 2 × 105 cells/well, grown overnight, and transfected by the FuGENE 6 method as described above with 1 µg of the pSNATCH-II vectors containing the Qr fragments. The first day after transfection, fresh culture medium was added. After a further 24 h, the medium was removed, and cells were washed twice with 1.5 ml of ice-cold phosphate-buffered saline. The cells were collected in 0.5 ml of ice-cold phosphate-buffered saline/well, transferred to a microcentrifuge tube, and pelleted by centrifugation. The phosphate-buffered saline was removed, and cells were harvested for 20 min in 30 µl of passive lysis buffer. The cell debris was pelleted by centrifugation, and the supernatant, which contained the cellular proteins, was used in a Western blot assay. The protein concentration was measured with the Coomassie protein assay (Pierce). The yield was about 60 µg of protein, starting from 2 × 105 cells.

Western Blot Analysis-- Western blot analysis of cellular extract of COS7 cells, which were transfected with the pSNATCH-II plasmids containing the Qr fragments, was performed to confirm the expression of the Qr-VP16AD proteins. Ten µg of nuclear extract was diluted in 10 µl of sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 0.002% bromphenol blue, 4% SDS, 10% beta -mercaptoethanol), loaded on a 15% SDS-polyacrylamide gel (38), and analyzed by Western blotting (39). After transfer of the proteins onto Hybond P membranes (Amersham Biosciences), the membranes were incubated with the M2 anti-FLAG antibody (1/1000) (Stratagene) as the primary antibody and with a peroxidase-labeled rabbit anti-mouse antibody (1/10000) (Dako) as the secondary antibody. Immunodetection was performed by using an ECL chemiluminescence detection kit (ECL, Amersham Biosciences) and visualized by exposure to an X-Omat AR film (Eastman Kodak Co.).

GST Pull-down Assay-- Androgen receptor was transcribed and translated in vitro from the pSG5 expression vector in the presence of [35S]methionine in reticulocyte lysate (Promega) according to the manufacturer's protocol. GST fusion proteins were induced, purified, bound to Sepharose beads (Amersham Biosciences), and incubated with translated proteins as previously described (40) in NETN buffer (20 nM Tris, pH 8.0, 1 nM EDTA, 0.5% Nonidet P-40, 100 mM NaCl) in the presence or absence of 10-7 M mibolerone. After washing, samples were separated on 8% SDS-polyacrylamide gels which were fixed and dried and then visualized using Typhoon phosphorimaging.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SRC1e Is a Coactivator of the NTD of the AR-- Nuclear receptors contain two transcription activation functions, AF1 in the NTD and AF2 in the LBD. Two SRC1 coactivator isoforms, a and e, diverge at their carboxyl termini and are functionally distinct in their abilities to enhance the activity of the ER (40). To analyze the capacity of SRC1e in stimulating the two activation functions of the AR, we performed a transient transfection experiment. Full-length SRC1e was cotransfected in COS7 cells with expression vectors for the full-length AR and for deletion constructs of the AR in the presence of a mouse mammary tumor virus (MMTV) luciferase reporter plasmid (Fig. 1). The deletion constructs NTD/DBD and DBD/LBD each retain the AR DNA binding domain but are missing the LBD and the amino-terminal domain, respectively. The activity of the full-length AR on the androgen-responsive reporter was enhanced 3-fold by the coexpression of full-length SRC1e in the presence of androgen. Transfection of the DBD/LBD fragment failed to demonstrate any measurable luciferase activity in the presence of ligand and in the presence of SRC1e. The NTD/DBD fragment transactivated the luciferase gene, and coexpression of SRC1e enhanced the reporter activity. This effect was ligand-independent, demonstrating that recruitment to and enhancement of AF1 by SRC1e is entirely separable from its recruitment to and effect on AF2.


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  		Fig. 1.   SRC1e is a coactivator of the NTD of the AR. The interaction of the full-length AR and of AR fragments with SRC1e was examined in a transient transfection assay. COS7 cells were transfected as described under "Experimental Procedures": 200 ng of MMTV-luc reporter plasmid, 20 ng of the expression vectors for the AR fragments, and 50 ng of SRC1e or the empty pGEM15 vector were added per well. Cells were incubated with or without 1 nM R1881. Gray bars show activity in the absence, and black bars show activity in the presence of hormone. The activity of the luciferase reporter was corrected for beta -galactosidase activity.

AR and ER Use Different p160 Interfaces-- To compare the recruitment of SRC1 to the activation functions of the AR with the recruitment to those of the ER, we tested different SRC1 fragments (Fig. 2A) for their ability to interact with the AR and the ER in a double-hybrid experiment. SRC1 fragments fused to a Gal4DBD were coexpressed with the LBD or the NTD of the AR or the ER fused to a VP16 activation domain (Fig. 2, B and C). For the 781-988 region, high luciferase activity was observed under all conditions (data not shown). This is due to the AD1 function of SRC1e (40). The 570-780 region is the nuclear receptor-interacting domain that contains the known LXXLL motifs (29, 33). This fragment interacts with the LBD of the ER in the presence of estrogen. This interaction is not seen for the LBD of the AR (Fig. 2B). The 1241-1441 region of SRC1a, which contains a LXXLL motif, interacts with the LBD of the ER and also with the LBD of the AR, albeit to a lower extent.


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  		Fig. 2.   AR and ER use different p160 interfaces. A, domains of p160 coactivators; schematic representation of the SRC1 fragments. Depicted are the basic helix-loop-helix and Per-Arnt-Sim domain (bHLH-PAS), the domain interacting with nuclear receptors (NID), the two activation domains (AD), and the Qr domain. B and C, Double-hybrid assay. COS7 cells were transfected as described under "Experimental Procedures": 50 ng/well of the SRC1 fragments fused to the Gal4DBD was cotransfected with 50 ng/well of the empty pSNATCH-II vector or with 50 ng/well of the pSNATCH-II vector containing the NTD or LBD of the AR or the ER. 20 ng of the Gal4-luc reporter plasmid was added per well. White bars show activity in the presence of the empty pSNATCH-II vector, gray bars show activity in the presence of the LBD (B) or NTD (C) of the ER, and black bars show activity in the presence of LBD (B) or NTD (C) of the AR. The activity of the luciferase reporter was corrected for beta -galactosidase activity.

A different domain of SRC1 interacts with the amino-terminal domain. It is mainly the 989-1240 fragment that has a high affinity for the NTD of the AR, but the interaction between the NTD of the ER and this fragment is barely detectable (Fig. 2C). A similar interaction was seen for the homologous fragment of the transcription intermediary factor 2 coactivator (data not shown). For the region 199-569, the strong interaction with the NTD of the AR could not be reproduced with the homologous transcription intermediary factor 2 fragment and was, therefore, not analyzed further in this work.

Analysis of the Qr Domain of SRC1e-- The previous results confirm our original observation that SRC1e interacts with the NTD of the AR via the region 989-1240 (33). This region is rich in glutamines and contains three amino acid motifs, A, B, and C, which are conserved between the p160 coactivator proteins (Fig. 3A). To map the residues within this Qr domain responsible for binding the NTD of the AR, we fused truncated fragments to the DNA binding domain of Gal4 (Fig. 3A). We tested whether these fragments were able to interact with the NTD of the AR using a mammalian double-hybrid system in COS7 cells as well as in CV1 cells (Fig. 3B). The AR NTD was fused to the VP16 activation domain. In the absence of the NTD/VP16 fusion, there was no activation of the luciferase reporter gene, implying that there is no activation function in the isolated Qr domain. When we cotransfected the NTD/VP16 vector, luciferase activity indicated binding to the Qr domain. Surprisingly, a 3-fold higher activity was seen for the construct with residues 1050-1185, the minimal region containing the three conserved motifs, when compared with the 989-1240 fragment. Deletion of the C motif (SRC1e 1050-1145) caused a decrease in luciferase activity. Further deletion on the carboxyl-terminal side abrogated the interactions. None of the conserved motifs, when tested separately, did interact with the AR NTD. We also tested the reverse double-hybrid assay in COS7 cells as well as in CV1 cells (Fig. 3C). SRC1e fragments were fused to the VP16 activation domain and cotransfected with an expression vector for the AR NTD/DBD fragment in the presence of a MMTV luciferase reporter plasmid. The NTD/DBD construct strongly stimulated the activation of the luciferase reporter gene due to the presence of the activation function AF1. The VP16 activation domain fusion with SRC1e 1050-1185 or 1050-1145 interacted with the NTD, since they both enhanced the reporter activity. Similar to the earlier observations (Fig. 3B), truncations at the carboxyl-terminal side of the Qr domain abolished the interactions with the NTD. Western analysis confirmed the expression of similar levels of each SRC1e fragment.


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  		Fig. 3.   Analysis of the Qr domain of SRC1e. A, alignment of the p160 coactivator proteins in the glutamine-rich region; schematic representation of the different deletion fragments of the Qr domain of SRC1e. The A, B, and C motifs are depicted in bold. The lower panel depicts SRC1 fragments used in double-hybrid analysis. TIF2, transcription intermediary factor 2; RAC3, receptor-associated coactivator 3. B, double-hybrid assay. COS7 cells and CV1 cells were transfected as described under "Experimental Procedures": 50 ng/well of the SRC1 fragments fused to the Gal4DBD was cotransfected with 50 ng/well of the empty pSNATCH-II vector or with 50 ng/well of the pSNATCH-II vector containing the NTD of the AR. 20 ng of the Gal4-luc reporter plasmid was added per well. Gray bars show activity in the presence of the empty pSNATCH-II vector, and black bars show activity in the presence of the NTD-VP16AD. C, reverse double-hybrid assay. 50 ng/well of the expression vector for the NTD/DBD of the AR was cotransfected with 50 ng/well of the empty pSNATCH-II vector or with 50 ng/well of the pSNATCH-II vectors containing the Qr fragments. 200 ng of MMTV-luc reporter plasmid was added per well. The right-hand panel depicts a Western blot assay of the SRC1-VP16AD fragments. COS7 cells were transfected with 1 µg of the pSNATCH-II vectors containing the SRC1 fragments. The Western blot was assayed as described under "Experimental Procedures."

These results suggested that the minimal binding fragment covers motifs A and B. To verify whether both motifs are necessary for this interaction, we introduced point mutations in the fragments 1050-1185 and 1050-1145 (Fig. 4A). To investigate the interaction of these mutated fragments, we performed the double-hybrid assays in COS7 cells (Fig. 4, B and C). In the SRC1e 1050-1185 fragment, mutation of motif A or B or both impaired the interaction of this construct with the NTD of the AR. However, we observed residual activity when we cotransfected the single mutated fragments. The same results were obtained when mutations were analyzed in the SRC1e 1050-1145 fragment.


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  		Fig. 4.   Analysis of the Qr domain of SRC1e. A, representation of the sequence of the conserved A and B motifs. The amino acids that have been changed are depicted in bold, relative to the wild type. B, double-hybrid assay. COS7 cells were transfected as described under "Experimental Procedures": 50 ng/well of the mutated Qr fragments fused to the Gal4DBD was cotransfected with 50 ng/well of the empty pSNATCH-II vector or with 50 ng/well of the pSNATCH-II vector containing the NTD of the AR. 20 ng of the Gal4-luc reporter plasmid was added per well. Gray bars show activity in the presence of the empty pSNATCH-II vector, and black bars show activity in the presence of the NTD-VP16AD. C, reverse double-hybrid assay. 50 ng/well of the expression vector for the NTD/DBD of the AR was cotransfected with 50 ng/well of the empty pSNATCH-II vector or with 50 ng/well of the pSNATCH-II vectors containing the mutated Qr fragments. 200 ng of MMTV-luc reporter plasmid was added per well. The right-hand panel depicts a Western blot assay of the SRC1-VP16AD fragments. COS7 cells were transfected with 1 µg of the pSNATCH-II vectors containing the SRC1 fragments. The Western blot was assayed as described under "Experimental Procedures." D, yeast double-hybrid assay. LexA DNA binding domain fused to fragments of SRC1 as indicated was used as bait. The activity of the lacZ reporter gene in the presence of AR AF1 fused to the Gal4 activation domain represents interaction of the SRC1 fragment with AF1. Activity of the reporter gene in the absence of AF1 (Gal4 activation domain alone) was negligible. The average of three independent transformants is shown ±S.D. E, mutation of the A and B boxes abolishes in vitro interaction of AR with SRC1-(1050-1185). GST fusion proteins coupled to Sepharose beads were incubated with in vitro translated [35S]methionine-labeled full-length AR in the absence or presence of 10-7 M androgen. After extensive washing samples were boiled and run on an 8% SDS-polyacrylamide gel that was fixed and dried, and the bound labeled protein was visualized by phosphorimaging. F, mutation of the A-box abolishes coactivation by SRC1e. COS1 cells were transfected with AR, reporter, and various SRC1e constructs as indicated in the absence (black bars) or presence (gray bars) of 10 nM mibolerone. The hormone-dependent activity in the absence of added SRC1e was set at 100% for each experiment, and other values are expressed relative to this. Data represent the average ±S.D. of three experiments, each performed in duplicate and are normalized for transfection efficiency. WT, wild type.

Interaction of the Qr domain with the AF1 region of the AR was also analyzed in yeast double-hybrid assays. The different SRC1e fragments were fused to the lexA DBD, whereas AR AF1 was fused to the Gal4 activation domain (Fig. 4D). In yeast, similar results were obtained as in mammalian cells. The minimal binding fragment is the 1050-1145 SRC1e fragment that covers motifs A and B. Mutation of the A motif and deletion of the 1050-1123 region in the Qr domain impairs the interaction with AF1.

Using the GST pull-down system, we investigated the ability of the two proteins to interact in vitro. Full-length in vitro translated AR bound to SRC1-(1050-1185) fused to GST in the presence as well as in the absence of androgen (Fig. 4E). This interaction was not observed when the A and B motif of the SRC1 fragment were mutated.

We observed that although either the A or B box in isolation was inadequate to mediate the interaction with AF1 (Fig. 3B), mutation of a single motif was sufficient to severely impair the interaction in double-hybrid experiments (Fig. 4, B-D). We tested the effect of a single mutation on the ability of SRC1e to coactivate the AR. On two different reporters, wild type SRC1e coactivated the AR, since the induction factors increased 2-3-fold. However, point mutations in the A-box of full-length SRC1e renders it unable to enhance ligand-dependent transcription from the androgen-responsive promotors, implying that functional in vivo recruitment of SRC1e to the AR requires an intact A-box (Fig. 4F). In contrast, mutation of the LXXLL motifs abolishes coactivation of the AR only on the MMTV promoter but not on the TAT-GRE-E1BTATA-luc promoter, since cotransfection of the M123 mutant results in a 2-fold increase of the androgen-responsiveness of the latter construct (Fig. 4F).

The Tau1 Domain Is Sufficient for the Activation of AF1 by SRC1e in the Full-length AR-- In a transient transfection experiment, we tested whether the isolated Qr domain of SRC1e is capable of interacting with the full-length AR and with deletion constructs of the AR (Fig. 5A). Full-length SRC1e or the Qr fragment fused to the VP16 activation domain were coexpressed in COS7 cells with deletion constructs of the AR in the presence of a MMTV-luciferase reporter plasmid (Fig. 5B). Deletion of most of the NTD (AR Delta 37-494) caused a loss of AR activity. When Tau5 (AR Delta 370-494) was deleted, there was still hormone-dependent activity, albeit lower than for the full-length AR. Deletion of Tau1 (AR Delta 1-360) resulted in an inactive receptor. These effects were reported earlier by Jenster et al. (10). The activity of the full-length AR as well as the Tau5-deleted form was enhanced 3-fold by the coexpression of full-length SRC1e. The Qr fragment of SRC1e was able to interact with the full-length as well as with the Tau5-deleted AR, and mutation of the A motif in the Qr fragment largely prevented this interaction. Thus, in the presence of the LBD, Tau1 seems sufficient for coactivation of AR activity by SRC1e via interaction with the Qr region.


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  		Fig. 5.   The Tau1 domain is sufficient for the activation of AF1 by SRC1e in the full-length AR. A, schematic representation of the full-length AR and of AR deletion fragments. B, transient transfection assay. COS7 cells were transfected as described under "Experimental Procedures": 20 ng/well of the expression plasmids for the full-length AR and for AR deletion fragments was cotransfected with 50 ng/well of the empty pGEM15 vector, 50 ng/well of the expression vector for SRC1e, or with 50 ng/well of the SRC1 fragment fused to the VP16 activation domain. Cells were incubated with or without 1 nM R1881. The gray bars show activity in the absence, and the black bars show activity in the presence of hormone. The activity of the luciferase reporter was corrected for beta -galactosidase activity. H, hinge region.

The Isolated Tau1 or Tau5 Domain Is Not Able to Interact with the Glutamine-rich Domain of SRC1e-- In the absence of the LBD, the Tau5 region mediates transcription activation (10). To investigate whether SRC1e is involved, we attempted to determine whether NTD fragments could interact with the glutamine-rich domain of SRC1e. We have examined this interaction in a double-hybrid system in COS7 cells (Fig. 6). All NTD fragments were expressed as demonstrated in the Western blot. SRC1e fragments fused to a Gal4DBD were expressed alone or together with NTD fragments fused to the VP16 activation domain in the presence of a Gal4-responsive luciferase reporter. Although the full-length NTD fused to the VP16 activation domain interacts with the Qr fragment 1050-1185 of SRC1e, we could not detect a significant interaction between the isolated Tau1-(1-360) or Tau5-(370-494) and the Qr domain.


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  		Fig. 6.   The isolated Tau1 or Tau5 domain is not able to interact with the glutamine rich domain of SRC1e. Shown is a double-hybrid assay. COS7 cells were transfected as described under "Experimental Procedures": 50 ng/well of the SRC1 fragments fused to the Gal4DBD was cotransfected with 50 ng/well of the NTD fragments fused to the VP16 activation domain. 20 ng of the Gal4-luc reporter plasmid was added per well. The activity of the luciferase reporter was corrected for beta -galactosidase activity.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Steroid receptor coactivator 1 is a transcriptional coactivator that is capable of potentiating the activity of nuclear receptors including the ERs and the AR (12-13). For the ERs, it is widely accepted that the ligand-dependent transcription activation capacity is mediated via activation function AF2 in the ligand binding domain (40). However, for the AR a different mechanism of activation seems to exist since the AF2 is very weak and the NTD harbors a strong transactivating function (7, 33). This correlates with our earlier observation that binding of SRC1 to the LBD of the AR is weak. Instead, SRC1 is primarily recruited by the NTD of the AR (7, 33).

The major transcription activation domains differ between the AR and the ER. This suggested that the mechanisms by which these receptors recruit SRC1 are also different. SRC1 interacts with the LBD of nuclear receptors via LXXLL motifs (where L stands for leucine, and X stands for any amino acid) (29). Mutation of these motifs to LXXAA (where A stands for alanine) destroys the ligand-dependent interaction of the ERalpha but not of the AR (33). To compare the interaction of SRC1 with the LBDs of the AR and the ERalpha , we tested different fragments of SRC1 in a double-hybrid assay. Our results confirm the hypothesis that an alternative pathway exists for recruitment of SRC1 to the AR. The region 570-780, with three LXXLL motifs, and the carboxyl-terminal region of SRC1a, with a fourth LXXLL motif, interact with the LBD of the ERalpha and to a lower extent with the LBD of the AR. In addition, SRC1 interacts with the NTD of the AR mainly via the region 989-1240. In comparison, the ERalpha NTD interaction with the Qr domain is barely detectable despite earlier reports by Webb et al. (41).

The SRC1e region 989-1240 is rich in glutamines and contains 3 amino acid motifs, A, B, and C, that are conserved between the p160 coactivator proteins. To map the residues within this Qr domain involved in the binding of the NTD of the AR, we made progressive carboxyl-terminal-deleted constructs and constructs that contain only one of the three conserved motifs. These fragments were tested in a double-hybrid system in mammalian cells and in yeast for their ability to interact with the NTD of the AR. The results demonstrate that the minimal region in the Qr domain of SRC1e for interaction with the NTD of the AR is between positions 1050 and 1145; this region covers motifs A and B. Point mutation analysis of the A and B motifs in the glutamine-rich region of SRC1e has shown that they are both required for the interaction with the NTD of the AR. The residual activity of the single-mutated fragments implies that both motifs independently interact weakly with the NTD. The binding to motif A and B could, therefore, be cooperative. However, multimers of the A or B motif did not interact with the NTD of the AR (data not shown).

Within the NTD, different transcription activation units (Taus) have been described. The observation that different Taus are used depending on the absence or the presence of the LBD is an indication that in the native receptor these domains communicate through direct or indirect interactions (7, 10). Full-size SRC1e interacts with the isolated NTD via the Qr domain. In the absence of the LBD, only the activation function Tau5 is active. There was a weak interaction between Tau5 and the Qr domain of SRC1e (Fig. 6 and Ref. 33), and this was abolished by mutation of the A motif.

Deletion of both Tau functions resulted in an inactive receptor, and overexpression of SRC1e is unable to restore full activity. The effect of SRC1e on Tau1, which is only active in the full-size AR, is more complicated to analyze because not only Tau1 and -5 are present but also the LBD, which might form secondary interactions with SRC1e via the LXXLL motifs. We therefore tested deletions of Tau1 and Tau5 in the AR for interactions with the Qr SRC1e fragment. Deletion of Tau1 abrogated Qr binding, whereas deletion of Tau5 had only a minor effect. From these experiments, one would conclude Tau5 is dispensable for AR activity, but in the context of the isolated NTD, Tau5 is necessary for the transactivation activity and for the interaction with the Qr domain. We, therefore, can conclude that both Tau1 and Tau5 interact with the Qr domain of SRC1e.

As postulated earlier, the activation function seems to shift in the NTD depending on the presence or the absence of the LBD. It has been described previously that on some promotors, the interaction between the NTD and the LBD of the AR is more important than on others (42); for example, on the MMTV promotor, a direct NTD/LBD interaction is apparently not required for full activity. In Fig. 4F, the transient transfection assay shows that mutation of either the LXXLL motifs (M123) or of the A-box abolishes coactivation. Thus, both interfaces between the AR and SRC1e are required. However, on the TAT-GRE-E1BTATA-luc promotor, mutation of the A-box but not the LXXLL motifs abolishes coactivation, implying that only the amino-terminal interaction is required. Possibly on this promoter the NTD and LBD interact directly, whereas on the MMTV enhancer more complex interactions take place.

In conclusion, we have shown that in contrast to ERalpha , the transcription activation capacity of the AR is mediated via a ligand-independent activation function AF1 in the amino-terminal domain. This AF1 interacts with p160 coactivators such as SRC1e via a glutamine-rich domain. Analysis of this Qr domain revealed that two conserved motifs are needed in this interaction. The Qr domain is sufficient for binding of SRC1e to the AR in eucaryotic cells. Tau1 seems to be involved, since deletion of Tau1 results in an inactive AR, and deletion of Tau5 results in an active AR. However, in the isolated NTD both Tau1 and Tau5 are necessary for Qr recruitment. Our results combined with earlier work point to a complex interplay between the two Taus, the LBD and different regions of the p160 coactivators. Another level of complexity originates from the observation that the AR interacts in two alternative conformations with its response elements (43-45). In Fig. 7, models of the intra- and intermolecular interactions between the androgen receptor and the p160 coactivators are shown (46-50). Note that no stoichiometric data are available on numbers of coactivator molecules per receptor dimer, although one p160 per receptor dimer was suggested (51). In addition, the AR and the p160 coactivators recruit different enzymes, histone acetyltransferases, CREB-binding protein/p300, p300/CREB-binding protein-associated factor, and histone methyltransferases, coactivator-associated arginine methyltransferase 1 (26-27, 52-53). Because these proteins interact with each other via overlapping domains, a detailed knowledge of the structure-function relations within the interaction domains will be instrumental in the further study of the molecular biology of the steroid responses in general and the androgen responses in particular.


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  		Fig. 7.   Model of the complex interplay between the androgen receptor and the p160 coactivators. Schematic representation of the functional domains of the AR and of the p160 coactivators. Intermolecular interactions between the androgen receptor and the p160 coactivators are shown; activation domains Tau1 and Tau5 in the NTD recruit the Qr. As reported earlier (33), the nuclear receptor-interacting domain of SRC1 interacts with the LBD. Intramolecular interactions within the AR are shown; Tau1 and the FQNLF motif within the NTD interact with the LBD (7, 10, 46-50). The activation domains AD1 and AD2 of SRC1 recruit CREB-binding protein (CBP) and coactivator-associated arginine methyltransferase 1 (CARM1) respectively.


    ACKNOWLEDGEMENTS

We thank H. Debruyn and R. Bollen for excellent technical assistance. We are indebted to P. Chambon, A. Brinkmann, M. Parker, and G. Jenster for plasmids.

    FOOTNOTES

§ These authors contributed equally to this work.

|| Supported by a grant from the Prostate Cancer Charity.

** Holders of a Postdoctoral Fellowship of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen.

Dagger Dagger To whom correspondence should be addressed. Tel.: 32-16-345770; Fax: 32-16-345995; E-mail: frank.claessens@med.kuleuven.ac.be.

Published, JBC Papers in Press, October 4, 2002, DOI 10.1074/jbc.M209322200

This work was supported by the Geconcerteerde Onderzoeksactie van de Vlaamse Gemeenschap and by grants from the Fonds voor Wetenschappelijk Onderzoek Vlaanderen and the Inter Universitaire Attractie Pool, Belgian State Prime Minister's Office, Federal Office for Scientific, Technical, and Cultural Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    ABBREVIATIONS

The abbreviations used are: AR, androgen receptor; DBD, DNA binding domain; LBD, ligand binding domain; NTD, amino-terminal domain; AF, activation function; ER, estrogen receptor; Tau, transcription activation unit; SRC1, steroid receptor coactivator 1; Qr, glutamine-rich; CREB, cAMP-response element-binding protein; GST, glutathione S-transferase; MMTV, mouse mammary tumor virus.

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DISCUSSION
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