Identification of a Highly Conserved Domain in the Androgen Receptor That Suppresses the DNA-binding Domain-DNA Interactions*

Guo-Zhen LiuDagger, Hua Wang, and Zhengxin Wang§

From the Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas

Received for publication, December 2, 2002, and in revised form, February 17, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The androgen receptor (AR) is a ligand-regulated and sequence-specific transcription factor that activates or represses expression of target genes. Here, we show that the N terminus of AR contains an inhibitory domain located in an 81-amino acid segment lying upstream of the DNA-binding domain (DBD). The inhibitory domain interacted directly with DBD and repressed DBD binding to the androgen response element. Mutations of the conserved amino acid residues (K520E and R538E) within the inhibitory domain decreased its inhibiting ability in vitro and increased AR trans-activation in vivo. These data demonstrate the existence of a novel inhibitory domain in the N-terminal part of AR, which might play important roles in the regulation of AR trans-activation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The androgen receptor (AR)1 mediates androgen functions in the differentiation and maturation of the male reproductive organs and in the development of male secondary sex characteristics (1). Mutations in the AR gene are associated with the androgen insensitivity syndrome (2, 3). Numerous somatic mutations in the AR gene have been reported among prostate cancer patients and as well as in prostate cancer cell lines and xenografts (3, 4). Most of these mutations have been detected in tumor tissues of late-stage prostate carcinoma, indicating that somatic mutation of the AR gene might be involved in the progression and aggressiveness of prostate cancer.

The AR is a member of the nuclear receptor superfamily (5). These receptors are characterized by distinct functional domains: an N-terminal part involved in ligand-independent transcription activation (AF1), a DNA-binding domain (DBD), and a C-terminal ligand-binding domain involved in ligand binding and ligand-dependent transcription activation (AF2) (6). As for other steroid receptors, ligand binding is generally believed to result in a conformational charge in AR with consequent dissociation of heat shock proteins/chaperones (7), dimerization, and binding to cognate androgen response elements (AREs) in target genes and (through its AF1 and AF2 domains) interactions with various coactivators that facilitate transcription by the general transcriptional machinery (8). The DBD encompasses two zinc finger-like modules and binds as dimers to two hexameric sequences orientated as direct or inverted repeats (9, 10). Although the DBD and the ligand-binding domain of steroid hormone receptors are highly conserved, there is much less homology among steroid hormone receptors in their N-terminal parts. The AR has a long N-terminal part with a strong autonomous AF1 and interacts directly with AF2 in the C-terminal part (11, 12). The N- and C-terminal interactions are important for androgen-induced gene regulation, and disruption of these interactions may be linked to androgen insensitivity syndrome (13, 14). The conserved FXXLF and WXXLF motifs within the N-terminal part seem to be involved in pairwise interactions between AF1 and AF2 (15). The N-terminal part contains stretches of glutamines (coded by CAG) and glycine (coded by GGN) (16). Expansion of the CAG repeats is associated with X-linked spinal and bulbar muscular atrophy (17). A shorter CAG repeat is associated with an increased trans-activation of AR (18, 19), but the biological role of GGN repeats is less clear.

In this study, we demonstrated that AR contains a highly conserved inhibitory domain within the N-terminal region. The inhibitory domain interacted directly with DBD and inhibited the DBD-DNA interactions. The mutations in the inhibitory domain resulted in decreased inhibitory ability and increased AR trans-activation activity, indicating that this domain might play important roles in the regulation of AR function.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production and Purification of Recombinant Proteins-- The human full-length AR was expressed in Sf9 cells via the baculovirus expression vector pVL1393 (BD Biosciences), and the recombinant AR was purified as described previously (20). All of the AR and glucocorticoid receptor (GR) cDNA fragments were amplified by PCR with specific oligonucleotides, cut with NdeI and BamHI, and subsequently cloned in the corresponding restriction sites of the vectors pET15d (Novagen), pGEX-2TL (Amersham Biosciences), and pcDNA3.1 (Invitrogen). The fragments were expressed as His6-tagged (via pET15d) or GST fusion (via pGEX-2TL) proteins in Escherichia coli BL21 and purified through nitrilotriacetic acid Ni2+-agarose or glutathione-Sepharose columns, respectively. Point mutations were generated by using the QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions and confirmed by DNA sequencing analysis. The mutated proteins were expressed and purified similarly.

Gel Shift Assay-- Two pairs of oligomers (5'-AGCTTTTGCAGAACAGCAAGTGCTAGCTG-3' and 5'-AAATTCAGCTAGCACTTGCTGTTCTGCAA-3'; 5'-AGCTTTTGCAGAATAGCAAATGCTAGCTG-3' and 5'-AAATTCAGCTAGCATTTGCTATTCTGCAA-3') derived from the prostate-specific antigen gene (-152 to -174) were synthesized, annealed, and subcloned into HindIII and EcoRI sites of the vector pBluescript II (Stratagene). The underlined bases were mutated from their corresponding bases in the wild-type prostate-specific antigen gene sequence. The wild-type and mutant ARE probes were made by cutting these constructs with XhoI and XbaI and purification of fragments from agarose gel. Probes were labeled with [alpha -32P]dCTP by a fill-in reaction with the Klenow enzyme. In gel shift assays, 20-µl reaction contains 20 mM HEPES, pH 7.9, 70 mM KCl, 1 µg of poly(dI-dC), 1 mM dithiothreitol, 0.1% Nonidet P-40, 100 µg/ml of bovine serum albumin, and various proteins. The reaction mixture was incubated for 20 min at room temperature, and the binding reaction was initiated by the addition of the labeled probes (20,000 cpm) and then incubated for an additional 30 min at room temperature. The reaction mixture was loaded directly onto a 4% (37.5:1, acrylamide:bisacrylamide) nondenaturing polyacrylamide gel with 0.25× Tris borate EDTA and run at 150 V for 2 h at room temperature.

Cell Culture and DNA Transfection-- PC3 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. Cells (5 × 105) were plated in each well of 24-well plates and transfected with 100 ng of 4xARE-E4-Luc reporter plasmid, 2.5 ng of control plasmid pRL-CMV, and various amounts of expression plasmids. Cells were grown in the presence of 10 nM R1881 for 48 h after transfection and harvested for dual-luciferase activity assay (Promega).

Protein-Protein Pull-down Assay-- GST and GST-DBD(AR537-644) were expressed in bacteria and immobilized on glutathione-Sepharose beads. Beads (10 µl) containing 100 ng of GST or GST-DBD proteins were incubated with 5 µl of transcription and translation coupled rabbit reticulocyte lysates containing 35S-labeled AR477-538 in BC150, 0.1% Nonidet P-40 for 2 h at 4 °C. After being washed with the incubation buffer, beads were boiled with SDS sample buffer and subjected to SDS-PAGE followed by autoradiography.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Full-length AR Interacts with the Androgen Response Element More Weakly than the DNA-binding Domain-- The ligand-dependent interaction of AR with the ARE has been demonstrated in vitro with crude AR-containing cell extracts (21). However, the AR-DNA interactions have not been studied with the highly purified recombinant AR. To this end, the FLAG epitope-tagged human AR was expressed in Sf9 cells and immunopurified under high salt conditions (500 mM KCl) to strip off heat shock proteins associated with the unliganded AR. The recombinant AR preparation is near homogeneity (Fig. 1B, lanes 2 and 3) and contains two bands that migrated near the 110-kDa position. The top band might be the phosphorylated form of AR (22). Two minor polypeptides (70 and 55 kDa, indicated by stars on the right) were recognized by the anti-FLAG monoclonal antibody (data not shown), indicating that they are degraded products of the full-length AR. A DNA probe containing the ARE derived from the prostate-specific antigen promoter (-152 to -174) (Fig. 1D) (23) was used for a gel shift assay. The recombinant AR (0.9 pmol) shifted the probe (Fig. 1C, lane 2) while there was no ligand (androgen) dependence (lane 3 versus lane 2). The band of the AR·ARE complex (indicated by an arrow on the left) is quite broad. However, mutations of the nucleotides in the probe that are critical for AR-ARE interaction (24) (Fig. 1D) dramatically decreased the density of the AR-ARE band (Fig. 1C, lanes 7 and 8 versus lanes 2 and 3), indicating that the shifted band is specific. The DBD of AR (amino acid residues 537-644) (Fig. 1A) was expressed as a His6-tagged fusion protein and purified through an nitrilotriacetic acid Ni2+-agarose affinity column (Fig. 1B, lane 4). In the same assay, 0.3 pmol of AR537-644 almost completely shifted the probe (Fig. 1C, lane 4). The band of the AR537-644·ARE complex (indicated by an arrow on the left) is much sharper (Fig. 1C, lanes 4 and 5), and mutations in the probe (Fig. 1D) completely diminished the formation of the AR537-644·ARE complex (Fig. 1C, lanes 9 and 10). The results indicate that the binding affinity of DBD to the ARE is much stronger than that of the purified full-length AR to the same ARE.


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Fig. 1.   The recombinant AR interacts with the ARE weaker than the DBD. A, diagram of domains and truncations of AR. B, SDS-PAGE analysis of the recombinant AR and DBD. Proteins of 100 ng (lane 2) and 200 ng (lane 3) of the purified recombinant AR expressed in Sf9 cells and recombinant His6-tagged truncations of AR expressed in bacteria (lanes 4-6) were subjected to SDS-PAGE with Coomassie Blue R250 staining. Lane 1 is standard protein markers (Bio-Rad). C, the gel shift assay was performed using a DNA probe containing the wild-type ARE (lanes 1-5) or the mutant ARE (lanes 6-10). 0.9 pmol of AR (lanes 2, 3, 7, and 8) and 0.3 pmol (lanes 4 and 9) or 0.6 pmol (lanes 5 and 10) of AR537-644 were used in the binding reactions. The synthetic androgen R1881 (100 nM) was included in the reactions in lanes 3 and 8, and lanes 1 and 6 are probes only. D, sequences of the wild-type (AREwt) and mutant (AREmt) ARE and the ARE consensus. The mutated bases in AREmt are underlined. PSA, prostate-specific antigen. M, standard molecular markers. Qn and Gn, stretches of glutamines and glycines, respectively. LBD, ligand-binding domain. AR+L, the gel shift assay performed in the presence of androgen R1881.

A Domain within the AR N Terminus Inhibits DBD-ARE Interactions-- A C-terminal extension of the DBD of AR was found to be required for specific and high affinity interactions of DBD with ARE (25). To investigate whether the sequences surrounding DBD would affect DBD-DNA interactions, AR537-662 and AR477-644 (Fig. 1A) were expressed in and purified from bacteria (Fig. 1B, lanes 5 and 6). AR537-662 strongly interacted with the probe, similar to AR537-644 (Fig. 2A, lanes 2-4). However, AR477-644 completely lost the ability to interact with the ARE probe even though much more protein (up to 1.6 pmol) was used in the binding reaction (lanes 5-7). The N-terminal extension of DBD (amino acid residues 477-558) was expressed and purified (Fig. 2B, lane 1). Its molecular mass as determined by SDS-PAGE (16 kDa) is much bigger than the calculated mass (10 kDa), and it was heavily degraded (Fig. 2B, lane 1). This region contains 20% charged amino acids and 16% proline residues, which may be responsible for this aberrant mobility of the protein. When AR477-558 was added to the binding reaction that contained the fixed amount (0.3 pmol) of AR537-644, the density of the DBD·ARE complex dramatically decreased (Fig. 2C, lanes 3-8). These results indicate that AR477-538 specifically inhibits the DBD-ARE interactions in trans as well as in cis. We noticed that different preparations of AR477-538 contained various amounts of the full-length protein and that amounts of the full-length protein (Fig. 2B, lane 1, indicated by the top arrow on the right) were correlated with the inhibition ability of AR477-538. As negative controls, the recombinant prostate apoptosis response-4 (26), 30-kDa Tat-interaction protein (27), and 39-kDa subunit of RNA polymerase C (28) expressed and purified similarly did not significantly affect the DBD binding to the ARE probe (Fig. 2D).


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Fig. 2.   AR477-558 blocks DBD binding to ARE. A, AR477-644 did not interact with the ARE. 0.15 (lane 2), 0.3 (lane 3), or 0.6 pmol (lane 4) of AR537-662 and 0.4 pmol (lane 5), 0.8 pmol ng (lane 6), or 1.6 pmol (lane 7) of AR477-644 were used in the binding reactions. Lane 1 is the probe-only control. B, SDS-PAGE of the purified recombinant wild-type (lane 1), K580E (lane 2), or R538E (lane 3) AR477-558. The full-length proteins (top arrow) and the main degraded products (bottom arrow) are indicated on the right. Nonspecific background bands are marked with a star on the right. C, AR477-558 inhibits AR537-643 binding to the ARE. The binding reactions contained 0.3 pmol of AR537-644 (lane 2) or 0.3 pmol of AR537-644 plus 0.0625 (lane 3), 0.125 (lane 4), 0.25 (lane 5), 0.5 (lane 6), 1 (lane 7), or 2 pmol (lane 8) of AR477-558. D, the purified recombinant PAR-4, TIP30, and RPC39 proteins do not affect the interaction of AR537-644 with ARE. The binding reactions contained 0.3 pmol of AR537-644 (lane 2) or 0.3 pmol of AR537-644 plus 0.225 (lane 3), 0.45 (lane 4), 0.9 (lane 5), or 1.8 pmol (lane 6) of prostate apoptosis response-4 (Par-4) (lane 8) or 1.25 (lanes 7 and 10), 2.5 (lanes 8 and 11), or 5 pmol (lanes 9 and 12) of 30-kDa Tat-interaction protein (TIP30) (lanes 7-9) or 39-kDa subunit of RNA polymerase C (RPC39) (lanes 10-12), respectively. WT, wild type.

The Inhibitory Domain Interacts with DBD and Inhibits AR Trans-activation-- The protein-protein pull-down assay was performed to investigate whether the inhibitory domain (ID) interacts directly with DBD. GST and GST-DBD(AR537-644) fusion protein were expressed in bacteria and immobilized on glutathione-Sepharose beads (Fig. 3A, lanes 2 and 3). The in vitro translated 35S-labeled AR477-558 (lane 4) bound to GST-DBD (lane 6) and not to GST (lane 5). This result indicates that the inhibitory domain interacts directly with DBD.


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Fig. 3.   AR477-558 interacted directly with DBD in vitro and inhibited AR trans-activation in vivo. A, AR477-558 interacted directly with DBD(AR537-644) in vitro. SDS-PAGE analysis of GST (lane 2) and GST-DBD (lane 3) expressed in bacteria and immobilized on glutathione-Sepharose 4B beads is shown. Bands corresponding to GST and GST-DBD fusion protein are indicated by arrows on the right. The immobilized GST-DBD pulled down 35S-labeled AR477-558 (lane 6). Lane 4 is 10% 35S-labeled AR477-558 input (IP) for the pull-down assay. B, AR477-558 inhibited AR trans-activation in vivo. PC3 cells were transfected with 100 ng of the reporters PGL3-ARE-E4 or pGL3-GAL4-E4, 2.5 ng of the internal control reporter pRL-CMV, 20 ng of pcDNA-AR or pcDNA-GAL4-p53-(1-53), and 18.5 ng of pcDNA-AR477-538 as indicated. Cells were treated with 10 nM R1881 after transfection and harvested 48 h later for the dual luciferase assay. C, AR477-538 did not affect AR protein levels in the transfected cells. Western blot analysis of cells transfected with pcDNA3.1 (lane 1), pcDNA-AR (lane 2), or pcDNA-AR plus pcDNA-AR477-558 (lane 3) with the anti-AR antibody is shown.

We then investigated the effect of the ID on AR trans-activation by performing transient transfection assays. A luciferase reporter containing four tandem copies of the same ARE used for the gel shift assay upstream of the minimal adenovirus E4 promoter was cotransfected with expression vectors for AR, AR477-558, or both into prostate cancer PC3 cells in the presence of the synthetic androgen R1881. As shown in Fig. 3B, AR activated the reporter gene ~25-fold, and coexpressed AR477-558 showed a strong (62%) inhibition of this activity. Coexpression of AR477-558 did not influence reporter gene activity driven by p53, indicating that the inhibiting effect of AR477-558 was specific for AR. Western blot analysis revealed that the AR protein levels in the absence and presence of AR477-558 were comparable (Fig. 3C, lane 3 versus lane 2). On the basis of in vitro studies (Fig. 2), the ID inhibited AR trans-activation most likely by blocking the interaction of the AR with the ARE.

The Inhibitory Domain Is Specific for AR-- The DNA-binding domains of AR, GR, progesterone receptor, and mineralocorticoid receptor are highly conserved (29). Not surprisingly, they bind to the same consensus DNA site (GGTACANNNTGTTCT) and can be considered a subfamily of the nuclear receptor superfamily. However, the inhibitory domain of AR is not conserved in the other receptors (Fig. 4A). GR418-525 and GR358-525 were expressed and purified (Fig. 4B, lanes 1 and 2). Gel shift assay demonstrated that GR358-525 and GR418-525 bound the ARE probe similarly (Fig. 4C, lane 3 versus lane 2). The lower band (Fig. 4C, lane 3, indicated by a star on the right) might contain a monomer of GR358-525. These results indicated that the ID in AR is not conserved in GR; thus, the inhibitory domain is specific for AR.


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Fig. 4.   The DBD-containing fragments of GR bind to the ARE probe. A, sequence alignment of DBD and ID of AR with the corresponding regions of rat GR. B, SDS-PAGE analysis of the recombinant GR418-525 and GR358-525. 500 ng of His6-tagged GR418-525 (lanes 1) and GR 357-525 (lane 2) expressed in bacteria were subjected to SDS-PAGE with Coomassie Blue R250 staining. The bands corresponding to the full-length protein fragments are indicated by arrows on the right, and a nonspecific background band is marked by a star on the left. The standard protein markers (Bio-Rad) are indicated on the left. C, GR417-525 and GR357-525 bind to the ARE. 0.3 pmol of GR417-525 (lane 2) or GR357-525 (lane 3) was used in the binding reactions. Lane 1 is the probe-only control.

Mutations in the ID Enhance AR Trans-activation-- Sequence alignment shows that the ID of AR is highly conserved through evolution (Fig. 5A). To further characterize the biological effects of this region, we mutated two conserved residues (Lys-520 and Arg-538) in the ID and cDNAs encoding the mutated AR (K520E and R538E) were transiently transfected in PC3 cells with the luciferase reporter plasmid. The mutated AR had elevated trans-activation activity compared with the wild-type AR (Fig. 5B), although the mutated and wild-type AR were expressed at the same level in the transfected cells (Fig. 5C, lanes 2-4). The ID (AR477-558) from the mutated AR (K520E and R538E) were expressed and purified (Fig. 2B, lanes 2 and 3). The gel shift assay revealed that mutations of K520E and R538E decreased the inhibitory ability of ID (Fig. 5D, lanes 9-12 and 13-16 versus lanes 5-8). Ten nanograms of the wild-type AR477-558 almost completely blocked AR537-644 binding to the ARE probe (Fig. 5D, lane 5). However, the same amount of the mutated (K520E and R538E) AR477-558 inhibited the DBD-ARE interaction only 65 and 35%, respectively (Fig. 5D, lanes 9 and 13). Thus, the enhancement of AR trans-activation by mutations of K520E and R538E correlates with a decrease in the inhibitory effect of ID on DBD-ARE interactions.


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Fig. 5.   Mutations in ID enhanced AR trans-activation and decreased ID inhibitory activity. A, sequence alignment of ID and DBD of human (hAR), rabbit (rAR), mouse (mAR), and Xenopus (xAR) AR. Point mutations found in prostate cancer (PC) and in complete (CAIS), mild (MIAS), or partial (PAIS) androgen insensitivity syndrome patients are indicated by arrows on the top. B, mutations (K520E and R538R) enhanced AR trans-activation in vivo. PC3 cells were transfected with 100 ng of the reporter pGL3-ARE-E4, 2.5 ng of the internal control reporter pRL-CMV, or 10 ng of pcDNA-wild-type AR or pcDNA-mutant (K520E or R538) AR as indicated. Cells were treated with 10 nM R1881 after transfection and harvested 48 h later for the dual luciferase assay. Each value represents the mean ± S.D. of a representative experiment performed in triplicate. C, Western blot analysis of cells transfected with pcDNA3.1 (lane 1) or with wild-type (lane 2), K520E (lane 3), and R538E (lane 4) mutant AR. D, mutations of K520E and R538E decreased ID inhibitory ability on DBD-ARE interactions. The binding reactions contained 0.075 (lane 2), 0.15 (lane 3), or 0.3 pmol (lane 4) of DBD alone or 0.3 pmol of DBD plus 0.625 (lanes 5, 9, and 13), 0.125 (lanes 6, 10, and 14), 0.025 (lanes 7, 11, and 15), or 0.005 pmol (lanes 8, 12, and 16) of wild-type (lanes 5-8), K520E mutant (lanes 9-12) or R538E mutant AR477-558 (lanes 13-16). WT, wild type.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The N-terminal parts of nuclear receptors are the most divergent among members of this superfamily of proteins, suggesting that each receptor will take on a unique N-terminal conformation to determine its specificity. This paper describes a highly conserved novel inhibitory domain designated ID, which lies in N-terminal 81-amino acid residues upstream of the DBD of AR. ID interacts directly with DBD and strongly inhibits the DBD-ARE interactions in vitro and AR trans-activation in vivo.

Much of the work devoted to understanding regulation of transcription by the AR has focused on the N-terminal AF1 and the C-terminal AF2 (30). However, transcriptional inhibition may be equally important as a way of preventing activation. Studies that deal with inhibition of AR-dependent transcription have focused on silencing mechanisms through recruitment of corepressors to the target promoters and through receptor occupancy at one DNA site interfering with transcription by an activator at an adjoining site (5, 31). We have now demonstrated that negative function element exists in the AR molecule itself and markedly suppresses the DNA binding activity of DBD. The ID function is similar to that of the N-terminal region of TAF250 (the 250-kDa TATA box-binding protein-associated factor 1), which forms a DNA-like structure, interacts with the DNA-binding surface, and inhibits the DNA binding activity of TATA box-binding protein (32). In contrast, the direct interactions between the ID and DBD suggest that perhaps ID acts through intramolecular contacts. In this respect, ID is similar to p53, which exists in a latent DNA-binding form as a result of the C-terminal tail-DNA-binding domain interactions (33, 34). Phosphorylation of lysine residues in the C-terminal region leads to the disruption of interactions between the C-terminal domain and the core DBD, thus allowing the DBD of p53 to adopt an active conformation. It is important to know whether modifications in the ID of AR or interactions of this domain with the other proteins might regulate the DNA binding activity of AR. A study on the rat AR indicated that the unknown protein could enhance the DNA binding activity of the protein fragment containing the DBD in a gel shift assay (35). Another study has demonstrated that mutations on 668QPIF671 at the boundary of the hinge and ligand-binding domain of AR, resulting in receptors that exhibit 2-4-fold increased activity compared with the wild-type AR in response to dihydrotestosterone, and these mutations have been detected in prostate cancer patients (36). However, the molecular mechanism for this phenomenon is unclear.

Several mutations found in men with prostate cancer (37) and in men with the androgen insensitivity syndrome (38, 39) localize in ID (Fig. 5A). These mutations might change the function of ID, therefore affecting AR trans-activation. D528G mutation was detected in a patient with prostate cancer (37), and we found that AR with D528G mutation was more active (>3-fold) than the wild-type AR in transient transfection assays (data not shown). Currently, we are investigating whether the enhanced AR trans-activation is because of the decreased ID function. Thus, ID may play an important regulatory role in AR function, and dysfunction of ID may contribute to prostate cancer or androgen insensitivity syndrome in some men.

    ACKNOWLEDGEMENTS

We thank Michael S. Worley for critical editorial review and Liliana DeGeus for expert assistance in the preparation of the paper.

    FOOTNOTES

* This work was supported in part by U. S. Department of the Army Grant DAMS17-01-1-0097, the Association for the Cure of Cancer of the Prostate, and Cancer Center Support Core Grant CA16672 from NCI, National Institutes of Health.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.

Dagger Present address: Dept. of Infectious Diseases, XiangYa Hospital, Central-South University, Chang Sha 410008, People's Republic of China.

§ To whom correspondence should be addressed: Dept. of Cancer Biology, Unit 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030-4009. Tel.: 713-794-1035; Fax: 713-792-8747; E-mail: zhenwang@mdanderson.org.

Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M212229200

    ABBREVIATIONS

The abbreviations used are: AR, androgen receptor; AF, transcription activation; DBD, DNA-binding domain; ARE, androgen response element; GR, glucocorticoid receptor; GST, glutathione S-transferase; Luc, luciferase; ID, inhibitory domain.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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