Specific Androgen Receptor Activation by an Artificial Coactivator*

Xiaomei Sui, Kelli S. Bramlett, Michael C. Jorge, David A. Swanson, Andrew C. von Eschenbach, and Guido JensterDagger

From the Department of Urology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transcription activation of steroid receptors, such as the androgen receptor (AR), is mediated by coactivators, which bridge the receptor to the preinitiation complex. To develop a tool for studying the role of the AR in normal development and disease, we constructed artificial coactivators consisting of the transcription activation domains of VP16 or p65/RelA and the AR hinge and ligand-binding domain (ARLBD), which has been shown to interact with the AR N-terminal domain. The artificial VP16-ARLBD and ARLBD-p65 coactivators interacted with the AR N terminus and wild-type AR in an androgen-dependent and androgen-specific manner. VP16-ARLBD and ARLBD-p65 enhanced the AR transactivity up to 4- and 13-fold, respectively, without affecting the expression of the AR protein. The coactivators did not enhance the transcription activity of the progesterone receptor (PR) or the glucocorticoid receptor (GR), showing their specificity for the AR. In addition, to construct PR- and GR-specific coactivators, the VP16 activation domain was fused to the PR and GR hinge/ligand-binding domain. Although VP16-PRLBD and VP16-GRLBD interacted with the C-terminal portion of steroid receptor coactivator-1, they did not enhance the transcription activity of their receptor. The presented strategy of directing activation domains or other protein activities into the DNA-bound AR complex provides a novel means of manipulating AR function in vitro and in vivo.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Steroid receptors are hormone-dependent transcription factors that regulate the expression of a large variety of genes affecting cell growth, differentiation, development, and homeostasis. How these nuclear receptors activate or repress transcription of target genes has been the focus of much research in the past years. A major breakthrough in our understanding of these activities was the identification of so-called coactivators that bridge the receptors to the preinitiation complex (PIC) (1-4).1 These coactivators are recruited into the promoter-bound complex by the receptor and facilitate assembly of basal transcription factors into a stable PIC, likely via their activation domains. In addition to this bridging function, some coactivators, including steroid receptor coactivator-1 (SRC-1), cAMP response element-binding protein-binding protein (CBP), p300, and ACTR, can also remodel chromatin by acetylating histones (5-8). Moreover, the nuclear receptors, ACTR, SRC-1, CBP, and p300 can recruit the p300/CBP-associated factor (9), which also harbors an intrinsic histone acetyltransferase activity (6, 7, 10, 11). The model that emerges from these data takes the two activities of coactivators into account; the liganded steroid receptor binds as a homodimer to the hormone response element and recruits coactivators and p300/CBP-associated factor. These cofactors loosen the nucleosomal structure by targeted histone acetylation. The coactivators can then initiate the stable assembly of the PIC by their bridging function, which results in enhanced rates of transcription initiation by RNA polymerase II (9).

To perform their bridging function, coactivators need to harbor at least two domains: a receptor-interacting domain and a domain that contacts and stabilizes assembly of the PIC. This latter domain will almost certainly be a transcription activation domain that directly or indirectly binds proteins of the PIC. The receptor-interacting domain determines the binding specificity. Except for AR-associated protein 70 (12), none of the known coactivators is specific for a certain nuclear receptor (1, 3). In addition, SRC-1 and CBP can mediate transactivity of transcription factors other than nuclear receptors (13-15).

The objective of this study was to generate an AR-specific and very potent coactivator to manipulate AR function in vitro and in vivo. Such a coactivator could be used as a tool to study the role of the AR in normal development and disease. Moreover, this coactivator could form the blueprint for the construction of artificial corepressors that inhibit AR function in the presence of ligand, of cofactors that direct any other enzyme activity into the AR DNA-bound complex, and of coactivators that are specific for other nuclear receptors.

To form a coactivator that is AR-specific and more potent than the known coactivators, a domain had to be found that interacts only with the AR and not with other nuclear receptors. The first evidence for the existence of such a domain came from the observation that the AR ligand-binding domain (ARLBD) binds to the AR N terminus in an androgen-dependent manner (16-18). However, it was unknown whether the ARLBD could also interact with the wild-type AR and whether this binding is AR-specific.

The activation domains are important determinants of coactivator potency and are responsible for the direct interaction with proteins of the PIC and/or recruitment of additional coactivators. There are a few obvious choices for strong activation domains, including the activation domain from viral protein 16 (VP16) (19) and p65/RelA (20). Both have been shown to directly contact proteins of the PIC and associate with coactivators (21-30).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- R1881 (17alpha -methyltrienolone) and R5020 (promegestone) were purchased from NEN Life Science Products. Dexamethasone, estradiol, testosterone, hydroxyflutamide, cyproterone acetate, and dihydrotestosterone were purchased from Sigma. Casodex (bicalutamide, ICI-176, 334) was obtained from Zeneca Pharmaceuticals (Newark, DE).

Plasmids-- The construction of pAR0 (encoding the wild-type AR of 910 amino acids), pAR3, pAR5, pAR65, pAR106, pAR126, and pAR104 has been described previously (31, 32). The pcDNA-ARLBD was constructed by inserting the partial EcoRI 1-kilobase pair ARLBD fragment (encoding amino acids 605-910) from pAR65 into the pcDNA3.1HisB vector (Invitrogen, Carlsbad, CA) digested with EcoRI. A multiple cloning site (33) was introduced before the AR stop codon by polymerase chain reaction using the following primers: AR861A, 5'-GCGAGAGAGCTGCATCAGTTCAC-3', and AR910mcsB, 5'-CTAGGGCCCGTTAACCTCGAGACCGCGGACTGGGTGTGGAAATAGATGGG-3'. The polymerase chain reaction fragment was digested with BspEI and Bsp120I and inserted into pcDNA3.1His- ARLBD digested with the same enzymes. The resulting plasmid (pcDNA-ARLBDmcs) contains the unique SacII, XhoI, HpaI, and Bsp120I sites into which fragments can be inserted in frame with the ARLBD. pcDNA-VP16 was constructed by inserting the VP16 EcoRI fragment (encoding amino acids 411-490) from pRSET-VP16 into pcDNA3.1HisC linearized with EcoRI. pcDNA-ARLBDVP16 was constructed by in frame insertion of VP16 from pcDNA-VP16 (BamHI filled in with Klenow and Bsp120I-digested) into the pcDNA-ARLBD mcs vector (XhoI filled in with Klenow and Bsp120I-digested). pcDNA-VP16ARLBD was generated by inserting the ARLBD (Asp718 filled in with Klenow and XbaI-digested) into the pcDNA-VP16 vector (NotI filled in with Klenow and XbaI-digested). pcDNA-VP16ARLBDVP16 was constructed by inserting the Bsp120I/BspEI fragment from pcDNA-ARLBDVP16 into the pcDNA-VP16ARLBD vector digested with the same enzymes. pcDNA-ARLBDp65 was constructed by inserting the 1.3-kilobase pair Asp718-Klenow/SalI fragment of p65/RelA encoding amino acids 286-550 into the XhoI/HpaI-digested pcDNA-ARLBDmcs vector. pcDNA-VP16ARLBDp65 was constructed by inserting the HindIII/BspEI fragment from pcDNA-VP16ARLBD into the pcDNA-ARLBDp65 HindIII/BspEI-digested vector. pcDNA-p65 was constructed by inserting the 1.3-kilobase pair BamHI-Klenow/EcoRI p65 fragment into the XbaI-Klenow/EcoRI-digested pcDNA3.1HisC. pcDNA-AR0mcs was generated by inserting the MluI-Klenow/EcoRI AR sequence from pAR3 into KpnI-T4 DNA polymerase-blunted/EcoRI pcDNA-ARLBDmcs vector. pcDNA-AR0 was constructed by replacing the mcs sequence in pcDNA-AR0mcs (EcoRI/XhoI-digested) with the wild-type sequence from pAR0 (EcoRI/SalI fragment). pcDNA-VP16PRLBD was constructed by inserting the HincII-digested PRLBD fragment from pABGAL-PRLBD into the XhoI-digested, filled-in pcDNA-VP16 vector. pcDNA-p65GRLBD was generated by inserting the filled-in ClaI/DraI GRLBD fragment into the pcDNA-p65 XhoI-digested, filled-in vector. pcDNA-p65PRLBD was constructed by inserting the 1-kilobase pair XhoI PRLBD fragment from pcDNA-VP16PRLBD into the XhoI-digested pcDNA-p65 vector. pcDNA-PRLBD and pcDNA-GRLBD were generated by releasing the p65 fragment from pcDNA-p65PRLBD and pcDNA-p65GRLBD, respectively. pcDNA-VP16GRLBD was constructed by inserting the HpaI/Bsp120I GRLBD fragment from pcDNA-p65GRLBD into the NotI/Klenow/Bsp120I-digested pcDNA-VP16 vector. pABGAL-SRC-1(0.9) was constructed by inserting the HindIII-Klenow/BglII SRC-1(0.9) fragment from pVLGST-SRC-1 (6) into PvuII/BamHI-digested pABGAL1-147 (34). The reporter plasmid harboring two androgen/progesterone/glucocorticoid response elements and a TATA-box driving the luciferase gene ([ARE]2-E1b-luc) and the (UAS)4TATA-luciferase reporter have been described previously (9, 35). The pcDNA3.1His-LacZ plasmid (Invitrogen) was used as an internal transfection efficiency control. Correct nucleotide sequence of all constructs was verified by DNA sequencing, and correct protein expression in HeLa cells was determined by Western blotting analysis.

Cell Culture and Transient Transfections-- HeLa cells (human epithelial cervix carcinoma (American Type Culture Collection)) were maintained in minimal essential medium supplemented with 5% fetal bovine serum and antibiotics. LNCaP cells (American Type Culture Collection) were maintained in RPMI medium supplemented with 10% fetal bovine serum and antibiotics. 24 h before transfection, 105 cells were plated in each well of 12-well dishes in medium containing dextran-coated charcoal-stripped serum. Cells were transfected with 0.3 µg of reporter plasmid, 0.1 µg of pcDNA-LacZ, 30 ng of receptor construct, and 0.1 µg of coactivator plasmid per well using Lipofectin (Life Technologies, Inc.) according to the manufacturer's guidelines. 24 h later, cells were washed and fed with medium containing stripped serum and the indicated hormones. Cells were harvested 14 h later, and cell extracts were assayed for luciferase activity using the luciferase assay system (Promega). Luciferase values were corrected for the beta -galactosidase internal control. Experiments were performed in triplicate; data are presented as the means ± S.E. of at least three independent experiments.

Immunoblots-- Transfected HeLa cells were lysed in 40 mM Tris-HCl pH 7.0, 1 nM EDTA, 4% glycerol, 10 mM dithiothreitol, 2% SDS, and protease inhibitors. Protein concentration of the samples were determined by Bradford assay using 10 µg of protein loaded in sample buffer on a 7.5% SDS-polyacrylamide gel as described previously (36). Gels were blotted onto nitrocellulose, and the protein of interest was visualized with the AR-specific F39.4 antibody (37) or Xpress antibody (Invitrogen) and a goat-anti-mouse horseradish peroxidase secondary antibody using the ECL kit (Amersham Pharmacia Biotech). The pcDNA3.1His vectors that were used for the construction of almost all plasmids described above harbor the Xpress epitope tag to which the Xpress antibody is directed.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Artificial AR Coactivators-- To investigate whether the ARLBD interacts with the wild-type AR, various fusion proteins were constructed containing the ARLBD and the activation domains from VP16 and p65. The various constructs that were used in this study are depicted in Fig. 1. The wild-type AR (AR0) cotransfected with the (ARE)2E1b-luciferase reporter in HeLa cells was activated up to 50-fold by 1 nM synthetic androgen R1881 (Fig. 2A). Cotransfection with empty pcDNA3.1His vector, ARLBD, VP16 activation domain, or p65 activation domain did not significantly change the capacity of AR to activate transcription. However, fusion proteins of the ARLBD with VP16 or p65 greatly enhanced AR transactivity. The location of the VP16 activation domain with respect to the ARLBD did not influence the coactivation potential of the VP16-ARLBD and ARLBD-VP16 fusion proteins. We cotransfected all the different coactivators and their components with the reporter plasmid in the absence and presence of R1881 to test their potential effect on the transcription of the reporter in the absence of the AR. As expected, none of the coactivators or their components changed the basal transcription from the (ARE)2E1b-luciferase reporter (data not shown). To show that endogenous AR can also be superactivated by coactivators, VP16-ARLBD and ARLBD-p65 were transiently transfected with the reporter plasmid into the prostate cancer cell line LNCaP. LNCaP cells express the AR and are sensitive to androgens with respect to their growth. Transfection of the reporter alone showed that R1881 can activate the endogenous AR (Fig. 2B). Cotransfection of the ARLBD or p65 activation domain did not significantly affect AR activity. However, as was observed in the HeLa cell transfections, the ARLBD-p65 coactivator strongly enhanced the AR up to 9-fold.


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Fig. 1.   Schematic representation of constructs. Various steroid receptors, including AR, GR, PR, and AR mutants, and artificial coactivators consisting of the receptor hinge region with a LBD and activation domain of VP16 or p65/RelA were used in this study. In addition, the GAL4 DNA binding domain and a fusion with the receptor-interacting part of the SRC-1 coactivator were used as bait in the mammalian two-hybrid system. NT, N-terminal domain.


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Fig. 2.   Superactivation of the AR by artificial coactivators. A, AR transcription activity was examined by cotransfection of the AR expression plasmid and the (ARE)2E1b-luciferase reporter in HeLa cells. In addition, empty vector, a coactivator fragment (ARLBD, VP16, and p65) or a coactivator (ARLBD-VP16, VP16-ARLBD, and ARLBD-p65) were cotransfected. B, LNCaP cells, which express endogenous AR, were cotransfected with (ARE)2E1b-luciferase reporter and empty vector, coactivator fragment (ARLBD and p65) or ARLBD-p65 coactivator. Luciferase activity was determined from cell lysates of transfected cells, which were cultured for 16 h in the absence (-) or presence (+) of 1 nM synthetic androgen R1881. Activities were corrected for a pcDNA3.1His-LacZ internal control and are presented as the percentage of luciferase activity ± S.E. relative to the AR activity in the presence of R1881 (second lane from the left in both panels).

Lack of Effect of Artificial Coactivators and Their Components on AR Protein Expression-- To ensure that cotransfection of the AR with the artificial coactivators did not increase AR expression and consequently transcription activity from the reporter, HeLa cells were cotransfected, and the AR protein expression was determined by Western blot analysis. The AR expression was not affected by the coactivators or their components (Fig. 3).


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Fig. 3.   Western blot analysis of AR protein coexpressed with artificial coactivator or coactivator fragments. HeLa cells were cotransfected with AR expression vector, (ARE)2E1b-luciferase reporter, and empty vector, coactivator fragment (ARLBD, VP16, and p65) or coactivator (VP16-ARLBD, and ARLBD-p65). Protein extracts of transfected cells were analyzed after culturing for 16 h in the presence of 1 nM R1881. Proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted, and immunostained with the F39.4 monoclonal antibody.

The VP16-ARLBD and ARLBD-p65 Coactivators Are Specifically Androgen-dependent-- Because the artificial coactivators harbor the ARLBD, we tested whether the VP16-ARLBD and ARLBD-p65 fusion proteins enhanced the AR in a hormone-dependent manner. The AR5 mutant lacks the AR ligand-binding domain and is constitutively active (32). As expected, the addition of R1881 to the HeLa cells cotransfected with AR5 and the (ARE)2E1b-luciferase reporter did not affect AR5 activity (Fig. 4). The artificial ARLBD-p65 coactivator did not enhance AR5 transactivity in the absence of ligand but did superactivate transcription in the presence of 1 nM R1881. This result was repeated with the VP16-ARLBD fusion protein, showing that the coactivators containing the ARLBD depend on ligand for function. To investigate to which part of the AR N-terminal domain the ARLBD binds, we tested two different AR mutants harboring different fragments of the N terminus for enhanced transcription activation by ARLBD-p65. Both AR126 (amino acids 1-370) and AR106 (amino acids 360-528) were only partially enhanced by the artificial coactivator as compared with AR5, indicating the necessity for both fragments for full ARLBD-p65 interaction. In addition, the binding of the artificial coactivator to the AR DNA-binding domain and ligand-binding domain was analyzed using AR104, which completely lacks the N-terminal domain. The transcription activity of this mutant is very low (32), and in this study ARLBD-p65 did not enhance its transcription (Fig. 4).


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Fig. 4.   Transcription enhancement of AR mutants by artificial coactivators. HeLa cells were transfected with the AR mutant, (ARE)2E1b-luciferase reporter, and empty vector or ARLBD-p65 coactivator. Luciferase activity was determined from cell lysates of transfected cells, which were cultured for 16 h in the absence (-) or presence (+) of 1 nM R1881. Activities were corrected for a pcDNA3.1His-LacZ internal control and are shown as the percentage of luciferase activity ± S.E. relative to AR5 (second column from the left), AR126 (sixth column from the left), AR106 (tenth column from the left), or AR104 (fourteenth column from the left) in the presence of hormone.

We used this same experimental approach to determine whether the fusion of the ARLBD to VP16 or p65 changed ligand specificity. Various agonists (R1881, testosterone, and dihydrotestosterone), antagonists (Casodex, hydroxyflutamide, and cyproterone acetate), and other hormones (estradiol and the synthetic progestin R5020) were tested for their ability to induce ARLBD-p65 binding to AR5 (Fig. 5). Only the androgens testosterone, dihydrotestosterone, and R1881 significantly induced AR5 superactivation, showing that the artificial coactivator did not change ligand specificity and remained androgen-specific.


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Fig. 5.   Ligand specificity of the ARLBD-p65 coactivator. HeLa cells were transfected with AR5, (ARE)2E1b-luciferase reporter and empty vector or ARLBD-p65 coactivator. Luciferase activity was determined from cell lysates of transfected cells, which were cultured for 16 h in the absence (-) or presence (+) of 1 nM R1881, 1 nM testosterone (T), 1 nM dihydrotestosterone (DHT), 100 nM estradiol (E2), 100 nM synthetic progestin R5020, 100 nM antiandrogen Casodex, 100 nM antiandrogen hydroxyflutamide (OH-flu), or 100 nM antiandrogen cyproterone acetate (CA). Activities were corrected for a pcDNA3.1His-LacZ internal control and are presented as the percentage of luciferase activity ± S.E. relative to the AR5 activity in the absence of R1881 (first column from the left).

The VP16-ARLBD and ARLBD-p65 Coactivators Are AR-specific-- To determine whether the ARLBD would interact only with the AR and not with other steroid receptors, we analyzed the effect of the artificial coactivators on two of the most homologous nuclear receptors, the progesterone receptor (PR) and the glucocorticoid receptor (GR). Cotransfection of the PR or GR with ARLBD-p65 in the presence of R1881 and R5020 or dexamethasone for activation of the PR and GR, respectively, showed no superactivation (Fig. 6), indicating that the ARLBD-p65 coactivator is AR-specific. Note that R1881 is a potent progestin and is able to activate the PR (Fig. 6).


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Fig. 6.   Superactivation of the AR, PR, and GR by ARLBD-p65. HeLa cells were transfected with AR, PR, or GR, (ARE)2E1b-luciferase reporter and empty vector, or ARLBD-p65 coactivator. Luciferase activity was determined from cell lysates of transfected cells, which were cultured for 16 h in the absence (-) or presence (+) of 1 nM ligand or a mix of ligands for AR (R1881), PR (R5020), and GR (dexamethasone). Activities were corrected for a pcDNA3.1His-LacZ internal control and are presented as the percentage of luciferase activity ± S.E. relative to the activity of AR (second column from the left), PR (sixth column from the left), or GR (twelfth column from the left) in the presence of ligand.

Testing of Artificial PR and GR Coactivators-- To determine whether the hinge/ligand-binding domains of the PR and GR can also interact with their receptors, the VP16 activation domain was fused to the PRLBD and GRLBD. Although their construction was almost identical to that of VP16-ARLBD, neither VP16-PRLBD nor VP16-GRLBD enhanced transactivation of its full-length receptor (Fig. 7). For control purposes, we cotransfected the activation domain of VP16 by itself with the different receptors to analyze the effect of this potent activation domain on receptor transactivity. The activity of all receptors was reduced, probably because of sequestering of essential transcriptional cofactors. Interestingly, the GR activity was the most severely reduced (down to 25%). Cotransfection of the GR with VP16-ARLBD or VP16-PRLBD in the presence of the various ligands again showed the transcription-inhibiting effect of VP16 (Fig. 7). However, in the absence of the ligand that interacts with the VP16-ARLBD (R1881) or VP16-PRLBD (R5020), the GR activity was again higher. One explanation for this phenomenon is that in the unliganded VP16-ARLBD and VP16-PRLBD, the VP16 is shielded by the heat shock proteins that associate with the ligand-binding domain. In the presence of ligand, the heat shock proteins dissociate, and VP16 is able to squelch the GR transactivity.


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Fig. 7.   Functional analysis of VP16 and AR, PR, or GR ligand-binding domain fusion proteins for superactivation of their wild-type receptor. HeLa cells were transfected with AR, PR, or GR, (ARE)2E1b-luciferase reporter and empty vector, or VP16-ARLBD, VP16-PRLBD, or VP16-GRLBD fusion proteins. Luciferase activity was determined from cell lysates of transfected cells, which were cultured for 16 h in the absence (-) or presence (+) of 1 nM ligand or a mix of ligands for AR (R1881), PR (R5020), and GR (dexamethasone). Activities were corrected for a pcDNA3.1His-LacZ internal control and are presented as the percentage of luciferase activity ± S.E. relative to the activity of AR (second column from the left), PR (fifteenth column from the left), or GR (twenty-eighth column from the left) in the presence of ligand.

To check correct folding and the ability to bind hormone, we cotransfected VP16-PRLBD and VP16-GRLBD with the receptor interacting-part of the SRC-1 coactivator, which is fused to the GAL DNA-binding domain (DBD) (GAL-SRC-1[1139-1441]) (38, 39). None of the VP16 fusion proteins interacted with the GAL DBD (Fig. 8). However, VP16-ARLBD, ARLBD-VP16, ARLBD-p65, VP16-PRLBD, and VP16-GRLBD bound the GAL-SRC-1(1139-1441) in a hormone-dependent manner, showing correct expression, nuclear import, folding, and hormone binding.


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Fig. 8.   Mammalian two-hybrid analysis of binding of AR, PR, and GR ligand-binding domain to the receptor interacting part of SRC-1. HeLa cells were transfected with GAL DBD or GAL-SRC-1(1139-1441), (UAS)4TATA-luciferase reporter and empty vector, or VP16-ARLBD, ARLBD-VP16, ARLBD-p65, VP16-PRLBD, or VP16-GRLBD fusion proteins or fragments thereof. Luciferase activity was determined from cell lysates of transfected cells, which were cultured for 16 h in the absence (-) or presence (+) of 1 nM ligand for AR (R1881), PR (R5020), and GR (dexamethasone (dex)). Activities were corrected for a pcDNA3.1His-LacZ internal control and are presented as the percentage of luciferase activity ± S.E. relative to the activity of GAL-DBD or GAL-DBD-SRC-1(1139-1441).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our results show for the first time that the ARLBD can interact with the full-length AR but not with the PR or GR. The binding of the artificial coactivators occurred in the AR N-terminal domain and was diminished when the first 360 amino acids or the last 158 residues of the N terminus were deleted, confirming the previous observation that two independent domains (the first 36 amino acids and residues 370-494) are necessary for full ARLBD binding (40).

Binding of the hinge/LBD to the N terminus or full-length receptor has also been shown for the estrogen receptor alpha  (41) and the PR (42). However, VP16 fusion proteins with the PR and GR hinge/LBD did not enhance the transcription activity of their receptor, indicating that they did not bind the full-length receptor in our mammalian protein-protein interaction system. The folding, ligand binding, and nuclear import of VP16-PRLBD and VP16-GRLBD were correct because they interacted with the receptor-interacting domain of SRC-1 in a ligand-dependent manner (38, 39). The interaction of VP16-ARLBD and ARLBD-p65 with SRC-1(1134-1441) was very weak compared with the binding of VP16-PRLBD and VP16-GRLBD, confirming previously published data showing that the AR binding to SRC-1 is the weakest of all steroid receptors (39). The reason for the inability of VP16-PRLBD and VP16-GRLBD to bind to their cognate receptor is not clear. We speculate that the three-dimensional structure of the AR DNA-bound homodimer is different as compared with the PR and GR homodimers, allowing an additional ligand-binding domain to be recruited into the AR complex.

Previously, Nyanguile et al. (43) constructed a completely synthetic low-molecular-weight transcription activator by fusing FK506 to a short peptide with transactivating properties. FK506 interacts with the FK-binding protein FKBP12, which was fused to the GAL DNA-binding domain, thereby directing the transactivating peptide onto the reporter gene. As a complementary strategy, we show that the ARLBD, which binds the full-length AR also gives us the opportunity to direct proteins into the DNA-bound AR complex. In addition to the transcription activation domains described in this manuscript, the effects of transcription repression domains, DNA-modifying enzymes, and chromatin-remodeling proteins on AR function are currently being tested. These artificial cofactors provide a novel means of manipulating AR activity and AR target gene expression to investigate the role of AR function in normal and disease states.

    ACKNOWLEDGEMENTS

We thank Dr. J. Trapman for helpful discussions and careful reading of the manuscript, Drs. A. O. Brinkmann and J. Trapman for the F39.4 antibody, and Dr. B. W. O'Malley for the GAL-SRC-1 construct.

    FOOTNOTES

* This work was supported by the Physicians' Referral Service and Prostate Cancer Research Program of The University of Texas M. D. Anderson Cancer Center.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 To whom correspondence should be addressed: University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 026, Houston, TX 77030. Tel.: 713-792-8917; Fax: 713-792-4456; E-mail: gjenster{at}mdacc.tmc.edu.

    ABBREVIATIONS

The abbreviations used are: PIC, preinitiation complex; AR, androgen receptor; LBD, ligand-binding domain; PR, progesterone receptor; GR, glucocorticoid receptor; SRC-1, steroid receptor coactivator-1; CBP, cAMP response element-binding protein-binding protein; DBD, DNA-binding domain; VP16, viral protein 16; LNCaP, lymph node carcinoma of the prostate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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