RFG (ARA70, ELE1) Interacts with the Human Androgen Receptor in a Ligand-Dependent Fashion, but Functions Only Weakly as a Coactivator in Cotransfection Assays

Tianshu Gao, Ken Brantley, Erol Bolu and Michael J. McPhaul

Department of Internal Medicine The University of Texas Southwestern Medical Center Dallas, Texas 75235-8857


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Abnormalities of the human androgen receptor (hAR) cause a range of clinical defects in male development. A large proportion of these mutations are single amino acid substitutions in the hormone-binding domain (HBD) that alter AR function by interfering with the capacity of the AR to bind androgen or to form stable hormone-receptor complexes. Prior studies have suggested that the formation of such stable, active hormone-receptor complexes is a crucial step in the modulation of genes by the AR. It is presumed that these hormone-receptor complexes interact with other proteins to participate in the formation of active transcription complexes at the initiation sites of androgen-responsive genes.

Using a yeast two-hybrid screening method, we isolated a partial cDNA encoding the carboxy terminus of a protein that interacts with the hAR-HBD (amino acid residues 623–917) in a ligand-dependent fashion in a yeast two-hybrid assay. Sequence analysis of this clone revealed that it encoded a portion of a protein that had been previously characterized as RFG (RET Fused Gene). Using glutathione-S-transferase (GST) fusions of the hAR HBD and immunoprecipitation of the in vitro translated proteins, we have demonstrated that this interaction can be reproduced in vitro. To determine the capacity of this protein to modulate the activity of the AR in transfection assays, we expressed full-length RFG in the CV1 and DU145 cell lines, in combination with an AR expression vector and model androgen-responsive genes [mouse mammary tumor virus (MMTV) and PRE2-tk luciferase]. Our results demonstrate that RFG alters the induction of these reporter genes very weakly (no greater than 2-fold compared with transfections without the RFG expression plasmid). Thus, while our findings are in agreement with published reports which indicate that RFG interacts with AR-HBD in a ligand-dependent fashion, in our assays RFG does not exert major effects on the activity of the hAR in response to androgen or to other steroid hormones.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human androgen receptor (hAR) is a ligand-dependent transcription factor that modulates the actions of androgen. Mutations that impair the function of the AR lead to abnormalities of male sexual development (1, 2). Definition of the mutations in the AR that cause androgen resistance have demonstrated that a large proportion of these are caused by amino acid substitutions in AR protein, and the majority of these amino acid replacements are localized to the hormone-binding domain (HBD). Detailed studies of such mutant ARs have revealed that an impairment of ligand binding or an instability of ligand binding is a common characteristic of substitution mutations in the AR HBD (3). Furthermore, studies of the ARs expressed in individuals with complete testicular feminization caused by single amino acid substitutions in the HBD of the AR indicated that increased doses of hormone or increased frequency of hormone addition can restore near-normal receptor function (3).

Such studies have focused attention on understanding the conformational changes that the AR undergoes after the binding of androgen and the protein-protein interactions that such changes facilitate. To this end, in parallel with studies of many members of the nuclear receptor family, attempts have been made using a variety of techniques to define the mechanisms by which androgens activate the AR. Studies by two groups explored the conformational changes of the AR in response to androgen agonists and antagonists. Using a limited proteolysis technique, these researchers demonstrated that a 29- to 30-kDa fragment of the AR agonist complex showed an increased resistance to trypsin digestion. By contrast, proteolysis of AR complexed to antagonists identified a distinct 35-kDa fragment of hAR that was resistant to proteolysis (4, 5), indicating that the conformation change induced by androgen was distinct from that stimulated by antiandrogens. Such inferences are in agreement with similar studies using limited proteolysis conducted in the study of other members of the nuclear receptor family (6, 7, 8, 9) and consistent with the more detailed information derived from crystallographic studies (10, 11, 12, 13, 14, 15).

While providing information regarding changes in the conformation of the AR protein, such studies do not identify the components of the transcriptional apparatus that are contacted by the AR when bound to agonists or antagonists. Experiments employing both biochemical and genetic approaches have identified a number of proteins that appear to serve as bridges between members of a nuclear receptor family and the transcription apparatus (16, 17).

Recently, cDNAs encoding a 70-kDa human protein, termed ARA70, were reported by Yeh and Chang (18). Sequence analysis indicated that this protein shares 94% homology with a previously characterized protein, RFG (RET-fused gene) (19). In their studies, these investigators found that RFG interacted with AR in an androgen-dependent manner and increased the activation of a reporter gene 10-fold in a cotransfection assay. In the studies detailed here, we isolated cDNAs encoding proteins that interacted with the HBD of the hAR (amino acid residues 623–917) in a ligand-dependent fashion. We found that one of these encoded a fragment of the RFG protein. In the present report, we have analyzed the capacity of the full-length RFG protein to interact with the AR. Our experiments demonstrate that in cotransfection assays, RFG behaves only weakly as a coactivator for the AR in the CV1 and DU145 cell lines and does not alter the ligand responsiveness of the AR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The RFG Protein Interacts with the hAR in a Ligand-Dependent Fashion
To understand the mechanisms regulating the function of the hAR, we used a two-hybrid screening method to identify proteins that interacted with the HBD of the hAR (20). One of the clones () that we isolated corresponded to a protein previously identified by Santoro et al. (19) as a fusion to the RET protooncogene in a human papillary thyroid cancer (Fig. 1Go). This protein, which they termed RFG (RET-fused gene), at the time was not known to perform any function (19).



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Figure 1. Schematic Structures of the Plasmids

Using a yeast two-hybrid screening method, we isolated a partial cDNA encoding a protein fragment that interacted with the hAR-HBD in a ligand-dependent fashion (clone 23–5). Sequence analysis of this partial cDNA revealed a 99% homology with a cDNA isolated by Santoro and co-workers (19 ), which they termed RFG (RET-fused gene). Three nucleotide sequence differences were noted compared with the originally reported sequence. These differences predict three amino acid differences at residues 316, 317, and 364, as indicated (FL RFG). Yeh and Chang (18 ) also identified the same differences in their isolate, compared with the sequence published for RFG (19 ). As noted in Materials and Methods, complete sequence of the cDNA provided to us by Santoro et al. revealed these same alterations, suggesting that the differences from the sequence reported by these investigators represent inadvertent inaccuracies in the nucleotide sequence that was originally reported (Genbank accession number X77548).

 
Initial experiments focused on examining the interaction with the AR HBD that was inferred from the conditions in which the clone 23–5 had been isolated. To address the specificity of this interaction, we transformed yeast with plasmids encoding analogous fusion proteins of the glucocorticoid receptor (GR) and progesterone receptor (PR) HBDs in the pAS vector and incubated the cells in the presence of their respective cognate ligands. Measurements of ß-galactosidase activity indicate that RFG displays increased interaction with the AR HBD in the presence of androgen. Parallel incubations of yeast cells harboring pACT plasmids encoding the PR or GR HBD indicated a ligand-dependent interaction of the RFG fusion protein with the PR, but not with the GR. Similar experiments examining the interaction of steroid receptor coactivator 1 (SRC-1) with these ligand-binding domains (LBDs) were performed to contrast with the RFG results and demonstrated a strong hormone-dependent interaction with the liganded PR and a weaker association with the liganded AR (Fig. 2Go).



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Figure 2. Interactions of RFG and the Human Androgen, Progesterone, and Glucocorticoid Receptors Using the Yeast Two-Hybrid Assay

Partial cDNAs encoding analogous segments of the hAR (amino acid residues 623–917), human GR (amino acid residues 483–777), and human PR (amino acid residues 639–933) were generated by PCR and subcloned into pAS vector. These plasmids were transformed with the 23–5/pACT plasmid (encoding the segment of the RFG protein depicted in Fig. 1Go) into yeast strain Y190 to examine the strength and specificity of interaction occurring between the receptors and RFG. As shown, while RFG exhibits a strong interaction with AR HBD in the presence of androgen and the PR HBD in the presence of progesterone, no such ligand-independent interaction could be detected when the pAS plasmid encoding the HBD of the GR was employed. In parallel experiments, a ligand-dependent interaction was observed in experiments assessing the interaction of the PR HBD with the carboxy terminus of SRC-1. No such interaction was observed in experiments using the AR or GR HBDs, although a ligand-dependent interaction of fusion proteins containing the GR LBD and the carboxyl terminus of GRIP-1 could be detected (data not shown).

 
As the RFG protein bears no significant similarity to other transcriptional coactivators or corepressors that have been identified, we performed experiments to determine whether the two proteins interact directly. Our first experiments employed a GST pull-down assay to examine the interactions of these two proteins. As shown in Fig. 3AGo, beads to which the HBD of the AR has been bound displayed increased retention of the labeled RFG protein synthesized using an in vitro transcription/translation system. While the results of these experiments were suggestive of a direct interaction, this conclusion was tempered by concerns regarding the poor efficiency of synthesis of AR protein in bacteria that is capable of binding hormone (21). For this reason, we employed a second technique to examine whether a direct interaction was occurring. In these experiments, we in vitro transcribed and translated both the hAR and the RFG protein. As shown in Fig. 3BGo, antibodies that sequester the in vitro transcribed/translated hAR precipitate the RFG protein in parallel. By contrast, little or no RFG protein is precipitated by resin to which no anti-AR antibody has been linked.



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Figure 3. Physical Interactions of the RFG Protein and the hAR

A, Radiolabeled RFG was tested for its ability to bind to GST or to GST-AR-HBD fusion protein immobilized on glutathione-Sepharose beads. A sample of the input-labeled RFG protein is shown in lane 2. Aliquots of the supernatants after incubation with GST-AR or GST-alone beads are shown in lanes 3 and 4, respectively. After incubation, the amount of radiolabeled RFG released after incubation with the GST-AR-HBD fusion protein beads and four successive washes are shown (lanes 5–8). The proteins released by solubilization in loading buffer of the GST control beads (lane 9) and the GST-AR-HBD beads (lane 10) after the four washes are shown. The level of nonspecific binding of RFG to the glutathione beads is evident in lane 9. B, Samples of the labeled RFG and hAR proteins were prepared in vitro as described in Materials and Methods. Samples of the input-labeled AR and input-labeled AR/RFG mixture are shown in lanes 2 and 3, respectively. Supernatants resulting from the incubation of the labeled mixture with beads to which no antibody (control), antibody directed the AR amino terminus (U402), or at an internal epitope (U407) are shown in lanes 4–6, respectively. The radiolabeled proteins that remained absorbed to the beads to which the different anti-AR antibodies had been affixed are shown in lanes 8 and 9, respectively. Molecular weight markers are shown in lane 1, and the positions of the in vitro translated AR and RFG proteins are shown to the right.

 
Effects of RFG Expression on AR Function
To examine the effects of RFG expression on AR function, CV 1 cells were transfected with varying ratios of the expression plasmids encoding RFG and hAR. In these experiments, the quantities of DNA employed, particularly the cytomegalovirus (CMV) expression plasmid, were held constant. As detailed in Fig. 4Go, only small alterations of AR function could be observed and were similar whether expressed as fold induction or in terms of the level of reporter gene activity that was measured. These small effects were not limited to experiments in which the mouse mammary tumor virus (MMTV) promoter was used to drive the reporter plasmid (panel A). Experiments in which the PRE2-tk luciferase reporter plasmid was employed also demonstrated only small alterations of AR function (panel B).



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Figure 4. Effect of RFG on AR Function Assayed in the CV1 Cell Line Using the MMTV and PRE2-tk Luciferase Reporter Genes

To assess the effect of RFG expression on the functional responsiveness of an androgen-responsive reporter gene, expression plasmids encoding the normal hAR, RFG, and either the MMTV luciferase (column A) or PRE2-tk luciferase (column B) reporter plasmid were introduced into CV1 cells. In each instance the data are expressed as either the percent of fold induction (open bars) or the percent of stimulated activity (stippled bars), with the degree of responsiveness observed after transfection with the AR expression plasmid alone after stimulation with 2 nM 5{alpha}-dihydrotestosterone (DHT) (included in each experiment) set at 100%. Each of the data sets within columns A and B is derived from six separate transfected cell samples (three basal and three stimulated with hormone). The corrected basal and DHT-stimulated activities of the cells transfected with the AR expression plasmid (included in each experiment) are shown for each group in Table 1Go.

 
A prior report had suggested that RFG exerts potent effects on AR function in assays conducted in cells that do not express the RFG protein, such as has been reported for the DU 145 cell line (18). To examine this possibility, we performed an additional series of experiments using the DU145 cell line. Figure 5Go presents the results of experiments in which the recipient DU145 cell line was transfected with expression vectors encoding RFG and hAR. These experiments included conditions representing transfection with both small and large quantities of both expression vectors. Examination of this figure reveals similar levels of reporter gene activity in cells transfected with 50, 100, or 200 ng of AR expression plasmid. As expected, transfection with larger amounts of AR cDNA led to decreased levels of reporter gene activity. In general, when cells were transfected with a single level of the AR cDNA, transfections with increasing amounts of the RFG expression plasmid led to a gradual decline in the level of reporter gene activity that was observed. These patterns are similar whether expressed in terms of the absolute level of stimulated reporter gene activity or as the fold induction of reporter gene activity, as summarized in Fig. 6Go for the MMTV-luciferase reporter plasmid. Cotransfection assays using cDNAs encoding SRC-1 and GRIP-1 (GR interacting protein 1) yielded modest effects on AR function (data not shown).



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Figure 5. Effect of RFG on AR Function Assayed in the DU145 Cell Line Using the MMTV and PRE2-tk Luciferase Reporter Genes

To assess the effect of RFG expression on the functional responsiveness of an androgen-responsive reporter gene in the DU145 cell line, these cells were transiently transfected with an expression plasmid encoding the normal hAR, RFG, and either the MMTV luciferase (column A) or PRE2-tk luciferase (column B) reporter plasmids. In each panel, the data are expressed as either the percent of fold induction (open bars) or the percent of stimulated activity (stippled bars) after stimulation with 2 nM DHT, and the level of response observed is expressed relative to the level observed in cells transfected with the AR expression plasmid alone (as 100%). Each of the data sets is derived from six separate transfected cell samples (three unstimulated and three stimulated with hormone). The corrected basal and DHT-stimulated activities of the cells transfected with the AR expression plasmid (included in each experiment) are shown for each group in Table 1Go.

 


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Figure 6. Summary of Experiments Using the MMTV Luciferase Reporter Plasmid in the DU145 Cell Line

Because of the variability of the individual experiments depicted in Figs. 4Go and 5Go, the results from three separate experiments are summarized by relating the data to the activity of the reporter gene in cells transfected with 0.1 µg of AR expression plasmid after stimulation with DHT (included in each experiment). The data are presented as percentage of the stimulated value (panel A) or the fold induction (panel B) observed. Similar results were obtained using the PRE2-tk-luciferase plasmid as the reporter plasmid (data not shown).

 
Effect of RFG Expression on the Responsiveness of hAR
Yeh and Chang (18) have reported that the expression of the RFG protein in cells causes an alteration of the hormone responsiveness of the AR in functional assays. In their experiments, incubations of cells transfected with expression plasmids encoding RFG (ARA 70) and hAR displayed responsiveness to estrogen, which was not displayed when the RFG plasmid was omitted from the transfection mixture (22). To examine this issue, we conducted such experiments in both the DU145 and CV1 cell lines. As shown in Fig. 7AGo, while small increments of reporter gene activation were observed after treatment with estrogen when such experiments were conducted in CV1 cells, these increases were evident only when extremely high concentrations of estradiol were employed. In addition, the stimulation that occurred at these supraphysiological estradiol concentrations did not require the expression of the RFG plasmid, and no such increases were observed when the experiments were performed using lower, more physiological concentrations of hormone. Diethylstilbestrol (DES) was unable to stimulate this effect, even when used at high concentrations (1 µM; data not shown). When such experiments were repeated using the DU145 cell line (Fig. 7BGo), estrogen at low concentrations had a limited capacity to induce a model androgen-responsive gene, and the small changes of expression were not altered by the expression of the hAR or RFG expression plasmids. At supraphysiological estrogen concentrations, an approximately 2-fold increase of reporter gene activity was observed. This effect was not seen when DES (1 µM) was employed or when lower, more physiological concentrations of estradiol were employed.



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Figure 7. RFG Expression Does Not Alter the Ligand Responsiveness of the hAR

A, CV1 cells were transfected with expression plasmids encoding the hAR and the RFG, alone and in combination, and the MMTV luciferase reporter plasmid. After transfection, the cells were incubated with no hormone, DHT, or estradiol at the indicated concentrations. Reporter gene activity was measured 48 h later. The data are plotted as percent of fold induction (open bars) or percent of stimulated reporter gene activity (stippled bars), relative to the activation observed using saturating doses of DHT. In this experiment, the basal and DHT-stimulated activities were 3142 and 162,011 relative light units (RLU) (52-fold induction). No stimulation was observed when DES was used in place of estradiol (data not shown). B, The results of similar experiments conducted in the DU145 cell line using the MMTV luciferase reporter plasmid are shown. In this experiment, the basal and DHT-stimulated activities were 137 and 929 RLU (7-fold induction). As in panel A, the data are plotted relative to the reporter gene activity measured after stimulation with saturating doses of DHT. No stimulation was observed when DES was used in place of estradiol (data not shown).

 
Detection of the RFG Protein
The interpretation of the experiments described above requires that the RFG expression plasmid employed be capable of directing the expression of the RFG protein in the transfected cells. To examine this in the systems that we have employed, we performed immunoblots using an antiserum raised to segments of the RFG protein to detect the expression of the RFG protein in transfected cells. Figure 8Go presents the results from one such experiment. While no immunoreactivity is observed in untransfected cells (data not shown), a single band is observed in extracts of cells transfected with the RFG expression plasmid that migrates in SDS-polyacrylamide gels with an apparent molecular mass of 70 kDa. The detection of this protein can be blocked if the anti-RFG antibody is preincubated with the RFG fusion protein.



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Figure 8. Immunoreactive RFG Is Detected in Cells Transfected with the RFG Expression Plasmid

To examine whether the intact RFG protein could be detected in cells transfected with the RFG expressions, COS cells were transfected with either the RFG expression plasmid or the empty expression plasmid. Cell extracts were prepared after which immunoblots were prepared using antibodies raised against the RFG protein. The RFG protein is detected in the extracts of the cells transfected with the RFG expression plasmid and migrates with an apparent molecular mass of about 70 kDa as shown in panel A. This species is efficiently competed when the anti-RFG antiserum is preincubated with purified RFG protein before use in immunoblotting experiments (panel B). This immunoreactive band is also absent from extracts of COS cells that were not transfected with the RFG expression plasmid (data not shown).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our studies of AR containing a variety of amino acid substitutions within the HBD demonstrated that the inability to form a stable hormone-receptor complex was a common attribute of such mutant ARs. This observation emphasized the need to identify the proteins that interact with liganded androgen receptor to mediate its effects on the activities of responsive genes (3).

In our attempts to approach this problem, we employed a yeast two-hybrid screening method to identify proteins interacting with the liganded hAR HBD. In this screen, we isolated a partial cDNA encoding of protein previously identified by Santoro and co-workers (19) and termed RFG by these investigators. A cDNA encoding a part of the same protein was identified by Yeh and Chang (18) and termed ARA 70; it was shown to interact with the hAR HBD in a ligand-dependent fashion when expressed as a fusion protein in yeast. These latter investigators reported that RFG (ARA70) was capable of acting as a specific coactivator for AR function and capable of modifying the hormonal responsiveness of the AR (22).

We have conducted experiments in yeast in vitro and in mammalian cells to examine the capacity of this protein to interact with the AR and to modulate its function. In yeast, our experiments showed that the interaction of the AR HBD with the carboxy terminus of RFG was maximal in the presence of agonist, and that this interaction was greatly diminished when ligand was not bound to the AR HBD. This interaction was not specific for the hAR, as the RFG fusion protein was capable of interacting with the PR in a similar fashion in response to progesterone in the yeast two-hybrid assay. By contrast, while a strong interaction could be demonstrated between the LBD of the human PR and the carboxy terminus of the SRC-1 coactivator, our studies indicated that the ligand-dependent interaction of the LBD of the hAR with SRC-1 was substantially weaker.

To examine whether these findings were the result of a direct interaction between the AR and RFG proteins, we performed GST pull-down and immunoprecipitation experiments. Although GST pull-down experiments suggested that an interaction was occurring between the full-length RFG and the GST AR fusion protein, our interpretation of this result was tempered by the results of our previous studies in which we demonstrated that much of the AR fusion protein synthesized in this fashion is not capable of binding ligand. To address this potential shortcoming, we also performed immunoprecipitation experiments using labeled full-length RFG and AR proteins derived from in vitro translation results. The results of these experiments also suggested that the RFG and AR proteins interact directly.

Despite these indications that a ligand-dependent interaction occurs between these two proteins, our experiments testing the functional significance of this interaction did not demonstrate an important role of this protein in modulating gene activation by the hAR. In the cotransfection assays reported here, RFG exhibited only subtle effects on AR function, results that were similar whether performed in the DU 145 or CV1 cell lines. The results that we obtained were not dependent on the promoter plasmids employed and yielded similar results using both the MMTV and PRE2-tk luciferase reporter plasmids.

We were unable to detect an effect of RFG expression on the ligand responsiveness of the hAR. In our experiments, only small increases of luciferase activity could be observed in cells transfected with the hAR and stimulated with estradiol. These increases were observed only at high estradiol concentrations and did not require transfection with the RFG expression plasmid. Such observations are reminiscent of the activation of the AR that is effected by estradiol seen in cells that do not oxidize steroids at the 17-hydroxyl group (23). Since estradiol is capable of displacing androgen in binding assays when added at high concentrations (24), such a finding would be most consistent with activation occurring as the result of the binding of estradiol to the AR in a nonphysiological fashion.

In summary, we have demonstrated that the interaction of the AR with the RFG (ARA70) protein can be demonstrated in the presence of an AR agonist ligand in a variety of systems. This interaction appears to be direct, but is not specific for the AR, as a similar ligand-dependent interaction can also be demonstrated for the PR. Most importantly, although our studies confirm the predicted sequence difference detected by Yeh et al. (22) and the synthesis of the predicted protein product after transfection of the cDNA into mammalian cells, we are unable to demonstrate an important effect of this protein in modulating responsiveness of model androgen-responsive genes. At present, we are unable to reconcile our findings with those previously published on this subject.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Yeast Two-Hybrid Screens
The Gal4 DNA-binding domain fusion plasmid encoding the AR HBD was prepared by cloning a PCR-amplified fragment encoding amino acid residues 623–917 of hAR into the NdeI and BamHI restriction endonuclease cleavage sites of the pAS vector (25). Transformation of the pACT cDNA library into Y190 yeast cells (prepared from human prostate RNA by Dr. S. Elledge) was performed using the method of Rose et al. (26), except that 40 µg of plasmid DNA and 4.0 mg of carrier DNA were added to 800 µl of LISORB (100 mM lithium acetate, 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1.0 M sorbitol). This mixture was used to transform 10- to 100-µl aliquots of competent cells. After transformation, the cells were plated on YNB Trp (-) Leu (-) His (-) plates containing 3-amino-1,2,4-triazole and mibolerone.

Expression of the AR Fusion Protein in Bacteria
The GST fusion protein encoding the AR HBD has been described previously (27). After induction with isopropylthiogalactoside, the bacteria were lysed and the soluble fraction was incubated with [3H]mibolerone at 4 C for 20 h. After labeling, the extract was passed over a glutathione-Sepharose column and washed extensively. The bound fraction was eluted with reduced glutathione, pooled, and dialyzed before use in the experiments examining the interaction with labeled RFG.

Plasmids
The RFG expression plasmid was constructed by subcloning an RFG cDNA (obtained from M. Santoro, Universita degli Studi di Napoli, Naples, Italy) into the eukaryotic expression plasmid CMV5. In vitro transcription/translation experiments employed the same cDNA inserted into the Bluescript plasmid (pBSKS M13+). An expression plasmid of full-length SRC was provided by B. O’Malley (Baylor College of Medicine, Houston, TX) and was inserted into the expression plasmid CMV5. The plasmids GR HBD/pAS and PR HBD/pAS were constructed by subcloning analogous fragments of GR (amino acid residues 483–777) (28) and PR (amino acid residues 639–933) (29), which were generated by PCR and cloned into the pAS vector (25).

GST Pull-Down Assay
RFG labeled with [35S]methionine was prepared using the full-length RFG contained in an expression vector (inserted in the expression vector pBSKS M13+) in a coupled in vitro transcription/translation system (Promega Corp., Madison, WI). Partially purified GST-hAR-HBD fusion protein (prebound to mibolerone, see above), was incubated overnight with a 20-µl aliquot of glutathione-Sepharose beads at 4 C. After the overnight incubation, unbound fusion protein was washed away and the beads were incubated with an aliquot of the in vitro transcribed translated [35S]methionine-labeled RFG protein at 4 C. Beads were washed four times with the binding buffer, resuspended in 50 µl of 2x SDS loading buffer, and analyzed by SDS-PAGE and autoradiography.

In Vitro Transcription/Translation and Immunoprecipitation Assays
The assay was performed as described previously (30) with modification: in vitro-translated and 35S-radiolabeled protein was obtained using a transcription and translation system (TNT-coupled reticulocyte lysate system, Promega Corp.). Crude lysate (40 µl) diluted at ratio 1:10 with modified CSK (mCSK) buffer (10 mM 1,4-piperazinediethane sulfonic acid, pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgCL2, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) containing 0.1 Triton X-100. Four 100-µl aliquots of mixed lysates were incubated overnight with 20 µl beads conjugated with no hormone, U402, U407, and R489 anti-AR antibodies, respectively, at 4 C (31). After the overnight incubation, the lysate was centrifuged for 1 min at 10,000 x g, and the beads were washed three times (1 ml mCSK buffer with 0.3 M NaCl). Both supernatant and washed beads were solubilized in 2x SDS loading buffer and analyzed by SDS-PAGE and autoradiography.

Transient Transfection Assay
The day before transfection, CV1 and DU145 cells were plated into six-well plates at a density of 2 x 105 cells per well. Twenty hours later, each plate was transiently transfected by the addition of a calcium phosphate precipitate containing the indicated amount of expression plasmid DNAs, the reporter plasmid MMTV or PRE2-tk luciferase (10 µg), and a control plasmid, CMV-ß-galactosidase (1 µg), in culture medium for 14–18 h. Twenty hours later, the medium was replaced with fresh medium containing 5% charcoal-stripped serum and either no hormone or a hormone at the indicated concentrations. Cells were harvested 48 h after hormone addition, and the levels of luciferase and ß-galactosidase activity were measured. In transfections in which the concentrations of plasmids (e.g. CMV RFG or CMV hAR) were varied, the quantity of CMV5 vector was adjusted so that the total amount of CMV5 expression plasmid remained constant.

Sequence Analysis
Nucleotide sequence analysis was performed using a PE Applied Biosystems (Norwalk, CT) automated sequencer that utilized the Sanger dideoxy method of DNA sequencing with a fluorescent. The sequence of the RFG provided by Dr. Santoro was determined on both strands over the complete cDNA fragment, as detailed in the figures and text. The nucleotide sequence of the partial RFG cDNA isolated in the pACT plasmid in the two-hybrid assay was also sequenced on both strands.

Anti-RFG Antiserum
Antibodies directed at RFG were generated by immunizing rabbits with fragments of the recombinant RFG expressed in bacteria (32). The resulting antiserum was purified using an affinity column to which the purified RFG peptides were covalently attached (K. Brantley, T. Gao, and M. J. McPhaul, unpublished data).

Note Added in Proof
The studies of Alen et al. (33) also demonstrated only minor effects of the RFG/ELE1/ARA70 protein on the transcriptional activity of the human AR as assayed in cotransfection experiments.


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Table 1. Basal and Stimulated Luciferase Activities

 

    ACKNOWLEDGMENTS
 
We wish to thank Judy Gruber and Diane Allman for superb technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Michael McPhaul, Internal Medicine, Room J6.110, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8857.

This work was supported by NIH Grant DK-03892 and by Welch Foundation Grant I-1090.

Portions of this work were reported in abstract form at the 75th Annual Meeting of The Endocrine Society (Minneapolis, MN, 1997).

Received for publication September 22, 1998. Revision received May 20, 1999. Accepted for publication June 25, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS 1995 Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 16:271–321[Medline]
  2. McPhaul MJ, Marcelli M, Zoppi S, Griffin JE, Wilson JD 1993 The spectrum of mutations in the androgen receptor gene that causes androgen resistance. J Clin Endocrinol Metab 76:17–23[Abstract]
  3. Marcelli M, Zoppi S, Wilson CM, Griffin JE, McPhaul MJ 1994 Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex. J Clin Invest 94:1642–1650[Medline]
  4. Kuil CW, Berrevoets CA, Mulder E 1995 Ligand-induced conformational alterations of the androgen receptor analyzed by limited trypsinization. Studies on the mechanism of antiandrogen action. J Biol Chem 270:27569–27576[Abstract/Free Full Text]
  5. Kallio PJ, Janne OA, Palvimo JJ 1994 Agonists, but not antagonists, alter the conformation of the hormone-binding domain of androgen receptor. Endocrinology 134:998–1001[Abstract]
  6. Allan GF, Leng X, Tsai SY, Weigel NL, Edwards DP, Tsai MJ, O’Malley BW 1992 Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation. J Biol Chem 267:19513–19520[Abstract/Free Full Text]
  7. Allan GF, Tsai SY, Tsai MJ, O’Malley BW 1992 Ligand-dependent conformational changes in the progesterone receptor are necessary for events that follow DNA binding. Proc Natl Acad Sci USA 89:11750–11754[Abstract]
  8. Beekman JM, Allan GF, Tsai SY, Tsai MJ, O’Malley BW 1993 Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain. Mol Endocrinol 7:1266–1274[Abstract]
  9. Keidel S, Lemotte P, Apfel C 1994 Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping. Mol Cell Biol 14:287–298[Abstract]
  10. Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the RAR-gamma ligand-binding domain bound to all-trans retinoic acid. Nature 378:681–689[CrossRef][Medline]
  11. Wagner RL, Apriletti JW, McGrath ME, West BL, Baxter JD, Fletterick RJ 1995 A structural role for hormone in the thyroid hormone receptor. Nature 378:690–697[CrossRef][Medline]
  12. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carl-quist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  13. Williams SP, Sigler PB 1998 Atomic structure of progesterone complexed with its receptor. Nature 393:392–396[CrossRef][Medline]
  14. Tanenbaum DM, Wang Y, Williams SP, Sigler PB 1998 Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains. Proc Natl Acad Sci USA 95:5998–6003[Abstract/Free Full Text]
  15. Wurtz JM, Egner U, Heinrich N, Moras D, Mueller-Fahrnow 1998 Three-dimensional models of estrogen receptor ligand binding domain complexes, based on related crystal structures and mutational and structure-activity relationship data. J Med Chem 41:1803–1814[CrossRef][Medline]
  16. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:1167–1177[Abstract]
  17. Shibata H, Spencer TE, Onate SA, Jenster G, Tsai SY, Tsai MJ, O’Malley BW 1997 Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52:141–164[Medline]
  18. Yeh S, Chang C 1996 Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells. Proc Natl Acad Sci USA 93:5517–5521[Abstract/Free Full Text]
  19. Santoro M, Dathan NA, Berlingieri MT, Bongarzone I, Paulin C, Grieco M, Pierotti MA, Vecchio G, Fusco A 1994 Molecular characterization of RET/PTC3; a novel rearranged version of the RET proto-oncogene in a human thyroid papillary carcinoma. Oncogene 9:509–516[Medline]
  20. Gao TS, McPhaul MJ, The conformation of androgen receptor hormone-binding domain complexed to androgens and androgen receptor antagonists can be distinguished by protein-protein interactions. Program & Abstracts of the 10th International Congress of Endocrinology, San Francisco, CA, 1996, p 189 (Abstract P1–217)
  21. Cooper B, Gruber JA, McPhaul MJ 1996 Hormone-binding and solubility properties of fusion proteins containing the ligand-binding domain of the human androgen receptor. J Steroid Biochem Mol Biol 57:251–257[CrossRef][Medline]
  22. Yeh S, Miyamoto H, Shima H, Chang C 1998 From estrogen to androgen receptor: a new pathway for sex hormones in prostate. Proc Natl Acad Sci USA 95:5527–5532[Abstract/Free Full Text]
  23. Deslypere J-P, Young M, Wilson JD, McPhaul MJ 1992 Testosterone and 5a-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene. Mol Cell Endocrinol 88:15–22[CrossRef][Medline]
  24. Tilley WD, Marcelli M, Wilson JD, McPhaul MJ 1989 Characterization and expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA 86:327–331[Abstract]
  25. Bai C, Elledge SJ 1997 Gene identification using the yeast two-hybrid system. Methods Enzymol 283:141–156[Medline]
  26. Rose MD, Winston F, Hieter P 1990 Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  27. Roehrborn CG, Zoppi S, Gruber JA, Wilson CM, McPhaul MJ 1992 Expression and characterization of full-length and partial human androgen receptor fusion proteins. Implications for the production and applications of soluble steroid receptors in Escherichia coli. Mol Cell Endocrinol 84:1–14[CrossRef][Medline]
  28. Misrahi M, Atger M, d’Auriol L, Loosfelt H, Meriel C, Fridlansky F, Guiochon-Mantel A, Galibert F, Milgrom E 1987 Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA. Biochem Biophys Res Commun 143:740–748[Medline]
  29. Hollenberg SM, Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, Thompson EB, Rosenfeld MG, Evans RM 1985 Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature 318:635–641[Medline]
  30. Fujita M, Kiyono T, Hayashi Y, Ishibashi M 1997 In vivo interaction of human MCM heterohexameric complexes with chromatin. J Biol Chem 272:10928–10935[Abstract/Free Full Text]
  31. Wilson CM, McPhaul MJ 1994 A and B forms of the androgen receptor are present in human genital skin fibroblasts. Proc Natl Acad Sci USA 91:1234–1238[Abstract]
  32. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW 1991 Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
  33. Alen P, Claessens F, Schoenmakers E, Swinnen JV, Verhoeven G, Rombauts W, Peeters B 1999 Interaction of the putative androgen receptor-specific coactivator ARA70/ELE1{alpha} with multiple steroid receptors and identification of internally deleted ELE1ß isoform. Mol Endocrinol 13:117–128[Abstract/Free Full Text]