Domain Interactions between Coregulator ARA70 and the Androgen Receptor (AR)

Zhong-xun Zhou, Bin He, Susan H. Hall, Elizabeth M. Wilson and Frank S. French

Departments of Pediatrics (Z-X.Z., S.H.H., E.M.W., F.S.F.) and Biochemistry and Biophysics (B.H., E.M.W.), and The Laboratories for Reproductive Biology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7500

Address all correspondence and requests for reprints to: Dr. Frank S. French, Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7500. E-mail: fsfrench{at}med.unc.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The coregulator function of AR-associated protein 70 (ARA70) was investigated to further characterize its interaction with the AR. Using a yeast two-hybrid assay, androgen-dependent binding of ARA70 deletion mutants to the AR ligand-binding domain (LBD) was strongest with ARA70 amino acids 321–441 of the 614 amino acid ARA70 protein. Mutations adjacent to or within an FxxLF motif in this 120-amino acid region abolished androgen-dependent binding to the AR-LBD both in yeast and in glutathione-S-transferase affinity matrix assays. Yeast one-hybrid assays revealed an intrinsic ARA70 transcriptional activation domain within amino acids 296–441. In yeast assays the ARA70 domains for transcriptional activation and for binding to the AR-LBD were inhibited by the C-terminal region of ARA70. Full-length ARA70 increased androgen-dependent AR transactivation in transient cotransfection assays using a mouse mammary tumor virus-luciferase reporter in CV1 cells. ARA70 also increased constitutive transcriptional activity of an AR NH2-terminal-DNA binding domain fragment and bound this region in glutathione-S-transferase affinity matrix assays. Binding was independent of the ARA70 FxxLF motif. The results identify an ARA70 motif required for androgen-dependent interaction with the AR-LBD and demonstrate that ARA70 can interact with the NH2-terminal and carboxyl-terminal regions of AR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE AR IS a hormone-activated transcription factor that mediates the differentiation, development, and maintenance of male reproductive function (1, 2). AR mutations identified in the androgen insensitivity syndrome exemplify the essential role of AR in these processes (3, 4, 5, 6). AR belongs to the nuclear receptor superfamily that comprises receptors for steroid and thyroid hormones, vitamin D, retinoids, peroxisome proliferator, and orphan receptors (7, 8, 9, 10, 11, 12). Nuclear receptors have a conserved structural arrangement of the DNA and ligand binding domains (13, 14, 15, 16, 17, 18, 19, 20), whereas the NH2-terminal domains differ markedly in length and sequence (7, 21).

Hormone-bound nuclear receptors bind DNA-responsive elements and trigger a cascade of transcriptional events. To initiate transcription, nuclear receptors interact in a ligand-dependent fashion with coactivators and components of the transcriptional machinery such as transactivation factor IIB (TFIIB), TATA-binding protein, TATA-binding protein-associated factor, or TFIIH (22, 23, 24, 25, 26, 27). A family of proteins, termed the p160 coactivators, includes steroid receptor coactivator 1 (SRC-1), nuclear receptor coactivator 1 (28, 29, 30, 31); SRC-2, transcriptional intermediary factor 2 (TIF2), GR-interacting protein 1 (GRIP1), nuclear receptor coactivator 2 (32, 33); and SRC-3, p300/CBP cointegrator associate protein, receptor-associated coactivator 3, activator of the thyroid and RAR, amplified in breast cancer 1, thyroid receptor activator molecule-1 (34, 35, 36, 37, 38, 39). These p160 coactivators interact with nuclear receptors in a ligand-dependent manner (16, 40, 41) through LxxLL motifs that make up the nuclear receptor box (42, 43, 44). X-ray crystallographic studies revealed that nuclear receptor box peptides bind the hydrophobic cleft formed by nuclear receptor ligand-binding domain (LBD) helices 3, 4, 5, and 12 (45, 46, 47). Multiprotein complexes of nuclear receptor coactivators and chromatin remodeling factors gain access to target DNA and activate transcription (26, 31, 48, 49, 50, 51, 52).

AR-associated protein 70 (ARA70) was isolated from human brain (53) and prostate (54) cDNA libraries by its binding to the human AR-LBD and enhanced the transcriptional activity of AR in cotransfection assays. Alen et al. (55) reported that ARA70 bound TFIIB and p300/CBP-associated factor in vitro and suggested that ARA70 acts as a bridging factor between steroid receptors and components of the transcription initiation complex. Herein we further characterize ARA70 as an AR coactivator by analysis of its interactions and functional domains. A sequence motif required for androgen-dependent binding to the AR-LBD is identified. In addition, regions containing intrinsic transcriptional activation and inhibitory domains are delineated. We demonstrate also that ARA70 can interact with the NH2-terminal region of AR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of a Partial ARA70 cDNA and Hormone Requirements for Interaction with the AR-LBD
Yeast two-hybrid screening was used to identify human AR-LBD interacting proteins expressed by a random primed cDNA library from an androgen-stimulated LNCaP (lymph node-derived human prostate carcinoma) cell line. The AR-LBD (amino acids 624–919) included the hinge region and LBD. Approximately 8 x 107 yeast transformants of the VP16 library were screened. Among 18 positive yeast clones, four clones with identical restriction maps were sequenced and compared with data in GenBank. ARA70 coding sequence for amino acids 321–499 was identified and contained Gly 364 as in ARA70 (53) rather than Ala 364 as reported in RET-fused gene (RFG) (57). In yeast two-hybrid assays, a dose-dependent interaction of the AR-LBD with ARA70 321–499 was observed with dihydrotestosterone (DHT), T, or the synthetic androgen R1881 (methyltrienolone) (Fig. 1AGo).



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Figure 1. ARA70 Interaction with AR

A, Effects of DHT, T, and methyltrienolone (R1881) on the interaction of ARA70 amino acids 321–499 with AR-LBD in a yeast two-hybrid assay. Yeast L40 was cotransformed with expression plasmids pLexA-AR-LBD and VP ARA70 321–499 and grown on Ura-, Trp-, Leu- plates for 48 h. Colonies were picked and grown in liquid culture in the presence of increasing concentrations of steroid. ß-Gal activity was measured in yeast cell extracts of liquid cultures. Liquid assay results are plotted in units as defined in Materials and Methods. Shown are the mean and SD of assays on three independent transformants. B, Effect of ARA70 on AR transactivation. pSG5 expression vectors coding for ARA70, and full-length AR (0.1 µg) were cotransfected into 6-cm dishes of CV1 cells with MMTV-LUC (2.5 µg) and incubated in the absence and presence of 0.1 nM DHT. Control incubations included AR and MMTV-LUC alone or together with the indicated amounts of pSG5 empty vector DNA (molar equivalents of the pSG5ARA70) and pBR322 to make the same total weight of DNA. Incubations and analysis of luciferase were as described in Materials and Methods.

 
ARA70 Enhances AR Transactivation
To investigate coregulator function of ARA70, CV-1 cells were cotransfected with AR and ARA70 expression vectors and the mouse mammary tumor virus (MMTV)-luciferase reporter plasmid (Fig. 1BGo). The full-length ARA70 cDNA construct used in these experiments had sequence identity to that of Yeh and Chang (53). Transfection of pSG5ARA70 DNA resulted in increases in DHT-dependent transcription above the level with AR and reporter gene alone or in the presence of an equimolar amount of pSG5 vector DNA. ARA70 had little effect on background activity in the absence of DHT and no effect on the transcriptional activity of a constitutive reporter vector, psLuc2 (data not shown). The level of endogenous ARA70 mRNA detected by northern hybridization in CV1 cells was similar to that in COS cells and HeLa cells and 2–3 times higher than in the prostate cell lines DU145, PC3, and LNCaP (data not shown).

ARA70 Sequence That Binds the AR-LBD
To determine the region of ARA70 that interacts with the AR-LBD, a yeast two-hybrid assay was used. Yeast strain L40 was transformed with LexA-AR-LBD and VP16-ARA70 deletion mutants, and binding was measured by lift and liquid ß-galactosidase (ß-gal) assays in the presence of 10 nM DHT. Yeast transformed with LexA-AR-LBD and VP16 served as a negative control.

LexA-AR-LBD interacted with VP-ARA70 1–614, and binding increased 4-fold with deletion of the carboxyl-terminal region (VPARA70 1–499 and 1–441) (Fig. 2AGo). VPARA70 321–499 was 6-fold higher than VPARA70 291–614 or 320–614 mutants that contained carboxyl-terminal sequence. VPARA70 321–441 had the highest AR-LBD binding activity. NH2- and carboxyl-terminal deletions of VPARA70 321–441 resulted in a gradual decline of binding activity. ARA70 NH2-terminal fragments in VPARA70 1–321, 1–295, or 1–118 did not bind the AR-LBD despite the presence of a LxxLL sequence (42, 43, 44) at residues 92–96. Thus, 321–441 contained the amino acid sequence with highest binding activity for the AR-LBD. The increase in binding activity with deletion of the ARA70 carboxyl-terminal region suggested the presence of an inhibitory sequence within residues 499–614. This apparent repression domain within ARA70 may influence protein folding or interaction with a repressor protein.



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Figure 2. ARA70 Sequences That Interact with the AR-LBD

A, Expression plasmids coding for fusion proteins of LexA-AR-LBD (AR amino acids 624–919) and pVP16AD-ARA70 were analyzed in the yeast two-hybrid interaction assay. Numbers on the bars indicate amino acid residues of ARA70 in the fusion protein. Yeast containing an integrated ß-gal reporter gene controlled by LexA binding sites was transformed with the different expression plasmids. After transformation, yeast was grown on Ura-, Trp-, and Leu- plates for 48 h, replica plated to similar plates containing 10 nM DHT for 16 h for lift assays, or grown in liquid medium for liquid assays. Liquid assay results are expressed in units as defined in Materials and Methods. Results are representative of three independent experiments. B, Immunoblot analysis. Equal amounts of cell extract (1 x 108 cell equivalents) from transformed yeast L40 were immunoblotted with a monoclonal antibody against VP16 amino acids 1–21 to measure relative expression levels of the VP16 ARA70 fusion proteins. C, In vitro interaction of ARA70 deletion mutants with the carboxyl-terminal region of AR. Equal amounts of whole Sf9 cell extracts expressing AR 507–919 in the presence or absence of 10 nM DHT were incubated with glutathione-Sepharose beads containing bound GST-ARA70 1–614, GST-ARA70 321–499, GST-ARA70 321–441, (lanes 3–8), or GST alone (lanes 1 and 2). Beads were washed and bound protein was eluted and analyzed by Western blot using AR52 rabbit polyclonal anti-AR peptide antiserum (21 93 ). D, In vitro interaction of full-length ARA70 and ARA70(2KA) mutant with AR-LBD. [35S]ARA70 and [35S]ARA70(2KA) were synthesized in vitro and incubated in the presence or absence of DHT, 1 µM, with glutathione-Sepharose beads bound with GST-AR-LBD (AR 624–919). Control beads contained GST alone. Beads were washed and bound [35S]-labeled proteins were eluted and analyzed by SDS-PAGE and autoradiography. The input lane contained 15% of the total radioactivity added.

 
Immunoblot analysis using an antibody against the VP16 transcriptional activation domain indicated that the VP16-ARA70 fusion proteins were expressed at similar levels (Fig. 2BGo).

The ARA70 region that binds AR-LBD was further investigated using glutathione-S-transferase (GST)-fusion proteins. GST-ARA70 1–614, 321–499, or 321–441 bound to baculovirus expressed AR DNA-binding domain-LBD fragment, AR 507–919 (Fig. 2CGo). These fragments did not bind to GST protein alone, but bound AR 507–919 in the presence of DHT, confirming that ARA70 residues 321–441 contain a site for androgen-dependent interaction with the AR-LBD.

ARA70 K327A/K329A Abolished Androgen-Dependent Binding to the AR-LBD
ARA70 residues 321–441 contain basic amino acids, R322, K327, and K329, and five evenly spaced cysteines at residues 398, 404, 410, 416, and 422, each separated by five amino acids. No similar sequence was identified in the GenBank database. Missense mutations were created to test individual amino acid requirements for ARA70 321–441 interaction with the AR-LBD. R322A decreased activity by 20% whereas the double mutation K327A/K329A [ARA70(2KA)] reduced binding to an undetectable level in the yeast two-hybrid assay (Fig. 2AGo). C398A/C404A or C410A/C416A/C422A decreased binding by about 30%. The results suggested that a sequence associated with the two basic residues (K327, K329) has a key role in ARA70 binding to the AR-LBD.

Binding of full-length ARA70(2KA) to AR-LBD was measured in GST affinity matrix assays (Fig. 2DGo). [35S]-labeled full-length ARA70 and ARA70(2KA) were synthesized in vitro and incubated with GST-AR-LBD in the presence or absence of DHT. ARA70 binding to GST-AR-LBD was increased about 2-fold in the presence of DHT while there was no increase in ARA70(2KA) binding in the presence of DHT.

To test the effect of the 2KA double mutation K327A/K329A on coactivator activity, full-length ARA70(2KA) or wild-type ARA70 was cotransfected into CV-1 cells with AR and MMTV-luciferase (MMTV-LUC) reporter plasmids (Fig. 3AGo). Transfection of 2.9 µg of pSG5ARA70 DNA resulted in an increase in luciferase activity relative to AR and reporter gene alone or an equimolar amount of pSG5 vector DNA balanced with pBR322. AR coactivation with the mutant ARA70(2KA) was reduced compared with wild-type ARA70.



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Figure 3. Interaction of ARA70 and Mutant ARA70(2KA) with the AR NH2-Terminal Region

A, pSG5 expression vectors coding for full-length AR, 0.1 µg, and either ARA70 or ARA70(2KA), 2.9 µg, were cotransfected into CV1 cells with MMTV-LUC, 2.5 µg, and incubated in the absence and presence of 0.1 nM DHT. Control incubations contained AR and MMTV-LUC alone or together with 2.0 µg pSG5 empty vector DNA (the molar equivalent of 2.9 µg ARA70) and 0.9 µg pBR322 to make the same total weight of DNA. B, Expression plasmids encoding an AR NH2-terminal-DNA binding domain fragment, AR 1–660, 15 ng, and ARA70 or ARA70(2KA) mutant, 7.25 µg, were cotransfected into CV1 cells with MMTV-LUC, 2.5 µg. Control incubations contained AR 1–660 and MMTV-LUC alone or together with 5 µg pSG5 empty vector DNA [the molar equivalent of 7.25 µg ARA70 or ARA70(2KA)] and 2.25 µg pBR322 to make the same total weight of DNA. C, In vitro interaction of ARA70 or ARA70(2KA) mutant with AR NH2-terminal-DNA binding domain fragment, GST-AR 1–660. [35S]ARA70 and [35S]ARA70(2KA) mutant were synthesized in vitro and incubated with glutathione-Sepharose beads bound with GST-AR 1–660. Control beads contained GST alone. Beads were washed, and bound [35S]-labeled proteins were eluted and analyzed by SDS-PAGE and autoradiography. The input lane contained 15% of the total radioactivity added.

 
ARA70 Interacts with the AR NH2- Terminal Domain
The finding that ARA70(2KA) lacked DHT-dependent binding to the AR-LBD, yet retained partial function as a coactivator with full-length AR, raised the possibility that ARA70 interacts with the AR NH2-terminal region. The AR NH2-terminal-DNA binding domain fragment (AR 1–660) contains the AF1 region and has constitutive transcriptional activity. We tested the effects of ARA70 and ARA70(2KA) on transactivation by AR 1–660 (Fig. 3BGo). Expression plasmids encoding AR 1–660 and ARA70 or ARA70(2KA) were cotransfected into CV-1 cells with the MMTV-LUC reporter. ARA70 increased the transcriptional activity of AR 1–660 relative to control levels while the increase with ARA70(2KA) was less than that of the wild-type ARA70. In affinity matrix assays, both [35S]-labeled wild-type ARA70 and ARA70(2KA) bound to GST-AR 1–660 (Fig. 3CGo), indicating that ARA70 interacts with the AR NH2-terminal-DNA binding domain fragment.

FxxLF Is Required for Androgen-Dependent Binding of ARA70 to the AR-LBD but Not for Interaction with the AR NH2-Terminal Domain
After the discovery that the FxxLF motif is required for androgen-dependent binding of the AR NH2-terminal region to the AR-LBD (58), it was recognized that ARA70 321–441 contains a FxxLF sequence (328 FKLLF 332). To determine whether the FxxLF motif is required for ARA70 binding to the AR-LBD, the sequence FKLLF in full-length ARA70 was mutated to AKLAA(AxxAA), and binding of [35S]ARA70(AxxAA) to GST-AR-LBD was analyzed in the affinity matrix assay (Fig. 4AGo). ARA70(AxxAA) lacked-DHT dependent binding to AR-LBD while the binding of wild-type ARA70 in the presence of DHT was about 2 times higher than the no steroid control. A second affinity matrix assay was performed using [35S]AR-LBD (AR 624–919) to compare the binding of GST-ARA70 321–407 with binding to the same ARA70 protein fragment containing the AxxAA mutation (Fig. 4AGo). Binding in the absence of DHT was similar to that of the GST control. DHT had no effect on [35S]AR-LBD binding of the ARA70 321–407(AxxAA) mutant while it increased binding to the ARA70 321–407 fragment with wild-type sequence several fold.



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Figure 4. Role of FxxLF in ARA70 Interactions with the AR-LBD and the AR NH2-Terminal Domain

A, Effect of the ARA70 AxxAA mutation on androgen-dependent ARA70 binding to the AR-LBD. [35S]ARA70 and mutant [35S]ARA70(AxxAA) were synthesized in vitro and incubated with glutathione-Sepharose beads bound to GST alone or GST-AR-LBD (AR 624–919) either in the presence or absence of DHT. Beads were washed, and the bound [35S]-labeled proteins were eluted and analyzed by SDS-PAGE and autoradiography. The input lane contained 15% of the total radioactivity added. In the lower gel, [35S]AR-LBD was synthesized and incubated in the presence or absence of DHT with glutathione-Sepharose bound to GST alone, GST-ARA70 321–407(AxxAA), or GST-ARA70 321–407 containing wild-type sequence, and the bound [35S]-labeled protein was analyzed as above. The input lane contained 10% of the total radioactivity added. B, Effect of the ARA70 AxxAA mutation on ARA70 coactivation with full-length AR. pCMVhAR, 25 ng, and either pSG5ARA70 or ARA70(AxxAA), in the amounts indicated, were cotransfected into CV1 cells with MMTV-LUC, 2.5 µg, and incubated in the absence and presence of 0.1 nM DHT. Control incubations contained either AR and MMTV-LUC alone or together with 1, 2, or 5 µg pSG5 empty vector DNA (the molar equivalents of 1.45, 2.9, and 7.5 µg ARA70) and 0.45, 0.9, or 2.25 µg pBR322 to make the same total weight of DNA. C, Effect of the ARA70 AxxAA mutation on ARA70 coactivation with the AR NH2-terminal-DNA binding domain. Expression plasmids pCMVhAR 1–660, 10 ng, and pSG5ARA70 or ARA70(AxxAA), 7.25 µg, were cotransfected into CV1 cells with MMTV-LUC, 2.5 µg. Control incubations contained AR and MMTV-LUC alone or together with 5 µg pSG5 empty vector DNA (the molar equivalent of 7.25 µg ARA70 expression vector) and 2.25 µg pBR322 to make the same total weight of DNA. D, Binding of the ARA70 AxxAA mutant to the AR NH2-terminal-DNA binding domain. [35S]ARA70(AxxAA) and [35S]ARA70 were synthesized in vitro and incubated with glutathione-Sepharose beads bound to GST alone or to GST-AR 1–660. Beads were washed and the bound [35S]-labeled proteins were eluted and analyzed by SDS-PAGE and autoradiography. The input lane contained 15% of the total radioactivity added.

 
Since the ARA70(2KA) double mutation, K327A, K329A, flanks F328 in the FxxLF motif, it likely altered the binding function of the motif even though the consensus sequence FxxLF was unchanged. The results indicate that the ARA70 FxxLF motif mediates DHT-dependent binding to the AR-LBD.

In cotransfection assays (Fig. 4BGo), transcriptional activity of full-length AR with full-length ARA70(AxxAA) was less than that induced by wild-type ARA70. With ARA70(AxxAA), there was no change in luciferase activity with increasing amounts of transfected DNA, while wild-type ARA70 caused a dose-dependent increase in luciferase activity.

Cotransfection of ARA70(AxxAA) with AR 1–660 increased the constitutive transcriptional activity of this AR NH2-terminal fragment-DNA binding domain fragment (Fig. 4CGo), although it was less effective than wild-type ARA70. [35S]ARA70(AxxAA) binding to GST-AR 1–660 in the affinity matrix assay was similar to that of wild-type [35S]ARA70 (Fig. 4DGo), indicating that FxxLF is not essential for ARA70 binding to the AR NH2-terminal region. The reduced coactivation of ARA70(AxxAA) with AR 1–660 relative to that of wild-type ARA70 may have resulted from disruption of the ARA70 activation domain as indicated below.

Transcriptional Activation Domain in ARA70
Intrinsic activation was investigated by fusing regions of ARA70 to the pLexA DNA binding domain and measuring transcriptional activity of a ß-gal reporter gene. pLexA-lamin served as a negative control. Full-length ARA70 1–614 lacked activity; however, the carboxyl-terminal deletion mutant 1–499 induced ß-gal activity (Fig. 5AGo), and LexA-ARA70 1–441 activity was 2-fold higher, suggesting that residues carboxyl terminal to amino acid 441 suppress the transcriptional activation region. LexA-ARA70 1–382 activity was lower while 1–321 was similar to 1–441 as measured in both plaque lift and liquid ß-gal assays. LexA-ARA70 1–296 was inactive. NH2-terminal deletion mutants containing amino acids 291–614 had low activity whereas 321–499 was 5-fold higher, consistent with a repressor function in the carboxyl-terminal region. LexA-ARA70 366–499 and 384–499 had only 20% of the activity of 321–499. Western blots probed with monoclonal antibodies to the LexA DNA binding domain demonstrated that the LexA ARA70 fusion proteins were expressed at similar levels (Fig. 5BGo). Results of the yeast assays indicated the presence of an ARA70 transcriptional activation domain within the region of ARA70 amino acids 296–441.



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Figure 5. Transcriptional Activation Domain in ARA70

A, Analysis in yeast cells. L40 yeast cells containing an integrated ß-gal reporter gene controlled by LexA binding sites were transformed with expression plasmids coding for LexA DNA binding domain fused with full-length ARA70 or with deletion mutants of ARA70. Numbers on the bars indicate amino acid residues of ARA70. Transformed yeast were grown on Ura-, Trp- plates for 48 h and used for lift and liquid assays. On the right is summarized ß-gal activity of the liquid assays. Results are expressed as in Fig. 2Go and are representative of at least three independent experiments. B, Immunoblot analysis of expression in yeast. Equal amounts of cell extract (1 x 108 cell equivalents) from transformed L40 yeast were immunoblotted with monoclonal antibody against LexA DNA binding domain to measure relative expression levels of the various LexA-ARA70 fusion proteins. C, Effects of ARA70 and ARA70({Delta}AD) mutant on AR transcriptional activity. pSG5 expression vectors coding for full-length AR, 0.1 µg, and ARA70, 2.9 and 5.8 µg, or ARA70({Delta}AD), 2.7 and 5.4 µg, were cotransfected into CV1 cells with MMTV-LUC, 2.5 µg, and incubated in the absence and presence of 0.1 nM DHT. Control incubations contained AR and MMTV-LUC alone or with equimolar weights of pSG5 empty vector DNA, 2 or 4 µg, and 0.7 or 1.4 µg pBR322 to make the same total weight of DNA. D, Relative effects of TIF2 and ARA70 on transcriptional activation of the AR DNA-binding domain-LBD fragment. The expression vector pCMVhAR 507–919 coding for the AR DNA-binding domain-LBD (50 ng) and MMTV-LUC (2.5 µg) were cotransfected into CV1 cells with pSG5TIF2 or pSG5ARA70 and incubated in the absence and presence of 0.1 µM DHT. Weights of the two transfected coactivator expression vectors were kept equimolar. Control incubations contained an equimolar weight of pSG5 empty vector DNA with pBR322 to make the same total weight of DNA.

 
Deletion of amino acids 296–441 from ARA70 created ARA70({Delta}AD) for testing with AR in transient cotransfection assays using CV1 cells (Fig. 5CGo). Transfection of 2.9 or 5.8 µg wild-type pSG5-ARA70 DNA resulted in increases above that with AR and reporter gene alone or balanced with equimolar pSG5 empty vector and pBR322. Under the same conditions, ARA70({Delta}AD) induced substantially less transcriptional activity than did wild-type ARA70 and had an effect similar to that of the ARA70(AxxAA) mutant.

To assess the strength of the ARA70 activation domain, we compared its effect on the transcriptional activity of the AR DNA binding domain-LBD fragment AR 507–919 with that of the p160 coactivator TIF2 (Fig. 5DGo). The expression vector pCMVhAR 507–919 containing the AF2 domain was cotransfected with pSG5-TIF2 or pSG5-ARA70 and MMTV luciferase reporter. Androgen-dependent activity of AR 507–919 was minimal in the absence of cotransfected coactivator but greatly enhanced by TIF2, as reported previously (58, 59). However, transcriptional activity was increased less than 2-fold by ARA70, suggesting that ARA70 and TIF2 have different mechanisms of coactivation.

ARA70 Activation Domain Is Not Required for ARA70 Binding to the AR NH2-Terminal Domain but Is Necessary for Full Transactivation
[35S]ARA70({Delta}AD) was synthesized and binding to GST-AR 1–660 tested in an affinity matrix assay (Fig. 6AGo). ARA70({Delta}AD) bound GST-AR 1–660 but not GST-glutathione-Sepharose alone. The amount of bound ARA70({Delta}AD) relative to the input fraction was similar to that of wild-type [35S]ARA70.



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Figure 6. Mutant ARA70({Delta}AD) Interaction with AR NH2-Terminal Domain

A, Binding to the AR NH2-terminal-DNA binding domain fragment. [35S]ARA70 and ARA70({Delta}AD) were synthesized in vitro and incubated with Sepharose beads containing bound GST-AR 1–660. Beads were washed, and bound protein was eluted and analyzed by SDS-PAGE and autoradiography. The input lane contained 15% of the total radioactivity added. B, Coactivation with the AR NH2-terminal domain. Expression plasmids encoding the AR NH2-terminal-DNA-binding domain fragment pCMVAR 1–660, 15 ng, and pSGARA70, 7.25 µg, or ARA70({Delta}AD), 6.75 µg, were cotransfected into CV1 cells together with MMTV-LUC, 2.5 µg. Control incubations contained 5 µg pSG5 empty vector DNA [the molar equivalent of 7.25 µg pSGARA70 or 6.75 µg pSGARA70 ({Delta}AD)] and 2.25 µg pBR322 to make the same amount of total DNA.

 
Investigation of the involvement of this activation domain in ARA70 coactivation with AR is complicated by the presence of the FxxLF motif within the activation domain. Deletion of the activation domain also deleted the FxxLF motif and abolished androgen-dependent binding to the AR-LBD. However, since FxxLF was not required for ARA70 binding to NH2-terminal-DNA binding domain fragment AR 1–660, the ARA70 transcriptional activation domain within amino acids 296–441 could be tested for its effect on the constitutive transcriptional activity of AR 1–660. Cotransfection assays in CV-1 cells were performed with ARA70 or ARA70({Delta}AD) and MMTV-LUC reporter (Fig. 6BGo). ARA70 increased luciferase activity relative to AR 1–660 alone or balanced with equimolar pSG5 empty vector and pBR322. Under the same conditions, ARA70({Delta}AD) stimulation was about 50% of wild-type ARA70. Since the binding of ARA70({Delta}AD) to AR 1–660 was similar to that of wild-type AR, the reduced coactivation with ARA70({Delta}AD) is consistent with the presence of an activation domain in the region between amino acids 296–441. Similar reduced coactivation by ARA70(2KA) and (AxxAA) mutants with AR 1–660 suggested that these mutations interfered with the function of the ARA70 activation domain.

ARA70 Coactivation with AR Is Dependent on Androgen Activation of AR at Physiological Concentrations of Hormone in the Human Male
Ligand specificity of ARA70 coactivation was tested in CV-1 cells cotransfected with AR and the MMTV-LUC reporter in the presence of DHT, E2, or progesterone (P) (Fig. 7Go). At a concentration of 1 nM DHT, ARA70 increased luciferase activity several fold above that of AR balanced with pSG5 vector and pBR322. E2 or P at a concentration of 1 nM showed little or no increase in luciferase activity either in the presence or absence of ARA70 (Fig. 7AGo). When steroid concentrations were increased to 10 nM E2 or P, a concentration well above the blood levels found in human males, ARA70 increased AR transcriptional activity (Fig. 7BGo). The results indicate that within the physiological range of circulating free steroid concentrations in the human male, ARA70 coactivation of AR is androgen specific. Similar findings were observed with the AR coregulator, protein inhibitor of activated STAT1 (60), and the p160 coactivator thyroid receptor activator molecule-1/SRC3 (39), suggesting that steroid-induced AR activation is a prerequisite for coactivation. In the human male, total E2 levels are highest in testicular venous blood and in caput epididymis (see Ref. 61 for review) but much lower than T and would have relatively little influence on AR activation. Cotransfection results with 10 nM E2 in CV1 cells are in agreement with those of Yeh et al. (62), who performed CAT assays in DU145 cells, but contrast with those of Gao et al. (54), who did not observe substantial ARA70 coactivation with AR in the presence of E2 using MMTV-LUC assays either in CV1 or DU145 cells.



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Figure 7. Comparison of DHT, E2, and P Inducement of ARA70 Coactivation

pSG5 expression vectors coding for ARA70, 2.9 µg, and full-length AR, 0.1 µg, were cotransfected into CV1 cells with MMTV-LUC, 2.5 µg, and incubated in the absence and presence of 1 nM (panel A) and 10 nM (panel B) concentrations of the indicated steroids. Control incubations contained 2 µg pSG5 empty vector DNA (the molar equivalent of 2.9 µg pSGARA70) and 0.9 µg pBR322 to make the same amount of total DNA.

 
ARA70 Coactivation with PR and GR
Receptor specificity of ARA70 coactivator function was investigated in CV-1 cells cotransfected with human PR or GR with the MMTV-LUC reporter (Fig. 8Go). Steroid concentrations were 10 nM to optimize for activation of PR and GR. Transfection of 5.8 µg of pSG5-ARA70 increased transcriptional activity of both PR and GR. These results indicate ARA70 is not a specific coactivator for AR and are in agreement with results of others (54, 55, 62). ARA70 is also reported to be a coactivator with the PPAR{gamma} (63).



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Figure 8. Effects of ARA70 on Human AR, PR, and GR Transcriptional Activity

pSG5 expression vectors coding for ARA70, 5.8 µg, and the different steroid receptors, 0.1 µg, were cotransfected into CV1 cells with MMTV-LUC, 2.5 µg, and incubated in the absence and presence of 1 nM DHT or 10 nM P or dexamethasone. Control incubations contained 4 µg pSG5 empty vector DNA (the molar equivalent of 5.8 µg pSGARA70) and 1.8 µg pBR322 to make the same amount of total DNA.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We mapped the region of ARA70 required for androgen-dependent interaction with the AR-LBD, identified an intrinsic transcriptional activation domain (ARA70 296–441), and found that the carboxyl-terminal region of ARA70 has an inhibitory effect on these ARA70 functional domains in yeast. The sequence exhibiting maximum AR-LBD binding was ARA70 amino acids 321–441. These amino acids were contained within the sequence isolated by two-hybrid screening of a cDNA library using the AR-LBD as bait. Androgen-dependent binding of ARA70 321–441 to the AR-LBD was demonstrated also in cell-free assays in vitro. A double mutation, K327A, K329A, which changed KFKLLF to AFALLF in an FxxLF motif within this ARA70 region, eliminated androgen-dependent ARA70 binding to the AR-LBD in yeast and in the GST affinity matrix assays. Mutating FxxLF to AxxAA also eliminated androgen-dependent binding to the AR-LBD, indicating this motif is required for the interaction. Since coactivation by ARA70(2KA) or ARA70(AxxAA) mutants with full-length AR was not abolished, the possibility remained for another site of ARA70 interaction with AR outside the AR-LBD. We observed that ARA70 binds the AR NH2-terminal-DNA binding domain fragment and enhances its constitutive transcriptional activity. Binding to the AR NH2-terminal region was not dependent on an intact FxxLF motif or the presence of an activation domain (ARA70 296–441). However, deletion of the activation domain diminished ARA70 coactivation with the AR NH2-terminal-DNA binding domain fragment. This result in a mammalian cell line supported the presence of an ARA70 activation domain as defined in yeast. Our results are in agreement with existing evidence (53, 54, 55) that ARA70 is an AR transcriptional coactivator and indicate further that ARA70 coactivation results from interactions with both the NH2-terminal and LBDs of AR.

In pull-down assays, Alen et al. (55) observed that both GST-ARA70/ELE1{alpha} and GST-ARA70/ELE1ß bound full-length [35S]AR in an androgen-independent manner. ELE1{alpha} is identical to full-length ARA70 while ELE1ß contains a deletion of amino acids 238–566. Our results indicate that ELE1ß would not be expected to bind the AR-LBD in an androgen-dependent manner since the ELE1ß lacks the FxxLF motif. The results of Alen et al. (55) could be explained by binding to the AR NH2-terminal domain; however, binding to this region was not detected in their assay.

Like other nuclear receptors AR NH2-terminal and LBDs contain transcriptional activation subdomains designated activation function 1 (AF1) and AF2, respectively (1, 23, 64, 65, 66, 67). When AR is activated by androgen binding, it dimerizes on androgen response element DNA through an interaction of the NH2- and carboxyl-terminal regions referred to as the N/C interaction (67, 68, 69, 70, 71). As an isolated domain, the AR NH2-terminal activation function, AF1, is stronger than AF2 in the carboxyl-terminal region (56, 64, 70). Recent studies indicated the AR NH2-terminal domain recruits p160 coactivators while AF2 is the major contact site for the N/C interaction (59). The N/C interaction is dependent on the presence of an FxxLF motif in the AR NH2-terminal domain, and it is this motif that interacts with the AR AF2 (58). Because ARA70 contains an FxxLF motif, it likely also interacts with AF2 in the AR-LBD. The NH2-terminal region of ARA70 contains an LxxLL motif. This sequence motif is known to be important in the binding of p160 coactivators to AF2 of other nuclear receptors (42, 43, 44, 45, 46, 47). However, ARA70 sequences containing this motif (amino acids 1–118, 1–295, and 1–321) did not bind the AR-LBD in the yeast assay. These results are in agreement with recent studies showing that the AR AF2 region has higher affinity for the FxxLF motif than it does for the LxxLL motif (71A ).

Studies with TIF2 (72), GRIP1 (73), and SRC1a (74) point out the importance of the AR NH2-terminal domain in p160 coactivator-stimulated gene transcription. p160 coactivator mutants with deleted LxxLL motifs bound the AR NH2-terminal region and retained coactivator function despite the lack of binding to AF2 (72, 74). AF1 of the ER bound to sequences near the p160 coactivator carboxyl terminus (75). SRC-1 interacted with both NH2- and carboxyl-terminal regions of the PR (29), and GRIP1 stimulated the transcriptional activation of both AF1 and AF2 in the ER (75). Tremblay et al. (76) demonstrated that ligand-independent phosphorylation of MAPK sites in AF1 of ERß stimulated its interaction with SRC-1. In the AR NH2-terminal region, phosphorylation sites that include serines 81, 94 (77), and 515 (78) may be modulators of the ARA70 interaction.

Molecular interactions of the ARA70 transcriptional activation domain remain to be determined, and the precise relationship between the FxxLF motif and the activation domain is not yet known. The partial coactivation maintained by the ARA70 activation domain deletion mutant (deletion of amino acids 296–441) with the full-length AR and its reduced activity with the AR NH2-terminal domain suggest the possibility of a second activation domain. ARA70/ELE{alpha} and ARA70/ELEß binding to p300 CBP-associated factor and TFIIB reported by Alen et al. (55) may involve a second activation domain since the domain within amino acids 296–441 is deleted in ARA70/ELEß. The p160 coactivator TIF2 contains two distinct activation domains AD1 and AD2. Transcriptional activity of AD1 results from direct interaction with cAMP response element binding protein while the mechanism mediating AD2 appears independent of cAMP response element binding protein but remains undefined (32). Deletion of AD1 from GRIP1 had little effect on its enhancement of AR transactivation, indicating it has a minor role in AR coactivation (73). With regard to inhibition of the ARA70 intrinsic activation domain by its own carboxyl-terminal region, it may be relevant that AD2 activity of the SRC-1a isoform was suppressed by the 56 amino acids that are unique to its carboxyl-terminal region, thereby reducing its ability to enhance ER-mediated transcription (79). Whether this region suppressed AD2 by masking it directly or through interaction with another protein remains to be established. In ARA70, carboxyl-terminal inhibition of androgen-dependent binding to the AR-LBD in addition to inhibition of intrinsic transactivation might be an indication of the close proximity of these functional domains within amino acids 296–441.

Spermatogenesis is an androgen- and AR-dependent process (3). Expression of ARA70 in testis could modulate AR regulation of spermatogenesis. Since ARA70 is not AR specific, it may also be a coactivator with other nuclear receptors in testis both in Sertoli cells and spermatogenic cells. In Sertoli cells ARA70 is one of several potential AR coregulators. Others include the p160 family (39), protein inhibitor of activated STAT-1 (PIAS-1) (60), and related members of the PIAS family (80, 81). SNURF, a ring finger protein (82), and ANPK, serine/threonine kinase (83), add to the growing number of potential AR coregulators that may be important in spermatogenesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The following cells and reagents were used: monkey kidney CV1 cells from the American Type Culture Collection (Manassas, VA); DMEM with high glucose with or without phenol red from JRH Biosciences (Lenexa, KS); bovine calf serum from HyClone Laboratories, Inc. (Logan, UT); Enhanced Chemiluminescence Western Blotting Detection Kit from Amersham Pharmacia Biotech (Piscataway, NJ); unlabeled deoxynucleotide triphosphates, glutathione Sepharose 4B, and pGEX (GST gene fusion) vector; deep-Vent polymerase, T4 DNA ligase, and restriction endonuclease from New England Biolabs, Inc. (Beverly, MA); T4 polynucleotide kinase, MMLV RNA reverse transcriptase, RNase H, and Escherichia coli DNA polymerase I from Promega Corp. (Madison, WI); oligo(dT) cellulose columns and XL2-Blue MRF' supercompetent cells from Stratagene (La Jolla, CA); X-gal and o-nitrophenyl-ß-D-galactopyranoside from Sigma (St. Louis, MO); prestained protein mol wt standards from Life Technologies, Inc. (Gaithersburg, MD); X-OMAT-AR diagnostic x-ray film from Kodak (Rochester, NY); D-luciferin from Analytical Luminescence Laboratory (San Diego, CA) ; cell lysis buffer from Ligand Pharmaceuticals, Inc. (San Diego, CA); Immobilon from Millipore Corp. (Bedford MA); buffers and chemicals from Fisher (Suwanee, GA), EM Science (Ft. Washington, PA), and Sigma. psLuc2 was from S. K. Nordeen, University of Colorado Health Sciences Center (Denver, CO). Full-length cDNA of RFG containing a stop codon within the coding region was from M. Santoro, Universita Adegli studi di Napoli (Naples, Italy) (57). Yeast vectors and strains from S. Hollenberg, Oregon Health Sciences University (Eugene, OR) (84) included pLexA, the LexA DNA binding domain fusion vector; pVP16, the library vector for expressing fusion protein with the transcriptional activation domain of the herpes simplex virus VP16; and controls pLex-lamin, pLex-da, and pVP16MyoD. Yeast strains were L40 [MaTa (trp1–901 his3D 200 leu2–3, 112 ade2 LYS2:: (lexAop)4 -HIS3 URA3:: (lexAop)8 -lacZ GAL4] and AMR70 [MaTa his3 lys2 trp1 leu2 URA3:: (lexAop)8 -lacZ GAL4].

Construction of Plasmids
Plasmids expressing the indicated amino acids of ARA70 as fusion proteins were constructed using a cDNA with sequence identical to that of ARA70 (53). VP16-ARA70 and pGEX (GST-ARA70) fusion plasmids were constructed by PCR of the modified RFG using 5'-BamHI and 3'-EcoRI-stop primers or by site-directed mutagenesis (85, 86). Products were digested (BamHI /EcoRI) and cloned into pVP16 or pGEX vectors. pLexA-ARA70 fusion plasmids were created by PCR of the modified RFG. Products were blunt-ended with T4 DNA polymerase, digested (BamHI) and cloned into pLexA digested with PstI, blunt-ended, and digested with BamHI. pSG5-ARA70 and mutants pSG5-ARA70(2KA), (amino acids 327KFK329 changed to 327AFA329, pSG5-ARA70(AxxAA) (amino acids 328FKLLF332 changed to 328AKLAA332, or pSG5-ARA70({Delta}AD) (amino acids 296–441 deleted) were constructed by site-directed mutagenesis (85, 87). TIF2 construct was provided by H. Gronemeyer (32). AR 1–660 was as described previously (64). pLexA-AR-LBD, GST/AR-LBD (both contain AR amino acids 624–919), and GST-AR 1–660 were derived by PCR of pCMV5hAR. Deep-Vent polymerase was used to minimize PCR errors. PCR conditions for 500-bp regions were: 1 cycle 94 C for 5 min, 55 C for 2 min, 72 C for 3.5 min; 11 cycles of 95 C for 1.5 min, 55 C for 2 min, 72 C for 3 min; and 1 cycle of 95 C for 1.5 min, 55 C for 2 min, 72 C for 8 min. PCR-amplified constructs were verified by sequencing.

Library Screening and Analysis of ARA70 Functional Activity
Random primed LNCaP cell cDNA library and two-hybrid screening were performed as described by Hollenberg and associates (84) with modifications. LNCaP cells were treated for 4 d with 5 nM T. Total RNA was extracted using guanidinium isothiocyanate and cesium chloride. Poly(A)RNA was purified on oligo(dT) cellulose. First-strand cDNA was synthesized using random hexamers and reverse transcriptase at 42 C and second strand with E. coli DNA polymerase I and RNase H (Promega Corp.). DNA ends were blunted with T4 DNA polymerase and ligated with 500-fold molar excess of NotI adaptors. cDNAs of 350–800 nucleotides in length were purified on agarose gels and PCR amplified. cDNAs were digested with NotI, repurified, and ligated into pVP16 in NotI. E. coli were transformed with the ligation mixture to produce the cDNA fusion library. For the two-hybrid screen, coding sequence for the human AR hinge region and LBD residues 624–919 was cloned into pLexA to create LexA-AR-LBD.

Before screening the library, we tested the extent of nonspecific activation of the reporter gene by bait protein. Yeast strain L40 containing an integrated ß-gal reporter gene controlled by eight LexA binding sites was transformed with pLexA-AR-LBD and pVP16 and plated on Ura-, Trp-, Leu-, and His- yeast complete medium containing 1 nM dihydrotestosterone (DHT) and the histidine antimetabolite 3-aminotriazol (20 mM) and incubated for three d. The transformants grew and had detectable ß-gal activity in this yeast strain as reported previously (56). To minimize transcriptional activity of the AR-LBD, transformants were plated on Ura-, Trp-, Leu- plates without DHT for 2 d and replica plated to the same plates containing 10 nM DHT. ß-gal Activity was determined after 16 h using the lift assay. Under these conditions, yeast transformed with lexA-AR-LBD and pVP16 did not turn blue within 3 h. Yeast strain L40 was cotransformed with LexA-AR-LBD and the above random-primed cDNA fusion library using lithium acetate (84) and 10% dimethylsulfoxide. Transformants were plated in Ura-, Trp-, Leu- medium at 30 C for 2 d and replica plated to Ura-, Trp-, Leu- medium containing 10 nM DHT and incubated for 16 h. About 8 x 107 transformants were assayed by the filter lift method for ß-gal activity (84). Results were considered positive if the blue color developed within 3 h at 25 C. pLexA-lamin with pVP16 MyoD or pLexA-AR-LBD with pVP16 were negative controls. pLexA-da with pVP16 MyoD or pLexA-AR-LBD with AR NH2-terminal residues 1–583 in pVP16 were positive controls. Positive colonies were further analyzed after eliminating LexA-AR-LBD. Bait plasmids were introduced by mating with AMR70 containing pLexA-AR-LBD (84).

Sequence requirements of ARA70 interaction with the AR-LBD (amino acids 624–919) were determined using pLexA-AR-LBD and pVP16-ARA70 with wild-type or mutant ARA70 sequence. The ARA70 transcriptional activation domain was determined using ARA70 fragments cloned into pLexA. Yeast strain L40 containing an integrated ß-gal reporter gene controlled by eight LexA binding sites was transformed with the different fusion protein expression vectors. ß-gal Activity was measured by lift and liquid assays.

Liquid ß-Gal Assays
L40 yeast were transformed and plated in Ura-, Trp-, and Leu- media and incubated for 2 d at 30 C. Triplicate colonies were grown overnight in Leu- and Trp- liquid medium at 30 C. Cultures were diluted with fresh medium to OD600 0.02 in the presence or absence of 10 nM DHT and grown for 16 h to late log phase (OD600 0.6–0.8). Cells were permeabilized with three cycles of freeze/thaw and incubated at 30 C with o-nitrophenyl-ß-D-galactopyranoside. Activity was calculated as ß-gal = 1000 x [OD420/(t) (v) (OD600)] where OD420 is the absorbance of hydrolyzed o-nitrophenol; t, time of reaction in min; v, volume of culture in milliliters; and OD600, cell density at the start of assay (88).

Immunoblots
Yeast extracts were prepared by a urea-SDS method (89). Extracted proteins were fractionated on a SDS-polyacrylamide gel and transferred to nitrocellulose. Blots were probed either with monoclonal antibody Vp16 1–21 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) against the Vp16 transcriptional activation domain of the different Vp16 ARA70 fusion proteins or with monoclonal antibody LexA 2–12 (Santa Cruz Biotechnology, Inc.), against the LexA-DNA binding domain of the LexA-ARA70 fusion proteins. Primary antibodies were detected with goat-antimouse IgG coupled to horseradish peroxidase and detected with the enhanced chemiluminescence system.

ARA70-AR Interaction in Vitro
Glutathione S-transferase fusion protein expression, purification, and binding were described previously (86). In brief, overnight E. coli cultures transformed with pGEX expressing different regions of ARA70, AR-LBD (AR 624- 919), or the AR NH2-terminal-DNA binding domain fragment (AR 1–660) were diluted in LB medium and incubated at 37 C for 1.5 h. GST fusion protein expression was induced with 0.1 mM isopropylthiogalactoside (Sigma) at 30 C for 4 h. Bacterial cultures were pelleted at 5,000 x g for 5 min at 4 C and resuspended in 0.1 volume NENT (20 mM Tris, pH 8.0, 0.1 M NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing the proteinase inhibitors 0.5 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, and 0.1 µM aprotinin. Bacteria were sonicated and centrifuged at 12,000 x g for 5 min at 4 C. Supernatants were incubated for 30 min at 4 C with 30 µl glutathione-Sepharose and washed three times with NENT. Sepharose with bound GST-ARA70 1–614, GST-ARA70 321–499, or GST-ARA70 321–441 was incubated for 45 min at 4 C with equal amounts of the AR DNA binding-LBD fragment (AR 507–919) from Sf9 cell extracts (68) as determined by immunoblot analysis, washed five times with NENT, and eluted with SDS. Bound protein was separated on SDS-acrylamide gels and immunoblotted using AR52 antibody (21, 90).

In an affinity matrix assay using radiolabeled proteins, glutathione-Sepharose bound to GST alone, GST-AR 624–919, or AR 1–660 was incubated for 90 min at 4 C in the absence or presence of 1 µM DHT with 3–5 µl [35S]methionine-labeled full-length wild-type ARA70 or mutant ARA70(2KA), (AxxAA), or ({Delta}AD) synthesized using the TNT Coupled Reticulocyte Lysate System (Promega Corp.). Sepharose was washed repeatedly, the bound protein was eluted, and then fractionated by SDS-PAGE, and the radioactive protein was visualized by autoradiography. The input lane contained 15% of the total radioactivity added.

In a similar assay [35S]AR-LBD (AR 624–919) was incubated in the presence and absence of 1 µM DHT with glutathione-Sepharose bound to GST alone, GST-ARA70 321–407 wild-type sequence, or GST-ARA70 321–407 (AxxAA) mutant, and bound radioactive protein was measured as above. In this assay the input lane contained 10% of the total radioactivity added.

Cell Culture and Transfections
CV1 cells were maintained in 10% FCS in DMEM containing high glucose and antibiotics. Cells were plated at 4.5 x 105 cells per 6-cm dish. Expression vectors pSG5hAR (0.1 µg), pCMVhAR (0.025 µg), or pCMVAR 1–660 (0.015 µg) and MMTV-LUC (2.5 µg) were cotransfected with pSG5ARA70 or mutant pSG5ARA70 using calcium phosphate. Transfections with pSG5AR and MMTV-LUC alone were included as a control. As an additional control, equimolar amounts of the pSG5 empty vector were used to balance pSG5ARA70, and pBR322 was added to equalize the total transfected DNA. After 40 h with or without 0.1 nM or 10 nM DHT, cells were harvested and luciferase activity was assayed (91). Results are expressed as mean ± SD light units of three replicates and are representative of three or more experiments.

It was reported that cotransfection of pSG5 expression vectors in COS cells can inhibit the expression of AR from pSG5AR, presumably due to depletion of transcription factors (92), and we confirmed this observation by AR immunoblotting (our unpublished results). In contrast to COS cells that make multiple copies of pSG5 vector DNA, pSG5 is not amplified in CV1 cells. Nevertheless, we transfected equimolar amounts of pSG5 empty vector DNA in an attempt to maintain a balance of pSG5 in control and treated cells.


    ACKNOWLEDGMENTS
 
We thank De-Ying Zang, Michelle Cobb, and Raymond T. Johnson, Jr., for excellent technical assistance, and S. Hollenberg for yeast strains and plasmids. Cell culture and cotransfections were performed in the Cell Separation and Tissue Culture Core of the Laboratories for Reproductive Biology.


    FOOTNOTES
 
This work was supported by NIH Grants HD-04466 (F.S.F.) and HD-16910 (E.M.W.), Fogarty International Center D43TW/HD00627 Training and Health in Population and Research, by The Andrew W. Mellon Foundation, and by NICHD/NIH through cooperative agreement U54 HD-35041 as part of the Specialized Cooperative Centers Program in Reproduction Research.

Abbreviations: AF1 and AF2, Activation function 1 and 2; ARA70, AR-associated protein 70; DHT, dihydrotestosterone; ß-gal, ß-galactosidase; GRIP, GR-interacting protein; GST, glutathione-S-transferase; LBD, ligand-binding domain; LNCaP, lymph node-derived human prostate carcinoma cell line; MMTV-LUC, mouse mammary tumor virus-luciferase; P, progesterone; RFG, RET-fused gene; SRC, steroid receptor-coactivator; TF, transactivation factor.

Received for publication July 19, 2000. Accepted for publication October 8, 2001.


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 DISCUSSION
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