Mutational Analysis of the Androgen Receptor AF-2 (Activation Function 2) Core Domain Reveals Functional and Mechanistic Differences of Conserved Residues Compared with Other Nuclear Receptors

Thomas Slagsvold, Irene Kraus, Trine Bentzen, Jorma Palvimo and Fahri Saatcioglu

Biotechnology Centre of Oslo (T.S., I.K., T.B., F.S.), and Department of Biochemistry (T.S.), Department of Biology (I.K., T.B.), and Institute for Clinical Medicine (F.S.) University of Oslo 0349 Oslo, Norway
Department of Physiology (J.P.) Institute of Biomedicine University of Helsinki FIN-00014 Helsinki, Finland


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A short C-terminal sequence that forms the core of the activation function-2 (AF-2) domain is conserved in members of the nuclear receptor superfamily and is required for their normal biological function. Despite a high degree of sequence similarity, there are differences in the context and structure of AF-2 in different nuclear receptors. To gain deeper insight into these differences, we carried out an extensive mutational analysis of the AF-2 core in the androgen receptor (AR) and compared the changes in transcriptional activity with similar mutations that have previously been generated in other nuclear receptors. Mutagenesis of Met894 to Asp resulted in substantial decreases in both DNA and ligand binding activities and, consequently, a significant drop in ligand-dependent transcriptional activation. In contrast, substitution of Met894 with Ala did not affect DNA or hormone binding, and the transactivation potential was comparable to that of wild-type AR. Mutagenesis of Glu897 either with Val or Ala significantly impaired ligand-dependent activation that was not due to changes in DNA or ligand binding. There are both similarities and distinct differences between these findings compared with previous mutagenesis studies of the corresponding residues in other nuclear receptors. All mutants efficiently interfered with AP-1 activity, indicating that ligand-dependent activation of transcription and interference with AP-1 activity are separable functions in AR. For the Glu897 substitutions, the decrease in ligand-dependent transactivation could partially be reversed by overexpression of GRIP1 (GR-interacting protein 1) or CBP, putative coactivators for AR. However, there was no correlation between ligand-dependent in vitro or in vivo association with coactivators and the ability of the mutants to support ligand-dependent transactivation. This is in contrast to similar mutations in other nuclear receptors that lose interactions with putative coactivators concomitant with their loss of transcriptional activity. However, the Glu897 mutations disrupted the intramolecular interaction between the N-terminal domain and the ligand-binding domain of AR that was recently suggested to be required for normal AR function. We conclude that residues in the AF-2 core domain of AR make distinctly different contributions to its transcriptional activities compared with those of other nuclear receptors studied to date.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Androgens have a critical role in the development and maintenance of the male reproductive system (1, 2). The actions of androgens are mediated through the androgen receptor (AR), an intracellular receptor that belongs to the nuclear receptor superfamily (for reviews, see Refs. 3, 4, 5). Nuclear receptors are ligand-activated transcription factors that possess highly conserved DNA-binding domains (DBDs) and moderately conserved ligand-binding domains (LBDs), whereas they are quite divergent in the N-terminal domain (NTD) (for reviews, see Refs. 3, 4, 5). Transactivation functions of nuclear receptors are primarily mediated by sequences in both the NTD and a short region in the LBD, referred to as activation function 1 and 2 (AF-1 and AF-2) domains, respectively. Recent studies suggested that an interaction between the NTD and the LBD may play a role in the transcriptional activities of some nuclear receptors, including AR (6, 7, 8, 9).

The precise sequences and mechanisms that contribute to the AF-1 activity have not been conclusively established. One reason for this is that the N terminus of nuclear receptors is very divergent, both in sequence and length (3, 4, 5). It is also possible that some nuclear receptors do not possess AF-1 function due to their relatively short N terminus. In AR, AF-1 maps to two distinct regions and displays promoter and cell line specificity (8, 10, 11).

On the other hand, a short region that mediates AF-2 activity in the C terminus of nuclear receptors is highly conserved and has been studied in detail through mutational analyses (12, 13, 14, 15, 16, 17, 18). Recently, determination of the three-dimensional structures of the various apo- and holo-LBDs has greatly facilitated the study of AF-2 structure-function relationship (for a review, see Ref. 19). According to the crystal structures, the nuclear receptor LBDs display a common fold with 12 {alpha}-helices (H1–H12) and one ß-turn, together arranged as a three-layer {alpha}- helical sandwich (19). The core AF-2 is formed by H12 that is localized close to, but at variable distances from, the C terminus of the LBD in different nuclear receptors (for a review, see Ref. 19 and references therein).

The H12 is of varying length and flanked by different sequences in the nuclear receptors studied so far. Comparison of this structure in agonist- vs. antagonist-bound receptors, or an apo-receptor with a holo-receptor of different type, suggests that upon ligand binding and activation, H12 is substantially reorganized, contributing to the formation of a coactivator binding surface (19, 20, 21, 22, 23). The loss of transcriptional activation upon mutagenesis of H12 was interpreted as a loss of interaction with coactivators, which is supported by biochemical data (18, 20, 24, 25, 26, 27). These findings were recently confirmed by cocrystallization of liganded nuclear receptors and nuclear receptor interaction domains of coactivators (21, 22, 23).

The activity of nuclear receptors is modulated by interactions with other proteins. These could be mediated through heterodimeric interactions within the nuclear receptor superfamily, such as those between retinoid X receptors (RXRs) and thyroid hormone, retinoic acid, and vitamin D receptors (T3Rs, RARs, and VDRs) in which the heterodimer has an increased ability to activate transcription (for a review, see Ref. 28). On the other hand, AP-1 complexes, composed of either Jun homodimers or Jun-Fos heterodimers (for a review, see Ref. 29), interfere with ligand-dependent transactivation by some nuclear receptors including AR (for a review, see Ref. 30). Reciprocally, liganded nuclear receptors, as first described for the glucocorticoid receptor (GR), interfere with AP-1 activity (for a review, see Ref. 30). The molecular mechanisms of this cross-talk have not been definitively established but may involve competition for a common cofactor, such as CREB-binding protein (CBP) (30, 31, 32, 33). Nuclear receptors can also cross-talk with other transcription factors, but these have not been studied in as much detail (e.g. Refs. 34, 35).

Most recently, proteins that act as putative coactivators or corepressors and which physically interact with nuclear receptors have been identified (for reviews, see Refs. 36, 37, 38). Most of these cofactors are expressed ubiquitously and can interact with more than one type of nuclear receptor. Furthermore, it appears that multiple cofactors may regulate nuclear receptor function at any one time. Therefore, the exact contribution of these cofactors to the activities of different receptors in vivo is still not well understood.

Given the variations in the context, sequence, and length of the AF-2 core domains in various nuclear receptors and to gain a deeper insight into its functioning, we carried out a detailed mutational analysis of the AF-2 core in AR. This was based on information provided by the crystal structures of other nuclear receptors (19) and previous mutational studies on the AF-2 of T3R{alpha}, T3Rß, ER, and GR (12, 13, 14, 15, 16, 18). We aimed to target surface exposed residues that are likely to form recognition surfaces for AR regulatory proteins. Through this analysis, we identified various single amino acid substitutions that significantly compromised the transactivation potential of AR. There were both similarities and distinct differences regarding the importance of specific residues and the mechanisms involved in their loss of function when compared with identical or similar mutations of these conserved residues in other nuclear receptors, as well as a recent mutational analysis of AR H12 (50). In addition, none of the mutations that blocked transcriptional activation affected interference with AP-1 activity, indicating that transactivation and transrepression are mediated by different interaction surfaces in AR. These results indicate the presence of receptor-specific differences in the function of the AF-2 core that may contribute to differential activities of nuclear receptors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutagenesis of the Conserved C-Terminal Sequence of AR
We generated mutations within the conserved C- terminal region of AR, focusing on three residues that are expected to be located at the surface of the protein according to the crystal structures of liganded nuclear receptors that have been solved to date and their previous mutational analyses (12, 13, 14, 15, 16, 17, 18, 19). These residues, Met894, Ala896, and Glu897 (Fig. 1AGo), are therefore candidates for interacting with cofactors that may regulate AR function. The nonpolar residue Met894 was changed either to an acidic (Asp, M1) or a smaller nonpolar (Ala, M2) residue. The small nonpolar residue Ala896 was substituted with either Val (M3) or Leu (M4), both larger nonpolar residues. The conserved Glu897 was substituted with small nonpolar residues Val (M5) or Ala (M6).



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Figure 1. Detailed Mutational Analysis of AR AF-2

A, Domain structure of AR is depicted. The DBD and LBD are indicated. The sequence of the conserved AF-2 core region in the C-terminal end of the LBD (represented by a black band) was aligned with that of wild-type T3R{alpha}. For the AR mutants, only those residues that have been replaced are indicated. To the right of the figure are shown the dissociation constants (Kd) of the wild-type and mutant AR proteins, which were determined as detailed in Materials and Methods. The SD values are indicated in parentheses. B, Western analysis of the AR proteins. COS-7 cells were transfected with expression vectors encoding wild-type (WT) or mutant ARs (15 µg in 10-cm dishes). After 18 h, total cell extracts were prepared and used in Western analysis with an antiserum raised against the LBD of AR, as detailed in Materials and Methods. AR-specific band is indicated by an arrow. C, The wild-type and mutant AR proteins were translated in vitro and were used in the mobility shift assay. Proteins were preincubated with R1881 (10-6 M) for 20 min at room temperature and further incubated with the 32P-labeled ARE probe for 25 min in binding buffer as detailed in Materials and Methods. The binding reactions were run on a 5% nondenaturing polyacrylamide gel followed by autoradiography. The free probe (F) and the AR-specific complexes are indicated by arrows on the right of the figure. As a control, unprogrammed (UPL) lysate was also tested, which resulted in a band that is not AR-specific as indicated on the left (NS). The in vitro transcription/translation reactions used in the mobility shift assay contained equal amounts of input proteins (data not shown), ruling out that the decreased DNA binding by M1 was due to lower levels of M1 protein.

 
The wild-type and mutant ARs were first tested for proper expression. COS-7 cells were transiently transfected with expression vectors specifying the production of wild-type or mutant receptors, and whole cell extracts were prepared and assessed by Western analysis using an AR-specific antiserum. As shown in Fig. 1BGo, all mutants were expressed at approximately the same level as wild-type AR.

Since all known activities of AR are dependent on activation by androgens, we then tested the mutant proteins for proper ligand binding properties. COS-1 cells were transfected with expression vectors specifying the production of the wild-type or the mutant receptors, and the cells were incubated with increasing amounts of [3H]R1881. After the excess hormone was washed away, specifically bound [3H]R1881 was measured, and dissociation constants (Kd) were calculated. As shown in Fig. 1AGo, M2-M6 had Kd values that were comparable to those of wild-type AR, whereas M1 was significantly compromised in its Kd, being 6% of that of wild-type AR. M1 is anticipated to be saturated with hormone in the experiments that are described below since approximately 12-fold excess of hormone over the Kd was used in these experiments. Since mutagenesis of the corresponding residue in other nuclear receptors did not appreciably affect ligand binding [e.g. in T3R{alpha}, T3Rß, and estrogen receptor (ER) (12, 16, 18)], these results indicate that the contribution of specific residues in the conserved C-terminal region of AR to ligand binding may be different than the corresponding residues in other nuclear receptors.

As the transcriptional activity of AR depends on its ability to bind DNA, we next performed mobility shift assays to compare wild-type AR with the mutants for their DNA binding activities. Proteins were expressed by in vitro transcription/translation and then used in the mobility shift assay with the androgen response element (ARE) in the first intron of the rat C3 gene as probe (46). As shown in Fig. 1CGo, unprogrammed lysate resulted in a nonspecific band, whereas lysates containing wild-type AR resulted in a slower migrating band (depicted by an arrow). This slower migrating band is specific for AR as shown by a supershift experiment with an AR-specific antiserum and by competition studies using an excess of unlabeled ARE (T. Slagsvold, unpublished data, and Ref. 46). All the mutants displayed similar DNA binding activity compared with wild-type AR, except M1, which was severely impaired in its ability to bind to the ARE. Competition studies indicated that M2-M6 had similar DNA binding affinity, except for M3, for which binding affinity was lower than the other mutants (data not shown). In addition, M1 was defective in ligand-induced change in mobility compared with wild-type AR and the other mutants in the mobility shift assay (data not shown). These results indicate that in addition to a substantial decrease in ligand binding, M1 is also impaired in its DNA binding activity, whereas the other mutants are comparable to wild-type AR in these respects.

Divergent Effects of C-Terminal Mutations on Transcriptional Activities of AR
To assess the possible effects of the AF-2 core mutations to biological activities of AR, we first tested the wild-type and mutant receptors for their ability to stimulate androgen-dependent expression of -285PB-LUC (8) in which a deletion derivative of the rat probasin promoter drives expression of the luciferase (LUC) reporter gene. CV-1 cells were cotransfected with -285PB-LUC and either an empty expression vector or expression vectors encoding the wild-type or mutant ARs. After transfection, the cells were either left untreated or treated with R1881 for 18 h and LUC activities were determined. As shown in Fig. 2AGo, in the presence of R1881, wild-type AR activated transcription of -285PB-LUC by 5-fold. The mutants M2, M3, and M4 also stimulated transcription, but their transcriptional activities were reduced by 20%, 30%, and 10% compared with wild-type AR, respectively. In contrast, M1, M5, and M6 were inactive, as their activities did not significantly differ from that of the empty expression vector pSG5. In a similar experiment for the transcriptionally compromised mutants, M1, M5 and M6, qualitatively comparable results were obtained in HeLa cells, except that the mutants had approximately 20% activity compared with wild-type AR (Fig. 2BGo), indicating that the transcriptional defects of these mutants are not cell type specific.



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Figure 2. Transcriptional Properties of the AR AF-2 Mutants

A, Ligand-dependent transcriptional activation. CV-1 cells were cotransfected with the -285PB-LUC (0.25 µg) plasmid and expression vectors encoding wild-type (WT) AR, mutants M1 to M6, or the empty expression vector pSG5 (5 ng of each) by the calcium phosphate procedure. Cells were either left untreated or treated with R1881 (10-7 M) and harvested after 18 h, and LUC activities were determined. LUC activity in the presence of wild-type AR and R1881 is arbitrarily set at 100%. The results represent the average of at least five independent experiments with SD shown as error bars. B, Hela cells were transfected with -285PB-LUC and the indicated expression plasmids, and LUC activities were determined as described in Fig. 2AGo. The results are from five independent experiments done in duplicate. C, As in Fig. 2BGo, but the reporter used was 2XARE-LUC. Data shown are from two independent experiments done in duplicate. D, Ability of the AR mutants to interfere with AP-1 activity. Hela cells were cotransfected with -73Col-CAT and 1 µg of expression vectors encoding the wild-type or mutant ARs, or the empty expression vector pSG5. The cells were treated with TPA (10-7 M) in the presence or absence of R1881 (10-7 M). After 18 h, the cells were harvested and CAT activities were determined. The level of -73Col-CAT activity in the presence of wild-type AR and in the absence of R1881 was arbitrarily set at 100%. The results represent the average of at least three independent experiments with SD shown as error bars.

 
We also examined the activity of the transcriptionally compromised mutants from another reporter construct, 2XARE-LUC (33), in which two copies of the androgen response element in front of the thymidine kinase promoter drive expression of the LUC gene. HeLa cells were cotransfected with 2XARE-LUC and either an empty expression vector or expression vectors encoding the wild-type or mutant ARs. After transfection, the cells were either left untreated or treated with R1881 for 18 h, and LUC activities were determined. As shown in Fig. 2CGo, in the presence of R1881, wild-type AR activated transcription of 2XARE-LUC by 10-fold. Interestingly, all three mutants also stimulated transcription, but their activities were significantly reduced: M1 had 35%, whereas M5 and M6 had 50% and 60% activity, respectively, compared with wild-type AR. Similar results were obtained in CV-1 cells, although the activities were somewhat lower, or using another AR-dependent reporter construct, mouse mammary tumor virus (MMTV)-LUC (data not shown). These data suggest that M1, M5, and M6 may have variations in their transcriptional activity on different reporter constructs, but they are still significantly impaired compared with that of wild-type AR.

Another activity of AR is its ability to interfere with AP-1 transcriptional activity (for a review, see Ref. 30). We therefore tested the mutant proteins for their ability to interfere with AP-1. An AP-1-dependent reporter in which a deletion derivative of the collagenase promoter is fused to the chloramphenicol acetyltransferase (CAT) reporter gene, -73Col-CAT (39), was cotransfected into HeLa cells with expression vectors specifying the wild-type AR or the mutants M1-M6. After transfection, the cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) to maximize AP-1 activity and either left untreated or treated with R1881 for 18 h and CAT activities were determined. As was previously shown (32, 47), the liganded wild-type AR efficiently decreased -73Col-CAT expression (Fig. 2DGo). Interestingly, all mutants were similar to wild-type AR in their ability to inhibit -73Col-CAT expression, suggesting that the ability to activate transcription in response to R1881 is not required for AR to interfere with AP-1 activity, and that these two activities can be dissociated.

Interaction of AR and Its AF-2 Core Mutants with GRIP1 (GR-Interacting Protein 1) and CBP
Our earlier work with T3R{alpha} showed that mutations of the conserved Glu residue in the AF-2 domain inhibit interaction of T3R{alpha} with GRIP1 (42), a putative coactivator protein for a number of nuclear receptors, thus decreasing the ability of T3R{alpha} to activate transcription (18). To test whether this also applies to the AR mutants, we considered the possibility that there were alterations in the ability of the mutants to interact with GRIP1.

We first used the transient transfection assay to test the ability of GRIP1 to increase transcription by wild-type AR and the transcriptionally deficient mutants M5 and M6. The other transcriptionally compromised mutant M1 was not included in further analyses due to its severely impaired DNA and hormone binding properties. -285PB-LUC was transfected into HeLa cells with expression vectors encoding wild-type AR, M5, or M6 in the presence or absence of an expression vector for GRIP1. In the absence of GRIP1, the liganded wild-type AR activated -285PB-LUC expression by 3-fold, and this activity was further increased by 4-fold when GRIP1 was coexpressed (Fig. 3AGo). Interestingly, M5 and M6, which are severely defective in their R1881-dependent transactivation function (see Fig. 2Go, A–C), were significantly stimulated and thus were in part rescued by GRIP1 coexpression. In the presence of GRIP1, M5 had 125% and M6 had 150% of the activity elicited by the wild-type AR in the absence of GRIP1.



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Figure 3. Effect of GRIP1 and CBP on the Transcriptional Activities of AR and Its AF-2 Core Mutants

A, HeLa cells were cotransfected with the -285PB-LUC reporter and 1 ng of the AR expression vectors in the presence or absence of 20 ng of GRIP1 expression vector as indicated, and the cells were either left untreated or treated with R1881 (10-7 M). After 18 h, cells were harvested and LUC activities were determined. The results represent the average of at least five independent experiments with SD indicated as error bars. Note that suboptimal levels of AR vectors were used to facilitate the observation of the GRIP-1 effect in these experiments. All data shown are in the presence of R1881. -285PB-LUC activity in the presence of AR alone was set at 100%. B, GRIP1 (amino acids 415–812) was expressed as a GST fusion protein (GST-GRIP1) in E. coli and purified on glutathione-Sepharose beads. GST-GRIP1 or GST alone was then used in the GST pull-down assay with cell-free translated 35S-labeled ARs, in the presence (+) or absence (-) of R1881 (10-6 M) as detailed in Materials and Methods. Five percent of the input for the AR proteins are shown to the left of the figure. The migration of the AR-specific band is indicated by an arrow. Bands were quantitated by PhosphorImager analysis. A representative experiment repeated three times with similar results is shown. C, HeLa cells were cotransfected with the -285PB-LUC reporter and 10 ng of the AR expression vectors either in the presence or in the absence of 20 ng CBP expression vector as indicated. The cells were either left untreated or treated with R1881 (10-7 M). After 18 h, cells were harvested and LUC activities were determined. The results represent the average of two independent experiments done in duplicate with SD indicated as error bars. All data shown are in the presence of R1881. -285PB-LUC activity in the presence of AR alone was set at 100%. D, In vitro interactions between CBP and wild-type AR or the mutants, M5 and M6, were tested in the GST pull-down assay. CBP(1–452) was expressed in E. coli as a GST fusion protein, purified on glutathione-Sepharose beads, and was used in the GST pull-down assay as described in Materials and Methods. Ten percent of the input of wild-type AR, M5, and M6 is shown to the left of the figure. The AR-specific band is indicated by an arrow to the right of the figure. Bands were quantitated by PhosphorImager analysis.

 
We next tested the wild-type and mutant ARs for their ability to associate with GRIP1 in vitro. The previously characterized nuclear receptor interaction domain of GRIP1, corresponding to amino acid residues 415–812, was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein [GST-GRIP(415–812)] (42) and used in the GST pull-down assay with cell-free translated 35S-labeled wild-type and mutant ARs in the presence or absence of R1881. As shown in Fig. 3BGo, GST-GRIP(415–812) bound weakly to wild-type and mutant ARs in the absence of R1881. AR binding to GST alone was also weak and not enhanced by the addition of R1881. However, binding to GRIP1 was modestly increased (~2-fold) in the presence of R1881 for both wild-type AR and the mutants M5 and M6 resulting in similar levels of interaction. These results suggest that the decreased transcriptional activities of M5 and M6 were not due to their decreased ability to interact directly with GRIP1.

CBP was recently identified as a putative coactivator for AR (32, 33). We therefore tested whether there were any differences in the ability of CBP to increase stimulation of transcription by wild-type AR, M5, or M6. To that end, -285PB-LUC was transfected into HeLa cells with expression vectors encoding wild-type AR, M5, or M6 in the presence or absence of an expression vector for CBP. After transfection, the cells were either left untreated or treated with R1881 for 18 h and LUC activities were determined. In the absence of CBP, the wild-type AR activated -285PB-LUC expression approximately 4-fold that was increased a further 3.5-fold in the presence of R1881 and CBP (Fig. 3CGo). Interestingly, M5 and M6 were also stimulated by CBP expression: M5 had nearly 65% and M6 had 40% of the activity elicited by the wild-type AR in the absence of CBP. Thus, similar to the results obtained with GRIP1, M5 and M6 activity was partially rescued by ectopic expression of CBP.

We then tested the wild-type and mutant ARs for their ability to bind CBP in vitro. An N-terminal region of CBP (amino acid residues 1–452), which we have previously shown to bind AR (32), was expressed in E. coli as a GST fusion protein and used in the GST pull-down assay with cell-free translated 35S-labeled wild-type and mutant ARs in the presence or absence of R1881. As shown in Fig. 3DGo, AR binding to GST alone was weak and not enhanced by the addition of R1881. In contrast, wild-type AR efficiently bound to GST-CBP in the absence or presence of R1881. M5 and M6 also bound GST-CBP, although the binding was somewhat decreased compared with that of wild-type AR. These results suggest that the decreased transcriptional activity of M5 and M6 compared with wild-type AR was not due to their inability to physically interact with CBP.

AR Shows Significantly Diminished Ability to Bind GRIP1 in Vitro Compared with T3R{alpha}
In the GST pull-down experiments presented above, AR displayed weak binding to GRIP1, which was only modestly enhanced in the presence of hormone. In contrast, previous studies have indicated that the in vitro interactions between GRIP1 and some other nuclear receptors, such as T3R{alpha}, are significantly stronger than shown in this study for AR (e.g. Refs. 18, 27). We therefore compared the ability of AR and T3R{alpha} to interact with GRIP1 under the same experimental conditions. To that end, cell-free translated 35S- labeled wild-type AR or T3R{alpha} was used in the GST pull-down assay with GST-GRIP(415–812) in the presence or absence of R1881 or T3, respectively. As shown in Fig. 4AGo, T3R{alpha} did not significantly bind to GST either in the absence or presence of T3, but it displayed weak binding to GST-GRIP(415–812) in the absence of T3, which was further increased by 4-fold in the presence of T3. In contrast, AR bound to GST-GRIP(415–812) weakly, and the addition of hormone resulted in only approximately 2-fold increase in binding. The ability of T3R{alpha} to bind GST-GRIP(415–812) in the presence of hormone was approximately 5-fold stronger than that of AR. Addition of increasing amounts of GST-GRIP(415–812) in the binding reaction increased T3R{alpha} binding further (3- to 4-fold), but only modestly affected AR binding (data not shown). These data suggest that the AR-GRIP1 interaction in vitro is substantially weaker compared with interaction of GRIP1 with other nuclear receptors, such as T3R{alpha}.



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Figure 4. Comparison of AR and T3R{alpha} for GRIP1 Binding and Identification of GRIP1 Binding Domains of AR in Vitro

A, GRIP1 (amino acids 415–812) was expressed as a GST fusion protein in E. coli and purified on glutathione-Sepharose beads. GST-GRIP1 or GST alone were then used in the GST pull-down assay with cell-free translated 35S-labeled AR or T3R{alpha}, in the presence (+) or absence (-) of R1881 (10-6 M) or T3 (10-7 M), respectively, as detailed in Materials and Methods. Ten percent of the input for the AR and T3R{alpha} proteins is shown to the left. Even though T3R{alpha} input is slightly more than AR in this experiment, this difference does not make a significant contribution to the fold-difference in binding to GST-GRIP1 (data not shown). The migration positions of the AR- and T3R{alpha}-specific bands are indicated by arrows. The results of PhosphorImager quantitation of the specific bands are indicated below each lane. An experiment that was repeated three times with similar results is shown. B, The GST pull-down assay was performed as in Fig. 4AGo using 35S-labeled wild-type AR or its deletion mutants as indicated. Five percent of the input for the AR proteins is shown to the left of the figure. The migration position of the AR(1–333) deletion mutant, which has lost the ability to bind GST-GRIP1, is indicated by an arrow. Note that the migration position of the bands in the GST pull-down experiment are slightly lower than the input bands due to stretching in the right side of the gel. The specific bands were quantitated using PhosphorImager analysis as indicated below each lane. A representative experiment repeated three times with similar results is shown.

 
The weak binding of AR to GRIP1 suggested that the mechanisms of AR-GRIP1 interactions may be different and different regions of AR may be involved in interactions with GRIP1 compared with other nuclear receptors, such as T3Rs, in which the AF-2 core region plays a critical role (e.g. Refs. 18, 27). To test this hypothesis, we used C-terminal deletion mutants of AR (32) in a GST pull-down experiment with GST-GRIP(415–812). As shown in Fig. 4BGo, wild-type AR, AR(1–714), and AR(1–503), but not AR (1–333), efficiently bound to GST-GRIP(415–v812). Compared with wild-type AR, binding of AR(1–714) and AR(1–503) to GRIP1 was decreased by 25% and 40%, respectively. These data suggest that the N terminus of AR is sufficient for binding GRIP1, since the deletion of the C terminus and the DBD [as in AR(1–503)], although diminished, did not abrogate GRIP1 binding.

GRIP1 Can Activate the AR AF-2
We next tested whether the C terminus of AR is also sufficient for interacting with GRIP1 and studied the possible effects of AF-2 core mutations on this interaction by a different approach. To that end, we fused the LBD of wild-type AR, or M5 and M6, to the DBD of the yeast transcriptional activator GAL4 (43) (GAL4-AR-LBD-WT) and performed transfection assays using 5XGAL4-LUC (40) as reporter in which five copies of the GAL4 response element drive expression of the LUC gene. GAL4 DBD alone (data not shown), GAL4-AR-LBD-WT, GAL4-AR-LBD-M5, or GAL4-AR-LBD-M6 did not activate 5XGAL4-LUC in the presence or absence of R1881 (Fig. 5AGo). However, when GRIP1 was coexpressed with GAL4-AR-LBD in the presence of R1881, approximately 10-fold activation of 5XGAL4-LUC was observed, suggesting that the LBD of AR directly interacts with and is activated by GRIP1. Even though GAL4-AR-LBD-M5 and -M6 were inactive in the absence of GRIP1, they were activated by GRIP1 coexpression 36% and 63%, respectively, compared with the wild-type AR construct (Fig. 5AGo). These data suggest that the LBD of AR requires GRIP1, or a comparable cofactor, to activate transcription. In addition, these results indicate that Glu897 in the AF-2 core domain is responsible, at least in part, in mediating interactions between AR and GRIP1.



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Figure 5. Interactions between GRIP1 and AR LBD in Vivo

A, HeLa cells were transfected with the 5XGAL-LUC (0.25 µg) and 20 ng of the GAL4-AR-LBD expression vectors as indicated, either in the presence of the empty expression vector pSG5 (75 ng) or pSG5-GRIP1 (150 ng) expression vector. After transfection, cells were either left untreated or treated with R1881 (10-7 M). After 18 h, cells were harvested and LUC activities were determined. The activity of the reporter in the presence of GAL4-AR-LBD-WT, pSG5-GRIP1, and R1881 was arbitrarily set at 100%. The results represent the average of three independent experiments with SD indicated as error bars. B, HeLa cells were transfected with the 5XGAL-LUC (0.25 µg) and 20 ng of the GAL4-AR-LBD expression vectors as above, either in the presence of the empty expression vector pSG5 (75 ng) or 150 ng of VP16-GRIP1 expression vector. After transfection, cells were either left untreated or treated with R1881 (10-7 M). After 18 h, cells were harvested and LUC activities were determined. The activity of the reporter in the presence of GAL4-AR-LBD-WT and VP16-GRIP1 was arbitrarily set at 100%. The results represent the average of three independent experiments with SD indicated as error bars. Note that the bars for the GAL4-AR fusion constructs or VP16-GRIP1 alone are not visible at this scale since they are very small (<0.5%).

 
To further assess whether the above findings were a consequence of direct interactions between AR and GRIP1 in vivo, we used the mammalian two-hybrid system. A GRIP1 fragment containing the nuclear receptor interaction domain (amino acid residues 578–1,462) was fused to the activation domain of the viral activator VP16 (residues 412–480) to generate the fusion construct VP16-GRIP1. HeLa cells were transfected with the 5XGAL4-LUC reporter construct and VP16-GRIP1 or GAL4-AR-LBD-WT alone, or with the two expression plasmids together. As shown in Fig. 5BGo, there was no detectable activity over basal level of the reporter when VP16-GRIP1 or GAL4-AR-LBD was transfected alone. In contrast, when VP16-GRIP1 and GAL4-AR-LBD were coexpressed in the presence of R1881, 5XGAL4-LUC was activated by approximately 200-fold. This suggests that AR LBD and GRIP1 physically associate in vivo. When the GAL4-AR-LBD-M5 and GAL4-AR-LBD-M6 were tested in a similar way, they showed 20% and 85% of the activity of the wild-type AR, respectively, in response to GRIP1 coexpression (Fig. 5BGo). These results suggest that the AF-2 core of AR is involved in physical contacts with GRIP1 in vivo.

Mutations in the AR AF-2 Core Disrupt Interactions between the LBD and the NTD
The LBD of AR has recently been shown to interact with the NTD in a strictly hormone-dependent fashion (6, 8, 9). To assess the possible role of the AF-2 core region in this interaction, HeLa cells were transfected with the 5XGAL4-LUC reporter and GAL4-AR-LBD-WT, or the GAL4 fusions of the mutants M5 and M6, in the presence or absence of an expression vector specifying the NTD of AR [AR(1–566)]. As shown in Fig. 6Go, GAL4-AR-LBD-WT did not affect activity of the reporter construct in the presence or absence of R1881; similar results were obtained for the mutants M5 and M6 (data not shown). Coexpression of AR(1–566), specifying expression of the NTD, with GAL4-AR-LBD-WT did not activate 5XGAL4-LUC in the absence of R1881; however, there was 16-fold activation of the reporter in the presence of R1881. In contrast to GAL4-AR-LBD-WT, no significant reporter gene activation was observed in comparable experiments using GAL4-AR-LBD-M5 or GAL4-AR-LBD-M6. These results indicate that AF-2 core region in AR has an important role in mediating the intramolecular interactions between the LBD and NTD.



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Figure 6. The AF-2 Core Domain of AR Is Required for Interaction between the LBD and the NTD

HeLa cells were transfected with the 5XGAL-LUC (0.25 µg) and 20 ng of the GAL4-AR-LBD expression vectors in the presence of either the empty expression vector pSG5 (150 ng) or 300 ng of AR(1–566) expression vector as indicated. After transfection, the cells were either left untreated or treated with R1881 (10-7 M) and were harvested at 18 h and LUC activities were determined. The activity of GAL4-AR-LBD-WT in the presence of AR(1–566) is arbitrarily set at 100%. The results represent the average of three independent experiments with SD indicated as error bars.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Helix 12 of the LBD is of variable length in different liganded nuclear receptors: 6 amino acid residues in T3R{alpha}, 8 residues in RAR{gamma} and PPAR{gamma} (peroxisome proliferator-activator receptor {gamma}), 9 residues in ER, and 15 residues in PR (22, 48). Additionally, the length of the remaining part of the C terminus also varies among different nuclear receptors. Therefore, it is important to study the contribution of specific residues in this region to transcriptional activity in the context of different nuclear receptors. Here, we present such an analysis for AR based on information from mutational analysis and crystal structures of other nuclear receptors.

Substitution of Met894 with Asp (M1) significantly impaired the ability of AR to activate from AREs (Fig. 2Go, A–C). This is in line with previous work that has shown that the same mutation in the corresponding residue in T3R{alpha} (L398D) prevented transcriptional activation (18). However, the mechanisms for the loss of transactivation in these receptors appear to be quite different: AR-M1 is significantly reduced in its ability to bind either ligand or to DNA (Fig. 1Go, A and C), in contrast to the corresponding L398D mutation in T3R{alpha}, which did not significantly affect either of these functions (18). In addition, the characteristic shift in mobility of AR-ARE complexes in a band shift experiment in response to ligand binding (e.g. Ref. 46) is lost in M1 (data not shown). These data suggests that Met894, although conserved as a hydrophobic residue in most nuclear receptors, has a different role in the context of AR compared with in T3R{alpha} and other nuclear receptors. This hypothesis is supported by the finding that substitution of Met894 with Ala (M2) did not significantly affect AR activity, which is in stark contrast with the same mutation in the context of T3R{alpha} (18) and T3Rß (16) in which all transcriptional activity was lost. Furthermore, double mutants in ER (L543A/L544A) and GR (M758A/L759A) that include the corresponding mutation were incapable of transcriptional activation in response to ligand (12). None of these mutants, in the context of T3Rs, ER or GR, were significantly impaired in either DNA or hormone binding (12, 16, 18).

While this work was being prepared for publication, a mutational analysis of AR LBD was reported (50). In this study, mutagenesis of the hydrophobic pair Met894 and Met895 to alanine, resulted in the loss of all hormone-dependent transcription. Since mutagenesis of single amino acid residues was not performed, it is not possible to determine which of the methionine residues that were altered was responsible for the loss of activity. In our analysis, however, mutagenesis of Met894 into Ala (M2) did not significantly affect AR activity, suggesting that Met895 is the more crucial amino acid residue contributing to AR function. However, when Met894 was substituted to Asp (M1), transactivation ability of AR was lost due to substantially diminished DNA and hormone binding activities. Collectively, these results suggest that both Met894 and Met895 have important contributions to AR function, which may be mediated through different mechanisms.

In contrast to Met894, mutants of Ala896 and Glu897 (M3-M6) were similar to wild-type AR with respect to their hormone and DNA binding activities (Fig. 1Go, A and C). Substitution of Ala896 with either Val or Leu (M3 and M4) did not significantly affect AR transcriptional activity, which is consistent with the activities of similar mutations in T3R{alpha} and T3Rß (16, 18). Since Ala896 is less conserved in nuclear receptors and is substituted frequently with more distant amino acids, these data support the notion that Ala896 does not directly contribute to hormone-dependent transcriptional activity of AR.

Substitution of Glu897, which is completely conserved in different nuclear receptors, either with Val or Ala (M5 and M6), significantly diminished hormone-dependent activation by AR (Fig. 2Go, A–C). In contrast to our results, it was recently reported that the transcriptional activity of an AR double mutant, in which Glu893 is substituted along with Glu897, was not significantly altered (50). One possible explanation for this apparent discrepancy is that the substitution that was carried out at Glu897 in the double mutant was a glutamine compared with valine or alanine in our study. Thus, the differences in size and properties of the substituted residues could differentially affect the final structure of the AF-2 core region. Alternatively, the effect induced by substitution of Glu897 is neutralized by additional substitution at Glu893. More detailed mutagenesis will be required to assess these possibilities.

In the context of other nuclear receptors, the impaired transcriptional activity of the M5 and M6 mutants is consistent with the findings on similar mutations generated at this residue in T3R{alpha} and T3Rß (14, 16, 18), but different from that of ER{alpha} and GR in which mutants analogous to M5 were as active as the wild-type receptors (12). This suggests that the conformation, and perhaps function, of Glu897 in AR is more like that of the corresponding residue in T3Rs than in other steroid receptors, such as ER{alpha} and GR, to which AR is more closely related overall. Assessment of this hypothesis must await the delineation of the AR holo LBD crystal structure and its comparison with those of other nuclear receptors.

It was previously demonstrated that the loss in transactivation potential of many nuclear receptor AF-2 core mutants may be due to disruption of fruitful interactions with putative coactivator protein(s) (e.g. see Refs. 18, 25, 27). For example, the loss of ligand-dependent transactivation by the inactive AF-2 core mutants in T3R{alpha} could largely be reversed when GRIP1 was ectopically expressed (18). For the transcriptionally defective mutants of M5 and M6 of AR, we have made similar observations in vivo: GRIP1 significantly stimulated, and thereby rescued, M5 and M6 transactivation potential, yielding 125–150% of wild-type AR activity in the absence of GRIP1 (Fig. 3AGo). Similarly, CBP coexpression resulted in partial rescue of M5 and M6 activity, which was smaller (40–65%) than that obtained with GRIP1 (Fig. 3CGo).

The simplest explanation for these results is that the substitutions in M5 and M6 decrease the affinity of the AR for GRIP1, or another coactivator such as CBP, which in turn results in a significant loss of AR transactivation potential. Thus, when an excess of coactivator is available, this defect is in part overcome in that the ability of M5 and M6 to activate transcription significantly increases. Consistent with this model, we have earlier found an excellent correlation between T3-dependent in vitro association of GRIP1 with T3R{alpha} AF-2 mutants and their ability to support T3-dependent transcriptional activation (18). Similar observations were made with ER AF-2 mutants and GRIP1 binding in vitro (27). However, in contrast to these findings, AR-M5 and AR-M6 displayed in vitro GRIP1 and CBP binding characteristics that were comparable to those of wild-type AR (Fig. 3Go, B and D).

Recent findings demonstrated that interaction of AR with SRC-1, another putative coactivator, is mediated by the N terminus of AR and that the AF-2 core does not play an important role in this process (50). In addition, the transcriptional activity of AR N terminus is stimulated by GRIP1 and SRC-1 (54). These results suggested that the major interaction site of coactivators with AR is in the N terminus. This is consistent with our findings since for both wild-type and the mutant ARs, binding to GRIP1 in vitro was only modestly increased in response to ligand binding and did not change for CBP (Figs. 3BGo and 3DGo). This is in contrast to a substantial hormone-induced increase in interactions of other nuclear receptors, such as ER and T3R{alpha}, with GRIP1 (Fig. 4AGo, and Refs. 18, 27) and CBP (data not shown) studied under similar conditions. It is possible that additional factors are involved in the interaction between AR and coactivators in vivo, such as GRIP1, which give rise to these divergent results.

AR LBD did not activate transcription when fused to a heterologous DBD (Fig. 5AGo), indicating that the AF-2 function of AR is weak compared with other nuclear receptors (e.g. ER and GR), which is consistent with recent reports (50, 51). Ectopic expression of GRIP1 along with AR LBD fused to the GAL4 DBD resulted in hormone-dependent transactivation (Fig. 5AGo), indicating that an excess of a coactivator can evoke the activity of AR AF-2 in isolation, which is consistent with the findings summarized above and other recent findings (54).

Our mammalian two-hybrid data show that the wild-type AR LBD and GRIP1 [including the additional regions that were previously suggested to be required for AR interaction (52)] strongly interact in intact cells (Fig. 5BGo). In the same assay, M5 was significantly reduced in its ability to interact with GRIP1, whereas M6 was comparable to wild-type AR. Thus, although the decrease in the ability of M5 to interact with GRIP1 in vivo may explain, at least in part, its loss of transcriptional activation, this correlation does not hold for M6. Therefore, the latter results, coupled to the in vitro interaction data, suggest that additional mechanism(s) that could account for the loss of transactivation potential of these mutants should be at work in the cell.

It was recently shown that the interactions between N and C termini of AR are involved in receptor stabilization, reduction in ligand dissociation, and increase in DNA binding affinity (6, 8, 9, 53). The data we present in Fig. 6Go suggest that the disruption of fruitful intramolecular contacts between the LBD and NTD is responsible for the significant loss in transactivation potential of the M5 and M6 mutants. While this manuscript was being prepared for publication, similar findings on N/C interaction were reported using two mutants in the AR AF-2 (51, 54). Collectively, these data suggest that the AF-2 core domain mediates ligand-dependent intramolecular interactions between the LBD and the NTD, which are required for full transcriptional activation by AR.

Similar to many other nuclear receptors, an additional transcriptional property of AR is its ability to interfere with AP-1-mediated transactivation (32, 33, 47). Recent studies suggested that amino acid residues in or close to the AF-2 core domain may be involved in the cross-talk between AR and AP-1 (for a review, see Ref. 30). We found that all the mutants, regardless of their hormone-dependent transcriptional activation function, were similar to wild-type AR in efficiently inhibiting AP-1 activity (Fig. 2DGo). It is of note that the M1 mutant, which has substantially diminished ability to bind either hormone or DNA, can still interfere with AP-1 in a fashion that is comparable to wild-type AR. This suggests that the DNA binding activity is not critical for the ability of AR to interfere with AP-1, consistent with previous studies on the cross-talk between other nuclear receptors and AP-1 (for a review, see Ref. 30). These data are also consistent with the hypothesis that a cofactor, such as CBP, mediates the cross-talk between AR and AP-1 (32, 33), since all AR AF-2 mutants bound CBP as efficiently as the wild-type AR in vitro (Fig. 3DGo, and data not shown). These results support our earlier findings and suggestion (18) that transactivation and transrepression by nuclear receptors are mediated by two different interaction surfaces.

In summary, we identified important differences between the contribution of specific, conserved residues in the AF-2 core domain to AR activity compared with other nuclear receptors. These differences may reflect the fact that the structure of the AR AF-2 core domain, its function, its role in contacting the NTD, and the AR coregulatory proteins that it interacts with are different compared with other nuclear receptors. Such variations may be responsible for selective activation of different nuclear receptors by ubiquitous cofactors and thereby help determine specificity of transcriptional activation. Decisive assessment of this hypothesis must await the crystal structures of AR apo- and holo-LBD and AR NTD, as well as their functional characterization.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
Reporter plasmids -285PB-LUC (8), -73Col-CAT (39), and 5XGAL4-LUC (40), and expression vectors pSG5-AR (32), pSG5-T3R{alpha} (18), pSG5-GRIP1 (41), GST-GRIP1 (42). C- terminal deletion mutants of AR (32), CMV-CBP (55), and GST-CBP(1–452) (32) have been described. For the generation of the AR mutant constructs, single-stranded mutagenic primers were synthesized corresponding to amino acid residues from 890 to 902 in the putative AF-2 core region of AR (sequences available upon request). These primers and a primer corresponding to the SV40 polyadenylation sequence (nucleotides 1,109–1,130 in pSG5) were used in PCR with pSG5-AR as the template. Amplified fragments were digested with BspEI and BamHI, and exchanged with the corresponding fragment of wild-type pSG5-AR. All mutants were confirmed by sequencing. The GAL4-AR-LBD construct was generated by PCR amplification of the AR-LBD (amino acids 629–919) using primers containing BglII and BamHI restriction sites, which was inserted into the BamHI site of G4-CEA-C-M10 (17). The GAL4-AR-LBD-M5 and -M6 were generated by exchanging the NcoI-BamHI fragment of the wild-type with the corresponding fragments of pSG5-AR-M5 and -M6. To generate VP16-GRIP1, the VP16 activation domain (412–480) from pSGVP-GAL4-VP16 (43) as a StyI (blunt-ended)-EcoRI fragment was fused to the HindIII (fill-in)-EcoRI fragment of GRIP1 (amino acids 578–1,462). The fragments were then inserted into the EcoRI site of pSG5. Since the VP16 activation domain does not contain a Met start codon in its N terminus, a primer that contains an ATG with a consensus Kozak sequence was used together with a 3'-primer corresponding to the SV40 polyadenylation site to amplify the VP16-GRIP1 insert. The resulting PCR fragment was then cut with EcoRI and BglII and inserted into the EcoRI-BamHI sites in pSG5.

Cell Culture, Transient Transfection, LUC, and CAT Assays
CV-1, HeLa, COS-1, and COS-7 cells were maintained in DMEM supplemented with 5% newborn calf serum (NCS) or 5% or 10% FBS, respectively. The calcium phosphate coprecipitation method was used to transfect both the CV-1 and HeLa cells. CV-1 cells were transfected with 0.25 µg of the reporter plasmid and the indicated plasmids and pUC18 to a total of 0.67 µg DNA per well on 12-well plates. HeLa cells were transfected with 0.67 µg of reporter plasmid, indicated levels of expression vectors, and pUC18 to a total of 2 µg of DNA per well on six-well plates and similar to CV-1 cells when 12-well plates were used. After 5–6 h of incubation with the precipitates, CV-1 cells were washed once with PBS and then maintained in DMEM supplemented with 0.5% charcoal-treated NCS in the presence or absence of R1881 (10-7 M, NEN Life Science Products, Boston, MA). After transfection, HeLa cells were treated with 15% glycerol in PBS for 2 min, washed with PBS, and then maintained in DMEM supplemented with 0.5% charcoal-treated NCS in the presence or absence of R1881. In the experiments with -73Col-CAT, cells were treated with TPA (10-7 M) after transfection to maximize AP-1 activity. Sixteen hours after transfection, cells transfected with the luciferase reporter were washed once with cold PBS and harvested in Tris-MES solution (1 mM dithiothreitol, 0.5% Triton X-100, and 50 mM Tris-MES, pH 7.8), and the LUC activities were determined. The cells that were transfected with the CAT reporter were washed once with cold PBS, incubated with hypotonic buffer (25 mM Tris-HCl, pH 7.5, and 2 mM MgCl2) for 5 min, and then lysed in Triton lysis buffer (0.25 M Tris-HCl, pH 7.8, and 0.5% Triton X-100) to make the extracts. CAT activities in these extracts were determined as previously described (13).

R1881-Binding Assay
The calcium phosphate coprecipitation method was used to transfect COS-1 cells with 0.5 µg of expression vector per well in 12-well plates. After 4–6 h incubation, the cells were shocked with 15% glycerol for 2 min, washed once with PBS, and then maintained in DMEM supplemented with 0.5% charcoal-treated FBS. After 16 h, the cells were washed twice with PBS, and increasing amounts of [3H]R1881 (NEN Life Science Products) were added in serum-free DMEM. After incubation for 2 h, the cells were washed twice with cold PBS, harvested, and directly counted in scintillation fluid to determine the [3H]R1881 that remained bound in the cells. The dissociation constants were determined using GraphPad software (GraphPad Software, Inc., San Diego, CA).

Western Analysis
For Western analysis, the polyethylenimine (PEI) method (44) was used to transfect COS-7 cells with 15 µg of expression vectors specifying the production of wild-type or mutant ARs in 10-cm dishes. After incubation for 4–6 h, the cells were washed with PBS and maintained in DMEM supplemented with 10% FBS. Thirty six hours later, whole cell extracts were prepared. Briefly, cells were washed twice in cold PBS, resuspended in Dignam C solution (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM dithiothreitol) with protease inhibitors, and lysed by three cycles of freeze/thaw. The extracts were run on SDS-PAGE, blotted onto a nylon membrane, and probed with a polyclonal antiserum raised against the LBD of AR (45).

GST Pull-Down Assay
In vitro interactions between AR and GRIP1 or CBP were examined by the GST pull-down assay as described previously (18, 32). Briefly, GST and GST-GRIP1 (amino acids 415–812) (42), or GST-CBP(1–452) fusion proteins were expressed in E. coli and purified on glutathione-Sepharose beads (Amersham Pharmacia Biotech, Arlington Heights, IL). AR and its mutants and T3R{alpha} were translated in vitro using the TNT-coupled transcription/translation system (Promega Corp., Madison, WI) in the presence of [35S]methionine, 1.3 x 10-6 M ZnCl2, and in the presence or absence of R1881 (10-6 M) or T3 (10-7 M), for AR and T3R{alpha}, respectively. In the GST pull-down reactions, the translated proteins were incubated on ice for 20 min in the presence or absence of R1881 (10-6 M) or T3 (10-7 M) before the addition of fusion proteins. The reactions were incubated for 1–2 h on ice in NETN buffer (40 mM NaCl, 4 mM Tris-HCl, pH 8.0, 0.1% Nonidet P-40, and 0.2 mM EDTA) containing protease inhibitors and the same amounts of ligand as above with occasional mixing. The beads were then washed three times with NETN buffer, resuspended in sample buffer, and size fractionated on SDS-polyacrylamide gels. Labeled proteins visualized by fluorography were analyzed by using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).

Mobility Shift Assay
Wild-type AR and mutant proteins were prepared by in vitro transcription/translation as described above and tested for binding to a 32P-labeled androgen response element (ARE) of the first intron of rat C3 gene in the presence of R1881 (10-6 M) (46). Briefly, the translated proteins were incubated at room temperature for 15 min in binding buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM MgCl2, 0.4 mM EDTA, 10% glycerol, 0.05% Nonidet P-40, and 20 mg/ml BSA) in 10 µl volume. After hormone binding, 32P-labeled ARE was added and binding reactions were supplemented with dithiothreitol (2 mM), and 200 ng poly(dI-dC)2 were added, yielding a total volume of 20 µl, and the reaction mixtures were incubated for 25 min at room temperature. The binding reactions were then run on a 5% nondenaturating polyacrylamide gel (29:1) in 0.25x Tris-borate-EDTA buffer, dried, and visualized by autoradiography.


    ACKNOWLEDGMENTS
 
We are grateful to Drs. Richard Eckner, Steve Harris, Michael Stallcup, and Martin Zenke for generous gifts of plasmids, and to R. Miesfeld for the anti-AR antiserum.


    FOOTNOTES
 
Address requests for reprints to: Dr. Fahri Saatcioglu, Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125 Blindern, Oslo, Norway N-1317. E-mail: fahris{at}bioslave.uio.no

This work was supported by grants from the Norwegian Cancer Society, Norwegian Research Council, University of Oslo, The Odd Fellow and Jahre Foundations, the Medical Research Council of the Academy of Finland, the Finnish Foundation for Cancer Research, the Sigrid Jusélius Foundation, and Biocentrum Helsinki.

Received for publication February 3, 2000. Revision received July 13, 2000. Accepted for publication July 20, 2000.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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