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Originally published In Press as doi:10.1074/jbc.M002807200 on May 17, 2000

J. Biol. Chem., Vol. 275, Issue 30, 22986-22994, July 28, 2000
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FXXLF and WXXLF Sequences Mediate the NH2-terminal Interaction with the Ligand Binding Domain of the Androgen Receptor*

Bin He, Jon A. Kemppainen, and Elizabeth M. WilsonDagger

From the Laboratories for Reproductive Biology, Department of Biochemistry and Biophysics and Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599

Received for publication, April 3, 2000, and in revised form, May 16, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH2-terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motif-containing coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgen-dependent interaction with the AR NH2-terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted alpha -helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgen-dependent, NH2- and carboxyl-terminal interaction. FXXLF in the AR NH2-terminal domain with the sequence 23FQNLF27 mediates interaction with AF2 and is the predominant androgen-dependent interaction site. This FXXLF sequence and a second NH2-terminal WXXLF sequence 433WHTLF437 interact with different regions of the ligand binding domain to stabilize the hormone-receptor complex and may compete with AF2 recruitment of LXXLL motif-containing coactivators. The results suggest a unique mechanism for AR-mediated transcriptional activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear receptors facilitate ligand-dependent increases of gene transcription by direct interactions with nuclear coactivators. p160 coactivators have histone acetyltransferase activity (1) and interact with nuclear receptors through their ligand binding and NH2-terminal regions (2-4). Additional acetyltransferases p300/cAMP response element binding protein and p300/cAMP response element binding protein associated factor interact with the p160 coactivators (5-7). Nuclear receptors also interact with multiprotein complexes referred to as thyroid hormone receptor-associated proteins (8), activator-recruited cofactor (9), or vitamin D receptor-interacting protein complex (10). The p160 coactivators and at least one of the thyroid hormone receptor-associated proteins/activator-recruited cofactor/vitamin D receptor-interacting protein subunit 205 interact in a ligand-dependent manner with activation function 2 (AF2)1 in the ligand binding domain (LBD) of nuclear receptors through the consensus sequence LXXLL, where L is leucine and X is any amino acid (11-16). Crystal structures of nuclear receptor LBDs have shown that a hydrophobic cleft within a multilayered alpha -helical structure serves as the LXXLL coactivator binding surface AF2 (17, 18). In the estrogen receptor, agonist binding positions helix 12 over the binding cavity to complete the AF2 surface (19, 20), whereas binding of an antagonist such as 4-hydroxytamoxifen displaces helix 12 (21) causing an LXXLL-like sequence in helix 12 to mimic and thereby block coactivator binding (22).

It has become apparent that the AF2 region overlaps with regions that serve as the binding site for a variety of LXXLL-related sequences as recently shown for corepressor binding (23-25). Furthermore, we demonstrated that the AR AF2 region mediates an androgen-dependent NH2-terminal/carboxyl-terminal (N/C) interaction (26). Therefore we investigated the possibility that AF2 in the AR LBD interacts with an LXXLL-related sequence in the NH2-terminal domain. In this report, evidence is presented that sequences similar to but distinct from the LXXLL core sequence mediate a direct interaction between the NH2-terminal and carboxyl-terminal regions of AR. The FXXLF core sequence 23FQNLF27 in the AR NH2-terminal domain binds AF2 in the carboxyl-terminal region in an androgen-dependent manner. In addition, a second motif in the NH2-terminal region WXXLF with the sequence 433WHTLF437 binds to a region of the LBD outside of AF2. Interaction of these NH2-terminal sequences with the LBD slows the dissociation rate of bound androgen. Sequence specificity was indicated since FXXLF could not be functionally replaced by an LXXLL core sequence.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transcription Assays and Plasmids-- Mammalian two-hybrid N/C interaction assays were performed in Chinese hamster ovary (CHO) cells as described previously (27, 28). VPAR-(1-660) codes for the herpes simplex virus VP16 protein transactivation domain residues 411-456 expressed as a fusion protein with AR NH2-terminal and DNA binding domain residues 1-660. GALAR-(624-919) codes for a fusion protein of the Saccharomyces cerevisiae GAL4 DNA binding domain residues 1-147 and AR LBD residues 624-919. Deletions within VPAR-(1-660) (Delta 179-199, Delta 394-405, and Delta 429-439) were created by two polymerase chain reactions (PCR) using oligonucleotide primers with appropriate deletions. The reporter vector G5E1bLuc contained five GAL4 DNA-binding sites and the luciferase reporter coding region (29). CHO cells were plated at 0.425 × 106/6-cm dish and transfected using DEAE-dextran (27, 28). Cells were incubated for 24 h in the absence and presence of increasing concentrations of dihydrotestosterone (DHT) as indicated and harvested in lysis buffer (Ligand Pharmaceuticals). Luciferase activity was determined using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego).

For the assessment of AR transcriptional activity, monkey kidney CV1 cells maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), 20 mM Hepes, pH 7.2 (DMEM-H), and antibiotics were transiently transfected by plating 0.425 × 106 cells/6-cm dish. Wild-type and mutant pCMVhAR DNA (10-50 ng/dish) were precipitated with 5 µg of mouse mammary tumor virus promoter region (MMTV)-luciferase reporter vector (5 µg) using calcium phosphate (30). Cells were incubated for 24 or 48 h with the indicated concentrations of hormones and harvested and assayed in lysis buffer as described above. VPAR-(1-660) containing Delta 329-381, Delta 382-429, Delta 430-499, or Delta 429-439 was digested with AflII and KpnI, and the resulting inserts were ligated into pCMVhARL26A/F27A digested with the same enzymes. PCMVhARDelta 339-499, Delta 9-28 (27, 31), and AR-(507-919) (32) were previously described. PCR mutagenesis was used to create single and double amino acid mutations in the 23FXXLF27 and 433WXXLF437 domains. L26A/F27ADelta 339-499 was prepared by ligating pCMVhARDelta 339-499 digested with BglII/AflII with the insert from pCMVhARL26A/F27A using the same enzymes.

Androgen Binding and Dissociation Assays-- Apparent equilibrium binding affinity was determined in whole cell binding assays at 37 °C by plating monkey kidney COS1 cells (0.2 × 106/well of 12-well tissue culture plates) and transfecting 0.1 µg of pCMVhAR wild-type and mutant DNA using DEAE-dextran (26). Twenty four h after transfection, cells were incubated with increasing concentrations of [3H]R1881 from 0.1 to 5 nM in the presence and absence of a 100-fold excess unlabeled R1881. Cells were incubated at 37 °C for 2 h, washed, and harvested in 0.5 ml of 2% SDS, 10% glycerol, and 10 mM Tris, pH 6.8. Radioactivity was determined by scintillation counting. For determination of dissociation rates of [3H]R1881, COS cells were plated at 0.4 × 106 cells/well in 6-well plates and transfected with 2 µg of pCMVhAR DNA/well using DEAE-dextran. Cells were incubated for 2 h at 37 °C with 5 nM [3H]R1881 in the presence and absence of a 100-fold excess unlabeled R1881. Dissociation was started by the addition of 50 µM unlabeled R1881, and the cells were incubated for increasing times at 37 °C up to 3 h, washed once, and harvested in SDS buffer as described above, with radioactivity determined by scintillation counting.

In Vitro Protein Binding Assays-- Glutathione S-transferase (GST) fusion vectors GSTAR-(1-660) and GSTAR-(1-565) were prepared as described previously (26). pGEX5X-1AR-(1-660) was digested with XhoI (blunt) and SmaI and religated to make GSTAR-(1-36). pGEX5X-1AR-(1-660) was digested with AflII/XhoI, blunt-ended, and religated to make GSTAR-(1-173). pGEX5X-1AR-(1-660) was digested with SacI/XhoI, blunt-ended, and religated to make GSTAR-(1-333). pGEX-3XAR-(1-566) was digested with BamHI/AflII, blunt-ended, and religated to make GSTAR-(174-566). GALAR-(1-660)-L26A/F27A was digested with BamHI/AflII, and the insert was ligated into pGEX-5X-1AR-(1-660) digested with BamHI/AflII to make GSTAR-(1-660)-L26A/F27A. GSTAR-(1-660)-L26A/F27A was digested with KpnI/AflII and ligated with the insert from VPAR-(1-660)-Delta 429-439 digested with KpnI/AflII to make GSTAR-(1-660)-L26A/F27A-(Delta 429-439). AR NH2-terminal residues 344-566 were PCR-amplified from VPAR-(1-660) with the appropriate deletions, digested at BamHI/XhoI primer sites, and ligated into pGEX-4T-1 to make GSTAR-(344-566), GSTAR-(344-566)-Delta 339-381, GSTAR-(344-566)-Delta 382-429, GSTAR-(344-566)-Delta 430-499, and GSTAR-(344-566)-Delta 429-439. GSTTIF2 expressing the central TIF2 amino acid residues 624-1141 containing three LXXLL motifs was described previously (26). pcDNA3-AR-(624-919)-E897K, V716R, K720A, and V889M were prepared by digesting GALD-H containing the appropriate mutation with BamHI/XbaI and ligating the fragment into pcDNA3HA digested with BamHI/XbaI.

GST fusion protein binding studies were performed essentially as described previously (26). GST fusion proteins with different regions of the human AR NH2-terminal region were expressed from XL1-Blue Escherichia coli cells treated with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h during log phase growth. Glutathione-agarose beads (Amersham Pharmacia Biotech) were incubated for 1 h at 4 °C with sonicated bacterial supernatants containing the GST-AR fusion proteins. Beads were washed with 0.5% Nonidet P-40, 1 mM EDTA, 0.1 M NaCl, 0.02 M Tris, pH 8.0, and combined with 25 µCi of [35S]methionine (NEN Life Science Products)-labeled human AR LBD (AR amino acid residues 624-919) using TNT T7 Quick-coupled Transcription/Translation System (Promega) and incubated for 2 h at 4 °C in the absence or presence of 0.2 µM DHT. Beads were washed, eluted with SDS, and analyzed on 12% acrylamide gels containing SDS.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

FXXLF in the AR NH2-terminal Region-- The androgen-dependent interaction between the AR NH2-terminal and carboxyl-terminal (N/C) domains occurs in the regions of predicted alpha -helices 3, 4, and 12 that comprise AF2 of the LBD, overlapping with the binding site for p160 coactivators (26). Because LXXLL motifs mediate the interaction of p160 coactivators with AF2 of nuclear receptors (11-16), it raised the possibility that an LXXLL-like motif (13-15) in the AR NH2-terminal region has a similar function to mediate the N/C interaction. Sequence analysis of the AR NH2-terminal regions previously implicated in the N/C interaction (27) revealed four predicted amphipathic alpha -helices that resemble LXXLL core sequences at residues 21-34, 351-359, 395-405, and 432-434 with another predicted outside these regions (33) at residues 177-201. We tested wild-type, deletion, and single amino acid mutations of these alpha -helical regions in a variety of assays. These included the mammalian two-hybrid N/C interaction assay performed in CHO cells and functional assays that included the effects of the mutations on [3H]androgen dissociation rate and transcriptional activation. In vitro domain interactions were also directly tested using E. coli-expressed GST fusion proteins. For the mammalian two-hybrid assay, wild-type and mutant VPAR-(1-660) coding for the AR NH2-terminal and DNA binding domains were cotransfected with GALAR-(624-919) expressing the LBD (Fig. 1). Similar expression levels of wild-type and mutant VPAR-(1-660) vectors were verified by immunoblot analysis using AR52 antibody (data not shown). Also all of the VPAR-(1-660) constructs, when cotransfected with GALAR-(1-503) coding for just the NH2-terminal domain, resulted in 2.2-3.2-fold induction of luciferase activity indicative of the NH2-terminal/NH2-terminal AR interaction previously reported (27), thus confirming similar expression levels of the VPAR-(1-660) vectors.


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  		Fig. 1.   Identification of the 23FXXLF27 motif. Shown are schematic diagrams and two-hybrid interaction results between the AR LBD and wild-type and AR NH2-terminal mutants used to define the 23FXXLF27 motif in the AR NH2-terminal domain. Mutants were constructed in VPAR-(1-660) coding for the VP16 activation domain (VP16AD) expressed as a fusion protein with the AR NH2-terminal and DNA binding domain (DBD) residues 1-660. Wild-type (WT) and mutant VPAR-(1-660) were cotransfected with GALAR-(624-919) expressing the AR LBD residues 624-919 (AR LBD) as a fusion protein with the GAL4 DNA binding domain (GAL4DBD) and analyzed in CHO cells in the mammalian two-hybrid N/C interaction assay using the G5E1bLuc reporter vector as described under "Experimental Procedures." Shown is the mean fold induction and error of luciferase activity from at least three independent experiments determined in the absence and presence of 10 nM DHT. AR amino acid residue numbers are indicated at the top. Changes in individual amino acid residues in the NH2-terminal region are indicated at the bottom by arrows.

Wild-type and mutant VPAR-(1-660) coding for the AR NH2-terminal and DNA binding regions were coexpressed in the mammalian two-hybrid assay with GALAR-(624-919) coding for the AR LBD. The VPAR-(1-660) fragment with wild-type AR sequence induced 38 ± 19-fold luciferase activity relative to the no hormone control (Fig. 1). Deletion of NH2-terminal residues 179-199, 394-405, or 429-439 had no significant effect on the interaction (Fig. 1). In contrast, Delta 9-28 reduced the interaction to only a 1.5-fold increase over the no hormone control and Delta 339-499 to 10-fold relative to the control (Fig. 1) (27). These results suggested interactions between the LBD and residues 9-28 and 339-499. Because 23FQNLF27 lies within the 9-28 region and resembles the LXXLL core sequence, it was investigated further by mutagenesis. Changing phenylalanine 23 to alanine (F23A) or leucine 26 and phenylalanine 27 to alanine (L26A/F27A) reduced the N/C interaction to 1.6 ± 0.2-fold over background levels, whereas a flanking mutation of glutamine 28 to alanine (Q28A) resulted in an interaction similar to that of wild-type AR (Fig. 1). Changing both phenylalanine residues in 23FQNLF27 to leucine (F23L/F27L) to recreate a consensus LXXLL sequence resulted in only a 2-fold induction of luciferase activity (Fig. 1). These results indicated that an FXXLF motif was required in the N/C interaction. Specificity of the 23FXXLF27 interaction with the AR LBD was indicated by the greatly reduced interaction when FXXLF was substituted by LXXLL.

The requirement for the FXXLF motif in the N/C interaction was also investigated using AR NH2- and carboxyl-terminal fragments in transient transfection assays using the MMTV-luciferase reporter. As previously reported, the AR DNA binding and ligand binding domain fragment AR-(507-919) had negligible transcriptional activity in the presence of androgen (Fig. 2) indicative of the lack of AF2 transcriptional activity (32) and weak recruitment of p160 coactivators by the AR AF2 region (26). In cotransfection studies, wild-type NH2-terminal domain residues AR-(1-503) interacted with AR-(507-919) to stimulate an 18-28-fold increase in luciferase activity (Fig. 2). In contrast, NH2-terminal fragment AR-(1-503) with residues 14-150 deleted (Delta 14-150) or with the mutated sequence 22FQNAA27 (L26A/F27A) failed to interact with AR-(507-919), supporting an important role for 23FQNLF27 in the N/C interaction. Androgen-induced luciferase activity was 6-12-fold with deletion of NH2-terminal residues 142-337 comprising the AR transactivation domain (Delta 142-337) which could have resulted from reduced transactivation by AR rather than a decrease in the N/C interaction as previously suggested (27). Delta 339-499 also reduced the interaction but less effectively than did the L26A/F27A mutation (Fig. 2). The results support the requirement for 23FQNLF27 in the N/C interaction and the presence of a second interaction site between residues 339 and 499. Similar results using fusion proteins of the AR NH2-terminal region linked to the VP16 transactivation domain supported the role of these two regions in the N/C interaction (Fig. 2).


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  		Fig. 2.   N/C interaction using AR fragments and the MMTV-luciferase reporter vector. PCMVhAR-(507-919) (AR507-919, 50 ng/plate) coding for the AR DNA binding domain and LBD was coexpressed in CV1 cells without or with 0.5 or 1 µg of pCMVhAR-(1-503) (AR1-503) coding for the AR NH2-terminal domain with wild-type sequence or the indicated mutant sequence or with 1 or 5 µg of the VP16 activation domain-AR NH2-terminal domain fusion protein VPAR-(1-503) with wild-type sequence or with the indicated deletions. Assays were performed in CV1 cells using the MMTV-luciferase reporter (5 µg) in the presence and absence of 10 nM DHT. Parent expression vector control pCMV5 (p5) lacked AR sequence. LFAA indicates the L26A/F27A mutation (23FQNAA27). Fold induction of luciferase activity determined relative to the activity in the absence of DHT is indicated above the bars and is representative of at least three independent experiments.

Effect of the FXXLF Motif on Androgen Dissociation Kinetics-- To establish a functional effect of the NH2-terminal mutations on AR activity and to obtain additional evidence for the putative second interacting site, we measured the androgen dissociation rate using the synthetic radiolabeled androgen [3H]R1881. These studies were based on previous studies that certain mutations in AF2 of the AR LBD cause androgen insensitivity by disrupting the N/C interaction. Although the equilibrium androgen binding affinity was unaffected by these mutations, the dissociation rate of bound androgen increased suggesting a corresponding increase in association rate (26, 28). The results supported a role for the N/C interaction in slowing the androgen dissociation rate in wild-type AR (31).

Additional evidence that the N/C interaction influences AR ligand binding kinetics is that coexpression of the DNA binding domain and LBD fragment AR-(507-919) with NH2-terminal fragment AR-(1-660) slows the dissociation of [3H]R1881 by 2-fold (28). In contrast, no effect was seen by coexpression of the nuclear receptor coactivators transcriptional mediator/intermediary factor 2 (TIF2), vitamin D receptor-interacting protein 205, amplified in breast cancer-1 (AIB1) or protein inhibitor of activated signal transducer and activator of transcription-1 (PIAS1), each of which contains multiple consensus LXXLL motifs (10, 34-36). Dissociation of [3H]R1881 from the carboxyl-terminal AR-(507-919) fragment (half-time of dissociation t1/2 of 43 ± 3 min, see Fig. 4) was unchanged with t1/2 of 42 ± 4 min at 37 °C when coexpressed with each of these coactivators (data not shown). The results are consistent with a weak interaction of these coactivators with the AR AF2 region compared with the interaction with the AR NH2-terminal domain (26) and suggest a correspondingly higher apparent binding activity of the 23FXXLF27 core sequence for AF2 compared with the LXXLL motif.

The functional significance of the NH2-terminal FXXLF core sequence was also evident by increased dissociation rates of bound androgen with the introduction of mutations in the FXXLF region in full-length AR. Delta 9-28, F23A, or L26A/F27A increased the dissociation rate of [3H]R1881 at 37 °C to t1/2 of 72-74 min compared with t1/2 of 144 ± 24 min for wild-type AR (Fig. 3). In contrast, mutation of the flanking carboxyl glutamine (Q28A) had no effect on dissociation rate (t1/2 of 162 ± 24 min) (Fig. 3) or N/C interaction (Fig. 1). Furthermore, changing 23FQNLF27 to the consensus LXXLL sequence (F23L/F27L) increased the dissociation rate from t1/2 of 144 ± 24 min for wild-type AR to t1/2 of 84 ± 9 min (Fig. 3), which was similar to t1/2 74 ± 5 min for F23A and L26A/F27A, supporting that an LXXLL motif is much less effective than FXXLF in mediating the N/C interaction. None of these mutations or those described below changed significantly the apparent equilibrium binding affinity of [3H]R1881 that ranged from 0.3 to 0.8 nM (data not shown), suggesting a corresponding increase in association rate when the dissociation rate increased. The results indicate that the AR NH2-terminal FXXLF motif has an important role in the N/C interaction which results in a reduced rate of androgen dissociation.


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  		Fig. 3.   Effect of AR NH2-terminal motif 23FXXLF27 on androgen dissociation kinetics. Wild-type (WT) and AR mutants shown schematically were constructed in full-length pCMVhAR. Dissociation rates of [3H]R1881 were determined at 37 °C in transiently transfected COS cells as described under "Experimental Procedures." Mean dissociation half-times (t1/2 in min at 37 °C) and errors of at least three independent experiments are shown. The major AR domains are indicated by DNA binding domain (DBD) and ligand binding domain (LBD) and by amino acid residue number. Individual amino acid changes are indicated with arrows at the bottom.

WXXLF, the Second N/C Interaction Site-- Results from the two-hybrid interaction and androgen kinetic studies provided evidence for a second NH2-terminal site involved in the N/C interaction. The dissociation rate of [3H]R1881 from AR mutant Delta 9-28 (27) or 23FQNAA27 (L26A/F27A) of t1/2 of 72-74 min was faster than from wild-type AR (t1/2 of 144 ± 24 min, Fig. 3) but slower than that for AR-(507-919) with t1/2 of 43 ± 3 min which lacks the NH2-terminal region (Fig. 4). We therefore investigated further the location of a second interacting site that contributed to slowing the androgen dissociation rate. NH2-terminal deletions Delta 429-439 or Delta 339-499 alone did not increase the androgen dissociation rate relative to wild-type AR, with dissociation rates t1/2 of 152 ± 3 min and 140 ± 13 min, respectively (Fig. 3). However, when these mutations were combined with the mutation 23FQNAA27 (L26A/F27A), the dissociation rate increased to t1/2 of 49 ± 4 min for Delta 339-499L26A/F27A and t1/2 of 47 ± 1 min for Delta 429-439L26A/F27A (Fig. 4). These rates were similar to that for AR-(507-919) and slightly faster than when L26A/F27A was combined with Delta 430-499 (t1/2 of 57 ± 1 min, Fig. 4). On the other hand, no further increases in androgen dissociation rate were observed when L26A/F27A was combined with Delta 339-381 or Delta 382-429 with t1/2 of 90 ± 9 min (Fig. 4). The results suggest that residues 429-439 contribute to slowing the androgen dissociation rate and thus may be involved of the N/C interaction.


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  		Fig. 4.   Effect of the 433WXXLF437 motif on androgen dissociation kinetics. Wild-type (WT) AR and AR mutants shown schematically were constructed in full-length pCMVhAR without or with the NH2-terminal mutation 23FQNAA27 as indicated or AR-(507-919) expressing the DNA binding domain and LBD. Mean dissociation half-times (t1/2 in min at 37 °C) and errors of [3H]R1881 were determined from at least three independent experiments as described under "Experimental Procedures." Major AR domains are as described in Fig. 3 legend, and individual amino acid changes are indicated with arrows.

The LXXLL-like sequence 433WHTLF437 lies within NH2-terminal residues 429-439. To establish whether this putative WXXLF motif contributes to slowing the androgen dissociation rate, several additional mutations were combined with the mutant sequence 23FQNAA27 (L26A/F27A). With tryptophan 433 changed to alanine (W433A) or the mutation 433WHTAA437 (L436A/F437A) was combined with 23FQNAA27 (L26A/F27A), the dissociation rate increased to t1/2 of 52 ± 2 min (Fig. 4). However, no increase was observed when the flanking serine 432 was changed to alanine (S432A) and combined with L26A/F27A (t1/2 of 92 ± 3 min) relative to L26A/F27A alone (Fig. 4). When L436A/F437A was combined with E897K, a mutation that interferes with the N/C interaction (26), the androgen dissociation rate increased to 45 ± 2 min (data not shown). Thus NH2-terminal sequences 433WHTLF437 and 23FQNLF27 appear to act in concert to slow the androgen dissociation rate.

To test for possible functional equivalence between the FXXLF and WXXLF core sequences that might have depended on their position within the AR NH2-terminal sequence rather than the binding motif itself, we replaced phenylalanine 23 with tryptophan to thereby replace 23FXXLF27 with 23WXXLF27. The two-hybrid N/C interaction of this F23W mutant was only 1.9-2.2-fold compared with 35-59-fold for the wild-type control at 1 and 100 nM DHT. WXXLF therefore appears to have binding properties distinct from FXXLF such that the two motifs are not functionally equivalent. Whereas FXXLF appears to be the primary NH2-terminal interaction site for the AF2 region of the LBD, the core sequence WXXLF seems to contribute to the interaction with the AR LBD by further slowing the androgen dissociation rate.

Distinct Binding Regions of the Two NH2-terminal Motifs-- The AR NH2-terminal sequences that mediate the N/C interaction were investigated further using E. coli-expressed GST fusion proteins. AR LBD residues 624-919 were labeled with [35S]methionine and incubated in the presence of 0.2 µM DHT and GST-AR NH2-terminal fragments of increasing length. Similar expression levels of the GST AR NH2-terminal domain fusion proteins were confirmed by Coomassie staining of SDS-polyacrylamide gels (data not shown). AR NH2-terminal fragments 1-36, 1-173, and 1-333 bound to the AR LBD in the presence of 0.2 µM DHT and was reduced to near background levels by the L26A/F27A mutation (Fig. 5A, lanes 1-7), providing in vitro evidence that 23FQNLF27 mediates the N/C interaction. But surprisingly, using the extended NH2-terminal residues 1-660, the L26A/F27A mutation had only a minimal effect on the in vitro binding assay (Fig. 5A, lanes 8 and 9). Furthermore, AR NH2-terminal fragments 174-566 and 334-566 that lacked the 23FQNLF27 binding motif showed strong interaction with the AR LBD (Fig. 5A, lanes 10 and 11). The results support the role of the NH2-terminal FXXLF in mediating the N/C interaction and provide additional evidence for a second NH2-terminal interacting site located between residues 334 and 566. 


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  		Fig. 5.   In vitro GST fusion protein binding studies of AR interacting domains. GST fusion proteins either lacking AR sequence (GST AR 0) or with the indicated AR NH2-terminal regions with wild-type sequence, or with the L26A/F27A mutation (LFAA) (A), or the indicated deletions (B) were incubated in the presence of 0.2 µM DHT and 35S-labeled AR-(624-919) expressing the AR LBD residues 624-919 with wild-type sequence as described under "Experimental Procedures." 20% of the total radioactivity used in the binding reactions is shown in the input lanes.

To determine whether the second site was identical to 433WHTLF437 identified in the androgen kinetic studies described above, AR NH2-terminal deletions Delta 339-381, Delta 382-429, and Delta 430-499 were introduced into GST-AR-(334-556). As shown in Fig. 5B (lane 5), residues 430-499 were required for interaction of GSTAR-(344-566) with the AR LBD. Indeed, deletion of the predicted short alpha -helical region containing 433WHTLF437 in Delta 429-439 greatly decreased the LBD-GSTAR-(344-566) interaction in the presence of DHT (Fig. 5B, lane 6). More importantly, introducing the double mutations of the two putative interacting regions FXXLF and WXXLF (L26A/F27A and Delta 429-439) into AR-(1-660) reduced the N/C interaction to basal levels that were observed in the absence of androgen (Fig. 5B, lane 11). The results support the androgen kinetic studies that both 23FQNLF27 and 433WHTLF437 in the AR NH2-terminal region mediate the N/C interaction. In addition, they indicate that WXXLF interacts with the LBD to a greater extent than was evident in the two-hybrid assay.

The androgen dependence of the interaction of the two NH2-terminal sites with the AR LBD was further investigated in the GST fusion protein in vitro binding assay. Interaction of NH2-terminal fragment GSTAR-(1-333) containing the FXXLF binding motif 23FQNLF27 was dependent on the addition of androgen (Fig. 6, lanes 2 and 5) which agreed with the androgen dependence of the N/C interaction. However, surprisingly, binding of GSTAR-(334-566) containing the 433WXXLF437 motif to the 35S-labeled AR-(624-919) LBD fragment was independent of the presence of androgen (Fig. 6, lanes 3 and 6). The results raised the possibility that the FXXLF and WXXLF binding motifs interact with different regions of the AR LBD.


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  		Fig. 6.   Androgen dependence of AR NH2-terminal fragment interactions with the AR LBD. Androgen-dependent interactions were tested by incubating 35S-labeled AR-(624-919) expressing the AR LBD containing wild-type sequence together with GST-AR0 (lacking AR sequence), or with AR NH2-terminal fragments GST-AR-(1-333) or GST-AR-(174-566) in the absence (lanes 1-3) or presence (lanes 4-6) of 0.2 µM DHT. Incubations were performed as described under "Experimental Procedures." The input lane represents 20% of the total 35S-labeled AR-(624-919) used per reaction.

Previously, we showed that certain mutations in the AF2 region of the AR LBD disrupt the N/C interaction without changing the apparent equilibrium binding affinity yet require higher DHT concentrations to induce MMTV-luciferase activity relative to wild-type AR (26). We therefore determined whether one or both of the NH2-terminal sites were affected by these mutations in the LBD. In GST interaction assays performed in the presence of 0.2 µM DHT, LBD mutations E897K or V716R introduced into 35S-labeled AR-(624-919) greatly reduced the interaction between AR-(1-333) and the LBD (Fig. 7, lanes 13 and 18) as well as the interaction with the p160 coactivator fragment, TIF2-M (Fig. 7, lanes 15 and 20). However, interaction of these LBD mutants with AR-(174-566) which contained the second interacting motif 433WXXLF437 was similar to wild-type (Fig. 7, lanes 14 and 19). Similar results were observed using V889M (data not shown), a mutation in the AR LBD that causes nearly complete androgen insensitivity (31, 37). On the other hand, 35S-labeled AR-(624-919) containing K720A, a mutation that does not affect the N/C interaction or AR transcriptional activity (26), did not decrease the interaction with AR-(1-333) or AR-(174-566) but eliminated the interaction with TIF2-M (Fig. 7, lanes 7-10). The results suggest that glutamic acid 897 and valine 716 in AF2 and valine 889 preceding helix 12 of the LBD interact with 23FQNLF27 but not with 433WHTLF437. Because these residues are directly part (glutamic acid 897 and valine 716) or flanking (valine 889) the AF2 region, the results support the interaction of 23FQNLF27 with AF2 in the LBD and suggest that 433WHTLF437 interacts with another region of the LBD.


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  		Fig. 7.   LBD amino acids involved in interactions with AR NH2-terminal fragments. 35S-Labeled AR-(624-919) with wild-type sequence (WT) or with mutations K720A, E897K, or V716R were incubated in the presence of 0.2 µM DHT with GST0 lacking AR sequence, or with AR NH2-terminal fragments GST-AR-(1-333) or GST-AR-(334-566) as indicated, or with GST-TIF2-M (TIF2 residues 624-1141) as a positive control (26). Input lanes (I) represent 20% of the total 35S-labeled AR-(624-919) used per reaction. The major radiolabeled bands represent wild-type or mutant 35S-labeled AR-(624-919).

Transcriptional Activation-- The influence of mutations in the FXXLF and WXXLF core sequences on AR transcriptional activity by full-length AR was investigated in transient transfection assays using two different luciferase reporter vectors. The 23FQNAA27 mutation (L26A/F27A) either alone or combined with the mutation 433WHTAA437 (L436A/F437A) reduced the increase in transcriptional activity by at least 50%, whereas Delta 429-439 alone had less of an effect (Fig. 8A). By using an AR-specific probasin-luciferase reporter (38), mutations in the first or both NH2-terminal interacting sites also decreased transcriptional activity by about 50% (Fig. 8B). The results suggest that the N/C interaction facilitates an optimal transcriptional response.


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  		Fig. 8.   Transcriptional activation of wild-type AR and NH2-terminal domain mutants using luciferase reporter vectors. Transcriptional activity of full-length wild-type (WT) AR or with the indicated AR amino acid or deletion mutations were determined in CV1 cells at increasing DHT concentrations as described under "Experimental Procedures" using MMTV-luciferase (A) or probasin-luciferase reporter vectors (B). pCMVhAR DNA (25 ng) was transfected with 5 µg of reporter vector. Shown above the bars is the fold induction determined relative to the activity in the absence of DHT. The data are representative of at least three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we present evidence that the N/C interaction, referred to in the literature also as an interaction between AF1 and the LBD or AF1 and AF2 and initially described for AR (27, 39) and the estrogen receptor (40), is direct, androgen-dependent, and mediated predominantly by the core sequence FXXLF in the AR NH2-terminal region. Both mammalian two-hybrid interaction and GST fusion protein binding studies indicate that NH2-terminal sequence 23FQNLF27 mediates interaction with the AF2 region in the LBD in an androgen-dependent manner. This is substantiated by the observation that single amino acid mutations in either the NH2-terminal FXXLF motif or the LBD AF2 region eliminate the N/C interaction when the apparent androgen binding affinity is not altered. At least one functional consequence of the N/C interaction previously reported (28, 31) and shown here is a decreased dissociation rate of bound androgen. Mutations in 23FQNLF27 support this role of the N/C interaction since they increased the androgen dissociation rate.

A surprising finding of the study was that the FXXLF motif was not the only LXXLL-like motif contributing to the N/C interaction. Even though mutation of 23FQNLF27 essentially eliminated the N/C interaction in the two-hybrid assay and the interaction between the AR-(1-333) NH2-terminal fragment with the AR LBD in GST protein binding studies, mutations in 23FQNLF27 had only a marginal effect when the full NH2-terminal region AR-(1-660) was tested in the GST interaction assay. Further studies located the second interaction site between amino acid residues 429 and 439 in the NH2-terminal domain. [3H]R1881 dissociation studies implied that 433WHTLF437 within this region plays an important role in this interaction. However, although the interaction of 23FQNLF27 with the AF2 region was androgen-dependent and largely responsible for the N/C interaction in the two-hybrid assay, interaction with the AR LBD second site 433WXXLF437 located within the 429-439 region was androgen-independent. Furthermore, using the same AF2 LBD mutations that eliminated interaction with FXXLF, it became apparent that the WXXLF motif likely interacts with a region of the LBD outside of AF2. To account for these data and the effect of mutations in both the FXXLF and WXXLF interaction sites on increasing the androgen dissociation rate, it may be that in full-length AR, the second WXXLF site becomes available to bind the AR LBD subsequent to the androgen-dependent interaction of FXXLF with AF2. GST fusion protein interaction assays using AR NH2-terminal fragments may circumvent this requirement for androgen binding by releasing structural constraints within the NH2-terminal region. It suggests further that the binding region of the AR LBD that interacts with the WXXLF motif is not significantly altered by androgen binding as is the AF2 region in binding the FXXLF motif.

The AR NH2-terminal motifs FXXLF and WXXLF resemble the consensus core sequence LXXLL that mediates p160 coactivator interaction with AF2 in the LBD of nuclear receptors (26). Specificity for the 23FXXLF27 core sequence for interaction with AF2 was evident because substitution with either LXXLL or WXXLF essentially abolished the N/C interaction in the two-hybrid assay. The relatively weak AF2 interaction of the LXXLL motif when substituted for 23FXXLF27 is consistent with a weak interaction of the LXXLL-containing p160 coactivators with the AR LBD and with the low inherent AF2 activity of the AR LBD. The results reported here as well as previous evidence from GST fusion protein assays (26) support a direct interaction between the NH2- and carboxyl-terminal regions of AR and contrast with previous reports that the interaction is indirect and bridged (41, 42) or enhanced (43) by p160 coactivators such as steroid receptor coactivator 1.

Several models for the N/C and coactivator interactions of steroid receptors have been proposed that involve parallel versus antiparallel orientation of monomers and intra- versus intermolecular interactions (12, 27, 28, 40, 44). Binding of the AR NH2-terminal region to AF2 in the AR monomer or dimer may exclude binding of p160 coactivators as suggested from competition for different coactivator binding (45). The N/C interaction may also influence a proposed temporal sequence of coactivator binding (46) or create a new interacting surface to recruit coactivators (47). p160 coactivators interact with the AR NH2-terminal region (26) as reported for other nuclear receptors (33, 48). The greater apparent binding affinity of the AR N/C interaction with AF2 compared with p160 coactivator interaction with AF2 is supported by the slower ligand dissociation rate resulting from the N/C interaction but not from p160 coactivator binding. In striking contrast, p160 coactivator binding to the AF2 region slows the ligand dissociation rate from the estrogen receptor (49).

Ligand-dependent N/C interactions have been described for other nuclear receptors including the progesterone receptor, where it facilitates receptor dimerization (44, 50). In peroxisome proliferator-activated receptor-gamma , N/C interactions were ligand-independent and reduced ligand binding affinity presumably by modifying conformation of the unliganded receptor (51). The functional significance of the AR N/C interaction is supported by several naturally occurring spontaneous mutations that interfere with the N/C interaction and increase the androgen dissociation rate. These include mutations at valine 889 (28) and isoleucine 898 (26) that cause complete or nearly complete androgen insensitivity and at methionine 886 that causes oligospermic infertility (52). Similarly, the N/C interaction is inhibited by site-directed mutagenesis of LBD residues glutamic acid 893 (53), valine 716, and glutamic acid 897 (26). Whereas some of these mutations also interfere with p160 coactivator binding such as TIF2 to the AF2 region (52, 53), it became clear that a distinct yet overlapping binding site in AR AF2 acts preferentially as the binding site for the AR NH2-terminal region rather than for p160 coactivators (26). Lysine 720 in human AR corresponds to lysine 366 in the estrogen receptor that was required for AF2 activity (54). Mutations at AR lysine 720, while greatly reducing AR interaction of TIF2, did not decrease AR transcriptional activity significantly (26, 33). On the other hand, I898T, which causes complete androgen insensitivity, did not reduce interaction with TIF2 but greatly reduced the N/C interaction.

Sequence differences in the AF2 region of AR LBD compared with other nuclear receptors likely contribute to the selective binding of FXXLF versus LXXLL motifs (55). The AR AF2 sequence differs from other steroid receptors at several residues that are otherwise highly conserved. Mutating some of these residues to amino acids of other steroid receptors did not enhance p160 coactivator binding (26). The sequence differences unique to the AR LBD may redirect the function of AF2 toward higher binding affinity for the FXXLF binding motif in the AR NH2-terminal domain rather than for the LXXLL motifs in the p160 coactivators. Sequences flanking the LXXLL motifs of p160 coactivators (56, 57) apparently contribute to selective binding to AF2 of other nuclear receptors (45). The use of combinatorial phage display screening showed that in some cases sequences flanking the LXXLL motifs contribute to specific interactions with nuclear receptors (56). However, our study of 23FQNLF27 and 433WHTLF437 indicates that of the flanking AR NH2-terminal sequences tested, none had a predominant role in the N/C interaction. Mutation of glutamine 28 carboxyl-terminal to 23FQNLF27 or serine 432 NH2-terminal to 433WHTLF437 did not increase the androgen dissociation rate, an indicator of the N/C interaction.

Evolution of amino acid sequence in the AR NH2-terminal region and LBD seems to favor conservation of the N/C interaction sites. In the NH2-terminal region, both 23FQNLF27 and 433WHTLF437 and their flanking sequence are fully conserved among the primate AR even though other regions of the NH2-terminal domain are not conserved (58). In the rainbow trout AR alpha  and beta  forms, which have the strikingly low homology of 19% in the NH2-terminal amino acid sequence compared with human AR, structural conservation occurs at the androgen-dependent interaction site with the trout sequence 22FQNVF26 compared with 23FQNLF27 in human AR (59). Furthermore, whereas the primate LBD is fully conserved with respect to human amino acid sequence, rainbow trout AR alpha  and beta  forms have only 65 and 68% sequence similarity in the LBD, respectively (59), yet residues critical for the N/C interaction are conserved. Natural selection therefore seems to favor residues in AR domains that mediate the N/C interaction supporting their critical role in AR function.

The functional significance of the N/C interaction remains to be fully established. The N/C interaction appears to be required for AR function in vivo as suggested by androgen insensitivity mutations. It is, however, apparently not an absolute requirement for AR induction of MMTV reporter gene activity in transient transfection assays. The AR NH2-terminal and DNA binding domain fragment that lacks the LBD has strong constitutive transcriptional activity with the MMTV-luciferase reporter gene (32, 60), and only a 50% reduction in activity was observed with mutations in full-length AR that disrupt the N/C interaction using either the MMTV- or probasin-luciferase reporter genes. The N/C interaction may nevertheless contribute to androgen-specific gene induction since the AR shares the ability with other steroid receptors to bind simple consensus DNA response elements but has distinctly different physiological effects (61). Furthermore, ligands such as medroxyprogesterone acetate (Provera) that fail to promote the N/C interaction are weak androgens in vivo but strong androgen agonists in transient transfection assays (30). These observations support that the N/C interaction is important in mediating AR function in vivo and that transient transcriptional assays may not provide a good reflection of this in vivo requirement. The N/C interaction may increase the sensitivity of AR to low circulating androgen concentrations by slowing the dissociation rate of bound androgen. This function might be especially important during male sexual development in utero.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of K. Michelle Cobb and De-Ying Zang. We also thank Frank S. French for reviewing the manuscript and Robert J. Matusik for providing the probasin-luciferase reporter vector.

    FOOTNOTES

* This work was supported by NICHD Grant HD-16910 from the National Institutes of Health, through cooperative agreement U54-HD-35041 as part of the Specialized Cooperative Centers Program in Reproductive Research, and by the International Training and Research in Population and Health Program supported by the Fogerty International Center and the NICHD, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Laboratories for Reproductive Biology, CB 7500, Rm. 374 Medical Science Research Bldg., University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-5168; Fax: 919-966-2203; E-mail: emw@med.unc.edu.

Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.M002807200

    ABBREVIATIONS

The abbreviations used are: AF2, activation function 2; AR, androgen receptor; LBD, ligand binding domain; N/C, NH2-terminal and carboxyl-terminal; CHO, Chinese hamster ovary; MMTV, mouse mammary tumor virus; PCR, polymerase chain reaction; DHT, dihydrotestosterone; GST, glutathione S-transferase; TIF2, transcriptional mediator/intermediary factor 2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Spencer, T. E., Jenster, G., Burcin, M. M., Allis, C. D., Zhou, J., Mizzen, C. A., McKenna, N. J., Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1997) Nature 389, 194-198
2. Glass, C. K., Rose, D. W., and Rosenfeld, M. G. (1997) Cur. Opin. Cell Biol 9, 222-232
3. McKenna, N. J., Lanz, R. B., and O'Malley, B. W. (1999) Endocr. Rev. 20, 321-344
4. Webb, P., Nguyen, P., Shinsako, J., Anderson, C., Feng, W., Nguyen, M. P., Chen, D., Huang, S. M., Subramanian, S., McKinerney, E., Katzenellenbogen, B. S., Stallcup, M. R., and Kushner, P. J. (1998) Mol. Endocrinol. 12, 1605-1618
5. Chen, H., Lin, R. J., Schiltz, R. L., Chakravarti, D., Nash, A., Nagy, L., Privalsky, M. L., Nakatani, Y., and Evans, R. M. (1997) Cell 90, 569-580
6. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996) Cell 85, 403-414
7. Torchia, J., Glass, C., and Rosenfeld, M. G. (1998) Curr. Opin. Cell Biol. 10, 373-383
8. Fondell, J. D., Brunel, F., Hisatake, K., and Roeder, R. G. (1996) Mol. Cell. Biol. 16, 281-287
9. Naar, A. M., Beaurang, P. A., Zhou, S., Abraham, S., Solomon, W., and Tjian, R. (1999) Nature 398, 828-832
10. Rachez, C., Lemon, B. D., Suldan, Z., Bromleigh, V., Gamble, M., Naar, A. M., Erdjument-Bromage, H., Tempst, P., and Freedman, L. P. (1999) Nature 398, 824-828
11. Darimont, B. D., Wagner, R. L., Apriletti, J. W., Stallcup, M. R., Kushner, P. J., Baxter, J. D., Fletterick, R. J., and Yamamoto, K. R. (1998) Genes Dev. 12, 3343-3356
12. Nolte, R. T., Wisely, G. B., Westin, S., Cobb, J. E., Lambert, M. H., Kurokawa, R., Rosenfeld, M. G., Willson, T. M., Glass, C. K., and Milburn, M. V. (1998) Nature 395, 137-143
13. Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997) Nature 387, 733-736
14. Torchia, J., Rose, D. W., Inostroza, J., Kmei, Y., Westin, S., Glass, C., and Rosenfeld, M. (1997) Nature 382, 677-684
15. McInerney, E. M., Rose, D. W., Flynn, S. E., Westin, S., Mullen, T. M., Krones, A., Inostroza, J., Torchia, J., Nolte, R. T., Assa-Munt, N., Milburn, M. V., Glass, C. K., and Rosenfeld, M. G. (1998) Genes Dev. 12, 3357-3368
16. Voegel, J. J., Heine, M. J. S., Tini, M., Vivat, V., Chambon, P., and Gronemeyer, H. (1998) EMBO J. 17, 507-519
17. Bourguet, W., Ruff, M., Chambon, P., Gronemeyer, H., and Moras, D. (1995) Nature 375, 377-382
18. Feng, W., Ribeiro, R. C. J., Wagner, R. L., Nguyen, H., Apriletti, J. W., Fletterick, R. J., Baxter, J. D., Kushner, P. J., and West, B. L. (1998) Science 280, 1747-1749
19. Freedman, L. P. (1999) Cell 97, 5-8
20. Wurtz, J. M., Bourguet, W., Renaud, J. P., Vivat, V., Chambon, P., Moras, D., and Gronemeyer, H. (1996) Nat. Struct. Biol. 3, 87-94
21. Brzozowski, A. M., Pike, A. C. W., Dauter, Z., Hubbard, R. E., Bonn, T., Engstrom, O., Ohman, L., Greene, G. L., Gustafsson, J. A., and Carlquist, M. (1997) Nature 389, 753-758
22. Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937
23. Nagy, L., Kao, H. Y., Love, J. D., Li, C., Banayo, E., Gooch, J. T., Krishna, V., Chatterjee, K., Evans, R. M., and Schwabe, J. W. R. (1999) Genes Dev. 13, 3209-3216
24. Perissi, V., Staszewski, L. M., McInerney, E. M., Kurokawa, R., Krones, A., Rose, D. W., Lambert, M. H., Milburn, M. V., Glass, C. K., and Rosenfeld, M. G. (1999) Genes Dev. 13, 3198-3208
25. Hu, X., and Lazar, M. A. (1999) Nature 402, 93-96
26. He, B., Kemppainen, J. A., Voegel, J. J., Gronemeyer, H., and Wilson, E. M. (1999) J. Biol. Chem. 274, 37219-37225
27. Langley, E., Zhou, Z., and Wilson, E. M. (1995) J. Biol. Chem. 270, 29983-29990
28. Langley, E., Kemppainen, J. A., and Wilson, E. M. (1998) J. Biol. Chem. 273, 92-101
29. Lillie, J. W., and Green, M. R. (1989) Nature 338, 39-44
30. Kemppainen, J. A., Langley, E., Wong, C. I., Bobseine, K., Kelce, W. R., and Wilson, E. M. (1999) Mol. Endocrinol. 13, 440-445
31. Zhou, Z. X., Lane, M. V., Kemppainen, J. A., French, F. S., and Wilson, E. M. (1995) Mol. Endocrinol. 9, 208-218
32. Simental, J. A., Sar, M., Lane, M. V., French, F. S., and Wilson, E. M. (1991) J. Biol. Chem. 266, 510-518
33. Alen, P., Claessens, F., Verhoeven, G., Rombauts, W., and Peeters, B. (1999) Mol. Cell. Biol. 19, 6085-6097
34. Voegel, J. J., Heine, M. J. S., Zechel, C., Chambon, P., and Gronemeyer, H. (1996) EMBO J. 15, 3667-3675
35. Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X. Y., Sauter, G., Kallioniemi, O. P., Trent, J. M., and Meltzer, P. S. (1997) Science 277, 965-968
36. Tan, J. A., Hall, S. A., Hamil, K. G., Grossman, G., Petrusz, P., Liao, J., Shuai, K., and French, F. S. (2000) Mol. Endocrinol. 14, 14-26
37. De Bellis, A., Quigley, C. A., Marschke, K. B., El-Awady, M. K., Lane, M. V., Smith, E. P., Sar, M., Wilson, E. M., and French, F. S. (1994) J. Clin. Endocrinol. & Metab. 78, 513-522
38. Rennie, P. S., Bruchovsky, N., Leco, K. J., Sheppard, P. C., McQueen, S. A., Cheng, H., Snoek, R., Hamel, A., Bock, M. E., MacDonald, B. S., Nickel, B. E., Chang, C., Liao, S., Cattini, P. A., and Matusik, R. J. (1993) Mol. Endocrinol. 7, 23-36
39. Wong, C. I., Zhou, Z., Sar, M., and Wilson, E. M. (1993) J. Biol. Chem. 268, 19004-19012
40. Kraus, W. L., McInerney, E. M., and Katzenellenbogen, B. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12314-12318
41. Bevan, C. L., Hoare, S., Claessens, F., Heery, D. M., and Parker, M. G. (1999) Mol. Cell. Biol. 19, 8383-8392
42. McInerney, E. M., Tsai, M. J., O'Malley, B. W., and Katzenellenbogen, B. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10069-10073
43. Ikonen, T., Palvimo, J. J., and Janne, O. A. (1997) J. Biol. Chem. 272, 29821-29828
44. Tetel, M. J., Giangrande, P. H., Leonhardt, S. A., McDonnell, D. P., and Edwards, D. P. (1999) Mol. Endocrinol. 13, 910-924
45. Leers, J., Treuter, E., and Gustafsson, J. A. (1998) Mol. Cell. Biol. 18, 6001-6013
46. Glass, C. K., and Rosenfeld, M. G. (2000) Genes Dev. 14, 121-141
47. Müller, J. M., Isele, U., Metzger, E., Rempel, A., Moser, M., Pscherer, A., Breyer, T., Holubarsch, C., Buettner, R., and Schüle, R. (2000) EMBO J. 19, 359-369
48. Norris, J. D., Fan, D., Stallcup, M. R., and McDonnell, D. P. (1998) J. Biol. Chem. 273, 6679-6699
49. Gee, A. C., Carlson, K. E., Martini, P. G. V., Katzenellenbogen, B. S., and Katzenellenbogen, J. A. (1999) Mol. Endocrinol. 13, 1912-1923
50. Tetel, M. J., Jung, S., Carbajo, P., Ladtkow, T., Skafar, D. F., and Edwards, D. P. (1997) Mol. Endocrinol. 11, 1114-1128
51. Shao, D., Rangwala, S. M., Bailey, S. T., Krako, S. L., Reginato, M. J., and Lazar, M. A. (1998) Nature 396, 377-380
52. Ghadessy, F. J., Lim, J., Abdullah, A. A. R., Panet-Raymond, V., Choo, C. K., Lumbroso, R., Tut, T. G., Gottlieb, B., Pinsky, L., Trifiro, M. A., and Yong, E. L. (1999) J. Clin. Invest. 103, 1517-1525
53. Berrevoets, C. A., Doesburg, P., Steketee, K., Trapman, J., and Brinkmann, A. O. (1998) Mol. Endocrinol. 12, 1172-1183
54. Henttu, P. M. A., Kalkhoven, E., and Parker, M. G. (1997) Mol. Cell. Biol. 17, 1832-1839
55. Hong, H., Darimont, B. D., Ma, H., Yang, L., Yamamoto, K. R., and Stallcup, M. R. (1999) J. Biol. Chem. 274, 3496-3502
56. Chang, C. Y., Norris, J. D., Gron, H., Paige, L. A., Hamilton, P. T., Kenan, D. J., Fowlkes, D., and McDonnell, D. P. (1999) Mol. Cell. Biol. 19, 8226-8239
57. Mak, H. Y., Hoare, S., Henttu, P. M. A., and Parker, M. G. (1999) Mol. Cell. Biol. 19, 3895-3903
58. Choong, C. S., Kemppainen, J. A., and Wilson, E. M. (1998) J. Mol. Evol. 47, 334-342
59. Takeo, J., and Yamashita, S. (1999) J. Biol. Chem. 274, 5674-5680
60. Zhou, Z. X., Sar, M., Simental, J. A., Lane, M. V., and Wilson, E. M. (1994) J. Biol. Chem. 269, 13115-13123
61. Scheller, A., Hughes, E., Golden, K. L., and Robins, D. M. (1998) J. Biol. Chem. 273, 24216-24222

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