Ser-884 Adjacent to the LXXLL Motif of Coactivator TRBP Defines Selectivity for ERs and TRs

Lan Ko, Guemalli R. Cardona, Toshiharu Iwasaki, Kelli S. Bramlett, Thomas P. Burris and William W. Chin

Lilly Research Laboratories, Department of Gene Regulation, Bone and Inflammation Research, Eli Lilly & Co., Indianapolis, Indiana 46285

Address all correspondence and requests for reprints to: Dr. Lan Ko, Department of Gene Regulation, Bone & Inflammation Research, Lilly Corporate Center, Building 98/C, Drop Code 0434, Indianapolis, Indiana 46285. E-mail: kol{at}lilly.com


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand-dependent interaction of nuclear receptors and coactivators is a critical step in nuclear receptor-mediated transcriptional regulation. TR-binding protein (TRBP) interacts with nuclear receptors through a single LXXLL motif. Evidence suggested that the sequences flanking the LXXLL motif in a number of coactivators determine receptor selectivity. We performed mutagenesis studies at residues adjacent to the TRBP LXXLL motif and identified S884 of TRBP at the -3 position of the LXXLL motif as a key residue for receptor selectivity. Analysis of in vitro and in vivo receptor interactions with TRBP suggested that S884 allowed selective interactions for ERß, TR, and RXR vs. ER{alpha}. Transient transfection studies further confirmed that the LXXLL-binding affinity correlates with TRBP transcriptional activity. Consistent with the structural modeling, an E380G substitution within ER{alpha} altered the binding to TRBP mutants, demonstrating the direct contact between TRBP S884 and ER{alpha} E380, which is a residue that distinguishes receptor subclasses. Furthermore, S884 can be phosphorylated by MAPK in vitro, an event that significantly altered the binding of TRBP to ER and suggests a potential mechanism for regulatory interaction. As the differential recruitment of TRBP to ER{alpha} and ERß may rely on S884, our finding provides insight into estrogen signaling and may lead to the development of therapeutic receptor-selective peptide antagonists.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HORMONE-INDUCED GENE ACTIVATION mediated by nuclear receptors underlies many biological events such as cell proliferation, differentiation, reproduction, and development (1, 2, 3). Ligand-dependent interaction between nuclear receptors and their cofactors is a critical step required for gene activation. Previous biochemical and structural studies of many nuclear receptors indicate that the ligand-dependent interaction of the receptor with coactivators relies on the positioning of helix 12 located at the extreme carboxyl terminus of the conserved ligand-binding domain (LBD) (4, 5). Ligand binding induces a conformational change within the receptors that enables dissociation of corepressors and association with coactivators (6, 7, 8). A common LXXLL motif present in a number of coactivator molecules is necessary and sufficient to mediate the interactions between the coactivator and the nuclear receptor LBD (9, 10). The most extensively characterized LXXLL-containing coactivators are the three members of the SRC-1 family (11), SRC-1/NcoA-1 (12), TIF-2/GRIP-1/NcoA-2 (13, 14, 15), and pCIP/AIB-1/TRAM-1/ACTR/RAC-3 (16, 17, 18, 19, 20, 21). Other coactivators also possess LXXLL motifs and include CBP/p300 (22), PGC-1 (23, 24), CIA (25), RIP140 (26), and DRIP205/TRAP220/PBP (27, 28, 29). TRBP/ASC-2/RAP250/PRIP/NRC/AIB3 (30, 31, 32, 33, 34, 35) is another recently characterized coactivator that has a single functional LXXLL motif.

Crystal structures of the LBD of many nuclear receptors have been resolved (36, 37, 38, 39, 40, 41). The coactivator LXXLL motif binding site of ligand-bound ER{alpha} is a shallow hydrophobic groove on the surface of the LBD primarily formed by helices 3, 4, 5, and 12 (36). In antagonist-bound ER{alpha}, the coactivator-binding groove is obscured by unique positioning of helix 12. Alignment of helix 12 in a number of nuclear receptors with many coactivator LXXLL peptides revealed that the hydrophobic residues in helix 12 closely resemble the leucines in the LXXLL and appear to mimic functionally the LXXLL motif of the coactivator (37). Thus, helix 12 functions as an autoinhibitory peptide for antagonist-bound nuclear receptors.

It has been shown that the distinct LXXLL motifs display varying degrees of nuclear receptor selectivity, which appear to be specified by the amino acid residues immediately flanking the LXXLL motifs (42, 43, 44, 45, 46, 47). The biological significance of selective interactions between various nuclear receptors and coactivators is still largely unclear; however, selective interaction in vivo may play a role in tissue- and cell-specific effects (48). In this regard, characterization of the mechanism(s) of coactivator selectivity is important for understanding coactivator function in nuclear receptor-mediated gene regulation.

The coactivator TRBP has been shown to interact with a number of nuclear receptors in a ligand-dependent manner and enhance nuclear receptor-mediated transcriptional activation. TRBP is a high mol wt (2,063 amino acids) coactivator that associates with both CBP/p300 and the DRIP complexes (30, 31, 33). TRBP also coactivates multiple transcription factors including AP-1 and NF{kappa}B (30) and its gene has been demonstrated to be amplified in human breast cancers (31, 35). An RRM-containing coactivator CoAA was recently shown to interact with the carboxyl terminus of TRBP (49). In addition, the carboxyl terminus of TRBP has also been shown to interact with nuclear DNA-dependent protein kinase and poly (ADP-ribose) polymerase complexes that are implicated in transcriptional regulation (30). Interestingly, a single LXXLL motif is required for the interaction of TRBP with TR, RXR, ER, and many other receptors. However, the relative selectivity of these interactions between TRBP and these receptors has not been characterized. To define the residues flanking the TRBP LXXLL motif responsible for nuclear receptor selectivity, we performed random mutagenesis of specific amino acids at positions predicted to contact the surface of the nuclear receptor LBD. A mutation at S884 at the -3 position of the TRBP LXXLL motif was identified as a residue with the ability to alter TRBP’s selectivity for ER{alpha} vs. ERß both in vitro and in vivo. Mutations at this residue also dramatically increased TRBP binding to TR and RXR. We also show that the binding activities of TRBP mutants to nuclear receptors correlates with their transcriptional coactivation activities. Our results suggest that the interaction of nuclear receptors with TRBP can be selective and may be regulated.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of S884 in TRBP for Its Role in Receptor Binding Selectivity by Random Mutagenesis
We previously reported that the LXXLL motif of the TRBP coactivator is necessary and sufficient for the interaction with TRß in a strict ligand-dependent manner (30). In addition, this interaction is also completely AF-2 dependent. Several lines of evidence suggested that various LXXLL motifs in a number of coactivators display receptor selectivity in which the sequences flanking the LXXLL motif specify the degree of receptor selectivity (42, 43, 46). Many of these studies were conducted using small synthetic coactivator peptides for selective interaction with receptor LBD. We considered the possibility that a larger natural protein might be more physiologically relevant in this type of selection, since the global protein conformation may be influential.

To assess the importance of residues flanking the TRBP LXXLL motif in determining receptor selectivity, we performed random mutagenesis of TRBP (amino acids 714-1242) in which the -3, -4, +7, and +8 positions near the LXXLL motif were mutated. These four residues were chosen because the LXXLL motif is an {alpha}-helical structure in which the -3, -4, +7, and +8 positions are most likely aligned in the same orientation as the conserved hydrophobic leucines. Hence, they are likely to have direct contact with the receptor LBD. Degenerate PCR primers introduced mutations randomly at these four specific amino acid positions, and each mutated clone was screened for in vitro nuclear receptor binding activity.

As shown in Fig. 1AGo, 36 mutated TRBP (714–1,242) clones were screened for binding of the encoded proteins to the LBDs of TRß, RXR{alpha}, ER{alpha}, and ERß in the presence of their respective ligands. Compared with the wild-type control, most of the mutants displayed decreased binding, probably because of alteration of the natural coactivator protein conformation. By calculation, approximately 17% of the mutations would introduce stop codons and fail to produce full-length proteins. This was detected by the lack of an input band in the assay and was verified by sequencing of the mutations (Figs. 1Go and 2AGo). Our interest was primarily focused on the mutations that display increased binding activity. Among the positive clones, clone 6 had a surprisingly significant increase in binding to TR and RXR and also had marked increased binding to ERß but dramatically decreased binding to ER{alpha}. Interestingly, clone 33 had a similar pattern as clone 6, in that the binding to TRß, RXR{alpha}, and ERß was selectively higher than ER{alpha}, but to varying degrees. Sequence analysis of the clones revealed that a tyrosine residue at position -3 contributed to selectivity of TRBP binding of TRß, RXR{alpha}, and ERß vs. ER{alpha} (Fig. 2AGo). In particular, S884 at position -3 in clones 6 and 33 was replaced by a tyrosine. By contrast, most other mutants reduced TRBP-receptor interaction. Of note, clone 6 had the least number of alterations at other amino acid positions (S884Y and T883S), suggesting that the S884 position may significantly contribute to selective receptor binding.



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Figure 1. Randomly Mutated TRBP Clones Interact with TRß, RXR{alpha}, ER{alpha}, and ERß

A TRBP fragment (714–1242) containing the LXXLL motif was randomly mutated by PCR using degenerated primers that introduce mutations at amino acid residues -3, -4, +7, and +8 relative to the LXXLL motif. Thirty-six plasmids containing mutations were isolated, and the proteins were [35S]-methionine-labeled by in vitro translation. The GST-LBD fusion protein of each of the nuclear receptors (TRß, RXR, ER{alpha}, and ERß) was tested in pull-down assays in the presence of 1 µM of cognate ligand with the labeled TRBP proteins. Specific nuclear receptors are indicated on the left. Clones are numbered and shown at bottom of each panel. WT is wild-type TRBP protein. The input lane indicates 10% of the total input. A missing band indicates generation of a stop codon, which was verified by sequencing. The amount of GST protein used in assays for each receptor was carefully balanced.

 


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Figure 2. The S884 Residue of TRBP Displays an Important Role in Receptor Selectivity

A, Diagram of a TRBP LXXLL motif (882–895) is shown. Full-length TRBP with the LXXLL motif is depicted on top. The amino acids of the LXXLL motif are depicted in bold in both wild-type (WT) and mutant (Mut) TRBP. The bold X’s represent any amino acids at mutated -3, -4, +7, and +8 positions. Clones with mutated amino acids are shown; an asterisk indicates a stop codon. B, A computationally engineered TRBP peptide (882–895) shows that that the side chain of S884 (red) has the same orientation as the leucines (green) in the LXXLL motif. The side chain of S884 is near that of L887.

 
A TRBP peptide was computer generated to obtain detailed structural information of S884 at position -3 (Fig. 2BGo). Relative to the LXXLL motifs of other coactivators, the LXXLL motif of TRBP is unique. It contains a leucine at -1 position and a proline at the -2 position. Consistent with previous reports, these characteristics lead to greater binding to TRß (44, 45). As shown in Fig. 2BGo, the TRBP LXXLL (882–895) motif structure was adapted and computationally engineered based on the structure of the PPAR{gamma} AF-2 helix, which aligns with the coactivator LXXLL motifs and has a leucine at the -1 and a proline at the -2 positions (37). The orientation of the side chain of S884 in TRBP, due to Pro885, was found to align with an orientation similar to that of the other four hydrophobic leucines, L886, L887, L890, and L891 (Fig. 2AGo). The conformation of the TRBP LXXLL motif suggested that S884 might be involved in receptor binding, and this is consistent with our finding that mutation of S884 alters the binding of TRBP to the LBD.

S884Y Differentiates the Binding of TRBP with ER{alpha} and ERß, TRß, and RXR{alpha} in Vitro
To verify our data obtained in the random mutagenesis screen, the TRBP protein (714–1,242) with a S884Y mutation was tested in in vitro binding assays. Binding of fusion proteins consisting of glutathione-S-transferase (GST) fused to the LBDs of TRß, RXR{alpha}, ER{alpha}, and ERß to wild-type TRBP and the S884Y mutant were compared in the presence and absence of each cognate ligand (Fig. 3Go). Consistent with the observations from the screen, the S884Y mutant had a significant increase in binding to TRß, RXR, and ERß, but decreased binding to ER{alpha}. Notably, the increase was strictly ligand dependent for TRß and RXR, while S884Y increased ERß ligand-independent binding. These results confirm that the S884 position plays an essential role in determining receptor selectivity in vitro.



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Figure 3. Tyrosine Substitution of TRBP S884 Alters Nuclear Receptor Selectivity

Tyrosine mutation of S884 (S884Y) in TRBP fragment (714–1242) was compared with the wild-type (WT) for the binding of the nuclear receptor LBDs. GST-LBDs of TRß, RXR, ER{alpha}, and ERß were incubated with [35S]methionine-labeled TRBP and S884Y mutant in the presence and absence of 1 µM of each cognate ligand as indicated. The bound protein was quantitated, and results are shown as the mean values of triplicate determinations ± SE values.

 
Similar experiments were performed with ER{alpha} and ERß using various ER ligands including E2, diethylstilbestrol (DES), estriol, genestein, LY 117018, 4- hydroxytamoxifen, and ICI 182,780. The S884Y mutant of TRBP showed selective binding to ERß vs. ER{alpha}, although it displayed different profiles with each ligand (Fig. 4Go). In particular, the S884Y mutant favored estriol-bound ERß over E2- and DES-bound ERß. This selectivity for estriol was confirmed in transfection and peptide binding assays. Genestein has been previously shown to exhibit selectivity for ERß (50). The S884Y appeared to increase genestein’s selectivity for ERß over ER{alpha}, suggesting that the S884 residue may play a role in determining ER-coactivator complex responsiveness to various ligands. All together, these data further indicate that S884 plays an essential role in establishing TRBP-ER isoform selectivity.



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Figure 4. S884Y Increases TRBP Binding Affinity for ERß, But Not ER{alpha}, in Vitro

Similar experiments were done as in Fig. 3Go, except ER{alpha} and ERß were compared without (-) or with (+) different ER ligands: E2 (E2), DES, estriol (E3), genestein (Gen), LY 117018 (LY), 4-hydroxytamoxifen (OHT), and ICI 182,780 (ICI).

 
S884Y Mutation Differentiates the Binding of TRBP with ER{alpha} and ERß in Vivo
To confirm the effects of the S884Y mutation observed in vitro, we employed a mammalian two-hybrid assay using Gal4-TRBP (795–999) and ER{alpha}- or ERß LBD-VP16 fusions. The results suggest that the S884Y mutation leads to increased binding of TRBP with ERß and decreased binding with ER{alpha} (Fig. 5Go). However, the ligand-independent interaction with ERß was also significantly increased in the S884Y mutant. The S884E mutant, which has a negatively charged side chain, was not able to mimic the effect of the hydrophobic tyrosine and failed to interact. Alanine mutations (LA) of the conserved leucines in the LXXLL motif abolished TRBP interaction with the ER-LBD and was used as a negative control. These data indicate that the S884Y mutant was also able to display selective interaction with ER isoforms in a cellular content.



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Figure 5. The S884Y Mutation Increases TRBP Binding Affinity for ERß, But Not ER{alpha}, in Vivo

The mammalian two-hybrid system was used to detect protein-protein interactions in CV-1 cells. Plasmids encoding GAL-TRBP (WT) or its mutations at S884 (S884Y, S884E) were cotransfected with ER{alpha}-LBD-VP16 (top panel) or ERß-LBD-VP16 (bottom panel) together with the Gal-luciferase reporter. Experiments were performed without or with 100 nM E2 (E2) or estriol (E3). S884Y and S884E are tyrosine and glutamic acid substitutions at S884. LA represents alanine mutations at conserved leucines in the LXXLL motif. Relative luciferase activities were measured and shown as means of triplicate transfections ± SE values.

 
TRBP LXXLL Peptides with S884 Mutations Display Selectivity for ER{alpha} and ERß
Binding to ER{alpha} and ERß was further evaluated in the LXXLL motif peptide binding assay utilizing synthetic TRBP peptides (878–896) mimicking the different mutations at the S884 position. In this time-resolved fluorescence assay, synthetic TRBP LXXLL peptides are fluorescent labeled and then assessed for their ability to bind to recombinant ER{alpha} and ERß. Consistent with our previous data, the S884Y mutation significantly increased the interaction with ERß but not ER{alpha} (Fig. 6Go). A negative charge at S884 (S884E) abolished binding of TRBP to both ER{alpha} and ERß. A positive charge at the same position (S884R) did not decrease, but rather increased, TRBP peptide binding to ER. This effect was particularly apparent for ER{alpha}. As expected, a peptide harboring alanine mutations (LA) of the conserved leucines was not able to bind. In agreement with previous binding assays using large TRBP fragments, these findings using synthetic peptides suggest that the S884 of TRBP is an important residue responsible for the selective interactions with ER isoforms. The binding activity within the peptide-binding assay may not be entirely consistent with the activity of the large TRBP protein fragments. In fact, variations of efficacy and affinity profiles for each receptor were observed using the various assays. However, S884 was consistently demonstrated, both in in vitro and in vivo assays, to play an important role in ER{alpha} vs. ERß selectivity. These results indicate that the conformation of the side chain of S884 may contribute to the binding affinities of TRBP for ER{alpha} and ERß.



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Figure 6. Peptide Binding Analysis of S884 Mutations for Altered ER Selectivity

Synthetic TRBP peptides (878–896) and mutations at S884 were assayed with full-length baculovirus-expressed ER{alpha} (top panel) and ERß (bottom panel) using a time-resolved fluorescence assay as described in Materials and Methods. Experiments were performed with various concentrations of E2. Arginine, tyrosine, and glutamic acid mutations at S884 are indicated. LA represents TRBP with alanine mutations at Leu890 and Leu891, which was used as a negative control.

 
Transcriptional Activity of S884Y TRBP Correlates with Its Receptor Binding Affinity
The S884Y mutation of TRBP selectively altered the binding to nuclear receptors. This prompted us to examine whether the S884Y mutant would also affect the transcriptional activity of TRBP. Since the C- terminal region of TRBP possesses the major transcriptional activation domain, the S884Y mutant of the TRBP C-terminal region (714–2063) was constructed and compared with the wild-type TRBP in terms of their transcriptional activities. In transient transfection assays, TRBP S884Y increased ligand-dependent ERß-mediated transcription and decreased ligand- dependent ER{alpha}-mediated transcription (Fig. 7Go, A and B). Consistent with our data suggesting that this mutation increased ligand-independent interaction with ERß, the ligand-independent transactivation mediated by ERß was also increased. In the case of TRß, both thyroid hormone response element (F2)-reporter activities mediated by TRß1 and Gal-reporter activity mediated by the Gal4-TRß-LBD fusion protein were tested. In both cases, the ligand-dependent activity was increased with the TRBP S884Y mutant (Fig. 7Go, C and D). Taken together, these results show that S884 affects the selectivity of TRBP for nuclear receptor interaction, as well as its coactivation potential.



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Figure 7. Transcriptional Activity of the Mutant TRBP at S884 Correlates with Its Binding Activity

A and B, Coactivation functions of wild-type TRBP (714–2063)(WT), S884Y mutant (S884Y), and vector control (V) were tested with 2XERE luciferase reporters. CV-1 cells were transfected in 24-well plates with 10 ng full-length ER{alpha} or ERß, 200 ng TRBP or mutant, and 100 ng luciferase reporter. Cells were treated with or without 100 nM estriol (E3). Luciferase activity were measured and data were shown as means of triplicate transfections ± SE values. C, Similar transfection as those in panels A and B were performed except that the full-length TRß1 (10 ng) and F2/TRE luciferase reporter (100 ng) were used, and 100 nM T3 was used as ligand. D, A Gal-TRß-LBD fusion plasmid (100 ng) and 5XGAL-luc reporter (100 ng) were used for transfection together with 200 ng TRBP, mutant, or vector control. Other conditions were the same as in panel C.

 
S884 Has Close Contact with Helix 4 of ER{alpha} at Glu380
Using computer modeling of the crystal structure of ER{alpha}, ERß, and TRß with the TRBP LXXLL peptide, we obtained further structural evidence for the role of the S884 position in determining the interaction with the nuclear receptor LBD. The structure of the TRBP LXXLL (882–895) motif was engineered based on the PPAR{gamma} helix 12 (Fig. 2BGo). The engineered TRBP peptide docking was carried out by three-dimensional alignment with the TRBP peptide and the GRIP-1 peptide cocrystallized with ER{alpha} (36). The backbone and the leucine residues within the LXXLL motif of GRIP-1 were aligned with the backbone and the leucines within the TRBP LXXLL peptide. Side chain orientations were adapted from PPAR{gamma} helix 12 (40). As a result, the L887, L890, and L891 in TRBP LXXLL motif were found to insert into the hydrophobic coactivator-binding pocket of ER{alpha} between the charged clamp flanked by the conserved K362 in helix 3 and E542 in helix 12 of ER{alpha} (Fig. 8AGo). The L886 at the -1 position of the TRBP LXXLL motif also directly contacted the hydrophobic regions on the ER{alpha} surface. Importantly, the structure revealed that Ser884 of TRBP points toward the receptor as it had a similar orientation as the leucine residues (Fig. 2BGo). In addition, TRBP S884 may have close contact with E380 in helix 4 of ER{alpha}. This indicated that E380 of ER{alpha} might influence TRBP binding via the S884 residue. Interestingly, the surfaces around S884 in ERß and TRß are much different than in ER{alpha} (Fig. 8Go, B and C) (38, 39). There are more open spaces and the environment has more hydrophobic character. This may explain why the S884Y mutation resulted in a higher binding activity of the TRBP peptide for TRß and ERß than for ER{alpha}. Based on the model, it was also understandable why the negatively charged glutamic acid residue, but not positively charged arginine residue, at the TRBP S884 position completely abolished binding for both ER{alpha} and ERß (Fig. 6Go). Furthermore, when E380 of ER{alpha} was aligned with a number of nuclear receptors, amino acids at the E380 position of ER{alpha} showed conservation within nuclear receptor subclasses (Fig. 8CGo). Class I receptors including AR, GR, MR, and PR have glutamines (Q) at this position while ER{alpha} and ERß have glutamic acids (E), and class II receptors have lysine (K) or arginines (R). It is also noteworthy that the hydrophobic character of the coactivator binding pocket is conserved except for this charged residue within helix 4. This indicated that each class of receptor might share a degree of conformational similarity around this position, and that different coactivators might be able to distinguish among various receptor subclasses.



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Figure 8. The S884 of TRBP Has Close Contact with ER{alpha} E380

A, The engineered coactivator TRBP peptide bound to ER{alpha}-LBD is shown in a molecular surface view. Coordinates of DES/ER{alpha}-LBD are from 3ERD. The side chains of Leu886, Leu887, Leu890, and Leu891 of TRBP are shown in green, and the side chain of S884 is shown in red. The negatively charged (red) E380 in helix 4, E542 in helix 12, and positively charged (blue) K362 in helix 3 of ER{alpha} are also indicated. B, The surface view of ERß-LBD (1QKM) with TRBP peptide is shown. Side chains of residues in TRBP are labeled in an identical manner as above. The negatively charged (red) E332 in helix 4 and positively charged (blue) K314 in helix 3 of ERß are indicated. Helix 12 of ERß is not shown. C, A similar surface view with TRß-LBD is shown. TRß (1BSX) structure was obtained from the Protein Data Bank. The K306 (blue) in helix 4, conserved E457 (red) in helix 12, and K288 (blue) in helix 3 of TRß are indicated. D, Amino acid alignment of nuclear receptors within LBD helices 4 and 5. The numbers indicate the amino acids of ER{alpha}, ERß, and TRß. The aromatic amino acids are shown in brown, prolines in green, threonines and serines in magenta, positively charged residues in dark blue, negatively charged residues in red, cystines and methionines in yellow, glutamines and asparagine in light blue, and hydrophobic or small residues in gray. The conserved lysines in helix 3 (ER{alpha}/K362, ERß/K314, and TRß/K288) and aligned ER{alpha}/E380, ERß/E332, and TRßK306 in helix 4 are indicated by arrows.

 
To test the hypothesis that TRBP S884 might closely interact with this charged residue on helix 4, a mutation of ER{alpha} at E380 residue was generated with the substitution of a glycine residue (E380G). The ER{alpha} E380G was compared with the wild-type ER{alpha} and ERß for its binding with a number of TRBP S884 mutants (714–1242), in which the S884 is changed to a variety of residues including aromatic, positive, and negative charged residues (S884E, S884F, S884H, S884K, S884L, S884P, S884Q, S884W, S884Y). As shown in Fig. 9Go, the S884Y and S884F mutants had increased ERß binding, but decreased ER{alpha} binding relative to wild-type (S884), suggesting that the aromatic side chains of phenylalanine (F) and tyrosine (Y) are responsible for this selectivity. A mutant bearing tryptophan (W), which is also aromatic but maybe too bulky in its side chain structure, bound poorly to both ER{alpha} and ERß. However, when the interaction of the ER{alpha} E380G mutant with the TRBP S884 mutants was compared, all three aromatic residues including tryptophan at S884 resulted in a dramatic increase in their binding activities, indicating that the deletion of the E380 side chain was primarily responsible for the increased interactions of the aromatic residues. These results strongly suggest that the 884 in TRBP and 380 in ER{alpha} may be in close contact. Hydrophobic leucine (L) and acidic glutamine (Q) at 884 had much less binding activity than the aromatic residue mutants (Fig. 9Go). Positively charged histidine (H) and lysine (K) in that position in TRBP results in different binding activities to ER{alpha} and ERß. However, E380G of ER{alpha} did not improve binding of either, indicating that the aromatic structure, rather than charge, may play a major role. A proline (P) at 884 completely abolished the binding. Inasmuch as the 885 position of TRBP is already a proline in the wild-type protein, another proline immediately next to it may produce a TRBP peptide that is too rigid to accommodate an induced fit binding. As expected, the glutamate mutation (S884E) abolished binding, most likely due to the proximity of other negatively charged residues nearby on the surface of the LBD. The precise contact of TRBP S884 with receptors remains to be established. However, it is clear that each receptor has a unique conformation surrounding the coactivator binding pocket. The S884 of TRBP undoubtedly influences the binding selectivity for each of the receptors.



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Figure 9. TRBP Mutants Interact with ER{alpha}, ER{alpha} E380G, and ERß

A TRBP fragment (714–1242) containing the LXXLL motif was mutated by PCR using degenerated primers so that S884 was replaced by a glutamic acid (E), phenylalanine (F), histidine (H), lysine (K), leucine (L), proline (P), glutamine (Q), tryptophan (W), or tyrosine (Y). The mutant proteins were [35S]-methionine-labeled by in vitro translation. The GST-LBD fusion protein of each of the nuclear receptors (ER{alpha}, ER{alpha} E380G, and ERß) was tested in the pull-down assays in the presence of 1 µM E2 with the labeled TRBP wild-type or mutant proteins. Specific nuclear receptors are indicated on the left. TRBP mutants were labeled and shown at the bottom of each panel. TRBP S884 is the wild-type protein. The input lane indicates 20% of the total input. The amount of GST protein used in the assays for each receptor was carefully balanced. These samples were quantitated by scintillation counter, and relative counts are shown as a bar graph in the bottom panel.

 
S884 of TRBP Can Be Phosphorylated in Vitro by MAPK
The S884 is a proline-directed serine, which is predicted to be a MAPK phosphorylation site. In vitro phosphorylation studies with His-tagged TRBP (795–931) suggested that S884 can be phosphorylated by MAPK (ERK2) in vitro (Fig. 10AGo). Alanine mutation at S884 significantly reduced, but did not completely abolish, the phosphorylation, indicating S884 is a major phosphorylation site for MAPK within this 137-amino acid LXXLL-containing fragment of TRBP. To evaluate the binding by a phosphorylated TRBP to ER{alpha} and ERß, a synthetic phosphopeptide bearing a phos-S884 was tested. The results suggested that phosphorylation at S884 of TRBP dramatically reduced ligand-dependent TRBP interactions with both ER{alpha} and ERß when compared with the wild-type unphosphorylated form, although there was a significant increase of interactions with the unliganded receptor (Fig. 10BGo). It is currently unknown whether S884 can be phosphorylated in vivo. However, gene-specific transcriptional activation, at least in part, may be regulated by phosphorylation. The phosphorylation at S884 of TRBP is able to modulate receptor binding in vitro, suggesting the possible regulatory interactions between coactivator and nuclear receptors, if such phosphorylation events occur in vivo.



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Figure 10. In Vitro Phosphorylation of TRBP at S884

A, Recombinant his-tagged TRBP (795–931) was phosphorylated in vitro by MAPK as described in Materials and Methods. After washing, phosphorylated wild-type or S884A mutant proteins were resolved on SDS-PAGE and detected by autoradiography. Coomassie staining of His-tagged proteins is shown below to confirm that equal amounts of protein were used. B, Phosphorylation of S884 altered binding of TRBP to ER{alpha} and ERß. Biotin-labeled TRBP phospho-S884 (pS884) peptide was compared with the wild-type peptide for the binding of ER{alpha} and ERß in the presence or absence of E2 (1 µM). The bound peptides were quantitated with streptavidin-horseradish peroxidase and chemiluminescence detection reagents.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The coactivator TRBP interacts with nuclear receptors through its single LXXLL motif in a ligand- as well as AF-2-dependent manner (30, 31, 32, 33, 34). In this study, we identified the S884 residue at the -3 position of the TRBP LXXLL motif as a critical residue for nuclear receptor discrimination. A single S884Y mutation significantly increased binding to TR, RXR, and ERß and markedly reduced binding to ER{alpha}. In addition, we show that the S884Y mutant not only differentially interacted with ERß vs. ER{alpha} both in vitro and in vivo, but also displayed functional differences in ER-mediated transactivation in cells.

The interaction of the LXXLL motif with the nuclear receptor LBD is well documented. Early mutagenesis studies established the absolute requirement of the three leucines of the LXXLL motif as well as an intact AF-2 domain for functional interaction of the coactivator with the nuclear receptor (1, 9, 10). Subsequent detailed analysis, including peptide library screens of the LXXLL motif, revealed that each nuclear receptor has a distinct preference for the amino acid sequences adjacent to the motif (42, 43, 44, 45, 46, 47). Interestingly, many LXXLL peptides with hydrophobic residues at the -1 position, resulting in a {Phi}LXXLL motif, had higher binding affinity to nuclear receptors such as TR and ERß (44, 45). This character is also found in the majority of naturally existing LXXLL-containing coactivators, which includes most of the motifs of the three members of the SRC-1 family, both motifs of TRAP220, a number of motifs in RIP140, and the single motif in both PGC-1 and TRBP. More interestingly, a proline at the -2 position (P{Phi}LXXLL) confers the highest selectivity for TRß in a peptide library screen (45). This P{Phi}LXXLL motif is found naturally in both TRBP and TRAP220 and, interestingly, both of these coactivators were initially identified with high affinity for TR.

Several points were considered for the rationale to identify the amino acids near the LXXLL motif for residues that may be important for receptor selectivity. First, small synthetic coactivator peptides can sometimes yield high binding affinity, but may not reflect physiological situations in which binding may be regulated and optimal for in vivo function. We used a natural coactivator TRBP fragment (528 amino acids) to overcome this limitation. Our data also suggested that an even longer TRBP fragment (1,350 amino acids, Fig. 7Go) was also able to retain the selectivity. Second, amino acid residues immediately adjacent to the LXXLL motif were selected for mutation since they are likely to have close contact with the receptor and thus may be important for selectivity. Since the LXXLL motif is {alpha}-helical in structure, the -3, -4, +7, and +8 positions are considered to be the most immediate residues next to the conserved leucines that are likely to contact the LBD surface. Because a PCR method can be used for convenient introduction of mutations, we undertook this approach in which mutations were randomly generated only at these four positions in a 528-amino acid TRBP fragment containing the LXXLL motif. Among 64 (43) possible nucleotide combinations for each amino acid, three will be stop codons (TAA, TGA, TAG). In theory, 17.4% (1 - (64 - 3)4)/644) of the mutations will be stop codons, and a minimum of 22 would need to be screened to have each residue appear at least once, by chance, at each of the four positions.

It was expected that most mutations near the motif would reduce the binding to the receptor since it was most likely that alteration of the natural structure of the protein would result in deleterious effects on function. The results did confirm that the majority of the clones displayed less binding than wild type (Fig. 1Go). However, the limited mutants displaying increased binding provided an opportunity to identify the residues that may be important. Among these mutants, clones 6 and 33 had a significant increase in binding to TR and RXR. Notably, these two clones also selectively favored interaction with ERß over ER{alpha}. Comparison of these clones suggested that aromatic residues at the -3 position might correlate with the ERß vs. ER{alpha} selectivity, and positive residues at the -4 position (clone 12 and 14) might display selectivity for TRß vs. RXR. On the other hand, hydrophobic residues at +8 position in clone 11,19, 25, 26, and 27 almost abolished TR, RXR, and ERß binding, but not ER{alpha} binding. These data indicated that the structural determinants on the surface of TR, RXR, and ERß might share similarities that differ from ER{alpha}.

Crystallographic studies with several nuclear receptors suggested that most of the residues comprising the coactivator-binding groove are conserved among receptors. They are nonpolar except for two highly conserved charged residues, which form the charge clamp. A glutamic acid residue within helix 12 and an lysine residue in helix 3 have been demonstrated to be indispensable for LXXLL peptide binding since mutations at either of these sites abolishes coactivator-peptide interactions (50). Our modeling studies revealed a residue near this region, corresponding to E380 in ER{alpha}, while variable among members of the entire nuclear receptor superfamily, is conserved within receptor subfamilies. The steroid receptors, including AR, GR, MR, and PR, contain a glutamine residue at this position, whereas ER{alpha} and ERß contain a glutamic acid residue. The class II receptors have either lysine or arginines at this position (Fig. 8DGo). Because the -2 position of the TRBP LXXLL motif is a proline, which breaks the helix and results in the side chain of S884 to be directed toward helix 4 of the ER{alpha} LBD, it was not surprising that S884 is a critical residue when the peptide structure of the TRBP LXXLL motif was modeled with the ER{alpha} crystal structure. This structural model prompted us to predict, and further demonstrate, that the E380 residue closely interacts with S884 of TRBP. In the case of TRß, RXR, and ERß, this location is relatively open and composed of mostly hydrophobic residues. This may explain why a tyrosine substitution at the -3 position increased TRBP binding to TRß, RXR, and ERß but not to ER{alpha}. Because the side chain of the S884 residue of TRBP is predicted to be in close proximity to this variable site in the coactivator binding region of the nuclear receptor, it is likely that the unique chemical character, as well as the relative spacing displayed by the variable nuclear receptor residue, may play a role in TRBP selectivity and discrimination between various nuclear receptor subfamilies. Interestingly, the S884 residue can be phosphorylated by MAPK in vitro (Fig. 10AGo), and phosphorylation alters the interaction of TRBP to ER{alpha} and ERß (Fig. 10BGo). These data indicate that TRBP, when posttranslationally modified, may exhibit differential interactions with various nuclear receptors.

Characterization of the unique requirements for nuclear receptor-specific coactivator binding may be useful for the design of peptide antagonists that selectively block coactivator binding. This may be useful for the development of ER isoform-selective antagonists. The treatment and prevention of ER-related diseases and disorders among tissues such as breast, bone, urogenital system, cardiovascular system, and central nervous system rely on our understanding of ER- mediated transcriptional regulation (48, 52). ER{alpha} and ERß have unique tissue distributions as well as distinct biological functions. ER{alpha} is predominantly expressed in many tissues including the uterus, mammary gland, bone, brain, liver, heart, kidney, and pituitary, while ERß mRNA is significant in ovary and prostate (48, 51). Knockout studies have revealed that phenotype differences exist between ER{alpha}- and ERß-deficient animals, such as breast tissue development (48). Consistent with the observation that ER modulator-induced conformation changes on ER{alpha} and ERß are distinct (43, 53, 54), our work provides further evidence that coactivators might differentially interact with the two receptors. Thus, a better understanding of ER action, including the selective activation of ER{alpha} and ERß, may be helpful in the development of such therapeutic agents.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials and Plasmid Vectors
The ligands for ER including E2, DES, estriol, genestein, LY 117018 (a raloxifene analog), and 4-hydroxytamoxifen were purchased from Sigma (St. Louis, MO). ICI 182,780 was purchased from Tocris (Ballwin, MO). Full-length baculovirous-expressed human ER{alpha} and ERß was purchased from Pan Vera (Madison, WI). The N-terminal biotin-labeled TRBP peptides (878–896) with mutations at S884 were synthesized by Sigma-Genosys (Woodlands, TX). The human TRBP plasmid used in this study was previously described (30). The TRBP fragment (714–1242) containing the LXXLL motif was subcloned into pcDNA3 and used for in vitro translation. Nuclear receptor LBDs were inserted in frame into pGEX 4T-2 (Amersham Pharmacia Biotech, Piscataway, NJ) to produce the glutathione S-transferase (GST)-LBD fusion proteins (TRß, RXR, ER{alpha}, ER{alpha} E380G, and ERß). Full-length human TRß1, ER{alpha}, and ERß were subcloned into pcDNA3 (Invitrogen, San Diego, CA). F2/TRE and 2XERE luciferase reporters were previously described (30). The 5XGal-luciferase reporter pFR-luc was from Stratagene (La Jolla, CA). Mammalian two-hybrid plasmid vectors pM (Gal DNA-binding) and pVP16 were from CLONTECH Laboratories, Inc. (Palo Alto, CA). GAL-TRBP (795–999) mutants S884Y and S884E were produced by PCR and inserted in frame at the C terminus of GAL4 DNA-binding domain in the pM vector. The GAL-TRBP (795–999) alanine mutant (LA) was constructed by mutating the N889, L890, and L891 residues of TRBP to alanine.

Random Mutagenesis of the TRBP LXXLL Motif and Binding Analysis of Individual Clones
Mutations were randomly introduced at amino acid positions -3, -4, +7, and +8 of the TRBP LXXLL motif (LTSPLLVNLLQSDI) using PCR. The underlined amino acids indicate the positions of the residues that were randomly mutated. Two degenerate primers were synthesized and used for PCR, producing random mutations at designated locations within a 528-amino acid TRBP fragment (714–1242). The primers used were P1: TGTTGGTCAACTTATTGCAGNNNNNNATATCTGCAGGCCAT; and P2: AATAAGTTGACCAACAATGGNNNNNNTAGCGTGACATCCTT. PCR products that contained a mixture of all of the different mutations were subcloned into the pcDNA vector under the control of the bacterial T7 promoter. The plasmid DNA clones for each mutation clones were separated and isolated by plasmid minipreps, and individually in vitro translated and [35S]methionine labeled. The labeled TRBP proteins carrying a variety of mutations were incubated with GST-LBD of TRß, RXR, ER{alpha} and ERß in a binding assay. The binding assays were performed at room temperature for 1 h in binding buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% Triton X-100, 10% glycerol, 1 mM dithiothreitol). Bound proteins were washed three times with binding buffer and subjected to SDS-PAGE followed by autoradiography. Equal amounts of the GST-LBD proteins within each receptor group were used. The signal strength was optimized with the exposure time of film to obtain the best contrast within each receptor groups.

Recombinant Protein Binding Assays
The GST fusion nuclear receptor LBDs were produced in Escherichia coli BL21(DE3) and purified with glutathione-Sepharose resin (Amersham Pharmacia Biotech). In vitro binding assays were performed by incubating GST-LBD resin (15 µl, 2–4 µg) and [35S]methionine-labeled, in vitro translated TRBP mutant proteins (5 µl) produced by rabbit reticulocyte lysate (Promega Corp., Madison, WI). Proteins were incubated at room temperature for 1 h in binding buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% Triton X-100, 10% glycerol, 1 mM dithiothreitol). Bound proteins were washed three times with binding buffer and subjected to SDS-PAGE followed by autoradiography or quantitation using a scintillation counter. For the phosphopeptide binding assay, GST-LBD resins (15 µl, 2–4 µg) were incubated with the biotin-labeled peptides (3 µM, WT: KDVTLTSPLLVNLLQSDIS; pS884: KDVTLTpSPLLVNLLQSDIS) in the presence or absence of E2 (1 µM) and washed as described above. The GST-LBD beads were incubated with streptavidin-peroxidase (1 U/ml, Roche Molecular Biochemicals, Indianapolis, IN) and washed three times. The beads were then transferred to the 96-well plate, and bound peptides were detected by chemiluminescence with ECL detection reagents (Amersham Pharmacia Biotech).

Peptide Binding Assays
The details of methods used in the peptide binding assay was as previously described (55). Briefly, white FluoroNunc 96-well plates were coated overnight with 4 pmol/well of baculovirous-expressed full-length human ER{alpha} or ERß. The receptor-coated plates were blocked with 7.5% BSA in blocking buffer TBS (0.1 M Tris-HCl, 0.15 M NaCl, 20 µM diethylenetriamine-pentaacetic acid) at room temperature for 1 h. The plates were then washed three times with 0.1% Triton X-100 in TBS (TBST). Biotin-labeled TRBP peptides (6.6 nM) carrying mutations at S884 were preincubated with europium-conjugated streptavidin (0.83 µg/ml) on ice for 30 min. Peptide-europium conjugates (120 µl) were diluted with DELFIA assay buffer (Perkin-Elmer Corp., Gaithersburg, MD) into 10 ml and applied to the 96-well plate with 90 µl/well. E2 was then added at appropriate concentrations to each well, and plates were incubated for 1.5 h at room temperature followed by five washes with TBST. The signal was detected by adding 100 µl of DELFIA Enhancement Solution (Perkin-Elmer Corp.) with 5 min of gentle shaking followed by analysis using a Wallac, Inc. Victor II plate reader. The peptides used in this assay are as follows. WT: KDVTLTSPLLVNLLQSDIS; S884R: KDVTLTRPLLVNLLQSDIS; S884Y: KDVTLTYPLLVNLLQSDIS; S884E: KDVTLTEPLLVNLLQSDIS; LA: KDVTLTEPLLVNAAQSDIS.

Cell Culture and Transient Transfection
CV-1 cells were maintained in DMEM supplemented with 10% FBS and 0.1 µg/µl penicillin/streptomycin in 5% CO2 at 37 C. Cells were plated in 24-well plates for 2 d before transfection. CV-1 cells were transfected using lipofect- AMINE reagent according to the manufacturer’s protocol (Life Technologies, Inc., Gaithersburg, MD). Cells were incubated in fresh serum-free medium containing 100 µM of ligand for 16–24 h after transfection. Total amounts of DNA for each well were balanced by adding vector DNA pcDNA3 (Invitrogen). Relative luciferase activities were measured and shown as means of triplicate transfections ± SE values.

Crystal Structure Modeling with TRBP Peptide
TRBP peptide structure (882–895, LTSPLLVNLLQSDI) was computationally engineered based on the backbone of the PPAR{gamma} AF-2 helix (SLHPLLQEIYKLDL) (2PRG) (37). The AF-2 domains were shown to contact the LXXLL-binding pocket and structurally overlap with the LXXLL helices (1, 10, 36). In addition, the AF-2 helices in a number of receptors align with the coactivator LXXLL motifs (37). Since the PPAR{gamma} AF-2 has a leucine at the -1 and a proline at the -2 positions, the backbone of PPAR{gamma} AF-2 resembles the backbone of TRBP LXXLL peptide near the -3 position. Amino acid side chains were computationally engineered using the QUANTA program. The three hydrophobic leucines of the engineered TRBP peptide were aligned with the three leucines of GRIP-1 peptide in the three-dimensional crystal structure of ER{alpha} or TRß. In the case of ERß, the leucines of the TRBP LXXLL were aligned with the corresponding hydrophobic residues of helix 12 in ERß. Coordinates of ER{alpha}/DES/GRIP-1 (3ERD) (36), ERß/genestein (1QKM), and TRß/GRIP-1 (1BSX) were obtained from the Protein Data Bank (38, 39).

In Vitro Kinase Phosphorylation
In vitro phosphorylation of recombinant His-tagged TRBP (795–931) by MAPK was assayed using the SigmaTECT Protein Kinase Assay buffer system from Promega Corp. with modifications. Briefly, MAPK (ERK2, New England Biolabs, Inc., Beverly, MA) was incubated in supplied buffer system with 32P-{gamma}-ATP and His-tagged wild-type TRBP (795–931) or TRBP S884A (795–931) as substrates. Labeled His-tagged protein beads were washed, and the proteins were resolved by SDS-PAGE followed by autoradiography.


    ACKNOWLEDGMENTS
 
We thank Gary Krishnan for the human ERß plasmid, Faming Zhang for crystal structure modeling, and Andrew G. Geiser for editing of the manuscript.


    FOOTNOTES
 
This work was supported by Lilly Postdoctoral Research Fellowships.

Abbreviations: ACTR, Activator of thyroid receptor; AF-2, activation function 2; AIB, amplified in breast cancer; ASC-2, activating signal cointegrator-2; CBP, cAMP response element binding protein binding protein; CIA, coactivator independent of AF-2 function; CoAA, coactivator activator; DES, diethylstilbestrol; DRIP, vitamin D receptor-interacting protein; GRIP, GR-interacting protein; GST, glutathione-S-transferase; LBD, ligand-binding domain; NcoA, nuclear receptor coactivator; NRC, nuclear receptor cointegrator; PBP, PPAR {gamma} binding protein; PGC-1, PPAR {gamma} coactivator-1; RAC-3, receptor associate coactivator 3; RRM, RNA recognition motif; SRC, steroid receptor coactivator; TIF-2, transcriptional intermediary factor 2; TRAP, TR associate protein; TRBP, TR binding protein.

Received for publication May 17, 2001. Accepted for publication September 14, 2001.


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