Three Amino Acids Specify Coactivator Choice By Retinoid X Receptors

Gang Shao1, Richard A. Heyman1 and Ira G. Schulman1

Nuclear Receptor Discovery Ligand Pharmaceuticals, Inc. San Diego, California 92121


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Binding of agonists to nuclear receptors results in a conformational change in receptor structure that promotes interaction between activated receptors and coactivators. Receptor-coactivator interactions are mediated by the agonist-dependent formation of a hydrophobic pocket on the part of receptors, and short leucine-rich sequences termed LxxLL motifs or nuclear receptor boxes present in coactivators. RXR-PPAR{gamma} (retinoid X receptor-peroxisome proliferator-activated receptor-{gamma}) heterodimers play important roles in adipocyte and macrophage differentiation and have been implicated as therapeutic targets in diabetes, atherosclerosis, and cancer. Analysis of interactions between RXR-PPAR{gamma} heterodimers and coactivator nuclear receptor boxes suggests that RXR and PPAR{gamma} can distinguish among coactivators by recognizing distinct structural features of nuclear receptor boxes. The results also indicate that coactivator choice by RXR is mediated by three nonconserved amino acids of the nuclear receptor box. The ability of an optimized seven-amino acid nuclear receptor box to specifically interact with RXR and function as a selective inhibitor suggests the coactivator-binding pocket may serve as a new target for drug discovery.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors are hormone-dependent transcription factors that play essential roles in development, differentiation, cell proliferation, and metabolism by controlling the expression of complex genetic networks. Members of the nuclear receptor superfamily are characterized by a conserved structural and functional organization consisting of a heterogeneous amino-terminal domain, a highly conserved central DNA-binding domain, and a relatively large carboxy-terminal ligand-binding domain (LBD). The LBD is functionally complex and mediates ligand binding, receptor homo- and heterodimerization, repression of transcription in the absence of ligand, and ligand-dependent activation of transcription (1).

Crystal structures of several LBDs support molecular and biochemical studies indicating that ligand binding promotes a conformational change in receptor structure. What appears to be a relatively flexible conserved helix near the carboxy terminus (helix 12) occupies unique positions when structures of unliganded, agonist-occupied, and antagonist-occupied LBDs are compared. Importantly, mutagenesis experiments indicate that helix 12, referred to as activation function 2 (AF-2), is necessary for ligand-dependent transactivation by nuclear receptors (2, 3, 4, 5, 6). Recent work indicates the AF-2 helix contributes an essential surface to the formation of an agonist-dependent hydrophobic pocket that serves as a binding site for coactivators. The alternative positions occupied by the AF-2 helix in the unliganded or antagonist-occupied conformations preclude the formation of this binding pocket (2, 5, 6, 7, 8).

Three major classes of coactivators have been studied in the context of ligand-dependent transcription by nuclear receptors. CREB binding protein (CBP)/p300 and the steroid receptor coactivators (SRCs; also referred to as p160 proteins) were identified using two-hybrid and in vitro protein-protein interaction assays and function as coactivators in transfection and microinjection experiments (9, 10, 11, 12, 13, 14). The thyroid receptor activating proteins (TRAPs)/vitamin D interacting proteins (DRIPs) represent identical protein complexes that were purified based upon their ability to interact with thyroid hormone or vitamin D receptors and promote ligand-dependent transcription in vitro (for review see Ref. 15). Interestingly, the ability of CBP/p300, SRCs, TRAP/DRIP, and several other cofactors to interact with nuclear receptors is mediated by a common signature motif, LxxLL (L = leucine, x = any amino acid), present once to several times within coactivator sequences. Coactivator domains encompassing LxxLL motifs shown to be necessary for receptor interaction have been referred to as nuclear receptor (NR) boxes. When receptor-coactivator interactions are examined, individual receptors exhibit distinct but overlapping coactivator preferences (16, 17, 18, 19, 20, 21).

Retinoid X receptors (RXRs) occupy a central position in nuclear receptor-mediated gene expression by functioning as obligate heterodimeric partners for a large number of receptors including retinoic acid, thyroid hormone, vitamin D, and peroxisome proliferator-activated receptors (22). The observation that peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) plays essential roles in adipocyte and foam cell differentiation and that a class of antidiabetic drugs, the thiazolidinediones, are PPAR{gamma} agonists has stimulated great interest in RXR-PPAR{gamma} heterodimers (23, 24). In this regard, we have shown that this dimeric transcription factor can regulate gene expression in response to agonists that bind either RXR or PPAR{gamma} (25, 26). Interestingly, RXR and PPAR{gamma} exhibit distinct coactivator preferences. RXR makes a relatively strong interaction with SRC-1 compared with CBP, while PPAR{gamma} prefers CBP relative to SRC-1 (26, 27). In this work we show that RXR’s preference for SRC-1 is determined by three amino acids in and around the LxxLL motif and that RXR and PPAR{gamma} utilize different strategies to distinguish between SRC-1 and CBP. These different modes of NR box recognition suggest it will be possible to identify receptor-specific small molecule antagonists that function by blocking access to the coactivator binding pocket.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Interaction of RXR-PPAR{gamma} with Single NR Boxes
We and others have shown, using two-hybrid and in vitro protein-protein interaction assays, that RXR and PPAR{gamma} exhibit opposite preferences for the coactivators SRC-1 and CBP (26, 27). To determine whether the ability of RXR and PPAR{gamma} to distinguish between coactivators results from the recognition of single NR boxes, mammalian two-hybrid assays were used to examine interactions between NR boxes fused to the DNA-binding domain of GAL4 and the two receptors either individually or as heterodimers (Figs. 1Go and 2Go). Each NR box used in this study consisted of 20 amino acids (Fig. 1AGo); eight amino acids amino terminal to the LxxLL motif (position -8 to position -1), the five-amino acid LxxLL motif (+1 to +5), and seven carboxy-terminal amino acids (+6 to +12). The analyses in Fig. 1Go, B–D demonstrate that the RXR-specific agonist LGD1268 (black bars) promotes interactions between RXR and the three NR boxes derived from SRC-1 with a slight binding preference for NR box III (compare bars 3–5 in Fig. 1BGo, average of eight independent experiments). Little or no RXR agonist-dependent interaction is observed with the NR box derived from the amino terminus of CBP (bar 2). Gel shift analysis of nuclear extracts from transfected cells using a GAL4 binding site indicates that all GAL4 fusions are expressed at similar levels (data not shown). Importantly, the interactions between RXR and the SRC-1 NR boxes are completely dependent on agonist binding. Little or no interaction is detected in the absence of the RXR agonist LGD1268 (white bars in Fig. 1BGo) or in the presence of an antagonist (data not shown) compared with the GAL4 DNA-binding domain alone. The interaction between RXR and the SRC-1 NR boxes also requires a functional AF-2 domain on the part of RXR. A mutation in the AF-2 domain that destroys transcriptional activity but allows ligand binding [leucine 451 to alanine (28)] eliminates interactions with the SRC-1 NR boxes (Fig. 1CGo). To confirm the results of the two-hybrid analysis, interactions between RXR and SRC-1 NR box III or the CBP NR box were examined by far Western blot analysis. Figure 1DGo indicates that SRC NR box III directly interacts with RXR in an agonist-dependent manner that requires a functional AF-2 domain (compare lanes 6 and 7 with 9 and 10). As expected, little interaction is observed between RXR and CBP (lanes 12 and 15).



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Figure 1. Interaction between RXR and Individual NR Boxes

A, Amino acid sequences in one-letter code of NR boxes from CBP, SRC-1, and TRAP220. The LxxLL motif is boxed. The first leucine of the LxxLL motif is considered position +1. B, Mammalian two-hybrid assay. CV1 cells were transfected with constructs expressing the appropriate GAL4-fusion, and VP16-RXR LBD as described in Materials and Methods. After transfection, cells were cultured for 36 h in the absence (white bars) or presence of 100 nM LGD1268 (black bars). The activity relative to that observed with VP16-RXR LBD + the empty GAL4 fusion vector is reported. Gel shift analysis of nuclear extracts from transfected cells using a GAL4 binding site indicates that all GAL4 fusions are expressed at similar levels. C, Same as panel B except a transcription-inactive RXR AF-2 mutant (L451A) was used. D, Far Western blot, 3 µg of purified GST (lanes 2, 5, 8, 11, 14, and 17), GST-RXR LBD wild-type (lanes 3, 6, 9, 12, 15, and 18), or GST-RXR LBD L451A mutant (lanes 4, 7, 10, 13, 16, and 19) were resolved on 10% SDS gels, and either stained with Coomassie blue to visualize proteins (lanes 1–4) or transferred to polyvinylidene difluoride (PVDF) membranes, and renatured. Blots were probed with 35S-in vitro translated GAL4-SRC-1 NR box III (lanes 5–10), GAL4-CBP NR box (lanes 11–16), or full-length hRAR{alpha} (lanes 17–19) in the absence (lanes 5–7, 11–13, and 17–19) or presence of (lanes 8–10 and 14–16) 1 µM LGD1268.

 


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Figure 2. Interaction between RXR-PPAR{gamma} Heterodimers and Individual NR Boxes

A, Modified mammalian two-hybrid assay. CV1 cells were transfected with constructs expressing the appropriate GAL4-fusion, VP16-RXR LBD, and the PPAR{gamma} LBD, as described in Materials and Methods. After transfection, cells were cultured for 36 h in the absence (white bars) or presence of 5.0 µM BRL 49653 (PPAR{gamma}, striped bars), 100 nM LGD1268 (RXR-specific, black bars), or with the combination of 5.0 µM BRL 49653 +100 nM LGD1268 (dotted bars). The activity relative to that observed with VP16-RXR LBD + PPAR{gamma} LBD + the empty GAL4 fusion vector is reported. B, Same as panel A except the VP16-RXR LBD construct was omitted from the transfection. After transfection, cells were cultured for 36 h in the absence (white bars) or presence of 5.0 µM BRL 49653 (PPAR{gamma}, striped bars). The activity relative to that observed with PPAR{gamma} LBD + the empty GAL4 fusion vector is reported. C, Standard mammalian two-hybrid assay examining interactions between a VP16-PPAR{gamma} fusion protein and GAL4-NR box constructs. After transfection, cells were cultured for 36 h in the absence (white bars) or presence of 5.0 µM BRL 49653 (PPAR{gamma}, striped bars). The activity relative to that observed with VP16-PPAR{gamma} + the empty GAL4 fusion vector is reported. Note that the y-axis differs in panel A, differs from the y-axes in panel B, and panel C. Gel shift analysis of nuclear extracts from transfected cells using a GAL4 binding site indicates that all GAL4 fusions are expressed at similar levels.

 
To examine interactions between PPAR{gamma} and individual NR boxes, a modified version of the mammalian two-hybrid assay was used in which the LBD of PPAR{gamma} is coexpressed with a VP16-RXR LBD fusion protein (Fig. 2AGo) (26). In this analysis the PPAR{gamma} agonist BRL 49653 (striped bars) promotes a relatively strong interaction between RXR-PPAR{gamma} heterodimers and the CBP NR box and little or no interaction with the SRC-1 sequences. The advantage of the modified two-hybrid system is that it ensures that the response to PPAR{gamma}-specific ligands is mediated by a heterodimer of PPAR{gamma} with RXR (compare Figs. 2AGo and 2BGo, note the difference in the y-axes). Removing the VP16-RXR LBD from the system (Fig. 2BGo) eliminates the ability to detect interactions between PPAR{gamma} and the CBP NR box. Nonetheless, a similar preference of PPAR{gamma} for the CBP NR box is observed using a VP16-PPAR{gamma} fusion in a standard two-hybrid assay (Fig. 2CGo). Consistent with the results of Fig. 2Go, PPAR{gamma} interacts only with the CBP NR box in far Western analysis (data not shown). Taken together, the results of this analysis indicate that RXR and PPAR{gamma} can distinguish among related NR boxes.

The modified two-hybrid system was also used to examine the response of RXR-PPAR{gamma} heterodimers to the RXR-specific agonist LGD1268 (Fig. 2AGo, black bars). As observed with RXR alone, binding of agonist to RXR in heterodimers promotes interaction with the SRC-1 NR boxes while little interaction with the CBP NR box is promoted by treatment with LGD1268 alone. Although we cannot exclude the possibility that forms of RXR other than RXR-PPAR{gamma} heterodimers contribute to the response to LGD1268 in the modified two-hybrid system (e.g. VP16-RXR homodimers), there are several observations indicating that heterodimers are a major species. First, the affinity of RXR for PPAR{gamma} is significantly higher than the affinity of RXR for itself (29). Second, we and others have previously shown that RXR can interact with SRC-1 when dimerized with PPAR{gamma} (26, 30, 31). Third, reversing the assay system (VP16-PPAR{gamma} + RXR LBD) yields a similar preference of RXR for SRC-1 and PPAR{gamma} for CBP (data not shown). Finally, the combination of RXR- and PPAR{gamma}-specific agonists has a synergistic effect on interactions with the CBP NR box (Fig. 2AGo, dotted bars). The synergy observed with the two receptor-specific ligands requires the presence of both RXR and PPAR{gamma} and therefore must be mediated by heterodimers. The synergistic interaction observed between RXR-PPAR{gamma} heterodimers and the CBP NR box is also consistent with our earlier work using larger fragments of CBP (26).

Structures of cocrystals composed of receptor LBDs complexed to NR boxes, and mutagenesis experiments have defined an agonist-dependent coactivator-binding pocket (2, 5, 6, 7, 8). To further demonstrate the specificity of receptor-NR box interactions, the ability of overexpressed NR boxes to inhibit transcription were examined. In agreement with the results of Figs. 1Go and 2Go, overexpression of SRC-1 NR box III functions as a dose-dependent inhibitor of transactivation mediated by RXR homodimers (Fig. 3AGo, solid circles). The CBP NR box, however, does not inhibit RXR (Fig. 3A, open boxes). The converse is true for PPAR{gamma}. The CBP NR box is a more effective inhibitor of BRL 49653-dependent transcription by RXR-PPAR{gamma} heterodimers than SRC-1 NR box III (Fig. 3BGo). The correlation between NR box binding and inhibition suggests that inhibition of transcription by overexpressed NR boxes most likely involves competition with endogenous coactivators for receptor binding.



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Figure 3. NR Boxes Specifically Inhibit Receptor-Mediated Transcription

A, CV1 cells were transfected with a reporter for RXR homodimers (CRBPII-tk-LUC), an expression construct for human RXR{alpha}, and increasing amounts of plasmids expressing GAL4-SRC-1 NR box III (solid circles) or CBP NR box (open squares). After transfection, cells were cultured for 36 h in the absence or presence of 100 nM LGD1268 (see Materials and Methods). Percent activity was calculated by setting the fold induction observed in the presence of identical amounts of the parental GAL4 DNA binding domain plasmid (10- to 15-fold) as 100%. B, CV1 cells were transfected with a reporter for RXR-PPAR{gamma} heterodimers (PPREx3-tk-LUC), expression constructs for RXR{alpha} and PPAR{gamma}, and increasing amounts of plasmids expressing GAL4-SRC-1 NR box III (solid circles) or CBP NR box (open squares). After transfection, cells were cultured for 36 h in the absence or presence of 5 µM BRL 49653 (see Materials and Methods). Percent activity was calculated by setting the fold induction observed in the presence of identical amounts of the parental GAL4 DNA binding domain plasmid (9- to 12-fold) as 100%.

 
Recent studies have implicated the TRAP/DRIP complex in the activity of several transcription factors (15). TRAP220/DRIP205 contains two LxxLL motifs (Fig. 1AGo) and appears to be the member of the TRAP/DRIP complex that interacts directly with nuclear receptors. Interestingly, both LGD1268 (RXR-specific) and BRL 49653 (PPAR{gamma}-specific) promote similar interactions between RXR-PPAR{gamma} heterodimers and the two TRAP220/DRIP205 NR boxes (Fig. 4Go). Interestingly, as observed with the CBP NR box, the combination of receptor-specific ligands has a synergistic effect on interactions between RXR-PPAR{gamma} heterodimers and TRAP220 NR box II. Thus, the analysis of six NR boxes indicates that RXR and PPAR{gamma} exhibit distinct but overlapping coactivator preferences.



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Figure 4. RXR and PPAR{gamma} Interact with the NR Boxes of TRAP220

Modified mammalian two-hybrid assay. CV1 cells were transfected with constructs expressing the appropriate GAL4-TRAP220 fusion, VP16-RXR LBD, and the PPAR{gamma} LBD as described in Materials and Methods. After transfection, cells were cultured for 36 h in the absence (white bars) or presence of 5.0 µM BRL 49653 (PPAR{gamma}-specific, striped bars), 100 nM LGD1268 (RXR-specific, black bars) or with the combination of 5.0 µM BRL 49653 + 100 nM LGD1268 (dotted bars). The activity relative to that observed with VP16-RXR LBD + PPAR{gamma} LBD + the empty GAL4 fusion vector is reported. Gel shift analysis of nuclear extracts from transfected cells using a GAL4 binding site indicates that both GAL4 fusions are expressed at similar levels.

 
Three Amino Acids Allow RXR to Distinguish SRC-1 NR Box III from CBP
To decipher how RXR distinguishes SRC-1 NR box III from the CBP NR box, chimeric NR boxes were constructed and examined in a standard mammalian two-hybrid assay with VP16-RXR LBD. Replacement of Arg+2 and Tyr+3 of SRC-1 NR box III with Ser and Glu, respectively (Fig. 5AGo), results in a 92% decrease in interaction with RXR relative to SRC-1 NR box III (Fig. 5BGo, compare bars 4 and 5). Conversely, making the complementary change in the CBP NR box (Ser+2 to Arg/Glu+3 to Tyr) increases interaction with RXR 3-fold relative to the CBP NR box (Fig. 5BGo, compare bars 2 and 3). Nevertheless, CBP Ser+2Arg/Glu+3Tyr has only 27% the activity of the original SRC-1 NR box III (Fig. 5BGo, compare bars 3 and 5) indicating that while the +2 and +3 positions are important determinants, other positions play important roles in RXR recognition.



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Figure 5. Three Amino Acids Determine RXR-NR Box Specificity

A, NR box amino acid sequences from position -1 to +6. Amino acids necessary and sufficient for interaction with RXR are shaded. B and C, Two-hybrid assay. CV1 cells were transfected with constructs expressing the appropriate GAL4-fusion and VP16-RXR LBD as described in Materials And Methods. After transfection, cells were cultured for 36 h in the absence or presence of 100 nM LGD1268 (RXR-specific). D, Modified mammalian two-hybrid assay. Cells were transfected as above; however, a construct expressing the PPAR{gamma} LBD was also included. After transfection, cells were cultured for 36 h in the absence or presence of 5 µM BRL 49653 (PPAR{gamma}-specific). The fold induction relative to the activity in the absence of ligand is reported. Note that the y-axis differs in panels B, C, and D. Gel shift analysis of nuclear extracts from transfected cells using a GAL4 binding site indicates that all GAL4 fusions are expressed at similar levels.

 
To determine the additional RXR-specific determinants in SRC-1 NR box III, the eight amino-terminal amino acids of SRC-1 NR box III were replaced with the amino acids in those positions from the CBP NR box. This chimeric NR box (CBP (-8)–(-1)/SRC-1 (+1)–(+12)) decreased interaction with RXR by 89% (Fig. 5BGo, compare bars 5 and 6). Additional chimeras and point mutations identify Leu-1 as the critical position. Individually replacing Leu-1 of SRC-1 NR box III with Gln, the -1 residue in the CBP NR box, decreases interaction with RXR by 87% (Fig. 5BGo, bar 7). In contrast the complementary Gln-1 to Leu change in the CBP NR box produces a 5-fold increase in interaction with RXR (Fig. 5BGo, compare bars 2 and 8). To determine whether the -1, +2, and +3 positions are sufficient to allow interaction with RXR, a triple-point mutant with Leu, Arg, and Tyr at these positions in the context of the CBP NR box was examined (CBP Q-1L/S+2R/E+3Y, Fig. 5CGo). Indeed, the triple point mutant interacts strongly with RXR (Fig. 5CGo, bar 4), indicating these three amino acids are necessary and sufficient for specific recognition of SRC-1 NR box III by RXR.

The contribution of the -1, +2, and +3 positions to preferential recognition of the CBP NR box by PPAR{gamma} was tested by examining the same triple point mutant. If PPAR{gamma}, like RXR, uses these three positions to discriminate between SRC-1 NR box III and the CBP NR box, then replacing the -1, +2, and +3 positions of CBP with the amino acids in SRC-1 NR box III (CBP Q-1L/S+2R/E+3Y) should eliminate interaction with PPAR{gamma}. As shown in Fig. 5DGo, however, the triple-point mutant has no effect on the PPAR{gamma}-CBP NR box interaction (compare bars 2 and 3). The difference in sensitivity of RXR and PPAR{gamma} to the -1, +2, and +3 positions suggests that coactivator preference is determined by the ability of RXR and PPAR{gamma} to recognize distinct structural features of NR boxes.

The results of Fig. 5Go indicate that RXR preference for SRC-1 is mediated by three amino acids (-1, +2, and +3). To examine whether the structural requirements determined for individual NR boxes are relevant in the context of full-length coactivators, the -1, +2, and +3 positions of the CBP NR box were changed to the preferred SRC-1 NR box III residues. This chimeric NR box (CBP Q-1L/S+2R/E+3Y) was then introduced into the full-length CBP protein. Unfortunately, the strong constitutive activity of GAL4-CBP full length precluded a two-hybrid analysis. However, Fig. 6Go clearly shows that the GAL4-CBP full-length NR box mutant (CBP Q-1L/S+2R/E+3Y) acts as a relatively effective inhibitor of RXR transcriptional activity compared with the wild-type GAL4-CBP full-length fusion protein (compare bars 2 and 3). As expected, both the wild-type and mutant full-length fusions are potent inhibitors of PPAR{gamma} activity (data not shown). The ability of the full-length CBP Q-1L/S+2R/E+3Y mutant to act as a selective inhibitor of RXR is consistent with the idea that the -1, +2, and +3 positions make significant contributions to the interactions between RXR and coactivators.



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Figure 6. The -1, +2, and +3 Positions of the NR Box Are Important for Interaction with RXR in the Context of Full-Length Coactivators

CV1 cells were transfected with a reporter for RXR homodimers (CRBPII-tk-LUC), an expression construct for human RXR{alpha}, and 12 ng of the appropriate GAL4 fusion/well. After transfection, cells were cultured for 36 h in the absence or presence of 100 nM LGD1268 (see Materials and Methods). The fold induction observed in the presence of the parental GAL4 fusion plasmid (8.3-fold) was set at 100%.

 
Seven Amino Acids Are Sufficient for Interaction of NR Boxes with RXR
Interestingly, RXR exhibits a 4-fold stronger interaction with CBP triple-point mutant (Q-1L/S+2R/E+3Y) compared with SRC-1 NR box III (Fig. 5CGo, compare bars 3 and 4). Relatively strong interaction with RXR is also observed for a chimeric NR box that contains the first 13 amino acids from SRC-1 NR box III and the last 7 amino acids from the CBP NR box (SRC (-8)–(+5)/CBP (+6)-(+12); Fig. 7BGo, compare bars 2 and 3). To determine which amino acids of the CBP NR box are responsible for enhanced interaction with RXR, mutations were constructed that individually replaced positions +6 through +12 of SRC-1 NR box III with the corresponding amino acids from the CBP NR box. Mutation of Asp+6 to Arg resulted in a 4-fold increase in interaction with RXR relative to SRC-1 NR box III, while substitutions at other positions had little or no effect (Fig. 7BGo, compare bars 2–5; also data not shown). Analysis of the complementary Arg+6 to Asp change in the CBP NR box indicates that the identity of the +6 position also significantly contributes to the PPAR{gamma}-CBP NR box interaction (Fig. 7CGo, compare lanes 2 and 3). A similar modified two-hybrid assay was used to examine the influence of the +6 position on interaction with retinoic acid (RAR) and thyroid hormone (TR) receptors. Both RAR and TR interact in an agonist-dependent manner with SRC-1 NR box III, and, like RXR, the Asp+6 to Arg mutation enhances the interaction (compare bars 2 and 3 in Figs. 7DGo and 7EGo). Thus, the identity of the +6 position, immediately carboxy-terminal to the LxxLL motif, significantly contributes to interaction with multiple receptors.



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Figure 7. The +6 Position of the NR Box Influences the Affinity of Multiple Receptors

A, NR box amino acid sequences from position -1 to +7. Amino acids that contribute to high-affinity interaction with RXR are shaded. B, Two-hybrid assay. CV1 cells were transfected with constructs expressing the appropriate GAL4-fusions and VP16-RXR LBD as described in Materials and Methods. After transfection, cells were cultured for 36 h in the absence or presence of 100 nM LGD1268 (RXR-specific). C, Modified mammalian two-hybrid assay. Same as panel B except that a construct expressing the PPAR{gamma} LBD was also included. After transfection, cells were cultured for 36 h in the absence or presence of 5 µM BRL 49653 (PPAR{gamma}-specific). D and E, Modified mammalian two-hybrid assay. Same as panel B except that constructs expressing either the RAR{alpha} LBD (D) or the TRß LBD (E) were also included. After transfection, cells were cultured for 36 h in the absence or presence of 100 nM LGD1550 (RAR-specific) or 100 nM T3 (TR-specific). The fold induction relative to the activity in the absence of ligand is reported. Note that the y-axis differs in B, C, D, and E. Gel shift analysis of nuclear extracts from transfected cells using a GAL4 binding site indicates that all GAL4 fusions are expressed at similar levels.

 
The mutagenesis results identify four positions (-1, +2, +3, and +6) that contribute to high-affinity RXR-NR box interaction in addition to the three leucines of the LxxLL motif. To determine whether these seven amino acids are sufficient for agonist-dependent interaction with RXR, the sequence Leu-Leu-Arg-Tyr-Leu-Leu-Arg (Fig. 8AGo) was fused to GAL4 and tested in two-hybrid assays. Figure 8BGo demonstrates indeed this minimal NR box interacts with RXR (compare black bars 2 and 3), although quantitatively the interaction is only 34% of that observed with the 20-amino acid SRC-1 NR box III (Fig. 8BGo, compare black bars 3 and 4). In contrast, little or no interaction is observed between the minimal NR box and PPAR{gamma} (Fig. 8BGo, compare white bars 2 and 3), RAR, or TR (data not shown). The minimal NR box also acts as an inhibitor of RXR agonist-dependent transactivation but has little effect on PPAR{gamma} agonist-dependent transcription (compare bar 3 in Figs. 8CGo and 8DGo). Likewise, no inhibition of RAR- or TR-mediated transcription is observed (data not shown). The ability of this short peptide sequence (molecular mass < 1000 daltons) to specifically interact with and inhibit transcription by RXR suggests it should be possible to identify small molecule receptor-specific antagonists that function by blocking coactivator recruitment to activated nuclear receptors.



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Figure 8. RXR Interacts with an Optimized 7 Mer

A, Amino acid sequence (-1-+6) of the CBP NR box, SRC-1 NR box III, and optimized seven-amino acid minimal NR box. Amino acids unique to CBP and SRC-1 are in white and gray, respectively. B, Modified mammalian two-hybrid assay. CV1 cells were transfected with constructs expressing the appropriate GAL4-fusion, VP16-RXR LBD, and the PPAR{gamma} LBD as described in Materials and Methods. After transfection, cells were cultured for 36 h in the absence or presence of 100 nM LGD1268 (RXR-specific, black bars) or 5.0 µM BRL 49653 (PPAR{gamma}, white bars). The fold induction relative to the activity in the absence of ligand is reported. Gel shift analysis of nuclear extracts from transfected cells using a GAL4 binding site indicates that all GAL4 fusions are expressed at similar levels. C, CV1 cells were transfected with a reporter for RXR homodimers (CRBPII-tk-LUC), an expression construct for human RXR{alpha}, and 36 ng of the appropriate GAL4 fusion/well. After transfection, cells were cultured for 36 h in the absence or presence of 100 nM LGD1268 (see Materials and Methods). The fold induction observed in the presence of the parental GAL4 fusion plasmid (13.6-fold) was set at 100%. D, CV1 cells were transfected with a reporter for RXR-PPAR{gamma} heterodimers (PPREx3-tk-LUC), expression constructs for RXR{alpha} and PPAR{gamma}, and 36 ng of the appropriate GAL4 fusion/well. After transfection, cells were cultured for 36 h in the absence or presence of 5 µM BRL 49653. The fold induction observed in the presence of the parental GAL4 fusion plasmid (10.2-fold) was set at 100%.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The results of this study demonstrate that RXR and PPAR{gamma} can distinguish among highly related NR box sequences, and they do so by recognizing different structural features. RXR’s preference for SRC-1 NR box III relative to the CBP NR box is determined by three amino acids. Interestingly, two of the important positions for RXR-NR box interaction are at +2 and +3, the nonconserved xx residues of the LxxLL motif. A role for the +2 and +3 positions as specificity determinants is surprising, given that these residues appear to point away from the coactivator binding pocket in the crystal structures of LxxLL motifs bound to estrogen receptor (ER), TR, and PPAR{gamma} (2, 5, 6). Nevertheless, a similar observation has been made for the glucocorticoid receptor (2). Comparison of the available receptor LBD structures suggests that differences in the depth of the coactivator binding pocket among receptors could contribute to receptor-specific roles for the nonconserved +2 and +3 residues. Further mutagenesis studies will be needed to address this question.

The identity of the nonconserved -1 position, directly amino terminal to the LxxLL motif, also significantly influences NR box choice by RXR. RXR’s preference for a hydrophobic amino acid at -1 has been independently confirmed using phage display in the study of Chang et al. (32). Crystal structures of several LBDs complexed with NR boxes show that the -1 position projects into the coactivator binding pocket and makes direct contact with a conserved hydrophobic amino acid in helix 12 (Phenylalanine 450 in hRXR{alpha}) (2, 5, 6). The finding that hydrophobic amino acids occupy the -1 position in approximately 65% of the LxxLL motifs analyzed (24 of 37) further highlights the critical function of this position in receptor-coactivator interactions. Taken together, the results of this study indicate that subtle changes in NR box sequence can have large and receptor-specific effects on receptor-coactivator interactions. Additionally, the possibility that different ligands, trans-acting proteins, or receptor and coactivator posttranslation modifications influence receptor-NR box affinity must be noted (33).

In contrast to RXR, the identity of the amino acids at positions -1, +2, and +3 has little effect on the ability of PPAR{gamma} to distinguish the CBP NR box from the SRC-1 NR box III. Preliminary experiments suggest that positions both amino- and carboxy terminal to the LxxLL motif are required for specific recognition of CBP by PPAR{gamma}. The difference in sensitivity of the two receptors for substitution at -1, +2, and +3 suggests that RXR and PPAR{gamma} recognize different structural features of NR boxes. Unique NR box recognition may contribute to the ability of RXR- and PPAR{gamma}-specific agonists to activate transcription synergistically (26). As discussed below, this observation also has important implications for the discovery of drugs that target the coactivator binding pocket.

Although RXR and PPAR{gamma} appear to use different strategies to distinguish among NR boxes, an arginine at +6 is favored over an aspartic acid by both receptors. A similar preference for arginine at +6 is observed for RAR and TR. Analysis of receptor-coactivator structures suggests that the ability of arginine to act as a hydrogen bond donor to the +2 carbonyl group may account for its preference compared with aspartic acid (5). It has also been suggested that the +6 position may function as a helix capper (2, 5). Additional studies will be needed to dissect the contributions of hydrogen bonding and helix capping at this position. Interestingly, McInerney et al. (19) have recently demonstrated that mutation of the +6 position interferes with the ability of SRC-1 to support transactivation mediated by RAR or PPAR{gamma}. Thus, the amino acid directly carboxy terminal to the LxxLL motif (+6) may also be considered a critical element of the NR box for at least a subset of nuclear receptors.

Recent studies suggest that nuclear receptors interact cooperatively with multiple LxxLL motifs present in individual coactivators (21, 34). Our studies, which utilize GAL4-LxxLL fusions harboring the intact dimerization domain of GAL4, do not directly address the role of cooperativity. Nonetheless, coactivators appear to function as members of large multisubunit complexes that contain several proteins capable of interacting directly with receptors (35, 36). For instance, SRC-1 and CBP can directly interact with each other (13, 37, 38). As a result, it is not difficult to imagine cooperative interactions between RXR-PPAR{gamma} heterodimers and coactivator complexes with each receptor contacting either an independent subunit (e.g. SRC-1 and CBP) or independent NR boxes in the same subunit. A similar model of two transcription factors independently contacting the same coactivator complex has recently been proposed for synergistic activation of transcription by the transcription factors STAT3 (signal transducer and activator of transcription 3) and Smad1 (spinal muscular atrophy mothers against decapentaplegic 1) (1). Since it is possible, however, to detect interactions between receptors and synthetic peptides encoding single NR boxes, we cannot exclude models in which each member of the heterodimer recruits a unique coactivator complex (e.g. SRC-1 and TRAP/DRIP). In either model, increased recruitment of coactivators by liganding each subunit may contribute to synergistic activation of transcription.

An additional model for a synergistic response to RXR- and PPAR{gamma}-specific ligands arises from our previous observations that a transcriptionally silent (AF-2 mutant) RXR can mediate synergy (26). The AF-2-independent ability of RXR agonists to synergize with PPAR{gamma} ligands suggests that binding of ligand to RXR can indirectly influence the conformation and activity of PPAR{gamma}. It is likely that RXR makes both AF-2-dependent (direct coactivator recruitment) and AF-2 independent (indirect effects on PPAR{gamma}) contributions to synergistic activation.

We and others have shown that PPAR{gamma} makes a relatively stronger interaction with CBP compared with SRC-1 and other SRC family members (26, 27, 31). The preference of PPAR{gamma} for CBP is seemingly at odds with experiments that suggest CBP is recruited indirectly to nuclear receptors via its association with SRC family members and that the CBP NR box is dispensable for coactivator function (34, 39). There are several possible explanations to resolve this apparent paradox. First, the role of the CBP NR box has not been examined in the context of PPAR{gamma} transactivation. Therefore, it is possible that a direct PPAR{gamma}-CBP interaction may be critical for activity. Second, our data suggest that RXR and PPAR{gamma} recognize different structural features of NR boxes. We cannot conclude, however, which coactivator is important in a particular physiological context. For instance, the ability of PPAR{gamma} to interact with the TRAP/DRIP complex may be more important than direct interactions with CBP. Third, it is possible to detect direct interactions between PPAR{gamma} and SRC-1, although this interaction is relatively weaker than the PPAR{gamma} and CBP interaction (5, 27, 30, 31) (data not shown). In cells, additional factors such as high local concentrations of receptors and/or coactivators or posttranslational modifications may act to increase the relative affinity and functional importance of PPAR{gamma}-SRC-1 interactions.

Knowledge of the critical residues for RXR-NR box interaction allowed construction of a seven-amino acid sequence (LLRYLLR) that interacts with RXR in an agonist-dependent fashion and inhibits RXR-dependent transcription. The small size of this peptide suggests it may be possible to identify small molecules that mimic this peptide and function as receptor antagonists by blocking the access of endogenous factors to the coactivator binding pocket. Given the structural conservation of the coactivator binding pocket among nuclear receptors (2, 5, 6, 7, 8), one might predict that such drugs would have pleiotropic effects by binding to a wide range of receptors. The results of this and other studies (16, 17, 19, 21), however, indicate that there is significant specificity built into receptor-NR box interactions. Therefore, it should be possible to exploit this specificity in the identification and design of small molecule antagonists. Since many transcription factor-coactivator interactions are dependent on small hydrophobic patches, it may also be possible to more generally apply these same principles to other gene regulation systems.

Antagonists of the coactivator binding pocket would be expected to have properties that may be advantageous when compared with traditional antagonists that competitively bind to the ligand binding pocket. First, coactivator pocket antagonists should recognize a particular receptor conformation and may provide remarkable tissue and/or gene selectivity. Second, coactivator pocket antagonists would be predicted to bind only to receptors that are in an active conformation (i.e. the ligand-dependent coactivator pocket has formed). Specificity for activated receptors predicts that coactivator pocket antagonists would lack the undesirable partial agonist activity that has limited the use of drugs such as the estrogen receptor antagonist tamoxifen. Exploiting the uniqueness and specificity of the coactivator binding pocket opens the door to a new receptor pharmacology that should have both scientific and therapeutic benefits.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The receptor and reporter vectors used in this study have been previously described (26, 40, 41). GAL4-NR box fusions were made by synthesizing oligonucleotides with the appropriate sequences utilizing mammalian codon preferences followed by cloning into pCMX-GAL4 (40). The full-length GAL4-CBP construct was provided by D. Chakravarti (University of Pennsylvania. Philadelphia, PA). The full length GAL4-CBP (Q-1L/S+2R/E+3Y) mutant was made by site-directed mutagenesis using the Quickchange mutagenesis system (Stratagene, La Jolla, CA). All clones were confirmed by DNA sequencing.

Cell Culture and Transfection
CV1 cells were cultured in DMEM supplemented with 10% FBS. Before transfection, cells were seeded in 48-well plates (1.5 x 104 cells per well) in DMEM supplemented with 10% charcoal resin-split FBS. After 12–16 h growth at 37 C, cells were transfected with the DOTAP transfection reagent following the manufacturer’s instructions (Roche Molecular Biochemicals, Indianapolis, IN). For two-hybrid assays each well was transfected with 36 ng of luciferase reporter, 36 ng of the appropriate GAL4 fusion, 36 ng of VP16-RXR LBD, and as an internal control 60 ng of pCMX-ßgal. Where noted in the figure legends, 72 ng of PPAR{gamma} LBD, 36 ng of RAR LBD, or 36 ng of TR LBD were also transfected. For inhibition experiments, cells were transfected as described above with 12 ng of luciferase reporter, 12 ng of the appropriate receptor expression vectors, and increasing concentrations of GAL4 fusion expression vectors. As an internal control, 60 ng of pCMX-ßgal were also included. When necessary, the parental expression plasmid pCMX was added to ensure that equal amounts of DNA were transfected in each well. After 5 h at 37 C, the media were removed, the cells washed once, and 200 µl of fresh media were added with or without the ligands described in the legend to each figure. Cells were harvested after an additional 36 h of growth at 37 C. Luciferase activity of each sample was normalized by the level of ß-galactosidase activity. Each transfection was carried out in duplicate and repeated at least four times.

Far Western Blotting
Far Western blots were carried out as previously described (26). Each blot contained 3 µg of purified glutathione-S-transferase (GST), GST-RXR, and GST-RXR LBD.


    ACKNOWLEDGMENTS
 
We would like to thank M. Manchester and D. Chakravarti for comments on the manuscript and the medicinal chemistry department at Ligand Pharmaceuticals, Inc. for providing LGD1268 and BRL 49653.


    FOOTNOTES
 
Address requests for reprints to: Ira G. Schulman, X-Ceptor Therapeutics, 4757 Nexus Center Drive, Suite 200, San Diego, California 92121. E-mail: ischulman{at}x-ceptor.com

1 Present address: X-Ceptor Therapeutics, 4757 Nexus Center Drive, Suite 200, San Diego, California 92121. Back

Received for publication October 8, 1999. Revision received March 28, 2000. Accepted for publication April 13, 2000.


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