The Nuclear Receptor Corepressor (N-CoR) Contains Three Isoleucine Motifs (I/LXXII) That Serve as Receptor Interaction Domains (IDs)

Paul Webb, Carol M. Anderson, Cathleen Valentine, Phuong Nguyen, Adhirai Marimuthu, Brian L. West, John D. Baxter and Peter J. Kushner

Metabolic Research Unit University of California School of Medicine San Francisco California 94143-0540


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unliganded thyroid hormone receptors (TRs) repress transcription through recruitment of corepressors, including nuclear receptor corepressor (N-CoR). We find that N-CoR contains three interaction domains (IDs) that bind to TR, rather than the previously reported two. The hitherto unrecognized ID (ID3) serves as a fully functional TR binding site, both in vivo and in vitro, and may be the most important for TR binding. Each ID motif contains a conserved hydrophobic core (I/LXXII) that resembles the hydrophobic core of nuclear receptor boxes (LXXLL), which mediates p160 coactivator binding to liganded nuclear receptors. Although the integrity of the I/LXXII motif is required for ID function, substitution of ID isoleucines with leucines did not allow ID peptides to bind to li-ganded TR, and substitution of NR box leucines with isoleucines did not allow NR box peptides to bind unliganded TR. This indicates that the binding preferences of N-CoR for unliganded TR and p160s for liganded TR are not dictated solely by the identity of conserved hydrophobic residues within their TR binding motifs. Examination of sequence conservation between IDs, and mutational analysis of individual IDs, suggests that they are comprised of the central hydrophobic core and distinct adjacent sequences that may make unique contacts with the TR surface. Accordingly, a hybrid peptide that contains distinct adjacent sequences from ID3 and ID1 shows enhanced binding to TR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors are a large family of conditional transcription factors that include receptors for thyroid hormone (T3), vitamins A (retinoids) and D, steroids, various components of lipid metabolism, and a large number of orphan receptors whose ligands, if any, have not been identified (1, 2, 3, 4). The thyroid hormone receptors (TRs) bind to cognate DNA response elements both in the absence and presence of ligand, most commonly as a heterodimer with the retinoid X receptor (RXR) (5, 6, 7). Unliganded TRs repress basal promoter activity, and addition of ligand both relieves this repression and further enhances basal promoter activity.

Thyroid hormone-dependent enhancement of basal promoter activity involves recruitment of two types of coactivator proteins. The TRs bind to a closely related family of p160 coactivators, including GRIP1 (TIF2/NCoA-2), SRC-1 (NCoA-1), and ACTR (pCIP/Rac3/AIB1/TRAM-1) (8, 9, 10, 11). The p160s, in turn, bind to other coactivators, including the integrator molecule CBP/p300 and p/CAF, both of which possess histone acetyltransferase activity. The TRs also bind TRAP220 (12), a component of the TRAP (DRIP/ARC/SMCC) complex that potentiates TR-dependent transcription from naked DNA templates in vitro, but also regulates transcription from DNA templates that have been assembled into chromatin in vitro (13, 14, 15, 16, 17). Thus, liganded TRs activate transcription by recruiting large coactivator complexes, which work by either modifying chromatin or via unspecified effects upon general initiation factors. In contrast, unliganded TRs repress transcription by recruiting corepressor proteins, which are released upon hormone binding. The TRs bind to nuclear receptor corepressor (N-CoR) (RIP-13) and to the closely related protein SMRT (TRAC-2) (18, 19, 20, 21, 22, 23, 24). Both N-CoR and SMRT, in turn, bind a large complex that contains mSin3a, SAP30, c-ski, and histone deace-tylases (HDACs) (25, 26, 27) and also bind directly to class II HDACs (28, 29). Thus, unliganded TRs repress transcription by recruiting large corepressor complexes, which work, at least in part (30), by deacetylating histones.

Recent studies have focused on the structural basis of coactivator and corepressor recruitment. Like most nuclear receptors, TRs possess a strong ligand- dependent transactivation function (AF-2) that is located within the receptor ligand-binding domain (LBD) and serves as a docking site for p160 coactivators (31, 32). A combination of x-ray crystallography and site- directed mutagenesis has revealed that the residues that comprise AF-2 form a small hydrophobic cleft upon the surface of the T3-liganded TR-LBD (32, 33). Mutational analysis of the p160s and crystallographic analysis of nuclear receptor/p160 cocrystals revealed that AF-2 binds to short, conserved {alpha}-helical motifs (termed NR boxes, consensus LXXLL) (34, 35, 36, 37, 38, 39, 40, 41). Initial structure-function analysis of the TRs and retinoic acid receptors suggested that key residues for corepressor binding were located near the junction of the hinge and LBD (18). However, the TR-LBD crystal structure revealed that these residues were not on the surface of the molecule (33), suggesting that mutations of these residues affect corepressor binding indirectly. Other evidence indicated that coactivators and corepressors were in dynamic equilibrium on the nuclear receptor (8, 11, 42) and that key residues for transcriptional repression by v-erbA, an oncogenic viral homolog of TR, and for transcriptional repression and corepressor binding by the orphan receptors Rev-erbA{alpha} and RVR, actually resided within the AF-2 hydrophobic cleft (43, 44). This suggested that similar mechanisms might underlie both coactivator and corepressor recruitment and prompted us to search the N-CoR primary sequence for motifs that bear similarities to nuclear receptor boxes. We find, in agreement with recently published studies of others (45, 46, 47), that the N-CoR C terminus contains a repeated receptor interaction domain (ID) that contains the conserved hydrophobic core motif I/LXXII. More surprisingly, we find that N-CoR actually contains three of these IDs, rather than the previously reported two. We present several lines of evidence that the hitherto unrecognized motif (ID3) is a fully functional TR binding site and that the three IDs represent the totality of TR binding activity. We also show that the IDs are comprised of both a hydrophobic core and distinct adjacent sequences and that a hybrid peptide containing distinct adjacent sequences from ID3 and ID1 binds more tightly to TR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Three Receptor IDs in N-CoR
We initially asked whether N-CoR contains sequence motifs that resemble the p160 NR Box, consensus LXXLL. Previous studies of TR/N-CoR interactions revealed two regions of TR binding (amino acids 2,239–2,453 and 1,944–2,239) in the N-CoR C terminus (18, 19, 22, 24, 48) (Fig. 1AGo). The C-terminal binding region contained a single short hydrophobic motif that resembled the NR box (LEDII, amino acids 2,276–2,281). The N-terminal binding region contained two of these motifs (ICQII, amino acids 2,073–2,077; IDVII, amino acids 1,949–1,953). We refer to these motifs as ID1, ID2, and ID3 (from C-terminal to N-terminal). During the course of this study, ID1 and ID2 were also identified by other groups (45, 46, 47) but ID3 was not.



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Figure 1. Three Receptor IDs in the Carboxyl Terminus of N-CoR

A, Schematic of the structural organization of N-CoR showing the relative positions of the silencing domains (striped boxes) and nuclear receptor IDs (black boxes). The C-terminal portion of N-CoR is shown, below, on a larger scale. The extent of previously defined TR binding regions are marked with lines above, the positions of each ID motif are marked with a black box, and the amino acid coordinates of each ID motif are marked below. B, Sequences of the ID motifs. The sequences of the ID motifs are presented and compared with each other, and to those of SMRT. At the top, the p160 coactivator NR box consensus sequence is presented. Underneath, the hydrophobic core motif of each ID is also shown in a similar box. Homologies between N-CoR ID1 and SMRT ID1 or N-CoR ID3, N-CoR ID2, and SMRT ID2 are highlighted. Conserved residues are indicated with a thick line, and conservative substitutions, or residues displaced by a single position, are indicated with a thin line.

 
N-CoR ID1 and ID2 had counterparts at similar locations in the related corepressor SMRT (46). Interestingly, both N-CoR IDs showed greater homology to their SMRT counterparts than to each other (Fig. 1BGo). The strongest conservation between N-CoR and SMRT ID1 was C-terminal to the core motif, whereas the strongest conservation between N-CoR and SMRT ID2 was N terminal to the core motif. While N-CoR ID3 did not have an obvious counterpart within SMRT, it did resemble N-CoR ID2. In particular, ID3 and ID2 contained an IXXII core, a conserved Arg and the sequence ITØA N-terminal to the cores, and a conserved Thr immediately C terminal to the core. Because of these homologies, we elected to examine all three IDs as candidate TR binding sites.

The ID Motifs Mediate TRß/N-CoR Interactions
To test whether each of the three IDs represented a functional TR binding site, we synthesized short peptides that spanned the entire region of conservation between N-CoR and SMRT ID1 or N-CoR ID3 and N-CoR and SMRT ID2 (Fig. 2AGo). We then asked whether these short ID peptides would compete for TRß/N-CoR interactions in vitro (Fig. 2BGo). ID1 and ID3 showed half-maximal competition at 0.3–1 µg of peptide. ID2 was weaker, with half-maximal competition requiring 1–3 µg of peptide. Overall, the efficiency of ID peptide competition for TRß/N-CoR interactions was comparable to the efficiency of NR box peptide competition for TRß/GRIP1 interactions (37). Moreover, several mutant peptides, some of which conserved overall ID hydrophobicity, failed to compete for TR/N-CoR interactions (data not shown and see Figs. 6Go and 7Go). Thus, each of the three ID motifs could represent a functional TR binding site.



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Figure 2. The ID Motifs Interact with TRß in Vitro

A, Schematic of GST-N-CoR matrix and competitor peptides used in TRß binding assays. B, Inhibition of TRß binding to GST-N-CoR C terminus by synthetic peptides. An autoradiogram of SDS-PAGE gels showing radiolabeled TRß input protein and TRß protein retained upon bacterially expressed GST- or GST-N-CoR matrices, either in the absence or the presence of T3 or in the presence of increasing doses of competitor peptide. C, TR binds to N-CoR fragments that overlap ID1, ID2, and ID3. The panel is an autoradiogram of an SDS-PAGE gel showing radiolabeled TRß input protein and TRß protein bound to N-CoR fragments that overlap ID3 (amino acids 1,944–2,031), ID2 (amino acids 2,051–2,208), and ID1 (amino acids 2,218–2,453). The radiolabeled TR appears as a diffuse band in the ID1 lanes because the TR migrates to approximately the same location as the GST-ID1 fusion protein.

 


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Figure 6. Comparison of ID1 and NR Box Peptides

A, Schematic of GST-N-CoR and GST-GRIP1 fusion proteins and competitor peptides. B, Peptide competitions for TRß binding to GST-N-CoR. The left hand panel shows an autoradiogram of SDS-PAGE gels showing radiolabeled TRß input protein and TRß protein retained upon a bacterially expressed GST- or GST-N-CoR matrix, either in the absence or the presence of T3 or in the presence of 30 µg of each competitor peptide. The right hand panel shows a similar experiment in which we examined the abilities of different peptides to compete for the binding of TRß to bacterially expressed GST-GRIP1.

 


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Figure 7. Sequence Composition of the IDs

A, Analysis of ID3 mutant peptides. A schematic of different competitor peptides and GST-N-CoR is shown at the top. Autoradiogram of a SDS-PAGE gel showing radiolabeled TRß protein retained upon a GST-N-CoR matrix in the absence or presence of T3 and 1 µg of competitor peptides. B, A similar analysis of TR binding to GST-N-CoR (1,944–2,453) in the presence of 30 µg ID1 peptide or mutant derivative, with peptide sequences shown at top. C, Concentration-dependent inhibition of N-CoR/TRß interactions with an ID3/ID1 hybrid competitor peptide. A schematic of the GST-N-CoR fusion protein and competitor peptide is shown at the top. The arrows indicate the extents of the ID3 and ID1 sequences. Below is an autoradiogram of a SDS-PAGE gel, revealing the amount of radiolabeled TRß retained on the GST-N-CoR matrix.

 
We then asked whether N-CoR fragments that contained each of the isolated IDs would bind to TR. Figure 2CGo shows that, in agreement with previous results (22, 24, 45, 46, 47), N-CoR fragments overlapping isolated ID1 or ID2 bound the TR in the absence of ligand. A N-CoR fragment that overlapped the ID3 IXXII motif (amino acids 1,944–2,031) also showed significant binding to TR. TR binding to ID3 was robust, even though this N-CoR fragment lacks some of the conserved ID3 sequences that lie to the N terminus of amino acid 1,944. Thus, the N-CoR C terminus contains three distinct TR binding regions, each of which overlaps an ID motif.

To determine whether the ID motifs themselves were required for N-CoR/TRß interactions in vitro, we prepared a vector that expresses the N-CoR C terminus (amino acids 1,681–2,453) and introduced mutations into the hydrophobic core of each ID. Figure 3Go shows that the N-CoR C-terminal fragment (WT) bound strongly to TRß in the absence of ligand, and that binding was reduced by T3. Similar N-CoR fragments that retained either ID3+ID2 or ID3+ID1 showed small reductions in TRß binding in the absence of ligand, but an N-CoR fragment that retained only ID2+ID1 showed a larger reduction in TRß binding. Phosphoimaging of several experiments revealed that ID3+ID2 allowed more than 90% of the level of wild type N-CoR binding to TRß, ID3+ID1 allowed up to 70%, and that ID2+ID1 allowed 40–60%. Thus, significant N-CoR binding is obtained with any two IDs, but the combination of ID3+ID2 is preferred and the combination of ID2+ID1 is weakest. By contrast, single IDs were insufficient to allow significant binding to TRß. An N-CoR fragment that only contained ID3 did give some weak binding to TRß (~5% of wild type), but fragments that contained only ID2 or ID1, or no IDs failed to bind TRß. Taken together, these results point toward several conclusions. First, the N-CoR IDs are essential for TR binding. Second, because the N-CoR triple mutant failed to bind to TRß, there is no additional TR binding site within the N-CoR C terminus. Third, because each possible ID pair allows significant TRß binding, yet isolated IDs do not, the IDs must cooperate in TRß binding. Lastly, and most surprisingly, because mutation of ID3 leads to the largest reduction in TRß binding, and because isolated ID3, but not ID1 or ID2, is sufficient for weak residual binding to the TRß-LBD, ID3 may be the strongest of the three IDs for TRß binding in vitro.



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Figure 3. Mutations in ID Motifs Reduce N-CoR/TRß Interactions

A schematic for the GST-TRß fusion protein and radiolabeled N-CoR C-terminal fragment is shown at the top. Mutated IDs contain double Ala substitutions within the C- terminal Iles of the ID hydrophobic core (I/LXXII>I/LxxAA). The panels represent autoradiograms of SDS-PAGE gels showing radiolabeled input wild-type and mutant N-CoR proteins, and the amounts of each N-CoR protein retained by GST-TRß, either in the absence or the presence of thyroid hormone (T3). In each case, IDs indicated at the left are those remaining after mutation.

 
IDs Are Required For TRß/N-CoR Interactions in Vivo
Next, we asked whether the ID motifs were needed for N-CoR/TRß interactions in vivo. We first determined whether our N-CoR C-terminal expression vector, which contains the IDs but lacks active repression domains, would act as a dominant negative for transcriptional repression. We transfected chicken embryo fibroblasts (CEF) with a reporter containing a herpes simplex thymidine kinase promoter and two GAL4 response elements (GAL-TK) and then determined the level of transcription in the absence or presence of a yeast Gal4-TRß fusion protein (Gal-TR) and N-CoR. Figure 4Go shows that, as expected, Gal-TR repressed transcription in CEF cells by 3-fold in the absence of T3 (left panel) and enhanced transcription by 15-fold in the presence of T3 (right panel). The N-CoR C-terminus (WT) reversed the repression (left panel), but not the ligand-dependent activation (right panel). Thus, consistent with previous observations (24), the N-CoR C terminus interferes with the ability of unliganded TRß-LBD to repress transcription.



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Figure 4. The IDs Are Needed for N-CoR Dominant Negative Activity in Vivo

A schematic of the transfection components is shown at top. The graphs below represent fold repression by unliganded Gal-TRß (left panel, black bars), or fold activation by liganded TRß (right panel, gray bars), either in the absence or presence of expression vectors for N-CoR C terminus or its mutated derivatives. To determine fold repression or fold induction luciferase activities were normalized to ß-galactosidase, and then compared with those obtained in the absence of Gal-TRß, which is set at 1.

 
We then asked whether the IDs were required for this dominant negative activity. An N-CoR C-terminal fragment that retained ID3+ID2 retained most of its dominant negative activity (left panel). By contrast, fragments that retained either ID3+ID1 or ID2+ID1 were devoid of dominant negative activity. Fragments that either retained single IDs (ID3, ID2, and ID1), or no IDs, also lacked dominant negative activity. None of the N-CoR expression vectors affected transcriptional activation (right panel). Thus, the IDs are required for the dominant negative activity of the N-CoR C terminus. Moreover, an N-CoR fragment that retains ID3+ID2 showed significant dominant negative activity indicating that in vivo, as in vitro, TRß prefers ID3+ID2.

Next, we examined TRß binding to the IDs in two-hybrid assays in CEF cells (Fig. 5AGo). A N-CoR fragment that contained all three IDs (ID3, -2, and -1) recruited unliganded TRß-VP16, but not liganded TRß-VP16. By contrast, a similar construction in which all three ID motifs were mutated (mID3, -2, and -1) failed to recruit TRß-VP16, even when TRß-VP16 was overexpressed (not shown). Thus, the three IDs constitute all of the TR IDs within the N-CoR C terminus in vivo. In parallel, N-CoR fragments that contained isolated ID3 or isolated ID1 both recruited TRß-VP16, but their mutated equivalents did not. An N-CoR fragment that contained ID2 only showed weak interactions with TRß-VP16. However, the relative weakness of this interaction was overcome when TRß-VP16 was overexpressed (Fig. 5BGo). Thus, each ID binds TRß-VP16 in vivo, but ID3 and ID1 are strongest.



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Figure 5. The IDs Are Needed for N-CoR/TRß Interactions in Two-Hybrid Assays

A, A schematic of the transfection components is shown at the top. The transfection was performed with 10 ng of VP16-TRß fusion protein expression vector. The panel below shows a comparison of the efficiency of either the Gal-DBD (none) or different Gal-N-CoR fusions as baits. The regions of N-CoR encoded by each fragment were as follows: IDs 3,2,1: amino acids 1,925–2,308; ID3: amino acids 1,925–1,994; ID2: amino acids 2,049–2,091; ID1: amino acids 2,239–2,308. Mutant N-CoR fragments overlap the same regions but contain alanine substitutions within the ID C-terminal isoleucines (I/LXXII>I/LXXAA). The panel shows results from a single transfection experiment: each value is the average of luciferase activities determined from three individual wells and normalized to ß-galactosidase, the black bars from cells maintained in the absence of ligand, the gray bars in the presence of T3. B, Results of a similar transfection which contained 1 µg of VP16-TRß expression vector. Here, ID2 is seen to efficiently recruit TR.

 
Further examination revealed other aspects of behavior of the Gal-N-CoR fusion proteins. First, the N-CoR molecule that contained all three IDs recruited TRß-VP16 more efficiently than the isolated IDs, implying, once again, that the IDs cooperate in TRß binding. Second, TRß-VP16 showed strong T3-dependent release from all three IDs, but its release from ID1 was incomplete (see Fig. 5AGo, inset). This indicates that TRß/ID1 interactions possess both ligand-dependent and ligand-independent components. Despite these subtleties, our results suggest that all three IDs bind TRß in vivo, and that the binding of each ID motif to TRß recapitulates the pattern of N-CoR binding to TRß.

The IDs Are Composed of a Hydrophobic Core (I/LXXII) Along with Distinct Adjacent Sequences
While we originally identified the IDs on the basis of their resemblance to the coactivator NR box (LXXLL), it is clear that some mechanism allows N-CoR to bind preferentially to unliganded TRs and coactivators to bind preferentially to liganded TRs. We asked whether the different types of hydrophobic residues within the N-CoR motifs (Ile, I/LXXII) and the NR box motifs (Leu, LXXLL) were sufficient to account for this preference. We synthesized a mutant N-CoR ID1 peptide (ID1-LL), in which key Ile residues were substituted with Leu, so that it resembled a p160 NR box, and a mutant GRIP1 NR box2 peptide in which key Leu residues were substituted with Iles, so that it resembled an N-CoR ID motif (Fig. 6AGo).

Figure 6BGo confirms the observations, shown above, that the N-CoR ID1 peptide competed for the binding of unliganded TRß to N-CoR (left panel) and also shows that Box2, ID1-LL, and Box2-II did not. In parallel (right panel), the ID1 peptide failed to compete for the binding of T3-liganded TRß to GRIP1, but Box2 competed efficiently. Both ID1-LL and Box2-II did not. Thus, even Ile/Leu exchanges, which conserve the hydrophobicity of the peptides, abolished their respective abilities to compete for TRß/N-CoR and TRß/GRIP1 interactions. Moreover, the Ile/Leu exchanges failed to allow the N-CoR ID peptide to compete for TRß/GRIP1 interactions or the GRIP1 NR box peptide to compete for TRß/N-CoR interactions. This indicates that the binding preferences of N-CoR for unliganded TRß, and GRIP1 for liganded TRß, are not dictated solely by the identity of the conserved hydrophobic residues within their TRß binding motifs.

We next examined the sequence requirements for ID motif/TRß interaction. In the case of ID3, Ala substitutions within the core hydrophobic Ile residues (m1) abolished its ability to compete for TRß/N-CoR interactions (Fig. 7AGo). Likewise, Ala substitutions at different locations within the conserved ID3 N-terminal region either completely abolished (m2, m3, m5), or partially reduced (m4), the ability of ID3 to compete for TRß/N-CoR interactions. In the case of ID1, Ala substitutions within the Leu and Ile residues of the ID1 core, and a Leu residue within the conserved region of the ID1 C terminus, were sufficient to abolish competition, whether they were placed within the same peptide (m1), or within different peptides (m2–m4) (Fig. 7BGo). Substitution of the Glu residue within the ID1 core, which is conserved between N-CoR and SMRT, also abolished competition (m5), but substitution of Asp, which is not conserved, did not (m6). Thus, residues both within and outside of the hydrophobic core motifs are required for ID3 and ID1 peptides to compete for TRß/N-CoR interactions.

Finally, because the area of best conservation between N-CoR ID3 and N-CoR and SMRT ID2 lay N-terminal to the core motif and the area of best conservation between N-CoR and SMRT ID1 lay C terminal to the core motif, we prepared an ID3/ID1 hybrid peptide that contained both regions (Fig. 7CGo). Half-maximal competition was obtained with as little as 10–30 ng of hybrid peptide and complete competition was obtained with as little as 300 ng to 1 µg of peptide. In parallel, half-maximal competition for TRß/N-CoR interactions required 0.3–1 µg of the ID3 or ID1 peptides (see Fig. 2Go). Thus, the hybrid peptide competes for TRß binding to N-CoR more efficiently than the parental peptides, suggesting that the binding of the IDs to TRß is suboptimal and that the identity and position of the TR binding determinants outside the core motif differs between the IDs.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
N-CoR Contains Three ID Motifs, and a Hitherto Unrecognized ID (ID3) Is Important For TR Binding
In this study we examined the structural requirements for the interactions of the nuclear receptor corepressor, N-CoR, and the unliganded TRß. We, along with others (18, 19, 22, 24, 45, 46, 47, 48), noticed that two different regions of N-CoR that had been previously shown to bind nuclear receptors contained the conserved core sequence I/LXXII. However, unlike other groups, we found three of these ID motifs, rather than two. Several lines of evidence indicated that all three IDs bind to TRß. First, peptides corresponding to each ID motif compete for TRß/N-CoR interactions in vitro. Second, TR bound to short N-CoR fragments that overlapped each of the IDs. Third, mutation of the individual ID motifs reduces, and mutations of any two of the three ID motifs abolishes, N-CoR binding to TRß in vitro. Fourth, each ID motif plays a role in the dominant negative activity of an N-CoR C-terminal fragment in vivo. Finally, all three IDs act as bait for a TRß-VP16 fusion protein in two-hybrid assays. The previously unrecognized motif, ID3, shows TRß binding activity that is good as, or more potent than, ID1 and ID2 in each of these TRß binding assays. We therefore conclude that the C-terminal region of N-CoR contains three separate receptor IDs and that the previously unrecognized domain (ID3) is important for TRß binding.

Our data also indicate that isolated ID3 and ID1 are stronger than isolated ID2 and that any combination of two IDs is sufficient for strong TR binding, although the particular combination of ID3 and ID2 is preferred both in vitro (Fig. 3Go) and in vivo (Fig. 4Go). Nonetheless, there are some apparent discrepancies between assays that need to be resolved. For example, any pair of IDs was capable of binding TR in vitro, and isolated ID3 was sufficient for residual interactions with the TR (Fig. 3Go). However, the specific combination of ID2 and ID3 was required for N-CoR dominant negative activity in vivo, and other pairs of IDs or single IDs were not functional (Fig. 4Go). Furthermore, a GST-N-CoR fragment that only contains ID2 binds strongly to TR (Fig. 2CGo), even though isolated ID2 appeared to bind relatively weakly to TR in other assays. We suggest that these apparent discrepancies arise from differences in the sensitivity and linear range of different assays. Thus, glutathione-S-transferase (GST)-pull-down assays would detect relatively weak TR/N-CoR interactions, whereas the dominant negative interference assay would only detect strong TR/N-CoR interactions. In accordance with the notion that it is important to "tune" each assay for true quantitative comparisons, it was possible to detect differences between the IDs in mammalian two-hybrid assays in the presence of low levels of TR (Fig. 5AGo), but these differences disappeared when the TR was overexpressed (Fig. 5BGo).

Our studies also raise the question of whether N-CoR might contain yet more unrecognized IDs. We found that mutation of all three IDs completely abolishes the ability of full length N-CoR to bind to the TRß-LBD (not shown), suggesting that N-CoR lacks any unidentified strong TRß-LBD binding site. Thus, we presently favor the idea that N-CoR only contains three IDs. While we have not directly examined TR interactions with SMRT in this study, sequence comparisons failed to reveal an ID3 motif at a conserved position, or any other position, in the SMRT primary sequence, nor any obvious homologies between other surrounding ID3 residues and SMRT. We therefore suggest that SMRT only contains two IDs. We stress that this conclusion is based only on sequence comparisons and needs to be treated with caution. Strong interactions between SMRT and TR can be observed even in the absence of ID1 (46). While this binding may simply reflect the interactions of SMRT ID2 with TR, it is also possible that SMRT contains both an ID2 and additional sequences that bind to TR, and that these sequences are not recognizable from the sequence data alone.

The IDs Cooperate in TR Binding
Our studies indicate that the IDs cooperate in TR binding, both in vitro and in vivo, just as the p160 NR boxes cooperate in nuclear receptor binding (37). This cooperativity provides obvious advantages for the sensitivity of hormone response: binding of ligand to either receptor molecule within a homo- or heterodimer pair would result in complete corepressor release. The reasons why N-CoR would contain three distinct ID motifs, when two are sufficient for high-affinity TRß interactions, are less clear. TRß shows some preference for the pairing of ID3+ID2 (Figs. 3Go, 4Go, and 5Go), even though ID2 is relatively weak when examined in isolation (Figs. 3Go and 5Go). Other nuclear receptors show a preference for ID1 (45, 46, 47). This finding is reminiscent of the preference of different nuclear receptors for different p160 NR boxes (34, 35, 36, 37, 38, 39). Thus, one possible explanation for the presence of three IDs in the N-CoR C terminus is that they might allow for higher order interactions between separate nuclear receptors, e.g. a dimer and a monomer might be able to bind simultaneously to a single N-CoR molecule.

The IDs Are Composed of a Hydrophobic Core and Distinct Adjacent Sequences: Speculations on the Nature of the TR/N-CoR Interface
The same nuclear receptor hydrophobic cleft that mediates interactions with p160 coactivators also mediates interactions with corepressors (43, 44, 45, 46, 47, 49). Moreover, N-CoR and SMRT ID motifs contain the core consensus sequence I/LXXI/VI, which resembles the NR box consensus LXXLL. Despite these similarities, there must be some mechanism that allows only unliganded TRß to bind N-CoR and only liganded TRß to bind GRIP1. This preference cannot be accounted for by the nature of hydrophobic residues within the ID motif and NR box core motifs (Fig. 6Go). N-CoR and GRIP1 must therefore recognize distinct structural features of TRß that are regulated by ligand.

What are the mechanisms that N-CoR uses to recognize unliganded TR? Our results, and the results of others (45, 46, 47) suggest that the IXXII motif is important for recognition of the unliganded TR surface. However, homologies between N-CoR ID3, N-CoR ID2, and SMRT ID2 and N-CoR and SMRT ID1 extend beyond the conserved hydrophobic core (Fig. 1Go), and we, and others (45, 46, 47), found that mutations within these adjoining residues disrupted TR/N-CoR interactions. Thus, the IDs are composed of both the hydrophobic core and adjacent sequences. We also know that key residues for both coactivator and corepressor binding lie within the nuclear receptor hydrophobic cleft (43, 44, 45, 46, 47, 49) and that helix 12 forms a key part of the AF-2 surface (39, 40, 41), but is dispensable or inhibitory for corepressor interactions (19, 45, 46, 47, 50, 51). Thus, it is likely that the choice between coactivator and corepressor binding is regulated by ligand-dependent repositioning of helix 12. Indeed, in the unliganded RXR-LBD crystal structure (52), helix 12 extends away from the LBD rather than packing against the LBD as in the T3-liganded TR (33). We therefore propose that the conserved I/LXXII motif binds to a region of the cleft that lies under helix 12 and is exposed by the repositioning of helix 12 in the unliganded state and that TR/N-CoR interactions are stabilized by interaction of adjoining sequences from the IDs with the TRß surface.

Our results also give some indication that the structure of the N-CoR ID motif may be different from the structure of the NR box. First, the central hydrophobic residues of both motifs are important for TR binding, but adjoining ID sequences play a more important role than adjoining NR box sequences (39, 53). Second, while the NR boxes adopt a two- turn {alpha}-helical structure (39, 40, 41), the IDs are longer and the position of some of the TR binding residues is inconsistent with a location on one face of an extended {alpha}-helix. Lastly, one of the nonhydrophobic residues within the ID1 core motif (LDVII) is required for ID1 peptide competitions (Fig. 7Go). The nonhydrophobic residues of the NR box core do not play a role in TR binding (39). There may also be subtle differences in the way that the TR recognizes distinct IDs. N-CoR ID1, but not ID2 or ID3, shows weak residual hormone-independent binding to TR in two-hybrid assays. Moreover, the region of best homology between N-CoR ID3, ID2, and SMRT ID2 lies N-terminal to the core motif, and the area of best conservation between N-CoR and SMRT ID1 lies C terminal to the core motif (Fig. 1Go). Recombining these two areas of conservation creates an artificial hybrid peptide that competes very efficiently for TR/N-CoR interactions (Fig. 7CGo). Similar increases in TR binding efficiency have also been obtained when distinct IDs are recombined and tested in two-hybrid assays (45). Together, these results are consistent with the notion that distinct adjoining sequences from different IDs make distinct contacts with the TR surface. While we do not know what these contacts are, one possibility is that the Leu-Met pair at the C terminus of ID1 (KALM) could bind to the upper part of the hydrophobic cleft, with the LM in a similar position to the C-terminal leucines (LXXLL) of the NR box (39). This interaction could also have analogies to the way that estrogen receptor helix 12 folds into the same region of the cleft in the presence of antiestrogens (40, 54) and account for the weak ligand-independent component of TRß/ID1 interactions. It is likely that the full understanding of TRß/N-CoR interactions will require resolution of TRß/N-CoR crystal structures. We speculate that it may be possible to take advantage of synthetic peptides, including the ID3/ID1 hybrid described here, for this x-ray structural analysis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The N-CoR C terminus expression vector was derived from a pBK vector containing N-CoR sequences 1,629–2,453 (18). The dual mammalian/in vitro transcription-translation vector pSG5 (Stratagene, La Jolla, CA), was adapted with an oligonucleotide containing SacII and XhoI cloning sites. The N-CoR cDNA was isolated as a SacII/XhoI fragment and ligated into SG5. N-CoR C-terminal expression vectors containing mutated ID sequences were prepared by standard PCR- based site-directed mutagenesis (Quickchange, Stratagene). Oligonucleotides homologous to ID1, ID2, and ID3, but containing sequences that code for Ala substitutions within the Ile pair of each ID (L/IXXII>L/IXXAA), were used to generate the mutations. Double and triple mutants were prepared by subsequent rounds of mutagenesis. The vector encoding the GST-N-CoR ID3 fusion protein (amino acids 1,944–2,031) was prepared by introducing a SalI site after residue 2031 into a GST-N-CoR fusion protein encoding residues 1,944–2,453 (18). This allowed the deletion of residues 2,032–2,453. The GST-N-CoR ID2 fragment (amino acids 2,051–2,208) and ID1 fragments (amino acids 2,218–2,453) were isolated by PCR.

Gal-N-CoR expression vectors were derived from the pM expression vector for the yeast Gal4 DNA-binding domain (CLONTECH Laboratories, Inc. Palo Alto, CA). N-CoR fragments were amplified by standard PCR methods. All 5'- oligonucleotides contained an EcoRI site for cloning. For Gal-N-CoR/1,925–2,308 the 3'-oligonucleotide contained a SalI site. The others were generated with a 3'-oligonucleotide that contained a HinDIII site. Similar Gal-N-CoR expression vectors with mutated ID sequences were generated using pSG5-N-CoR ID triple mutant as a template. GAL-RE-TK-luciferase was prepared by cloning a double-stranded oligonucleotide containing two copies of the GAL4 dimer binding site upstream of the fusion reporter gene containing the Herpes simplex virus thymidine kinase promoter (-109/+48) linked to the firefly luciferase gene.

The following plasmids have been previously described: GST-GRIP1 (563–1,121) (37), GST-TRß (55), Gal-RE-e1b- luciferase (37), cytomegalovirus (CMV)-TRß (32). The following were gifts: GST-N-CoR fusions 1,944–2,453 and 1,744–2,453 from Dr. M. Lazar (University of Pennsylvania School of Medicine, Philadelphia, PA), VP16-TRß from Dr. R. Evans (University of California San Diego, San Diego, CA), Gal-TRß from Dr. D. Moore (Baylor College of Medicine, Houston, TX), actin-ß-galactosidase from Dr. M. Garabedian (New York University, New York, NY).

Protein-Protein Interaction Assays
Labeled proteins, peptides, and GST fusion proteins were prepared as previously described (37, 39). Peptide sequences were as follows, ID1:- N-ASNLGLEDIIRKALMGSFDD-C; ID2:- N-RTHRLITLADHICQIITQDFARNQ-C; ID3 N-RGKTTITAANFI-DVIITRQIASDK-C; ID3/1 hybrid peptide:- N-RGKTTITAA NFI- EDIIRKALMGSFDD-C.

Cell Culture and Transfections
Chicken embryo fibroblasts (CEF, UCSF Cell Culture Facility) were grown in DMEM/F-12 Ham’s modified mix, without phenol red, supplemented with 10% iron- supplemented newborn calf serum (Sigma, St. Louis, MO) and pen-strep. CEF cells were transfected, by electroporation (56), with 2 µg reporters, 1 µg of Gal fusion protein expression vector or CMV vector control and, where indicated, 5 µg of N-CoR or pSG5 control or 50 ng of TRß-VP16. After electroporation, the cells were resuspended in medium containing 10% T3-depleted newborn calf serum. Luciferase and ß-galactosidase activities were measured, using standard assays (Promega Corp., Madison, WI; and Tropix, Bedford, MA) at 36–48 h. Individual transfections (containing data from triplicate wells) were repeated three to six times.


    ACKNOWLEDGMENTS
 
We thank Dr. R. Evans (University of California, San Diego) for communicating unpublished results and for providing plasmids, Dr. M. Lazar (University of Pennsylvania School of Medicine, Philadelphia, PA), Dr. D. Moore (Baylor College of Medicine, Houston, TX) and Dr. M. Garabedian (New York University, New York, NY) for providing plasmids, Amber Boast for technical assistance, and Dr. F. Schaufele, Dr. R. Fletterick, Dr. R. Price, and R. Wagner (University of California, San Francisco) and Dr. B. Darimont (University of Oregon) for advice and helpful discussions.


    FOOTNOTES
 
Address requests for reprints to: Dr. Peter Kushner, Metabolic Research Unit 1119 HSW, University of California San Francisco School of Medicine, San Francisco, California 94143-0540. E-mail: kushner{at}itsa.ucsf.edu

Supported by NIH Grants DK-32129 and CA-30913 to P.J.K. Peter J. Kushner is a shareholder and Director of KaroBio AB, a company with commercial interests in this area of research. John D. Baxter has proprietary interests in, and serves as a consultant to and deputy director of, KaroBio AB.

Received for publication May 22, 2000. Revision received August 28, 2000. Accepted for publication August 30, 2000.


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