TR Surfaces and Conformations Required to Bind Nuclear Receptor Corepressor

Adhirai Marimuthu1, Weijun Feng, Tetsuya Tagami, Hoa Nguyen1, J. Larry Jameson, Robert J. Fletterick, John D. Baxter2 and Brian L. West1

Metabolic Research Unit (A.M., W.F., H.N., J.D.B., B.L.W.) and Departments of Biochemistry and Biophysics and Cellular and Molecular Pharmacology (R.J.F.), University of California San Francisco, San Francisco, California 94143; Center for Endocrinology, Metabolism, and Molecular Medicine (J.L.J.), Northwestern University Medical School, Chicago, Illinois 60611; and Clinical Research Institute, Center for Endocrine and Metabolic Diseases (T.T.), Kyoto National Hospital, Kyoto 612-8555, Japan

Address all correspondence and requests for reprints to: Dr. Brian L. West, Molecular Biology Department, Plexxikon, Inc., 91 Bolivar Street, Berkeley, California 94710. E-mail: bwest{at}plexxikon.com


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Residues of the TR that are critical for binding the nuclear receptor corepressor (N-CoR) were identified by testing more than 100 separate mutations of the full-length human TRß that scan the surface of its ligand binding domain. The primary inferred interaction surface overlaps the surface described for binding of p160 coactivators, but differs by extending to a novel site underneath which helix 12 rests in the liganded TR, rather than including residues of helix 12. Nonconservative mutations of this surface diminished binding similarly to three isolated N-CoR receptor interaction domains (RIDs), but conservative mutations affected binding variably, consistent with a role for this surface in RID selectivity. The commonality of this surface in binding N-CoR was confirmed for the RXRs and ERs. Deletion of helix 12 increased N-CoR binding by the TR modestly, and by the RXR and ER to a much greater extent, indicating a competition between this helix and the corepressor that regulates the extent of corepressor binding by nuclear receptors. When helix 12 was deleted, N-CoR binding by the ER was stimulated by tamoxifen, and binding by the TR was stimulated by Triac, indicating that helix 12 is not the only feature that regulates corepressor binding. Two additional mutationsensitive surfaces were found alongside helix 1, near the previously described CoR box, and above helix 11, nearby but separate from residues that help link receptor in dimers. Based on effects of selected mutations on T3 and coactivator binding, and on results of combined mutations of the three sites on corepressor binding, we propose that the second and third surfaces stabilize TR unliganded conformation(s) required for efficient N-CoR binding. In transfection assays mutations of all three surfaces impaired the corepressor-mediated functions of unliganded TR repression or activation. These detailed mapping results suggest approaches for selective modulation of corepressor interaction that include the shape of the molecular binding surface, the competitive occupancy by helix 12, pharmacological stimulation, and specific conformational stabilization.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR HORMONE RECEPTORS comprise a family of related proteins that regulate transcription. Prototypical receptors bind both DNA and hormone and can cause both positive and negative changes in gene expression, depending upon the target gene promoter. These receptors are single polypeptide chains containing an amino-terminal domain, a centrally placed DNA-binding domain (DBD), and a C-terminal ligand-binding domain (LBD) (1). Several coregulatory proteins interact with nuclear receptors and contribute to gene-regulatory effects (2). Coactivators are recruited to the receptors in response to agonist binding and can participate in gene activation or repression, depending on the response element encountered. By contrast, corepressors mediate the functions of unliganded receptors to repress the basal activity of promoters that contain TR binding sites, but also to stimulate the basal activity of certain promoters such as that for TSH (3).

Corepressors like nuclear receptor copressor (N-CoR) (4), also called RIP13 (5), and silencing mediator of retinoid and thyroid receptor (SMRT) (6), also called TRAC (T3 receptor associating cofactor) (7), were identified by their binding to the TR and RAR, but binding of varying strength has been detected to a number of different nuclear receptors including the RXR (5), VDR (8), PPAR (9, 10), ER (11, 12), PR (13, 14, 15), and the orphan receptors RevErb (16, 17), chicken ovalbumin upstream promoter transcription factor 1 (18), DAX-1 (19), steroidogenic factor-1 (20), and RVR (17). To understand corepressor function it is important to determine how they interact with receptors. Central to this is to define site(s) that bind these proteins. Initially it was proposed that the corepressor binds to a CoR box, which includes helix 1 of the TR LBD based on the observation that deletion of this helix abolished N-CoR binding to the TR (4). This notion received further support by the observations that combined mutation of three conserved residues (A228, H229, and T232 AHT) within the CoR box region and of a residue preceding helix 1 (P214 in the hTRß) blocked binding of N-CoR and SMRT (4, 6, 7). However, the AHT and P214R mutations are of residues that are buried in the liganded TR structure (21), and these mutations and deletion of helix 1 may affect corepressor binding through conformational changes near to or distant from the helix 1 region.

Alterations of nuclear receptors outside helix 1 have also been reported to diminish binding of corepressors. In the TR these include deletion of the helix 11 (22) and mutations located in residues of helices 3, 4, and 5 that are surface exposed in the ligand-bound structure (23, 24). Evidence for a role of helices 3 and 5 has also been reported for the orphan receptors RevErb and RVR (17) and helix 11 in RevErb (16). Hormone binding stabilizes particular conformations of helix 12 relative to helices 3 and 5 (21), and because mutations of helices 3 and 5 also diminish binding to corepressors, it has been postulated that conformational changes in helix 12 could also play a role in blocking corepressor binding when the receptor binds ligand (23, 24). In further support of the notion that helix 12 can affect corepressor were the observations with several nuclear receptors that deletion (25) or mutation (10) of helix 12 increases corepressor binding. However, the boundaries of the corepressor interaction surface have not been described, and the precise structural role of helix 12 in destabilizing corepressor binding has not been elucidated. Helix 12 has mostly been considered to be passive in unliganded nuclear receptors and to serve its main role in formation of the coactivator-binding surface. However, it is also possible that this helix is important in the unliganded state.

Two receptor interaction domains (RIDs) have been described within both SMRT and N-CoR (5, 16, 22, 26), and a third has been described for N-CoR (27). All of these contain motifs of IxxII that are important for binding (23, 24, 25, 27). The separate RIDs exhibit specificity in their binding to different nuclear receptors (18, 28), but the basis for this specificity is unknown. The IxxII motifs are likely present within larger protein structural domains that bind to the receptors. Differences between these domains outside the consensus sequences could dictate specificity for interacting with nuclear receptors, although there is no direct evidence for this idea. It is also unclear to what extent the separate RIDs recognize the same or different surfaces on TR or other nuclear receptors.

In this study we applied scanning surface mutagenesis to identify TR surfaces that interact with three fragments of N-CoR, each of which contains one of the RIDs. We introduced mutations over the entire surface of the TR LBD, using the x-ray structure of the hormone-bound TR LBD (21) as a guide for placing mutations. Even though corepressors bind unliganded TRs, we cautiously anticipated that use of the liganded TR LBD structure would be suitable, because the available evidence suggests that most of the overall fold of unliganded and liganded structures would be similar. In comparative studies we used x-ray structures of the unliganded human RXR (hRXR{alpha}) LBD (29) and the human ER{alpha} (hER{alpha}) LBD bound to the mixed-antagonist 4-hydroxytamoxifen (30), to place mutations in these receptors. We used these mutated receptors in glutathione-S-transferase (GST) pull-down assays to identify the surface regions important for binding to N-CoR, and we used a selection of the vectors encoding mutated TRs for mammalian cell transfection to study the effect of the mutations on the TR unliganded functions.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The TR LBD surface (Site 1) That Binds N-CoR Overlaps the Coactivator-Binding Surface, but Extends Underneath Helix 12
To define the corepressor-binding site, more than 100 different full-length hTRßs, each with single-point mutations distributed over the TR LBD solvent-accessible surface, were assayed for in vitro binding to GST-N-CoR fusion proteins. In most cases, a charged Arg or Lys was chosen to replace each native TR residue, but native Arg or Lys residues were mutated to Ala. All areas of the TR LBD surface were sampled, and surface areas where mutational effects were observed were characterized further by mutating neighboring residues more densely. Binding of the [35S]-labeled TRs were compared using three different N-CoR fragments linked to GST. These fragments separately contained RIDs I and II described previously (5), as well as a third receptor-binding N-CoR fragment (RID III) described more recently (27).

The TR area in which mutations had the strongest effects on N-CoR binding was on the surface formed by helices 3 and 5, overlapping the surface previously shown to bind coactivators (31). We refer to this as Site 1. Mutations T277R, I280K, V284R, K288A, I302R, and C309K all showed decreased in vitro binding by N-CoR (Fig. 1AGo). By contrast, mutations of residues of helix 12 that form part of the coactivator-binding surface (L454R and E457K) did not decrease binding to N-CoR, indicating that, unlike the case with coactivator, the N-CoR-binding surface does not include the outside of helix 12. Further, a natural mutation of the TR that deletes helix 12 [F451X (32)] increased TR binding to N-CoR by about 2.5-fold for both the RIDs I and II (Fig. 1AGo). Thus, it appears either that N-CoR does not contact the residues of helix 12 or that such contact does not appreciably stabilize the interaction.



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Figure 1. A TR LBD Surface (Site 1) That Overlaps the Coactivator-Binding Surface, but Extends Underneath Helix 12, Selectively Binds All Three RIDs of N-CoR

A, Binding of a selection of [35S]-labeled hTRß mutants to mouse N-CoR protein fragments containing RIDs I (2,231/2,321, solid), II (2,034/2,114, shaded), or III (1,888/2,031, open) in GST pull-down assays. Binding is expressed as the percentage of binding observed for the WT hTRß in the same assay (typically 12% of the input for RIDs I and II and 30% for RID III). Results are the average ± SD of duplicates and are representative of at least two experiments. B, As for panel A, except a selection of mostly conservative mutants was examined. C, Space-filling models of TRß LBD showing the inferred corepressor-binding surface. Residues that bind N-CoR normally after mutation are shaded gray. Mutation-sensitive residues are colored by residue type: hydrophobic, green; polar, orange; basic, blue; and acidic, red. Computer graphics prepared using MidasPlus (UCSF Computer Graphics Laboratory) (53 ). Left panel, View based on the TR{alpha} LBD structure (21 ), which contains the complete helix 12 (H12, line), but in which the terminal two amino acids (E460 andD461) present on WT hTRß are unstructured and therefore not visible. Right panel, Model of the natural F451X mutant, in which 11 residues (451–461) are absent and therefore show the portion of the corepressor-binding surface obscured by helix 12 in the ligand-bound state. The corepressor-binding surface is comprised of a cluster of hydrophobic residues (I280, V283, V284, I302, and C309) bordered by polar (T277 and T281) and charged (K288 and K306) residues. The TR mutations tested and found not to diminish binding to N-CoR include (with approximate locations): Helix 1: E213R, D216A, E217R, E220R, K223A, T224R, E227R, V230R, A231R; Helix 2: Q235R, S237R, K240A, Q241R, K242E; Strand 1: R243A; F245K; Loop: V256R; Helix 3: F269A, H271R, K274E, T281R, D285A, K289A; Helix 4: M292K; Helix 5: E298R, E299A; Helix 6: R316H; Strand 2/3: E324R; Strand 4: M334K; Helix 7: R338W; Helix 8: V348R, M358A; Helix 9: D382R, P384R; Helix 10: A387R, V389K, E390R, E393R, L400R, H413R; Helix 11: V414K; H416R, W418K, K420A, L422R, M423R, K424A, T426R, D427A, M430R, A433R, C434R, S437R, L440R, V444R; Loop: E449R, F451A; Helix 12: L454R, L456R, E457K. These views summarize experiments of panels A and B and also experiments not shown.

 
Two of the residues that form the inferred corepressor-binding surface, I280 and C309, lie mostly underneath helix 12 and are solvent inaccessible in the liganded TR-LBD structure, and a third, T277, is partially obscured. Each of these residues would be predicted to be fully solvent exposed when helix 12 is deleted, but because placement of helix 12 in the unliganded TR is unknown, it is possible that mutation of these residues might interfere with corepressor indirectly by pushing helix 12 to an abnormal position. Therefore, as a control we examined effects of the I280K and C309K mutations when helix 12 is deleted and observed decreased corepressor binding with these double mutations as well (Fig. 1AGo, I280K/F451X and C309K/F451X). The finding that residues obscured by helix 12 in the liganded TR contribute to the binding surface provides support for the role hypothesized for helix 12 in destabilizing corepressor binding by demonstrating a mechanism: helix 12 and corepressor compete for interactions with the same residues.

The inferred corepressor-binding surface is partially obscured in the x-ray structure model of the liganded TR LBD (Fig. 1CGo, left panel), and therefore it is more easily viewed when the 11 C-terminal residues are not displayed, so as to show a structure that might occur in the natural deletion mutation, F451X (Fig. 1CGo, right panel). This binding surface is relatively small and is comprised of a cluster of hydrophobic residues I280, V283, V284, I302, and C309) bordered by polar (T277 and T281) or charged (K288 and K306) residues. The portion of this surface that overlaps with the coactivator-binding surface includes residues V284, K288, I302, and K306 (31).

Surface Structure of Site 1 Regulates RID Selectivity
Helices 3 and 5 are part of a region of homology within the nuclear receptor family named "the signature motif" (33). The fact that residues from these helices appear to contact corepressor suggests that a large number of receptor family members may bind corepressors. Binding of wild-type (WT) TR occurs to all three N-CoR RIDs, but selectivity of other nuclear receptors for separate corepressor RIDs has been reported (18, 28). The basis for such selectivity is unknown. We investigated the effect of surface shape changes on selectivity by testing effects of conservative mutations in which residues of helices 3 and 5 are substituted with amino acids present in other nuclear receptors. Although binding of the WT TR occurred to each of the three RIDs, differential effects were found within this set of conservative mutants, either specifically increasing (T281I, T281L, T281V, V284I, and I302V) or decreasing (T281L, T281Q, T281V, V284I, and I302V) binding to one of the individual RIDs (Fig. 1BGo). Other mutants affected binding to all N-CoR RIDs similarly, resulting in molecules with decreased (I280M, V283M, V284A, I302A, I302M, C309A, and C309W) or little changed (V283I and L305I) binding (Fig. 1BGo). Because the tested mutations are local and conservative, these results suggest that variations in the shape of the interaction surface of different nuclear receptors regulate their ability to bind corepressors and to define different modes of binding.

Ligand Regulation of N-CoR Binding When Helix 12 Is Deleted
As demonstrated previously (25), we found that deletion of the C-terminal helix of human RXR{alpha} (equivalent to helix 12 of the TR) results in a large increase in N-CoR binding (Fig. 2Go, A and C). Strong binding occurred to N-CoR RID I, but very little binding occurred to RID III and almost no binding occurred to RID II (Fig. 2AGo), results that are consistent with previous reports (28). Mutations of RXR residues equivalent to those forming the coactivator-binding surface of TR helix 3 have been shown to decrease corepressor binding (25), and we extended this finding to show that mutation of a residue of the RXR (W305), whose equivalent in the hormone-bound TR (C309) would be buried by helix 12, markedly decreases N-CoR binding (Fig. 2CGo). Thus it appears that a surface of RXR similar to that of the TR is recognized by N-CoR and that N-CoR binding to this surface is regulated by the presence of helix 12 even in the absence of ligand.



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Figure 2. N-CoR Also Binds to Site 1 of the RXR and ER, and N-CoR Binding Is Increased by Ligand When Helix 12 Is Deleted in the ER and TR

Pull-down experiments examining the binding of labeled receptors to mouse N-CoR protein fragment containing RIDs I (2,231/2,321, solid) or II (2,034/2,114, shaded), or III (1,888/2,031, open). Binding is expressed as the percent of input labeled receptor. Results are the average ± SD of duplicates and are representative of at least two experiments. The control binding to GST is 0.3% (not shown). A, Binding of [35S]-labeled hRXR{alpha} deletion mutant spanning residues 126–447, containing the DBD and LBD, but lacking the N terminus and H12 (labeled DBD-LBD{Delta}H12). In all cases, duplicate samples are performed to allow the average values to be quantified after phosphorimaging. B, Binding of [35S]-labeled hER{alpha} deletion mutant spanning residues 250–536, containing the DBD and LBD, but lacking the N terminus and H12 (labeled DBD-LBD{Delta}H12). TAMOX, - or +, indicates the absence or addition of 10 µM tamoxifen during the binding reaction. C, Binding of [35S]-labeled hRXR{alpha} deletion mutants spanning residues 126–462, containing the DBD and LBD (labeled WT) or residues 126–447, which lack the C-terminal helix of the LBD (labeled {Delta}H12). The W305K mutation alters the hRXR{alpha} residue equivalent to residue C309 in the TR Site 1. D, Binding of [35S]-labeled hER{alpha} deletion mutants spanning residues 250–595, containing the DBD and LBD (labeled WT), or residues 250–536, which lack the C-terminal helix of the LBD (labeled {Delta}H12). The I358R, K362A, and V376R mutations alter the hER{alpha} residues equivalent to residues V284, K288, and I302 in the TR Site 1. TAMOX, - or +, indicates the absence or addition of 10 µM tamoxifen during the binding reaction. E, Binding of [35S]-labeled hTRß deletion mutants spanning residues 202–461, containing the LBD (labeled WT), or the 202–450 mutant, which lacks the helix 12 (labeled {Delta}H12). TRIAC, - or +, indicates the absence or addition of 50 µM Triac during the binding reaction.

 
Similar to the RXR, binding of the human ER{alpha} could be obtained using N-CoR RID I but not RID II (Fig. 2Go, B and D), but good binding occurred with RID III (Fig. 2BGo). Furthermore, binding of RID I to the ER was enhanced by deletion of helix 12 (Fig. 2DGo). However, binding of RID I to the helix 12-deleted ER also required addition of the mixed antagonist, tamoxifen (Fig. 2BGo). This ligand stimulation was not observed for binding to RID III (Fig. 2BGo). Three mutations of the ER (I358R, K362A, and V376R) showed diminished binding by the helix 12-deleted ER (Fig. 2BGo), thus confirming that N-CoR binding likely occurs to a surface similar to Site I of the TR. These three mutations are located in the portion of Site 1 where corepressor and coactivator binding overlap. A fourth mutation of ER (W382K), which would occur in the part of Site 1 that is buried by the hormone-bound helix 12, also abolished N-CoR binding (data not shown), but ER W382 likely is also important for the binding of tamoxifen (30). The ligand requirement for N-CoR RID I binding by the ER in the absence of helix 12 implies that mechanisms in addition to a conformation change in helix 12 exist for regulation of corepressor binding. Presumably, ligand binding causes conformation changes in parts of the nuclear receptor in addition to helix 12.

We found that the TR can also be regulated by ligand-induced conformational mechanisms. Although addition of Triac blocks binding of N-CoR to the WT TR LBD that contains helix 12, it stimulates binding of N-CoR to the helix 12-deleted TR LBD (Fig. 2EGo). This effect required a high concentration of Triac, consistent with the expected weak binding affinity of Triac for the helix 12-deleted TR. We determined that the F451X TR LBD binds with a dissociation constant (Kd) of 100 nM in saturation radioreceptor binding assays with [125I]T3, and that this binding is competed more effectively by T3 compared with reverse T3, and not competed at all by tamoxifen (data not shown). Therefore, ligand-induced stimulation of corepressor binding is likely a general property of nuclear receptors, suggesting that pharmacological approaches may be more generally useful than previously appreciated for regulating corepressor functions via the stimulation of corepressor recruitment by nuclear receptors.

Mutations in Two Additional Surfaces of the TR LBD Influence N-CoR Binding
To investigate the previously defined CoR box (4) region, we mutated every surface residue on helix 1, and assayed these for N-CoR binding. Confirming the previous report (4), simultaneous mutation of three buried residues of helix 1 (A228, H229, and T232, AHT) was defective in binding N-CoR RIDs I and II (Fig. 3AGo). Most of the surface mutations of helix 1 had no effect on N-CoR binding, although one surface mutation located at the beginning of helix 1 (W219K, Fig. 3CGo) had impaired binding to RIDs I and II (Fig. 3AGo), and RID III (data not shown). This suggests there is little contribution to N-CoR binding by most of the helix 1 surface but indicates the region near W219 may regulate binding.



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Figure 3. A TR LBD Surface Forming a Cavity Along Helix 1 (Site 2) Contains a Cluster of Mutation-Sensitive Residues Affecting N-CoR Binding

Binding of a selection of [35S]-labeled hTRß mutants to mouse N-CoR protein fragments containing RIDs I (2,231/2,321, solid) or II (2,034/2,114, shaded), or III (1,888/2,031, open) in GST pull-down assays. Binding is expressed as the percentage of binding observed for the WT hTRß in the same assay. Results are the average ± SD of duplicates and are representative of at least two experiments. A, Results with mutations located on the surface of helix 1 and the loop connecting helices 1 and 2 and the AHT triple mutation of the buried helix 1 residues A228G, H229G, and T232G. B, Results with mutations located on helices 6 (V319K) and 8 (S361R), the loop between helices 8 and 9 (N364R and D366R), and helices 9 (D367R) and 10 (Y406K, Y409K, and R410A), which cluster to form surface Site 2. C, Space-filling model of Site 2. The mutation-sensitive residues affecting N-CoR interaction are colored by atom type (green for hydrophobic, W219, V319, and Y406; blue for basic, R410; red for acidic, D366 and D367; and orange for polar, S361 and N364). Residues unresponsive to mutation are shown in gray (see Fig. 1CGo). The position of helix 1 (H1) is indicated by a line. This view summarizes experiments of panels A and B and experiments not shown. Site 2 is mixed nonpolar and polar in character. Computer graphics prepared using MidasPlus (53 ).

 
Examination of the region near residue W219 revealed eight additional mutations, V319K, S361R, N364R, D366R, D367R, Y406K, Y409K, and R410A, that impaired TR binding to N-CoR (Fig. 3BGo), although not as strongly as did some mutations of Site 1. Together with W219, these residues define a concave surface running alongside helix 1 (Fig. 3CGo), which we refer to as "Site 2." Site 2 contains hydrophobic, polar, and charged residues that derive from distant locations in the TR linear sequence. Because mutation of the surface-exposed residues of helix 1 that lie over the buried AHT mutation do not affect N-CoR binding but do define a border of Site 2, effects of the AHT mutation could be due indirectly to effects on Site 2.

Amino acids on helix 11 have also been implicated in corepressor binding, because deletions of the terminal portion of helix 11 affect both N-CoR and SMRT binding (16, 22). Helices 10 and 11 participate in formation of the surface for TR-TR homodimerization and TR-RXR heterodimerization, and mutation L422R has the strongest dimer destabilizing effect (34). Most surface mutations of helices 10 and 11, including L422R, and a buried mutation of helix 11 (L428R) did not diminish binding to N-CoR RIDs I and II (Fig. 4AGo). Two mutations on the surface of helix 11 (K424R and D427A) caused increases in binding to N-CoR RID II (Fig. 4AGo). Two mutations of helix 10 (Q396R and L401R) caused weak (average 25%) decreases, and one mutation of helix 11 (R429A) caused stronger (60%) decreases in N-CoR binding (Fig. 4AGo). The Q396R, L401R, and R429A mutations were also demonstrated to decrease the binding to N-CoR RID III (data not shown). Whereas this surface influences N-CoR binding, the structural pattern is discontinuous rather than clustered (Fig. 4BGo). Residue R429, the most sensitive to mutation, has its side chain pointed above helix 11 toward residue Q396 of helix 10. We refer to these two residues as "Site 3." Residue L401 does not contact residues 396 or 429; its side chain points above helix 10 toward where the DBD could be oriented in the full-length TR. In Fig. 4BGo, residues that give rise to decreased binding to all N-CoR RIDs are colored by residue type. Residues K424 and D427, which increase binding to N-CoR RID II, are colored gray; these two residues are directed below helix 11, toward helix 8 (Fig. 4BGo).



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Figure 4. A TR LBD Surface Above Helix 11 (Site 3) Contains Residues Mutation Sensitive for N-CoR Binding

A, Binding of a selection of [35S]-labeled hTRß mutants in helices 10 and 11 to mouse N-CoR protein fragments containing RIDs I (2,231/2,321, solid) or II (2034/2114, shaded) in GST pull-down assays. Binding is expressed as the percentage of binding observed for the WT hTRß in the same assay. Results are the average ± SD of duplicates and are representative of at least two experiments. B, Space-filling model of Site 3. The mutation-sensitive residues affecting N-CoR interaction are colored by atom type (green for hydrophobic, L401; blue for basic, R429; and orange for polar, Q396). Residues unresponsive to mutation are shown in gray (see Fig. 1CGo). Computer graphics prepared using MidasPlus (53 ). Two of the unresponsive mutations are labeled: W418 is the residue examined as a control in Fig. 6Go, and L422 is the residue known to block homo- and heterodimerization (34 ). The basic residue K306 is part of surface Site 1. The positions of helices 10 (H10) and 11 (H11) are indicated by lines. This view summarizes experiments of Fig. 4AGo and also experiments not shown.

 
Loss of Corepressor Binding Correlates with Loss of Unliganded Inhibition at Activating Thyroid Response Elements (TREs) and Unliganded Activation at the Inhibitory TSH Promoter
The predominant function reported for N-CoR bound to unliganded TR is repression of gene promoters that are activated by T3. To test effects of the mutations on this activity, we transfected CV-1 cells with vectors expressing each mutant along with a synthetic gene containing two copies of the F2 (35) TRE linked to the thymidine kinase (TK) gene promoter, with luciferase (LUC) coding sequences as the reporter. Cells were incubated in culture medium deficient in thyroid hormones. As compared with the level of expression using the vector without TR coding sequences (CMX), the TR-WT decreased expression of this reporter gene by 80%, and a selection of TR mutants quantitatively varied in this effect (Fig. 5AGo). When the amount of unliganded repression is plotted against the amount of N-CoR binding for each TR mutant, a significant correlation is obtained, with r2 values of 0.52, 0.59, and 0.62 for RIDs I, II, and III, respectively (Fig. 5CGo). The mutations tested include those from Sites I, II, and III, and therefore likely indicate that the sensitivity to mutations for in vitro N-CoR binding have functional significance in vivo. One mutation (L422R) had strong effects in vivo without a correlated defect in the in vitro binding assay. Because this mutation affects receptor dimerization (34), this could suggest this unliganded function requires in vivo formation of a TR dimer or some other association at this site.



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Figure 5. The TR Mutants That Have Decreased N-CoR Binding Exhibit Specific Correlated Defects in Unliganded TR Functions

A, Unliganded repression at an activating TRE. A reporter construct consisting of two copies of the F2 element from the chicken lysozyme gene (35 ) cloned upstream of the TK -109/+45 promoter, linked to the LUC gene was examined after transfection into HeLa cells. Compared with the empty CMX vector, cotransfection of a vector expressing the WT hTR causes repression of LUC expression. Cotransfection with vectors expressing mutant hTRß (as indicated) resulted in variable loss of basal repression. Additional mutations tested, but found to have no effect on unliganded repression include E213R, D216A, E217R, E220R, T224R, Q235R, K240A, Q241R, F245K, L266K, T281R, D285A, K289A, P291R, M292K, C294K, E295R, C298R, E299A, M334K, Y389K, H413R, and Y414K. B, Unliganded activation at a negatively regulated promoter. A reporter consisting of the TSH{alpha} gene promoter linked to LUC was transiently transfected into TSA-201 cells. Compared with the empty CMX vector, cotransfection of a vector expressing the WT hTR causes activation of LUC expression. Cotransfection with vectors expressing mutant hTRß (as indicated) resulted in variable loss of basal activation. C, Correlation between relative effects of mutations on binding to N-CoR RIDs I, II, and III on relative unliganded repression at the activating promoter (F2)2-TK109-LUC (from experiment of panel A, but with the activation of WT set to 100 and each mutant normalized accordingly). The correlation for each RID was significant, with r2 values of 0.52, 0.59, and 0.62 for RIDs I, II, and III, respectively. D, Correlation between relative effects of mutations on binding to N-CoR RIDs I and II and on relative unliganded activation at the repressing promoter TSH{alpha}-LUC (from experiment of panel B, but with the activation of WT set to 100 and each mutant normalized accordingly). The correlation for each RID was significant, with r2 values of 0.44, 0.66, and 0.56 for RIDs I, II, and III, respectively.

 
Corepressor-TR complexes are also thought to activate promoters whose expression is inhibited by liganded TRs, such as the promoter for the TSH gene (3). With the TSH promoter, overexpression of N-CoR increases unliganded activation, and the P214R and the AHT mutants, which block N-CoR binding, abolish unliganded activation (3). We examined functional properties of TR mutants in TSA-201 cells using the hTSH{alpha} promoter linked to LUC. The unliganded TR increased LUC expression from this promoter by 2.5-fold, and mutations that impair N-CoR binding also were deficient in this unliganded activation (Fig. 5BGo). There was a significant overall correlation between defects in binding and this function (r2 values of 0.44, 0.66, and 0.56 for RIDs I, II, and III, respectively), although again, the L422R mutation was an outlier (Fig. 5DGo). There was also a significant correlation when the relative TRE repression and relative TSH promoter activation results for the various mutants were compared (r2 value of 0.81, plot not shown), consistent with both functions requiring interactions with corepressor. Thus defects in N-CoR binding have functional importance in vivo, and regulation of the TSH{alpha} promoter by unliganded TR may require receptor dimers or some other partner using this surface.

Specificity of Conformation Defects by Mutation of Sites 2 and 3
It has been suggested that the AHT triple mutation of buried residues of helix 1 may decrease corepressor binding indirectly through a change in conformation of the unliganded TR (36). The AHT mutations are near the mutation-sensitive Site 2 alongside helix 1, and therefore mechanism(s) for decreasing N-CoR binding may be similar for all these mutations. Residues of Sites 2 and 3 are on the surface of the hormone-bound TR LBD (21), making it less likely that mutations at these sites cause strong global structural changes. However, no unliganded TR structure has been determined, and the disposition of the residues of Sites 2 and 3 could be different in the unliganded conformation required for corepressor binding. Therefore it is not obvious whether Sites 2 and 3 represent alternate direct binding sites for N-CoR or whether the effects of mutations at these sites involve structural changes that indirectly affect N-CoR binding to Site 1.

To test whether mutational defects in N-CoR binding might be mediated by global structural changes, we examined abilities of the AHT triple mutation and TR mutations from Site 1 (I280M and I302R), Site 2 (W219K), and Site 3 (R429A) to function in other capacities (Fig. 6Go). Parts of Sites 1, 2, and 3 occur in the TR LBD subdomain that does not bind hormone; therefore, as a comparative control, mutation W418K of a partially buried residue of helix 11 was created to intentionally destabilize this same subdomain. The W418K mutation (displayed separately in Fig. 6Go) did not diminish binding to N-CoR (Fig. 4AGo). All the mutants showed good binding of [125I]T3 (Fig. 6AGo), although all exhibited a decreased affinity relative to WT TR. Therefore, each mutation appears to have some global effect. The worst [125I]T3 binder was the AHT mutation with a Kd of 600 pM.



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Figure 6. Receptor Conformation Likely Does Regulate N-CoR Binding, but the Effects Are Specific; Control Mutations Can Perturb the Receptor and Not Affect N-CoR Binding

Six different TR mutants, including the I280M and I302R mutations of Site 1, the AHT and W219K mutations of Site 2, and the R429A mutation of Site 3, were compared in their effects to perturb receptor functions. The W418K mutation of helix 11 (Fig. 4BGo) is included as an example of a mutation that does not affect N-CoR binding. A, Binding of [125I]T3 to full-length TRs produced by in vitro translation. Results are expressed as the percentage of Ka observed for the TR WT. For each receptor mutant, the Kd values (pM ± SD) are: WT (35 ± 5), AHT (296 ± 30), W219K (71 ± 9), I280M (240 ± 13), I302R (51 ± 6), R429A (138 ± 12), W418K (137 ± 15). B, Binding of [35S]-labeled hTRß mutants to GRIP1 (residues 563–1,121). GST pull-down results are expressed as the percentage of binding observed for the WT hTRß in the same assay. Binding was performed in 30 µM T3, and the TR WT binding was 77% of input. Results are the average ± SD of duplicates and are representative of at least two experiments. C, T3 activation after transfection of TR WT and mutants vectors into HeLa cells. The reporter consisted of two copies of the F2 element from the chicken lysozyme gene (35 ) cloned upstream of the TK -109/+45 promoter, linked to the LUC gene. Results are expressed as the percentage of fold activation by 10 µM T3 of LUC expression for the WT (47-fold). Empty CMX vector fold activation was 1.5-fold (data not shown). Results are the average ± SD of triplicates and are representative of two experiments.

 
Effects of these selected mutations on binding to GR-interacting protein 1 (GRIP1) were also assayed (Fig. 6BGo), using excess hormone (50 µM). The I302R mutation, which participates in the p160 coactivator-binding surface (31), showed only background binding. By contrast, the I280M and I280K (data not shown) mutations that lie underneath helix 12 in the liganded TR conformation, and which strongly block N-CoR binding to Site 1 (Fig. 1BGo), showed almost no defect in GRIP1 binding. These results are consistent with a model in which hormone binding causes I280 to be covered by helix 12, so that the corepressor binding Site 1 is blocked, and the coactivator binding surface is created. Thus, mutation of the buried part of the corepressor surface allows the best discrimination between corepressor and the coactivator binding by the TR. Mutations from Sites 2 and 3 and the control mutation all showed similar partial defects in GRIP1 binding, implying that these mutations have similar global defects in recruitment of coactivator. A similar pattern was obtained for hormone activation by these mutant receptors in transfection assays (Fig. 6CGo); the I302R mutant was inactive, but other mutants from Sites 2 and 3 and the control W418K mutant, although capable of good activation, were similarly slightly deficient. Thus, the mutations in Sites 2 and 3 exhibit indications of global structural changes, but no more so than the control W418K mutation that had no effect on N-CoR binding.

Mutation of Site 2 Alters N-CoR Recognition by the TR LBD
Mutation of Site 1 diminished N-CoR binding to levels close to the control GST protein, whereas the mutations of Sites 2 and 3 only partially blocked binding. We asked whether binding that remains in the W219K (Site 2) mutant could be blocked by secondary mutations of Site 1 and found the secondary Site 1 mutations to be surprisingly ineffective (N-CoR RID I; Fig. 7Go). Although low binding was observed for some of the single Site 1 mutants, these same mutants showed higher binding when present as double mutants coupled with the W219K (Site 2) mutation. Thus, mutation of residue W219 may cause a conformation change that affects Site 1 recognition. A search was made for LBD residues outside of Site 1 that would more effectively block N-CoR binding in combination as double mutants with W219K. The most effective of these secondary mutations was R429A, which nearly completely blocked the remaining binding (Fig. 7Go and data not shown). When the Site 1 mutations were combined with the R429A (Site 3) mutation, there was either no change (V284R), a smaller increase (I280M), or increased (I302R) binding, indicating that recognition of Site 1 by R429A is somewhat altered, but mostly intact.



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Figure 7. Mutations in Sites 2 and 3 Alter the N-CoR Recognition of Site 1

Binding of full-length [35S]-labeled hTRß mutants to a mouse N-CoR protein fragment containing RID I (2,231/2,321) in GST pull-down assays. The first bar represents binding by the WT hTRß (typically 12% of the input), set to 100%. The remaining bars represent binding by TRs with combined mutations from Site 1 (I280M, V284R, and I302R), Site 2 (W219K), and Site 3 (R429A), as indicated, with each expressed as the percentage of binding observed for the WT hTRß. Results are the average ± SD of duplicates and are representative of at least two experiments. N-CoR binding is increased for all three Site 1 mutants when combined with the Site 2 mutation, and N-CoR binding is increased for the I302R mutant when combined with the Site 3 mutation.

 
Representative mutants of Sites 1, 2, and 3 were also tested in the context of the isolated TR LBD for effects of helix 12 deletion and Triac addition (Fig. 8Go). Deletion of helix 12 caused a 5-fold increase in binding to N-CoR RID I, larger than the 2.5-fold increase observed with full-length receptor, implying that the N terminus and/or DBD affect binding. These effects may be conformational because we could find no evidence of direct binding to the N terminus or DBD using a mutation (A234X) that deletes the LBD (data not shown). In the LBD the R429A (Site 3) mutation showed little of the defect observed in the full-length TR, again implying that the N terminus and/or DBD influence corepressor binding. In the LBD the W219K (Site 2) mutation remained defective as for the full-length, and deletion of helix 12 allowed little improvement, consistent with an effect of Site 2 mutations to alter the recognition of Site 1. The I302R (Site 1) mutation diminished binding of the LBD to background levels, and deletion of helix 12 showed no improvement, consistent with a requirement of residue I302 in contacting N-CoR RID I.



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Figure 8. Differential Effects on N-CoR Binding of Helix 12 Deletion and Ligand Addition in the TR-LBD Mutated at Sites 1, 2, and 3

Binding of [35S]-labeled hTRß LBD mutants to a mouse N-CoR protein fragment containing RID I (2,231/2,321) in GST pull-down assays. The first bar represents binding by the WT hTRß LBD, expressed as percentage of labeled input. The remaining bars indicate binding by TR LBDs, either WT or mutated in Sites 1 (I302R), 2 (W219K), or 3 (R429A), and with deletions of helix 12 (mutation to F451X) and treated with 50 µM Triac, as indicated.

 
Addition of Triac to the TRs with helix 12 deleted had different effects for the three mutant types. The R429A mutant was fractionally increased as for the WT TR, consistent with a lack of effect of this mutant on binding of the LBD to RID I. The I302R mutant was little changed by Triac addition, which would be predicted if residue I302 is required for RID I binding. However, binding to the W219K mutant was increased 6-fold, indicating that a portion of the decrease observed with this mutation is due to a conformational effect that can be corrected by ligand binding.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we examined the mutational sensitivity of surface residues of the TR LBD for impairment of in vitro binding of full-length TR to each of the three RIDs of N-CoR. We mutated residues representative of the entire LBD surface using the hormone-bound rat TR{alpha} LBD (21) as a structural model. The results imply a complex pattern of interaction between N-CoR and the TR, as reflected by the fact that we found not one, but three sites of mutational sensitivity (Sites 1, 2, and 3, Fig. 9Go). Site 1 overlaps partially the surface defined for coactivator binding (31) but also extends underneath where helix 12 rests in the ligand-bound conformation. Site 2 is separated from Site 1 by helices 1 and 3 and runs along the side of helix 1; Site 3 is separated from Site 1 by the turn between helices 9 and 10 and runs between helices 10 and 11. Mutations in Site 1 nearly abolished binding, from which we infer that Site 1 contacts corepressor directly. Mutations in Sites 2 and 3 had partial effects, which could indicate that they form weaker contacts to corepressor. Alternatively, Sites 2 and 3 could function to define and maintain the presentation and stability of the unliganded TR conformation required for corepressor binding.



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Figure 9. Model of the Nuclear Receptor LBD Indicating the Surfaces and Conformation Changes That Appear to Regulate Corepressor Binding

Ribbon diagram of the TR LBD. The residues of helices 3 and 5 comprising the inferred N-CoR interaction Site 1 are blue at their Ca atoms. Two different conformations of helix 12 are indicated; the conformation in the hormone-bound TR structure (21 ) is cyan, whereas the conformation modeled from the unliganded PPAR structure (40 ) is green. In the hormone-bound structure some of the residues of Site 1 are buried, whereas in the unliganded model, Site 1 is fully exposed. The mutation-sensitive surface residues of Site 2 occur behind helix 1 on the right side of this view. The mutation-sensitive surface residues of Site 3 occur above helix 11 on the left side of this view.

 
The current studies suggest that the mechanism of hormone-dependent release of corepressor involves direct binding competition between helix 12 and corepressor. This conclusion is derived from the observation that residues buried by helix 12 in the hormone-bound conformation form part of the corepressor-binding surface of Site 1. In this model binding of the ligand induces a conformation that favors occupancy by helix 12. This mechanism explains previous studies that suggested a link between helix 12 conformation and hormone-dependent corepressor release based upon the correlation of increased protease attack of helix 12 in TRs with point mutations that block the hormone-dependent release of corepressor (37), the conversion of the TR into a constitutive repressor by deletion of part of helix 12 (38), and the disruption of corepressor binding by surfaceexposed residues near helix 12 (23, 24, 25).

The data demonstrate that helix 12 can regulate corepressor binding by nuclear receptors in the unliganded state through its affinity for Site 1. This is based on the observation that deletion of helix 12 in the unliganded TR increases corepressor binding by N-CoR RIDs I and II and, in the RXR, has a profound effect on corepressor binding (25), confirmed here. In some receptors, competition by helix 12 might be strong enough to preclude the use of corepressors as coregulators in the cell. However, it is conceivable these nuclear receptors interact with corepressors in vivo if mechanisms exist that reduce the competitive effect of helix 12, such as the ability of helix 12 to be drawn away from one receptor by other receptor molecules in a complex (39), or in vivo proteolysis of helix 12.

Although binding of corepressor to Site 1 requires displacement of helix 12 from its liganded conformation, the mapping of Site 1 indicates that the extent of required displacement is not large. Complete extension of helix 12, as reported for unliganded RXR (29), is probably not required. Sliding of helix 12 down along helix 3, as reported for unliganded PPAR (40), would provide sufficient exposure, as illustrated in Fig. 9Go. Helix 12 could also be uncoiled in the absence of ligand, because, even when stabilized by ligand, the helix 12 of crystallized TR shows a B-factor higher than the average for the entire LBD (21). It has been predicted (24) that the IxxII motifs within corepressors that interact with receptors (23, 25) extend longer as amphipathic helices, compared with the LxxLL motifs of coactivators. This prediction is consistent with our observation that the corepressor-binding surface extends beyond that for coactivator.

The residues of Site 1 are homologous between members of the nuclear receptor superfamily, deriving from the signature motif sequences between helices 3 and 5 (33). This observation suggests that corepressors might dock to Site 1 of various nuclear receptors more ubiquitously than previously thought. This is supported by our observation that mutation of the residues of Site 1 in the ER and RXR, including those under helix 12, also impaired corepressor binding. Interestingly, previous mutation of this region in the orphan nuclear receptor RevErb was found to impair corepressor binding (17). This receptor lacks helix 12, and the role of helix 12 in restricting corepressor binding was not derived from this study. Site 1 may be used generally, so long as it has a sufficient exposure to, and affinity for, the corepressor.

Despite similarities between different nuclear receptors in Site 1, receptors also show selectivity for the separate corepressor RIDs. Our results indicate that the shape of the Site 1 surface is one determinant of this selectivity. Whereas the helix 12 deleted unliganded RXR and tamoxifen-liganded ER bound RID I, they did not bind RID II, demonstrating that features other than helix 12 define selectivity. By contrast, TR exhibited equal binding to N-CoR RIDs I and II. We further observed that conservative changes in the residues of TR Site 1, such as occur naturally between different nuclear receptors, affected binding uniquely to N-CoR RIDs I, II, and III, either decreasing or increasing the affinity of receptor binding. Thus, the residues of Site 1 partially determine the selectivity. However, none of the single point mutations of Site 1 conferred selectivity as large as that observed for the RXR or ER, and no mutation reversed the preference of TR to bind better to RID III than I or II, indicating that either more than one mutation of Site 1 or contacts to or changes outside of Site 1 are required to dictate the complete selectivity observed when the natural receptors are compared. When previous reports on receptor selectivity are compared with the current results, it should be noted that differences often exist in the boundaries defined for the LBD-interacting RIDs of N-CoR (27), and that additional contacting surfaces of receptors and corepressors may yet be elucidated in the future (41) that could influence binding results in an assay-specific fashion.

Our study adds insights about the CoR box, which was originally reported to be the TR site for interactions with corepressors (4). The current results extend those of previous reports (24, 42), which found that mutations of the surface residues of helix 1 lying directly above the CoR box did not affect corepressor binding, indicating that the CoR box, as postulated, does not bind corepressor. We extended these studies by testing mutations at every surface residue of helix 1 and found one mutation (W219K) that diminished corepressor binding. Residue W219 on helix 1 forms part of surface Site 2. Thus, the previous mutations supporting the existence of the CoR box may have perturbed the receptor structure in a way that affects Site 2.

The mechanisms for decreased corepressor binding due to mutations of Sites 2 and 3 remain unclear. Some residues of Sites 2 and 3 are unique to TR, but it is notable that residues D366 and R429 are relatively conserved within some nuclear receptor family members (33), consistent with roles in either supporting the TR structure or providing protein-binding interfaces. The Site 2 and 3 mutants showed only small defects in both ligand binding and coactivator binding that are comparable to defects of another TR mutant (W418K) that is not defective in N-CoR binding. These results are similar to previous reports that natural TR mutants found in patients with the syndrome of Resistance to Thyroid Hormone (43, 44, 45, 46, 47) generally show some ligand-binding defects, but no loss of corepressor binding. Thus we conclude that if the effects of mutations at Sites 2 or 3 on N-CoR binding are due to structural perturbations, these perturbations are specific. More evidence supporting a mechanism whereby Site 2 and 3 mutations are required to specifically maintain the unliganded conformation essential for presentation of Site 1 as the primary N-CoR interaction surface was revealed by the observations that mutations of Sites 2 or 3 can alter the mutational sensitivity of Site 1, and that under some conditions ligand can reverse the effects of the Site 2 mutation.

Although conformation mechanisms likely contribute to the corepressor-binding defects of Sites 2 and 3, a role for these surfaces as direct contact sites should also be considered. Site 2 is separated from Site 1 by one-third the circumference of the LBD (~30 Å), whereas Site 3 is closer (~10 Å distance between residues R429 and C309). The TR LBD has a molecular mass of 32 kDa, whereas the fragments of N-CoR used to make the fusion proteins for GST pull-down experiments are 9.1, 8.8, and 16.4 kDa for RIDs I, II, and III, respectively. It seems unlikely the smaller N-CoR fragments could contact Sites 1 and 2 simultaneously without making contacts to the intervening residues of helices 1 and 3 simultaneously. By contrast, it seems possible for the N-CoR fragments to contact Sites 1 and 3 simultaneously. Such a model would be consistent with our finding that the N-CoR binding activity remaining in the partially defective W219K (Site 2) mutant requires the integrity of residue R429A (Site 3) more than it requires the residues of Site 1. The N-CoR binding activity remaining in the R429A (Site 3) mutant is abolished by either the W219K (Site 2) mutation or by some of the Site 1 mutations. Thus, the Site 3 mutations could act through both conformational and direct binding mechanisms.

Conceivably, Sites 2 and 3 could be interaction sites for other molecules that modulate N-CoR binding through allosteric mechanisms. The [35S]-labeled TRs used for the binding were produced using reticulocyte lysates, and therefore numerous candidates for putative allosteric regulators would have been present in our pull-down assays. The effects of the Site 2 and 3 mutations primarily appear not to be mediated through effects on the N terminus or DBD, because deletion of these domains generally did not remove the effects of the Site 2 or 3 mutations. However, the R429A (Site 3) mutation failed to affect the binding to RID I in the context of the LBD, so some role of the N terminus or DBD cannot be ruled out.

Effects of the mutations on the in vivo functions of the full-length unliganded TR were assayed using both T3-activated and T3-repressed promoters. All mutations of Sites 1, 2, and 3 that diminished in vitro N-CoR binding also caused correlated defects in unliganded repression and unliganded activation, and these two functions were also highly correlated when compared across the set of mutants. These results imply that the binding defects measured in vitro are also responsible for defects in corepressor binding and action measured in vivo. Interestingly, one mutation that did not affect N-CoR binding in vitro, L422R, strongly diminished the unliganded TR in vivo functions. L422R strongly affects stability of both TR dimers and heterodimers with RXR (34). N-CoR binding by DNA-bound TRs has been reported to require homodimers (48). Thus it is possible that the in vivo defects observed with L422R indicate that formation of a TR dimer is essential for corepressor to function at these promoters. As the TSH promoter contains no strong binding site for the TR, the actions of such a putative dimer would likely occur off the promoter DNA in this case.

N-CoR binding by the helix 12-deleted TR and ER was surprisingly stimulated by ligand binding. N-CoR binding by the ER with helix 12 deleted was barely detectable unless tamoxifen was bound. In the case of TR with helix 12 deleted, the agonist Triac stimulated N-CoR binding by 30%. These observations provide evidence that influences on the receptor extrinsic to helix 12 can regulate N-CoR binding. Tamoxifen releases ER from its associated chaperone proteins that are present in the reticulocyte lysates used to produce the radiolabeled receptors (49). Thus, this release could free the receptor from inhibitory influences of these proteins and thereby stimulate N-CoR binding. However, the unliganded TR is not known to associate with proteins other than corepressors. Thus, an alternative explanation is that tamoxifen and Triac could stabilize the LBD in a manner that favors N-CoR binding. The result that binding by the W219K/F451X mutant to N-CoR RID I was stimulated 6-fold by Triac suggests the defect caused by the W219K (Site 2) mutation can be appreciably corrected by addition of ligand. Thus there could be a continuum of dynamic states of the TR with different capacities of corepressor binding. In such a model, specifically located mutations, such as those of Site 2, could perturb the receptor conformation to disable binding, but other influences, such as ligand binding or, conceivably, allosteric effects with other domains of the TR or with TR dimerization partners, could stabilize conformations to improve binding.

The current studies also suggest pharmacological approaches to modulate selectively nuclear receptor functions through regulating corepressor binding. Ligands that bind to the combined coactivator and corepressor surface might impair or stimulate both corepressor and coactivator action. Other ligands that bind to the corepressor-binding surfaces might selectively impair or stimulate corepressor function. The fact that mutations such as C309K and I280M have a much more profound affect on corepressor than coactivator binding, despite their localization in a region where they could impair packing of helix 12, implies that such an approach might be successful. Ligands that bind in the hormone-binding cavity might also be used for selective regulation. In addition to the tamoxifen-stimulated binding of corepressors, it has been reported that different retinoids vary in their capacity to release corepressors (50). These results, coupled with our finding that ligands can enhance corepressor binding by helix 12-deleted TRs, suggest that in some contexts ligands that range in effect from corepressor recruitment to release for the same receptor may be possible.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutagenesis
The pCMX vector was used for expression of the full-length hTRß and hRXR{alpha}, and the pSG5 vector was used for expression of the full-length human ER{alpha} (hER{alpha}). Mutations within these nuclear receptor-encoding sequences were created using the methods described in the quick-change kit (Stratagene, La Jolla, CA). The same mutagenesis methods were used in protocols to engineer the Escherichia coli expression vectors encoding fragments of N-CoR and TR. For the GST-N-CoR RID I, the sequences encoding amino acids 2,231–2,321 were cloned in frame to the C terminus of the 26-kDa GST protein of the GEX-2T vector (Pharmacia Biotech, Piscataway, NJ) between modified linker sequences encoding His-Met and encoding Val-Ala-His-His-His-His-His-His. An NdeI site encodes the His-Met and a SalI site encodes the Val-Ala. The appropriate NdeI and SalI sites were introduced into the mouse N-CoR cDNA sequence using sequential mutagenesis steps. The same approach was used to make the GST-N-CoR RID II (encoding residues 2,034–2,114) and GST-N-CoR RID III (encoding residues 1,888–2,031). Verification of the target sequences as well as the flanking sequences was performed using both Sequenase kits (Stratagene), and by automated DNA sequence (UCSF Cancer Center Sequencing Facility).

GST Pull-Down Assay
The vectors encoding the GST-N-CoR-His fusion proteins of the three N-CoR RIDs were used to transform the bacterial strain BL21, using selection with ampicillin. One-liter cultures were grown using 2x LB medium to an OD600 of 0.6, and expression was induced with room temperature shaking by addition of 0.5 mM isopropyl-ß-D-thiogalactopyranoside, for 3 h. Cells were harvested by centrifugation and after storage at -80 C, extracted with 20 ml TST buffer [50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20, with 0.02% monothioglycerol and 20 µM phenylmethylsulfonyl fluoride (PMSF) added]. The lysate was centrifuged for 1 h at 17,000 rpm in a JA20 rotor (Beckman Coulter, Inc., Palo Alto, CA), and the lysates were mixed for 1 h at 4 C with 0.4 ml Talon Metal affinity resin (CLONTECH Laboratories, Inc., Palo Alto, CA) in the presence of 2 mM imidazole. Beads were collected by low-speed centrifugation and washed twice batchwise with 10 ml buffer S (50 mM NaPO4, pH 8, 300 mM NaCl, and 10% glycerol, with 2 mM imidazole added). Beads were then transferred to a 5 ml Econo column and washed with 2 ml buffer S containing 2 mM imidazole. The GST-N-CoR-His proteins were then eluted with 2 ml buffer S containing 100 mM imidazole. Metal affinity-purified GST-N-CoR His-tagged fragment (1.5 mg) was mixed for 1 h with 150 µl of glutathione Sepharose (Pharmacia Biotech), then washed three times with 1 ml TST buffer with 1 mM dithiothreitol added, and brought up to 150 µl of a 50% slurry, typically containing 5 mg/ml protein.

For studies of binding to the GST-N-CoRs, [35S]-Metlabeled nuclear receptors were made using 1 µg of each receptor-encoding plasmid in 20 µl reactions using TNT kits (Promega Corp., Madison, WI). For each experiment, equivalent amounts (15 fmol) of labeled receptors were diluted in 150 µl binding buffer (20 mM HEPES, pH 7.9, 150 mM KCl, 25 mM MgCl2, 10% glycerol, 0.1% Triton X-100, 0.1% NP-40, and 0.01% dithiothreitol), of which duplicate 10-µl portions (20% of the binding reaction inputs) were quantified by SDS-PAGE and phosphorimaging (Molecular Dynamics, Inc., Sunnyvale, CA). Binding buffer (100 µl) containing 4 µl of the glutathione Sepharose-bound GST-N-CoR-His was added to duplicate 50-µl portions of the diluted labeled receptors and mixed by mechanical inverting for 2 h at 4 C. The beads were washed three times with 1 ml binding buffer with brief microcentrifugation in between. The washed beads were boiled 3 min in SDS sample buffer and quantified by SDS-PAGE and phosphorimaging.

Mammalian Cell Culture and Transfection
The unliganded functions of the TR were analyzed by comparing the effects of a CMX vector encoding the WT and mutant receptors with the effects observed for the empty parent CMX vector. For unliganded repression studies, a LUC reporter plasmid was cotransfected that contained two copies of the F2 TRE from the chicken lysozyme gene (35) linked to the herpes simplex virus TK promoter sequences extending from -109 to +48 of the transcription start site. For these studies of the F2-TK109 promoter, CV-1 cells were harvested from DMEM-H21 supplemented with 10% FBS and plated at 7,000 cells per well. The cells were washed 2 h later with 2 ml PBS, and new medium was added containing serum stripped of thyroid hormone using Dowex AG X-8 ion exchange resin (Bio-Rad Laboratories, Inc., Hercules, CA) (51). One and one-half hours later, 0.5 ml suspension of DNA vectors precipitated with calcium phosphate was added [1.7 µg LUC reporter, 80 ng CMX, or CMX-TR expression vector, and 500 ng control ß-galactosidase vector]. After 15 h cells were washed twice with 2 ml DME-H21 and fresh DME-H21 containing stripped serum was added. After an additional 24 h, cells were harvested in 200 ml lysis buffer and assayed for LUC (kit from Promega Corp.) and ß-galactosidase (kit from Tropix, Inc., Bedford, MA) activities.

For unliganded activation studies, a reporter consisting of the TSH{alpha} gene promoter linked to LUC (3) was transfected by the calcium phosphate method (3) into TSA-201 cells and grown in DMEM (Nikken Biomedical Laboratory, Kyoto, Japan) with 10% Dowex resin-stripped FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). After exposure to the calcium phosphate-DNA precipitate for 8 h, DMEM with 10% Dowex resin-stripped FBS was added. Cells were harvested after 40 h for measurements of LUC activity.

For T3 activation studies, a LUC reporter plasmid was used containing two copies of a F2 thyroid response element linked to the herpes simplex virus TK -109/+48 promoter. HeLa cells were cultured and transfected as described previously (31).

T3 Binding Assay
Affinity measurements for 125I-T3 binding to hTRß mutant proteins produced by in vitro translation were performed as described previously (52).


    FOOTNOTES
 
This work was supported in part by a grant-in-aid for scientific research (no. 11671103) from the Ministry of Education, Japan (to T.T.), and by NIH Grants DK-53417, DK-51281, and DK-41842.

1 Current address for A.M., H.N., and B.L.W.: Plexxikon, Inc., 91 Bolivar Street, Berkeley, California 94710. Back

2 J.D.B. has proprietary interests in and serves as a consultant and Deputy Director to Karo Bio AB, which has commercial interests in this area of research. Back

Abbreviations: DBD, DNA-binding domain; GRIP, GRinteracting protein; GST, glutathione-S-transferase; h, human; LBD, ligand-binding domain; LUC, luciferase; N-CoR, nuclear receptor corepressor; RID, receptor interaction domain; SMRT, silencing mediator of retinoid and thyroid receptor; TK, thymidine kinase; TRE, thyroid response element; WT, wild type.

Received for publication February 26, 2001. Accepted for publication October 3, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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