A role for helix 3of the TRbeta ligand-binding domain in coactivator recruitment identified by characterization of a third cluster of mutations in resistance to thyroid hormone
1998; Springer Nature; Volume: 17; Issue: 16 Linguagem: Inglês
10.1093/emboj/17.16.4760
ISSN1460-2075
AutoresTrevor N. Collingwood, Richard Wagner, Clare Matthews, Roderick Clifton‐Bligh, Mark Gurnell, Odelia Rajanayagam, Maura Agostini, Robert J. Fletterick, Paolo Beck‐Peccoz, Walter Reinhardt, Gerhard Binder, Michael B. Ranke, Ad R. Hermus, R.-D. Hesch, John H. Lazarus, P. G. Newrick, Vernon Parfitt, P R Raggatt, Francis de Zegher, Krishna Chatterjee,
Tópico(s)Thyroid Disorders and Treatments
ResumoArticle17 August 1998free access A role for helix 3 of the TRβ ligand-binding domain in coactivator recruitment identified by characterization of a third cluster of mutations in resistance to thyroid hormone Trevor N. Collingwood Trevor N. Collingwood Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Richard Wagner Richard Wagner Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA Search for more papers by this author Clare H. Matthews Clare H. Matthews Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Rory J. Clifton-Bligh Rory J. Clifton-Bligh Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Mark Gurnell Mark Gurnell Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Odelia Rajanayagam Odelia Rajanayagam Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Maura Agostini Maura Agostini Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Robert J. Fletterick Robert J. Fletterick Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA Search for more papers by this author Paolo Beck-Peccoz Paolo Beck-Peccoz Istituto Clinico Humanitas, Milan, Italy Search for more papers by this author Walter Reinhardt Walter Reinhardt Abteilung fur Endokrinologie, Universitatsklinikum, Essen, Germany Search for more papers by this author Gerhard Binder Gerhard Binder Kinderklinik, Universitaet Tuebingen, Germany Search for more papers by this author Michael B. Ranke Michael B. Ranke Kinderklinik, Universitaet Tuebingen, Germany Search for more papers by this author Ad Hermus Ad Hermus Department of Endocrinology, University Hospital Nijmegen, The Netherlands Search for more papers by this author Rolf D. Hesch Rolf D. Hesch Seestrasse1, Konstanz, Germany Search for more papers by this author John Lazarus John Lazarus Department of Medicine, University of Wales College of Medicine, Cardiff, UK Search for more papers by this author Paul Newrick Paul Newrick Department of Medicine, Kidderminster General Hospital, Kidderminster, UK Search for more papers by this author Vernon Parfitt Vernon Parfitt Department of Endocrinology, Royal South Hants Hospital, Southampton, UK Search for more papers by this author Peter Raggatt Peter Raggatt Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UK Search for more papers by this author Francis de Zegher Francis de Zegher Department of Paediatrics, University of Leuven, Leuven, Belgium Search for more papers by this author V.Krishna K. Chatterjee V.Krishna K. Chatterjee Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Trevor N. Collingwood Trevor N. Collingwood Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Richard Wagner Richard Wagner Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA Search for more papers by this author Clare H. Matthews Clare H. Matthews Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Rory J. Clifton-Bligh Rory J. Clifton-Bligh Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Mark Gurnell Mark Gurnell Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Odelia Rajanayagam Odelia Rajanayagam Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Maura Agostini Maura Agostini Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Robert J. Fletterick Robert J. Fletterick Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA Search for more papers by this author Paolo Beck-Peccoz Paolo Beck-Peccoz Istituto Clinico Humanitas, Milan, Italy Search for more papers by this author Walter Reinhardt Walter Reinhardt Abteilung fur Endokrinologie, Universitatsklinikum, Essen, Germany Search for more papers by this author Gerhard Binder Gerhard Binder Kinderklinik, Universitaet Tuebingen, Germany Search for more papers by this author Michael B. Ranke Michael B. Ranke Kinderklinik, Universitaet Tuebingen, Germany Search for more papers by this author Ad Hermus Ad Hermus Department of Endocrinology, University Hospital Nijmegen, The Netherlands Search for more papers by this author Rolf D. Hesch Rolf D. Hesch Seestrasse1, Konstanz, Germany Search for more papers by this author John Lazarus John Lazarus Department of Medicine, University of Wales College of Medicine, Cardiff, UK Search for more papers by this author Paul Newrick Paul Newrick Department of Medicine, Kidderminster General Hospital, Kidderminster, UK Search for more papers by this author Vernon Parfitt Vernon Parfitt Department of Endocrinology, Royal South Hants Hospital, Southampton, UK Search for more papers by this author Peter Raggatt Peter Raggatt Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UK Search for more papers by this author Francis de Zegher Francis de Zegher Department of Paediatrics, University of Leuven, Leuven, Belgium Search for more papers by this author V.Krishna K. Chatterjee V.Krishna K. Chatterjee Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK Search for more papers by this author Author Information Trevor N. Collingwood1, Richard Wagner2, Clare H. Matthews1, Rory J. Clifton-Bligh1, Mark Gurnell1, Odelia Rajanayagam1, Maura Agostini1, Robert J. Fletterick2, Paolo Beck-Peccoz3, Walter Reinhardt4, Gerhard Binder5, Michael B. Ranke5, Ad Hermus6, Rolf D. Hesch7, John Lazarus8, Paul Newrick9, Vernon Parfitt10, Peter Raggatt11, Francis de Zegher12 and V.Krishna K. Chatterjee1 1Department of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ UK 2Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA 3Istituto Clinico Humanitas, Milan, Italy 4Abteilung fur Endokrinologie, Universitatsklinikum, Essen, Germany 5Kinderklinik, Universitaet Tuebingen, Germany 6Department of Endocrinology, University Hospital Nijmegen, The Netherlands 7Seestrasse1, Konstanz, Germany 8Department of Medicine, University of Wales College of Medicine, Cardiff, UK 9Department of Medicine, Kidderminster General Hospital, Kidderminster, UK 10Department of Endocrinology, Royal South Hants Hospital, Southampton, UK 11Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UK 12Department of Paediatrics, University of Leuven, Leuven, Belgium The EMBO Journal (1998)17:4760-4770https://doi.org/10.1093/emboj/17.16.4760 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Resistance to thyroid hormone (RTH) has hitherto been associated with thyroid hormone β receptor (TRβ) mutations which cluster in two regions (αα 310–353 and αα 429–461) of the hormone-binding domain and closely approximate the ligand-binding cavity. Here, we describe a third cluster of RTH mutations extending from αα 234–282 which constitute a third boundary of the ligand pocket. One mutant, T277A, exhibits impaired transactivation which is disproportionate to its mildly reduced ligand affinity (Ka). T3-dependent recruitment of coactivators (SRC-1, ACTR) by mutant receptor–RXR heterodimers was reduced in comparison with wild-type. Cotransfection of SRC-1 restored transactivation by T277A. In the TRβ crystal structure this helix 3 residue is surface-exposed and is in close proximity to residues L454 and E457 in helix 12 which are known to be critical for coactivator interaction, suggesting that they all constitute part of a receptor–coactivator interface. The transcriptional function of other mutants (A234T, R243W/Q, A268D, Δ276I, A279V, R282S) in this cluster correlated with their reduced Ka and they inhibited wild-type TRβ action in a dominant negative manner. DNA binding, heterodimerization and corepressor recruitment were preserved in all mutants, signifying the importance of these attributes for dominant negative activity and correlating with the absence of natural mutations in regions bordering the third cluster which mediate these functions. Introduction In keeping with other members of the nuclear receptor family, TRβ is a ligand-inducible transcription factor with a modular structure comprised of a central DNA-binding (C) domain flanked by a C-terminal (D/E/F) region that encompasses ligand binding, dimerization and hormone-dependent transactivation functions and an N-terminal (A/B) region implicated in ligand-independent transactivation (Mangelsdorf and Evans, 1995). In many target genes, TR interacts with regulatory DNA response elements typically consisting of hexanucleotide motifs oriented as direct, inverted or everted repeats. TR functions primarily as a heterodimer with the retinoid X receptor (RXR) but can function as a homodimer in some contexts (Lazar, 1993). In the absence of ligand, TR actively represses basal gene transcription by recruiting a complex containing corepressor proteins (SMRT or NCoR) (Chen et al., 1995; Hörlein et al., 1995) associated with a histone deacetylase (HDAC) via an intermediary factor (Sin3) (Heinzel et al., 1997; Nagy et al., 1997). Receptor occupancy by T3 results in dissociation of this corepressor complex and derepression, followed by recruitment of coactivators and associated cofactors (e.g. RIP140, SRC-1, TIF-2, CBP, ACTR, p/CAF) exhibiting histone acetyltransferase activity, as well as other associated factors, subsequently leading to transcriptional activation (Cavailles et al., 1995; Chen et al., 1997; Oñate et al., 1995; Chakravarti et al., 1996; Kamei et al., 1996; Voegel et al., 1996). Residues at the extreme C-terminus in many nuclear receptors have been shown to be critical for coactivator recruitment, and recent crystallographic studies indicate that these residues lie within a solvent-exposed amphipathic α-helix which may undergo a conformational change in response to ligand (Renaud et al., 1995). However, the role of other regions of the receptor which might play a part in this recruitment remains to be determined. Over 70 different natural TRβ mutations have been identified in the syndrome of resistance to thyroid hormone (RTH), the hallmark feature of which is elevated circulating free thyroid hormones (thyroxine, T4 and triiodothyronine T3) together with non-suppressed levels of thyroid stimulating hormone (TSH). These mutations all occur within the C-terminal ligand-binding domain (LBD) (Refetoff et al., 1993) and characterization of their properties has made a valuable contribution to the understanding of structure–function relationships in TR. With a few exceptions, a consistent abnormality with natural receptor mutants is a significant reduction in their ligand-binding affinity resulting in impaired hormone-dependent transcriptional activity (Meier et al., 1992; Collingwood et al., 1994). In addition, the mutant receptors inhibit the action of their wild-type counterparts in a dominant negative manner (Sakurai et al., 1990; Chatterjee et al., 1991), a property which is central to the pathogenesis of the RTH phenotype and consistent with their dominant mode of inheritance. This inhibitory effect is dependent on their ability to bind to DNA (Nagaya et al., 1992), heterodimerize with RXR (Nagaya and Jameson, 1993; Collingwood et al., 1994) and recruit corepressor (Yoh et al., 1997). These findings correlate with the observation that no natural RTH mutations have been identified in the DNA-binding domain (DBD) of TRβ and, moreover, that the majority of mutations occur in two clusters (αα 310–353; αα 429–461) wherein the intervening region contains a putative dimerization interface implicated in receptor interaction with RXR (Forman and Samuels, 1990). We have also described RTH mutants that interact aberrantly with transcriptional cofactors: a natural mutant, L454V, with relatively preserved ligand- and DNA-binding and dimerization functions (Collingwood et al., 1997) was impaired transcriptionally as a result of attenuated interaction with putative coactivators (RIP140, SRC-1); another mutant, R383H, was predominantly impaired for negative transcriptional regulation and hormone-dependent corepressor release (Clifton-Bligh et al., 1998). Here, we report a mutant (T277A) within a third natural mutation cluster (αα 234–282), which is impaired transcriptionally with reduced coactivator binding. We suggest that helix 3 of the LBD which encompasses this residue, together with helix 12, may constitute part of a wider receptor–coactivator interface. Other natural mutants in this cluster show variably impaired ligand binding and transcriptional function and inhibit wild-type TR action in a dominant negative manner, but retain DNA- and corepressor-binding and heterodimerization functions. These observations may explain the absence of naturally occurring mutations in receptor domains (α-helical extension of the DBD, signature motif, CoR box) bordering this third cluster. Results Genotype and clinical phenotype of RTH cases Thyroid function tests and receptor gene abnormalities in affected members of 14 different RTH families are shown in Table I. Most individuals exhibited biochemical abnormalities characteristic of RTH with elevated serum-free T4 and T3 levels together with an inappropriately normal TSH. Three patients (IV, IX, XIII.I) had undergone a previous subtotal thyroidectomy, disrupting the pituitary–thyroid feedback axis, resulting in variably elevated TSH levels with raised (IV, IX) or normal (XIII.I) serum-free T4 concentrations. Sequencing of the TRβ gene indicated that each affected individual was heterozygous for either a single nucleotide substitution corresponding to a missense mutation or, in one case, a three nucleotide in-frame codon deletion (Δ276I). Five novel mutations (A268D, Δ276I, T277A, A279V, R282S) have not been described previously in RTH. In addition, we have identified and characterized more extensively mutations (V264D, A234T, R243W, R243Q) which have been documented as case reports (Behr and Loos, 1992; Adams et al., 1994; Onigata et al., 1995; Pohlenz et al., 1996). In one instance (II), a de novo R243W mutation exists in the proband but not in either parent, supporting the hypothesis that these identical mutations have arisen independently in unrelated families. Table 1. Biochemical and genetic data from 14 RTH families Kindred Age/sex FT4 FT3 TSH Nucleotide change Codon changea Kab mean ± SEM I 13 F 26 13.8 3.0 985 GCC to ACC A234T 0.66 (0.04) I.I 45 F 28 12.6 2.0 985 GCC to ACC A234T IIc 7 M 27 12.0 11 1012 CGG to TGG R243W 0.19 (0.03) III 27 F 68 16.0 3.5 1012 CGG to TGG R243W III.I 67 M 28 9.5 0.4 R243W IVc 40 M 35 11.0 2.7 1012 CGG to TGG R243W IV.I 4 M 38 15.0 4.5 1012 CGG to TGG R243W V 70 F 28 9.5 0.5 1013 CGG to CAG R243Q 0.25 (0.03) VI 13 F 40 11.1 1.5 1013 CGG to CAG R243Q VI.I 47 M 49 9.7 0.9 1013 CGG to CAG R243Q VII 56 F 34 10.0 2.1 1013 CGG to CAG R243Q VIII 20 M 29 15.0 2.5 1076 GTT to GAT V264D IXc 40 M 31 16.6 10.4 1088 GCC to GAC A268D <0.2 X 20 M 55 20.6 2.1 1088 GCC to GAC A268D XId 7 F 32 2.1 1111-3 Δ ATC Δ276I BND XII 31 F 27 10.3 1.6 1114 ACA to GCA T277A 0.46 (0.04) XIII 26 F 27 9.7 1.5 1121 GCA to GTA A279V <0.2 XIII.Ic 30 F 17 7.4 14.9 1121 GCA to GTA A279V XIV 31 F 22 8.2 3.4 1131 AGA to AGT R282S 0.47 (0.04) XIV.I 37 M 29 13.5 3.3 1131 AGA to AGT R282S XIV.II 34 F 24 11.1 1.4 1131 AGA to AGT R282S XIV.III 60 M 20 10.0 3.4 1131 AGA to AGT R282S XIV.IV 10 F 33 15.4 2.9 1131 AGA to AGT R282S Normal range 9.0–20 pM 3.0–7.5 pM 0.4–4.0 mU/L 1.48 × 1010 (0.09) M−1 a Codon nomenclature based on a predicted protein sequence of 1–461 residues (Beck-Peccoz et al., 1994); b BND, binding not detected; c subtotal thyroidectomy; no thyroid hormone replacement therapy; d treated with TRIAC (interferes with FT3 assay). RTH mutant receptors show variably impaired transactivation function In keeping with their location in the TR LBD, the initial characterization of mutant receptors consisted of measurement of the ligand-binding affinities (Ka) of in vitro synthesized proteins. All showed variably reduced T3 binding, with a few (A279V, A268D, Δ276I) having affinities too low to quantitate (Table I). To evaluate their transcriptional properties, wild-type or mutant receptors were cotransfected with reporter genes containing one of three different types of thyroid response element (TRE) into JEG-3 cells with increasing concentrations of T3. All mutants were impaired transcriptionally on the direct repeat (MAL-tkLUC), everted repeat (F2-tkLUC) and palindromic (PAL-tkLUC) TREs (Figure 1A–C) and, as expected, those mutants with greatly reduced ligand binding (A279V, A268D, Δ276I) showed the greatest transcriptional impairment, demonstrating submaximal responses even in the presence of saturating concentrations (1000 nM) of T3. For most mutants with measurable ligand-binding affinities (A234T, R243Q, R243W, R282S), the extent of their transcriptional impairment when compared with wild-type receptor correlated negatively with their Ka (Figure 1D–F). However, a single mutant, T277A, exhibited submaximal transactivation on MAL-tkLUC and F2-tkLUC, despite saturating T3 concentrations (Figure 1A and B), but not on PAL-tkLUC (Figure 1C). Furthermore, the T3 concentration required for T277A to attain 50% wild-type receptor activity on the direct repeat and everted response elements was markedly discordant with its Ka (Figure 1D and E). This pattern of transcriptional impairment suggested that the T277A mutation might disrupt some function of the receptor other than ligand binding. Figure 1.(A–C) T3-dependent transcriptional activation by mutant thyroid hormone receptors on different configurations of TRE. JEG-3 cells were cotransfected with wild-type or mutant TRβ expression plasmids, along with the specified reporter construct (PAL-tkLUC, MAL-tkLUC or F2-tkLUC) an internal control plasmid (BOS-βgal), and incubated in the presence of increasing amounts of T3. Hormone-dependent activation was normalized against the internal control and expressed as the percentage of maximum wild-type receptor response. These data are the mean of at least three separate experiments performed in triplicate. The error for each point is <10%. (D–F) Correlation of transcriptional function of wild-type and mutant receptors with measurable hormone binding affinities (boxes: WT, A234T, R243W, R243Q, R282S; cross: T277A) with their respective Ka on direct repeat, everted and palindromic response elements. In each case, the concentration of T3 required to attain 50% of the wild-type maximal response [denoted as EC50 (wild-type)] is plotted against log Ka. Download figure Download PowerPoint Reduced transactivation by the T277A mutant is due to impaired coactivator recruitment The attenuated maximal transactivation attained by the T277A mutant on MAL-tkLUC and F2-tkLUC was analogous to the activation profiles on these reporters of a number of mutants of a proline residue (P453A/H/S/T) which precedes the C-terminal amphipathic α-helix of TR (Collingwood et al., 1994). Subsequent studies have shown that the P453S mutant receptor impairs hormone-dependent corepressor release to a greater extent than predicted from its reduced ligand-binding affinity (Yoh et al., 1997). Hence, we examined whether the impaired transcriptional function of T277A might also be accounted for by abnormal corepressor release. Wild-type TR dissociated from GST–SMRT in response to T3 with an EC50 of 4 nM (Figure 2). In contrast, the T277A mutant showed impaired ligand-dependent corepressor release such that its EC50 was 20 nM (Figure 2). However, the R282S mutant receptor, which has an almost identical Ka in comparison with T277A (Table I), yet transactivates fully (Figure 1A–C), showed a similar alteration in corepressor release (Figure 2). We conclude that the delayed corepressor release profiles exhibited by these two mutant receptors are in keeping with their reduced Ka and do not account for the diminished transcriptional function shown by T277A. Figure 2.Using a pull-down assay, bacterially expressed GST–C-SMRT was incubated with equal amounts of in vitro translated wild-type or mutant (T277A, R282S) TRβ in the presence of increasing amounts of T3. Wild-type TRβ (–○–), T277A mutant (●, hatched line) and R282S mutant (●, dashed line) dissociation was quantitated using a phosphorimager. Each point represents the percentage of receptor protein bound to SMRT relative to the maximum bound by that receptor. Data represent the mean ± SE of four separate experiments. Download figure Download PowerPoint We have shown previously that another RTH mutant with markedly impaired transactivation, L454V, exhibited reduced coactivator recruitment in vitro (Collingwood et al., 1997). Accordingly, we next examined the ability of wild-type or T277A mutant receptors to recruit coactivator proteins when bound to DNA as a heterodimer with RXR. Wild-type TR–RXR complexes, when bound to the direct repeat TRE, recruited glutathione S-transferase (GST) fusion proteins containing the central receptor-interacting domains of either SRC-1 (GST–SRC′; residues 570–780) or ACTR (GST–ACTR′; residues 621–821) in a T3-dependent manner (Figure 3A and B). In contrast, recruitment of GST–SRC′ by T277A–RXR heterodimers on this response element was diminished (Figure 3A), with maximal coactivator recruitment reduced to 60% that of wild-type TR when quantitated (data not shown). Hormone-dependent recruitment of GST–ACTR′ by T277A was also markedly impaired in comparison with wild-type (Figure 3B). These results suggest that reduced coactivator recruitment by T277A in vitro might account for its impaired transactivation in vivo. Indeed, we hypothesized that if impaired recruitment of endogenous coactivators was limiting the maximal transcriptional response of the T277A mutant, then overexpression of such factors might alleviate this defect. Accordingly, wild-type or T277A mutant receptors were cotransfected with Mal-tkLUC and increasing amounts (0–100 ng) of SRC-1 expression vector (Figure 4). Whereas the basal promoter (RSV) activity in mock-transfected cells or hormone-dependent activation by wild-type receptor were not affected significantly by co-expression of SRC-1, the transcriptional response of the T277A mutant was enhanced to wild-type levels. Figure 3.Differential recruitment of coactivators by wild-type TRβ and T277A when bound to DNA as a heterodimer with RXR. Using an electrophoretic mobility supershift assay, in vitro translated TR (wild-type or mutant T277A) and RXR were coincubated with the malic enzyme DR+4 TRE, along with either bacterially expressed GST–SRC′ (2.4 μg) (A) or GST–ACTR′ (0.6 μg) (B) in the presence of increasing concentrations of T3. Complexes were resolved by native PAGE. Download figure Download PowerPoint Figure 4.Restoration of T277A activity by overexpression of SRC-1. JEG-3 cells were cortransfected with 50 ng of RSV-TR wild-type or T277A mutant or empty vector, along with increasing amounts of pSG5-SRC1, as well as 500 ng of reporter MAL-TKLUC and 100 ng of the internal control Bosβgal. Empty pSG5 vector was added to maintain the amount of total DNA transfected. Cells were incubated in the presence or absence of 1 μM T3. Data are the mean ± SE of three separate experiments performed in triplicate and activity is expressed relative to wild-type T3-dependent activation at 100 ng SRC-1. Download figure Download PowerPoint Dominant negative activity of mutant receptors in the third cluster It has been suggested that mutant receptors inhibit the action of wild-type receptor in a dominant negative manner to manifest the RTH phenotype (Collingwood et al., 1994; Yen and Chin, 1994). We therefore examined the dominant negative potency of RTH mutants by co-expressing equal amounts of each with wild-type TR, again using three reporter genes containing different configurations of TRE in the presence of low (1 nM) or high (1000 nM) levels of T3 (Figure 5A–C). At 1 nM T3 all mutants showed marked dominant negative inhibition of wild-type receptor function on all three TREs. Reversal of this inhibition for some mutants (R243Q, R243W, R282S) at higher (1000 nM) T3 concentrations was in keeping with their ability to attain wild-type transactivation levels with saturating levels of ligand. Other mutants (A268D, Δ276I, A279V, T277A, A234T) retained dominant negative activity at high T3 levels, presumably as a consequence of marked impairment either in hormone binding and/or transactivation function. Figure 5.Dominant negative inhibition of wild-type receptor activity by mutant receptors on different TRE configurations. JEG-3 cells were cotransfected with equal amounts (100 ng) of wild-type plus each mutant TRβ expression plasmid, along with the specified reporter constructs (A) PAL-tkLUC, (B) F2-tkLUC or (C) MAL-tkLUC and an internal control plasmid (BOS-βgal). The transfected cells were incubated in the presence of either low (1 nM; solid bars) or high (1000 nM; white bars) concentrations of T3. Hormone-dependent activation was normalized against the internal control and expressed as the percentage of maximum wild-type receptor response at each concentration of T3. These data are the mean ± SE of at least three separate experiments performed in triplicate. Download figure Download PowerPoint DNA binding and heterodimerization with RXR is preserved for all RTH mutants One proposed mechanism of dominant negative inhibition by mutant receptors suggests that they form transcriptionally defective heterodimers with RXR which bind to DNA and compete with wild-type TR–RXR complexes at target gene TREs (Yen and Chin, 1994). Accordingly, we used an electrophoretic mobility shift assay to examine these properties for each RTH mutant using the three different TRE configurations utilized previously to test their transcriptional function (Figure 6A–C). All the RTH mutants bound DNA and heterodimerized with RXR comparably with wild-type receptor on palindromic (PAL), everted repeat (F2) or direct repeat (malic enzyme) TREs. In addition, both wild-type and mutant receptors bound DNA as homodimers with the everted repeat TRE (Figure 6B). In response to ligand (10 nM T3) the wild-type TR homodimer dissociated from DNA as described previously (Collingwood et al., 1994), whereas the variable dissociation of the RTH mutant homodimers was concordant with their reduced ligand binding. Figure 6.DNA binding, T3-dependent homodimer dissociation and dimerization of wild-type and mutant receptors with RXR on different configurations of TRE. In an electrophoretic mobility shift assay equal amounts of in vitro translated TRβ mutants were incubated with RXR, along with 32P-labelled DNA representing three different configurations of TRE. (A) Direct repeat element from the malic enzyme gene, (B) everted repeat element (F2) from the chicken lysozyme and (C) palindromic element derived from the rat growth hormone gene, in either the presence (+) or absence (−) of 10 nM T3. Complexes were resolved by native PAGE. Download figure Download PowerPoint Mutant receptors recruit corepressor and repress basal transcription Recent studies have suggested that the ability to silence basal transcription and recruit corepressor are important attributes of RTH mutants and that the introduction of additional artificial mutations which abolish corepressor binding abrogates their dominant negative activity (Yoh et al., 1997). We therefore examined the ability of each mutant to repress basal transcription by co-expressing fusions of wild-type or mutant TR LBDs linked to the DNA-binding domain (DBD) of Gal4 with a reporter gene containing Gal4-binding sites. In the absence of ligand, the wild-type Gal4–TR fusion repressed basal transcription to ∼15% of that observed with the Gal4 DBD alone. In comparison, each RTH mutant repressed to a similar degree, suggesting that corepressor recruitment was preserved (Figure 7A). In contrast, two artificial mutants (P214R, L428R) exhibited markedly impaired silencing function. The P214R substitution
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