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Endocrinology Vol. 142, No. 2 653-662
Copyright © 2001 by The Endocrine Society

ARTICLES

Impaired Interaction of Mutant Thyroid Hormone Receptors Associated with Human Hepatocellular Carcinoma with Transcriptional Coregulators1

Kwang-huei Lin and Yi-hsin Wu shen-liang chen

Department of Biochemistry, Chang-Gung University, Taoyuan, Taiwan 333, Republic of China

Address all correspondence and requests for reprints to: Kwang-Huei Lin, Department of Biochemistry, Chang-Gung University, 259 Wen-hwa 1 Road, Taoyuan, Taiwan, Republic of China. E-mail: khlin{at}mail.cgu.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormone (T3) exerts its many biological activities through interaction with specific nuclear receptors (TRs) that function as ligand-dependent transcription factors at genes that contain a thyroid hormone response element (TRE). Mutant TRs have been detected in human hepatocellular carcinoma cell lines and tissue, but their contribution to carcinogenesis has remained unclear. The interaction of four such mutant TRs (J7-TR{alpha}1, J7-TRß1, H-TR{alpha}1, and L-TR{alpha}1) with transcriptional coregulators has now been investigated. With the exception of J7-TR{alpha}1, which in the absence of T3 exhibited transcriptional silencing activity with a TRE-reporter gene construct in transfected cells, the mutant TRs had little effect (compared with that of wild-type receptors) on transcriptional activity of the reporter gene in the absence or presence of T3, of the transcriptional corepressors SMRT, NCoR or of the transcriptional coactivator SRC. Electrophoretic mobility-shift assays revealed that, in the presence of T3, the J7-TRß1 mutant did not interact with SRC, whereas J7-TR{alpha}1 and H-TR{alpha}1 exhibited reduced abilities to associate with this coactivator and L-TR{alpha}1 showed an ability to interact with SRC similar to that of wild-type TR{alpha}1. The dominant negative activity of the mutant TRs in transfected cells appeared inversely related to the ability of the receptors to interact with SRC. Whereas J7-TRß1, H-TR{alpha}1, and L-TR{alpha}1 did not interact with SMRT, and NCoR. J7-TR{alpha}1 bind to corepressors but failed to dissociate from them in the presence of T3. These aberrant interactions between the mutant TRs and transcriptional coregulators may contribute to the highly variable clinical characteristics of human hepatocellular carcinoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID hormone (T3), through interaction with its intracellular receptors (TRs), influences various physiological functions, including general metabolism, development, growth, and reproduction (1, 2, 3). Two principal types of TRs, which are encoded by genes on human chromosomes 17 (TR{alpha}) and 3 (TRß), have been identified; transcripts of each of these genes undergo alternative splicing to generate TR{alpha}1 and TR{alpha}2 as well as TRß1 and TRß2 receptor isoforms. Like other nuclear hormone receptors, TRs are ligand-dependent transcription factors and contain domains that mediate binding of hormone, binding to DNA, receptor dimerization, and interaction with various other transcriptional factors. TRs regulate the transcription of target genes by binding to specific DNA sequences, known as thyroid hormone response elements (TREs), in the promoter regions of these genes. In the absence of T3, TRs repress the activity of target promoters, a phenomenon known as transcriptional silencing that is thought to be mediated by interaction of the ligand binding domain of the receptor with nuclear hormone receptor corepressors, such as SMRT (silencing mediator of retinoic acid and thyroid hormone receptor). Ligand binding is thought to induce dissociation of TRs from corepressors and to result in the recruitment of coactivators such as SRC (steroid receptor coactivator) and in the consequent activation of transcription from target promoters (4, 5, 6, 7, 8, 9, 10). Thus, whereas SRC does not bind to wild-type TRs in the absence of T3, it binds to these receptors with increasing avidity as the T3 concentration is increased. The corepressors NCoR (nuclear receptor co-repressor) and SMRT were recently shown to repress transcription by interacting directly with class II histone deacetylases (11).

The role of TRs in neoplastic transformation is largely unknown (3). To evaluate the potential contribution of TR genes to the pathogenesis of human hepatocellular carcinoma (HCC), we previously examined the expression and regulation of these genes in nine human hepatoma cell lines (12, 13). Overexpression of TRß1 was detected in three of the nine cell lines. In addition, the degree of differentiation of the cell lines was inversely correlated with the abundance of TRß1 protein. These observations suggested that changes in expression of the TRß gene might contribute to hepatocarcinogenesis. We subsequently cloned the complementary DNAs (cDNAs) for TR{alpha}1 and TRß1 from the HCC cell line J7 and showed that the corresponding recombinant proteins exerted a dominant negative effect on the transactivation activity of wild-type TRs (14). The isolation of cDNAs for TR{alpha}1 from tumor tissue of two individuals with HCC also revealed that the encoded proteins acted in a dominant negative manner (15). Furthermore, a high prevalence (>60%) of point mutations has been detected in the TR genes of tumor tissue from individuals with HCC (16).

To characterize further the properties of naturally occurring TR mutants, we have now investigated the interactions of four such HCC-associated mutant proteins (three TR{alpha}1 and one TRß1) with transcriptional coregulators. All TR mutants identified in individuals with the syndrome of resistance to thyroid hormone (RTH) have been derived exclusively from the TRß gene (17, 18). However, our previous studies have shown that mutations in the TR{alpha} gene also occur naturally (14, 15), and we now show that TR{alpha}1 and TRß1 mutants exhibit similar aberrant interactions with transcriptional coregulators.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
Plasmids that encode glutathione S-transferase (GST) fusion proteins containing amino acids 570 to 780 of human SRC-1 or amino acids 1086 to 1449 of human SMRT or amino acid 2063 to 2300 of human NCoR were kindly provided by M. G. Parker (Imperial Cancer Research Fund, London, UK), M. Privalsky (University of California, Davis, CA), and A. N. Hollenberg (Harvard Medical School, Boston, MA) respectively. The encoded fusion proteins were expressed in bacteria BL-21 and purified as described (19). The mammalian expression vectors pBK-CMV-SRC1 and pCMX-SMRT were kindly provided by M. J. Tsai (Baylor College, Baylor, TX) and R. M. Evans (The Salk Institute, La Jolla, CA), respectively.

Determination of the transactivation activity of TRs
The effects of SRC and SMRT on the silencing activity and T3-dependent transactivation activity of TRs were examined by transfection of COS-1 cells (5 x 105 cells in a 60-mm dish), with the use of Lipofectamine (Life Technologies, Inc., Gaithersburg, MD), with expression vectors encoding mutant or wild-type TRs, SRC or SMRT, and ß-galactosidase (as a control for variability in transfection efficiency) as well as with a TRE-containing luciferase reporter plasmid (J. L. Jameson. Northwest University, Chicago, IL). After incubation for 24 h in the absence or presence of T3, the cells were harvested and lysed in 250 µl of lysis buffer and a portion of the cell lysate (20 µl) was assayed for luciferase and ß-galactosidase activities as described previously (20). The luciferase activity was normalized on the basis of the protein concentration and ß-galactosidase activity of the lysates.

Electrophoretic mobility-shift assay (EMSA)
32P-Labeled oligonucleotides corresponding to various TREs (F2, derived from the chicken lysozyme gene and consisting of two inverted repeats of the half-site binding motif separated by six nucleotides; DR4, two direct repeats of the half-site motif separated by four nucleotides; or Pal, palindromic TRE) were prepared as described (15). TR proteins were synthesized by in vitro transcription and translation with the use of a TNT-coupled reticulocyte lysate kit (Promega Corp.); their concentrations were determined by measuring the intensity of the corresponding 35S-labeled bands after SDS-PAGE. For EMSA, identical amounts of TRs were incubated with 32P-labeled TRE oligonucleotide in the absence or presence of retinoid X receptor {alpha} (RXR{alpha}) and T3 as described previously (14). Supershift analysis was performed by addition of GST-SRC or GST-SMRT or GST-NCoR. After electrophoresis, TR homodimers and heterodimers were visualized by autoradiography and quantified with a BAS2000 image analyzer (Fujifilm, Tokyo, Japan) (14).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of SMRT and SRC on the silencing and T3-dependent transactivation activities of TR mutants
The human TR mutants studied were as follows: H-TR{alpha}1 (mutant H) contains a Val -> Ala point mutation at codon 390; L-TR{alpha}1 (mutant L) contains Glu -> Lys and Pro -> Ser mutations at codons 350 and 398, respectively; J7-TR{alpha}1 contains a Met -> Ile mutation at codon 259; and J7-TRß1 contains a Met ->Val mutation at codon 334 (Fig. 1Go). The first two of these mutations were identified in individuals with HCC (15), and the latter two were identified in the HCC cell line J7 (14). Except J7TR{alpha}1, mutant L, which bound T3 with reduced affinity, the others had no T3 binding activity. The SRC, SMRT, NCoR interaction regions in the TR are indicated (Fig. 1Go) (21, 22).



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  		Figure 1. Schematic representation of the mutation sites of the four TR mutants used in the present study. The various domains and SRC, SMRT, and NCoR binding regions of the receptors, as well as the locations of two mutation hot spots of TRß1 associated with RTH (17 18 ), are indicated.

 
The effects of SMRT and SRC on the silencing and ligand-dependent transactivation activities of the mutant TRs were investigated by transfection of COS-1 cells with expression vectors encoding TRs and coregulators as well as with a reporter plasmid containing the luciferase gene under the control of the F2-TRE. Expression of wild-type TR{alpha}1 or TRß1 in COS-1 cells incubated in T3-depleted (Td) medium reduced the extent of basal transcription to 44 and 50% of control values, respectively (Fig. 2AGo). In contrast, in the presence of 50 nM T3, TR{alpha}1 or TRß1 increased luciferase activity by factors of approximately 3.8 and 4.8, respectively. Although the J7-TR{alpha}1 mutant exhibited silencing activity, none of the other mutants induced marked effects on the transcription of the reporter gene in the absence or presence of T3. In cells cotransfected with the SMRT vector, wild-type TR{alpha}1 or TRß1 proteins further repressed basal transcriptional activity to 21 and 40%, respectively, of control values (Fig. 2BGo). SMRT had little effect on the transactivation activity of the wild-type receptors in the presence of T3, or on the extent of transcription in cells expressing the mutant receptors either in the absence or presence of T3. In the presence of T3, SRC enhanced the transactivation activities of wild-type TR{alpha}1 and TRß1 (luciferase activities were increased by the receptors by factors of 4.6 and 6.3, respectively) (Fig. 2CGo). SRC had no substantial effect on the silencing activity of the wild-type receptors in the absence of T3, or on the extent of transcription in cells expressing the mutant receptors either in the absence or presence of T3. However, SRC enhanced the transactivation activity of J7-TR{alpha}1 in the presence of T3 by factor of 2.2. Similar results were obtained with the Pal-TRE and DR4-TRE as with the F2-TRE (data not shown).



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  		Figure 2. Effects of SMRT and SRC on the silencing and ligand-dependent transactivation activities of wild-type and mutant TRs. COS-1 cells were transfected with expression vectors encoding wild-type or mutant TRs (0.5 µg), a ß-galactosidase expression plasmid (0.5 µg), and an F2-TRE luciferase reporter construct (1 µg), in the absence (A) or presence of SMRT (B) or SRC (C) expression vectors (0.5 µg). Cells were incubated for 24 h in Td medium or in the presence of 50 nM T3, after which cell lysates were prepared and assayed for luciferase and ß-galactosidase activities. Normalized luciferase activity was expressed as a percentage of that of control cells that were transfected with the reporter construct and empty TR expression vector and were incubated in Td or T3 medium. Data are means ± SEM from at least three independent experiments.

 
Effects of SRC on the DNA binding activities of TR mutants
We next examined the effects of SRC, SMRT, and NCoR on the DNA binding activity of mutant TRs with the use of EMSA analysis. Production of the two wild-type TRs and four mutant receptors by in vitro transcription and translation yielded proteins of the expected size (49 and 55 kDa for TR{alpha}1 and TRß1, respectively); the preparations also contained various truncated proteins (three in the case of TR{alpha}1 and two for TRß1), which were likely the products of internal translational initiation (16). GST-SRC, GST-SMRT, GST-NCoR, and GST proteins were produced in and purified from bacteria BL-21; the identity of these proteins was confirmed by immunoblot analysis (data not shown). In the absence of T3, wild-type TR{alpha}1 bound to a Pal-TRE oligonucleotide as a heterodimer with RXR{alpha}, a homodimer, and a monomer (Fig. 3AGo). Addition of GST-SRC in the absence of T3 had no effect on the pattern of DNA-protein interaction. However, in the presence of 1 nM T3, a supershifted complex (GST-SRC-TR{alpha}1-RXR{alpha}) was detected (Fig. 3Go, A and C); further increasing the T3 concentration (up to 100 nM) did not greatly increase the amount of this complex, and a similar complex was not formed when GST was added in place of GST-SRC. In contrast, GST-SRC did not interact with the heterodimer formed by J7-TR{alpha}1 and RXR{alpha} in the presence of 1 nM T3; at T3 concentrations of 10 and 100 nM, small amounts of a supershifted complex were apparent. Whereas GST-SRC had virtually no effect in the supershift assay with TR{alpha}1 mutant H at any T3 concentration, it interacted with TR{alpha}1 mutant L to an extent similar to that observed with the wild-type receptor (Fig. 3Go, A and C). GST-SRC interacted in a T3-dependent manner with the heterodimer formed by wild-type TRß1 and RXR{alpha}; however, no supershifted complex was detected when GST-SRC was added to J7-TRß1 and RXR{alpha} in the presence of T3 (Fig. 3Go, B and C). Results similar to those shown in Fig. 3Go were obtained with oligonucleotides corresponding to the F2-TRE (Fig. 4Go) or the DR4-TRE (Fig. 5Go).



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  		Figure 3. EMSA analysis of the interaction of mutant TRs bound to the Pal-TRE with the coactivator SRC. Wild-type or mutant TR{alpha}1 (A) or TRß1 (B) proteins were preincubated in the presence of RXR{alpha}, 32P-labeled Pal-TRE oligonucleotide, and the indicated concentrations of T3 before the addition of GST (1 µg) or GST-SRC (1 µg) as indicated. SS, supershifted complex; HD, TR-RXR{alpha} heterodimer, D, TR homodimer; and M, TR monomer. C, The proportion of the amount of TR-RXR{alpha} heterodimer formed in the absence of GST-SRC that was supershifted by GST-SRC at each concentration of T3 was quantified by image analysis. Data are from an experiment that was repeated at least three times with similar results.

 


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  		Figure 4. EMSA analysis of the interaction of mutant TRs bound to the F2-TRE with the coactivator SRC. Analysis was performed as described in Fig. 3Go, with the exception that the Pal-TRE oligonucleotide was replaced by an F2-TRE oligonucleotide.

 


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  		Figure 5. EMSA analysis of the interaction of mutant TRs bound to the DR4-TRE with the coactivator SRC. Analysis was performed as described in Fig. 3Go, with the exception that the Pal-TRE oligonucleotide was replaced by a DR4-TRE oligonucleotide.

 
Dominant negative activities of TR mutants
To examine the relation between the affinity of TR mutants for SRC and the dominant negative activity of the receptors, we evaluated the inhibitory effects of the mutants on the transactivation activity of the wild-type proteins in transfected COS-1 cells. At a wild-type/mutant vector ratio of 1:1, the dominant negative activities of J7-TR{alpha}1 and J7-TRß1 were greater than were those of the TR{alpha}1 mutants H and L when tested with the F2-TRE (Fig. 6AGo) or the Pal-TRE (Fig. 6BGo). At a wild-type/mutant vector ratio of 1:5, the dominant negative activities of each of the mutant receptors assayed with the F2-TRE or the Pal-TRE were increased (Fig. 6Go). When cells were transfected with equal amounts of mutant and wild-type TR expression plasmids, the rank order of dominant negative activity was J7-TR{alpha}1 ~ J7-TRß1 > H-TR{alpha}1 > L-TR{alpha}1. Thus, TR{alpha}1 mutant L, which interacted with SRC to an extent similar to that observed with wild-type TR{alpha}1 ( Figs. 3–5GoGoGo), showed the lowest dominant negative activity, whereas the impaired interaction of the other three mutant receptors with SRC was associated with a greater dominant negative activity.



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  		Figure 6. Dominant negative activity of mutant TRs. COS-1 cells were transfected with an expression vector encoding wild-type (WT) TR{alpha}1 or TRß1 (0.5 µg), a TR mutant vector (0.5 or 2.5 µg), a ß-galactosidase expression plasmid (0.5 µg), and an F2-TRE (A) or Pal-TRE (B) luciferase reporter construct (1 µg). Cells were incubated for 24 h in Td medium or in the presence of 50 nM T3, after which cell lysates were prepared and assayed for luciferase and ß-galactosidase activities. Luciferase activity was normalized on the basis of protein concentration and ß-galactosidase activity of the lysates. Data are expressed as the percentage inhibition by each mutant receptor of the T3-dependent transactivation activity of the corresponding wild-type receptor, and are means ± SEM of three independent experiments.

 
Effects of SMRT, NCoR on the DNA binding activities of TR mutants
The ability of the mutant receptors to interact with the corepressor SMRT, NCoR and the ability of T3 to induce the dissociation of such complexes were examined by EMSA analysis. As expected, heterodimers of wild-type TR{alpha}1 or TRß1 with RXR{alpha} that were bound to a DR4-TRE (Fig. 7Go) or to an F2-TRE (Fig. 8Go) oligonucleotide interacted with GST-SMRT in the absence of T3, and T3 induced the dissociation of GST-SMRT from these complexes in a concentration-dependent manner. However, GST-SMRT did not bind to H-TR{alpha}1, L-TR{alpha}1, or J7-TRß1 mutants complexed with RXR{alpha} and a TRE; the corepressor did bind to a reduced extent to the ternary complex formed by J7-TR{alpha}1, but the resulting quaternary complex was not dissociated by T3 (Figs. 7Go and 8Go). Similar results were observed when GST-NCoR protein was used, except it supershifted the J7-TR{alpha}1-RXR heterodimer intensively in the absence of T3 by using DR4-TRE or F2-TRE (Figs. 9Go and 10Go). But the complex did not dissociate by T3 (Figs. 9Go and 10Go).



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  		Figure 7. EMSA analysis of the interaction of mutant TRs bound to the DR4-TRE with the corepressor SMRT. Analysis was performed as described in Fig. 3Go, with the exception that GST-SRC was replaced by GST-SMRT (1 µg) and that the Pal-TRE oligonucleotide was replaced by a DR4-TRE oligonucleotide.

 


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  		Figure 8. EMSA analysis of the interaction of mutant TRs bound to the F2-TRE with the corepressor SMRT. Analysis was performed as described in Fig. 3Go, with the exception that GST-SRC was replaced by GST-SMRT (1 µg) and that the Pal-TRE oligonucleotide was replaced by an F2-TRE oligonucleotide.

 


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  		Figure 9. EMSA analysis of the interaction of mutant TRs bound to the DR4-TRE with the corepressor NCoR. Analysis was performed as described in Fig. 3Go, with the exception that GST- SRC was replaced by GST- NCoR (1 µg) and that the Pal-TRE oligonucleotide was replaced by a DR4-TRE oligonucleotide.

 


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  		Figure 10. EMSA analysis of the interaction of mutant TRs bound to the F2-TRE with the corepressor NCoR. Analysis was performed as described in Fig. 3Go, with the exception that GST- SRC was replaced by GST- NCoR (1 µg) and that the Pal-TRE oligonucleotide was replaced by a F2-TRE oligonucleotide.

 
In summary, the J7-TR{alpha}1 mutant, which bind T3 weakly, formed a complex with RXR{alpha} and SRC at the TRE, but the interaction of the mutant with SRC required higher concentrations of T3 than did the interaction of wild-type TR{alpha}1 with SRC ( Figs. 3–5GoGoGo); furthermore, T3 did not induce dissociation of the J7-TR{alpha}1–RXR{alpha}-SMRT or J7-TR{alpha}1–RXR{alpha}-NCoR complexes ( Figs. 7–10GoGoGoGo). TR{alpha}1 mutant H, which shows no T3 binding activity, did not form a TR-RXR{alpha}-SRC complex at the DR4-TRE in the presence of T3 (Fig. 5Go) but did exhibit a reduced ability to form such a complex at the Pal-TRE (Fig. 3Go); and the F2-TRE (Fig. 4Go). TR{alpha}1 mutant L, which exhibits a reduced T3 binding activity, formed a TR-RXR{alpha}-SRC complex in the presence of T3 with all three TREs tested to an extent similar to that observed with wild-type TR{alpha}1 ( Figs. 3–5GoGoGo). The J7-TRß1 mutant, which does not bind T3, did not interact with SRC in the presence of RXR{alpha}, T3, and any of the TREs tested ( Figs. 3–5GoGoGo). Mutants H-TR{alpha}1, L-TR{alpha}1, and J7-TRß1 did not form TR-RXR{alpha}-SMRT or TR–RXR{alpha}-NCoR complexes in the absence of T3 with the two TREs tested ( Figs. 7–10GoGoGoGo). Finally, the impairment in the interaction of TR mutants with SRC appeared directly related to the dominant negative activity of these receptors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To shed light on the role of TRs in hepatocarcinogenesis, we previously characterized the TRs in 16 HCC specimens (16) and detected several point mutations in both TR{alpha}1 and TRß1. Most of the TR{alpha}1 mutations were located within two hot spots: amino acids 209–228, and residues 245–256. However, no hot spot was detected in TRß1. The mutant proteins showed either a partial impairment or a complete loss of DNA binding activity. The high prevalence of TR mutations detected in the tumors of individuals with HCC suggests that the mutant receptors might play an important role in liver carcinogenesis. We have now characterized the interactions of four mutant TRs with the coregulators SRC, SMRT, and NCoR. All of them are abundant in liver (23). The interactions of all four naturally occurring mutant receptors with the coregulators were impaired.

The mechanisms by which SMRT and SRC regulate the expression of T3-responsive genes have been characterized (24, 25, 26). In the case of positively regulated genes, SMRT binds to the unliganded form of TRs and this complex mediates the silencing of basal transcription (27, 28). On binding of T3, TRs dissociate from SMRT and are converted from transcriptional repressors to transcriptional activators. However, maximal transactivation activity is only achieved on interaction of the ligand-bound TRs with a coactivator such as SRC. In contrast, in the case of genes that are negatively regulated by T3, nuclear receptor corepressors activate rather than inhibit basal transcription (29).

The mutant TRs examined in the present study either did not bind to SMRT or NCoR (H-TR{alpha}1, L-TR{alpha}1, and J7-TRß1), or bound to the corepressor but failed to dissociate from it on addition of hormone (J7-TR{alpha}1). The failure of J7-TR{alpha}1 to disscociate from SMRT or NCoR in the presence of T3 might be due to its reduced affinity for the hormone. Indeed, the M259I mutation in J7-TR{alpha}1 results in almost loss all of hormone binding activity (14). The observation that the TR{alpha}1 mutants H (V390A) and L (E350K, P398S) as well as J7-TRß1 (M334V) did not bind to SMRT suggests that the mutated residues, which are all located in the COOH-terminal region of the receptors, are important for corepressor binding. Two mutation hot spots have been identified in the hormone binding domain of TRß1 in individuals with RTH (18, 30). Many TRß1 mutants isolated from such individuals are able to repress gene transcription but are unable to activate genes in response to T3 or T4 (31). The interaction of such mutant receptors with corepressors is thought to be aberrant (31). The mutation site for J7-TRß1 is located within the first hot spot for RTH mutations. Furthermore, alignment of the sequences of the hormone binding domains of TR{alpha}1 and TRß1 indicates that the mutated residue (Met259) of J7-TR{alpha}1 corresponds to Met313 of TRß1, which is also located in the first hot spot of RTH mutations. The mutation site for TR{alpha}1 mutant H (Val390) and one of the mutated residues of TR{alpha}1 mutant L (Pro398) both correspond to residues located in RTH hot spot 2 of TRß1 (17, 18).

Our results indicate that association of SMRT with mutant TRs is not required for their dominant negative actions. Thus, all four TR mutants examined in the present study exhibited dominant negative activities, but only J7-TR{alpha}1 interacted with SMRT or NCoR. In contrast, Yoh et al. (31). showed that corepressor association appears to be required for the dominant negative action of TRs associated with RTH. These researchers suggested that an altered interaction with corepressors likely plays an important role in the dominant negative action of RTH-associated TR mutants and may contribute to the variable phenotype of this disorder (31). Our results do not exclude the possibility that the mutant TRs studied associate with corepressors other than SMRT. However, with the exception of J7-TR{alpha}1, the mutants examined in the present study did not exhibit silencing activity in transfected cells. The silencing activity exhibited by J7-TR{alpha}1 is consistent with the binding of this mutant to SMRT or NCoR.

The splice variants TR{alpha}1 and TR{alpha}2 are identical for the first 370 amino acids, but the remaining COOH-terminal regions of the two proteins are completely different. This sequence divergence renders TR{alpha}2 unable to transactivate TRE-containing genes. Tagami et al. (32). showed that TR{alpha}2 acts as a weak antagonist because it is deficient in interactions with the nuclear receptor corepressors NCoR and SMRT. Cohen et al. (33) reported that the TRß1 mutant R429Q does not recruit NCoR. However, the R429Q-RXR heterodimer is able to recruit the SMRT. Besides, the TRß1 mutant {Delta}G337T binds NCoR more strongly than SMRT. This observation, together with our data, indicates that the COOH-terminal region of TRs is important for corepressor binding and, consequently, for silencing activity. However, we did not observe the two corepressors SMRT and NCoR differentially interacted with mutant TRs. In other words, SMRT and NCoR interact similarly with those four TR mutants at least in our experimental conditions.

The dominant negative effects of various TR mutants have been shown to correlate with the abilities of these receptors to bind NCoR but not with the impairment in coactivator binding (34), suggesting that the defective release of corepressors, together with unimpaired dimerization and DNA binding activities, is important for the inhibitory action of mutant TRs. The mutants examined in the present study all retained DNA binding and heterodimerization activities. However, compared with those of the wild-type receptors, the homodimerization activities of J7-TR{alpha}1 and J7-TRß1 appeared reduced, whereas those of H-TR{alpha}1 and L-TR{alpha}1 appeared increased, in EMSAs performed with the Pal-TRE (Fig. 3Go) or DR4-TRE (Fig. 5Go). The homodimerization activity thus appears inversely related to dominant negative activity for these receptors, the latter of which decreases in the rank order J7-TR{alpha}1 ~ J7-TRß1 > H-TR{alpha}1 > L-TR{alpha}1. The dominant negative mutants R316H and R338W of TRß1 show selective loss of homodimerization activity with preservation of the ability to form heterodimers with RXR{alpha} (35). Mutations in the hinge region of TRs selectively affect T3 binding when the receptors are complexed with DNA as well as prevent NCoR dissociation (36).

An inability to interact with coactivators such as SRC is also a determinant of dominant negative activity of mutant TRs associated with RTH (37). Consistent with this notion, we have shown that the extent of the impairment in the interaction of the mutant TRs with SRC appears related to the dominant negative activity of the receptors. The GST-SRC fusion protein that we used for EMSA analysis contains amino acids 570 to 780 of SRC. The interaction of hormone-dependent coactivators such as SRC or glucocorticoid receptor-interaction protein 1 (GRIP1) with nuclear hormone receptors is mediated by the AF-2 domain of the receptors, which contains a hydrophobic cleft (38). The interaction of a naturally occurring transactivation mutant (L454V) of TRß1 with SRC was also shown to be impaired (39), suggesting that the affected amino acid is important for this interaction. The mutation site for H-TR{alpha}1 and the downstream mutation site for L-TR{alpha}1 are located in a region corresponding to that of TRß1 containing residue 454.

In summary, our data indicate that the association of mutant TRs with SMRT or NCoR is not required for dominant negative activity but is required for silencing activity. Moreover, our observations suggest that the ability of mutant TRs to interact with SRC is inversely related to their dominant negative activity.


   Footnotes
 
1 This work was supported by grants from Chang-Gung University (CMRP 737, CMRP893, NMRP 407) and the National Science Council of the Republic of China (NSC 87-2316-B-182002). Back

Received May 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cheng SY 1995 New insights into the structure and function of the thyroid hormone receptor. J Biomed Sci 2:77–89[CrossRef][Medline]
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