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Endocrinology Vol. 138, No. 4 1456-1463
Copyright © 1997 by The Endocrine Society

ARTICLES

Differential Sensitivity of Thyroid Hormone Receptor Isoform Homodimers and Mutant Heterodimers to Hormone-Induced Dissociation from Deoxyribonucleic Acid: Its Role in Dominant Negative Action

Xu-Guang Zhu, Peter McPhie and Sheue-yann Cheng

Laboratory of Molecular Biology (X.-G.Z., S.-y.C.), Division of Basic Sciences, National Cancer Institute, and Laboratory of Biochemical Pharmacology (P.M.), National Institute of Diabetes, Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892-4255

Address all correspondence and requests for reprints to: Dr. Sheue-yann Cheng, Building 37, Room 2D24, 37 Convent Drive MSC 4255, Bethesda, Maryland 20892-4255. E-mail: sycheng{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
General resistance to thyroid hormone is an inheritable disease with resistance of peripheral tissues to elevated levels of thyroid hormone. Genetic studies have shown that it is due to interference in the functions of wild-type thyroid hormone nuclear receptors (wTRs) via the dominant negative effect of mutant TRs (mTRs). The present study compared the heterodimerization of the two TR isoforms, TRß1 and TR{alpha}1, with mutant TRs to understand if mTRs had isoform-dependent dominant negative action. Using electrophoresis gel mobility shift assay, we have demonstrated that mutant PV, S, ED, and OK form heterodimers with wTR{alpha}1 and {Delta}TRß1 (in which the A/B domain of wTRß1 has been deleted), on the F2-thyroid hormone response element (TRE). In the presence of T3, both homo- and heterodimer complexes are dissociated in a T3 concentration dependent manner. The ED50 for {Delta}TRß1 homodimers was 3-fold higher than that of wTR{alpha}1 homodimers. ED50s for {Delta}TRß1/mTR heterodimers were 10- to 40-fold higher than the corresponding wTR{alpha}1/mTR heterodimers. Mutant ED and OK homodimers were only partially dissociated at the highest T3 concentrations used (100 nM), whereas no dissociation could be detected for PV and S homodimers, indicating differential sensitivity of the F2-bound TR dimers to the T3-induced dissociation. We presented a model that indicates the dissociation of any particular TR dimer from F2 is determined by competition of T3 for both of its constituent TRs. By transfection assays, we showed that the potency of the dominant negative action of PV on TR{alpha}1 and TRß1 inversely correlated with the sensitivity of the appropriate mTR/wTR heterodimer to T3-induced dissociation from F2. The differential dominant negative action of mutants on the two TR isoforms could play an important role in the heterogeneity of tissue-specific manifestations in patients with resistance to thyroid hormone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RESISTANCE TO thyroid hormone (RTH) is an inheritable disorder characterized by an inappropriately normal or elevated level of TSH accompanied by resistance of the pituitary and peripheral tissues to the action of elevated circulating levels of thyroid hormones (1, 2). These may accompanied by one or several phenotypic features with variable degrees of tissue resistance to thyroid hormones (1, 2, 3). Classical features include goiter, decreased IQ, dyslexia, tachycardia, delayed bone maturation, attention-deficit hyperactivity disorder, and short stature. There is considerable heterogeneity in the degree of severity and the affected organs among individuals and kindreds with RTH (1, 2).

Genetic analyses of the affected kindreds indicate that the clinical manifestations of RTH patients are due to the interference of the functions of the wild-type thyroid hormone nuclear receptors (wTRs) via the dominant negative effect of the mutant TRs (mTRs). wTRs are ligand-dependent transcription factors that are the products of two genes, {alpha} and ß. Each gene gives rise to two subtypes, {alpha}1, {alpha}2, ß1, and ß2, by alternative splicing of the primary transcripts. wTRs regulate the functions of thyroid hormone target genes by binding to the specific DNA sequences known as the thyroid hormone response elements (TREs). Four domains can be assigned to each TR, domains A/B, C, D, and E. Domain C contains two zinc fingers and is involved in binding of the receptors to TREs. Domains D and E are structurally linked, in so far as part of domain D is required for the biological function of domain E, which is to bind to the thyroid hormone, T3 (4, 5). Domains D and E are also involved in binding to corepressors and dimerization, respectively (6, 7). Except for one recent report in which a naturally occurring mutation in TR{alpha} gene was detected in a human hepatocarcinoma cell line derived from a patient (8), the mutations detected so far in RTH patients only occur in the TRß gene. Most of the mutations are missense mutations that are clustered in two hot spots: one is in the middle of the T3 binding domain, and the other is close to the C-terminus. The functional impairments of mTRß1 include variably reduced or absent T3 binding, reduced or absent transactivation capacity, and dominant negative potency (1, 2, 9, 10).

Three nonmutually exclusive mechanisms have been proposed to account for the dominant negative action of mTRß1: 1) competition of wTRs and mTRß1 for binding to TREs; 2) competition by wild-type and mutant receptors for limiting amounts of TR-associated proteins or other nuclear factors essential for transcription (11, 12); 3) formation of inactive wild-type/mutant heterodimers that may limit access by transcriptionally relevant TR complexes to DNA and thus contribute to the dominant negative effect of mutant receptors. To date, this third mechanism has been the least studied, probably due to the difficulty of a convincing demonstration of the formation of wild-type/mutant receptor heterodimers. Recently, however, we have circumvented this problem by using a truncated form of TRß1 ({Delta}TRß1) in which the A/B domain was deleted. Deletion of A/B domain has no effect on the binding of wTRß1 to T3 and TREs (13). Using mutant PV that has a frame-shift mutation in the last 16 amino acids, we showed clearly the formation of wTRß1/PV heterodimers by electrophoretic mobility gel shift assay (EMSA). Furthermore, we showed that there is a strong correlation between the formation of wTRß1/PV heterodimers and the dominant negative potency of PV (13).

However, it is still unknown whether wTRß1 also forms heterodimers with other mutants. More importantly, at present, the role of wTR{alpha}1/mTR heterodimers in the dominant negative effect of mutants is unknown because formation of wTR{alpha}1/mTR heterodimers has not yet been demonstrated. We therefore examined the interaction of four mutants with wTRß1 and wTR{alpha}1 on F2-TRE in which the half-site binding motifs are arranged in an everted-repeat. We found that both isoforms form heterodimers with four mutants on F2. Furthermore, the sensitivity of the F2-bound heterodimers to T3-induced dissociation was mutant- and isoform-dependent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture media were purchased from BioWhittaker (Walkersville, MD). FCS and lipofectamine transfection reagent were obtained from GIBCO-BRL (Grand Island, NY). 5'-[{alpha}-32P]-dCTP (6000 Ci/mmol) was obtained from Amersham Life Science (Arlington Heights, IL). [14C]-Chloramphenicol and 3'-[125I]-T3 were purchased from DuPont New England Nuclear (Boston, MA). TNT-coupled reticulocyte lysate in vitro translation kits were from Promega (Madison, WI).

Plasmids
The T7 expression plasmid pJL08 encoding a truncated wTRß1 in which the A/B domain (amino acids 1–105) was deleted has been described previously (5). The T7 expression plasmids pCLC13 and pCJ3 encoding wTR{alpha}1 and wTRß1, respectively, were prepared as described (14, 15). The reporter plasmids, pF2-thymidine kinase promoter-chloramphenicol acetyltransferase (pF2-TK-CAT) was a gift from Dr. G. Brent. The plasmids pGEM3Z-PV, pGEM3Z-S, pGEM3Z-OK, pGEM3Z-ED encoding mutant PV, S, OK and ED, respectively, were gifts from Dr. C. A. Meier.

Transfection assay
CV1 cells were plated 24 h before transfection in DMEM containing 10% T3-depleted FCS in 60-mm Petri dishes at a density of 4 x 105 cells/dish. The cells were transfected by the appropriate plasmids using lipofectamine according to the manufacturer’s instructions. After 24 h, the medium was replaced with fresh DMEM containing 100 nM of T3. After incubation for an additional 24 h, cells were harvested and lysed. The transfection efficiency was monitored by ß-galactosidase activities, and the CAT activity was normalized to the protein concentration of the lysates.

Electrophoresis gel mobility assay (EMSA)
Two complementary oligonucleotides containing F2 sequences (shown below) were annealed and the recess 3'-end filled with DNA polymerase (Klenow fragment) in the presence of [{alpha}-32P]-dCTP.



The labeled oligonucleotides were separated on a 12% polyacrylamide gel and purified by electroelution. The gel mobility shift assay was carried out as below: in vitro translated TR receptor proteins were incubated with the labeled oligonucleotides in the binding buffer (25 mM HEPES, pH 7.5, 5 mM MgCl2, 4 mM EDTA, 10 mM DTT, 0.11 M NaCl and 0.4 µg single-stranded DNA). After incubation for 30 min at 25 C, the reaction mixture was loaded onto a 5% polyacrylamide gel and electrophoresed at 4 C for 2–3 h at a constant voltage of 250 V. The gel was dried and autoradiographed. The intensities of the retarded bands and free oligonucleotides were also quantified by PhosphoImager (Molecular Dynamics, CA). The binding data were analyzed based on the equations and methods described by Zhu et al. (13).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Both wTRß1 and wTR{alpha}1 can form heterodimers with mutants
We have recently shown that wTRß1 forms heterodimer with mutant PV on TREs (13). Among the three TREs in which the half-site binding motifs arranged in three different orientations, wTRß1 binds to F2 with highest affinity as a homodimer and as a heterodimer with mutant PV (13). PV has a frame-shift mutation in the last 16 amino acids of TRß1 and has virtually lost its T3 binding activity (10; Table 1Go). Because it is not known whether the formation of heterodimers between the TRß1 and mutant is only limited to PV or it is a more general phenomenon, we evaluated whether other mutants also form heterodimers with TRß1. We chose three other mutants as representatives (Table 1Go). Mutants S and ED (A317T) are located in the first hot spot. Mutant S has a codon deletion on 337 ({Delta}337) and like mutant PV, has completely lost T3 binding activity (10). Mutant OK (M442V), like PV, is located in the second hot spot. Both mutants, OK and ED, have a missense mutation, resulting in a reduction of 78% of T3 binding activity. Therefore, these four are representatives of mutants in terms of the location of hot spots and T3 binding affinity.


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  		Table 1. Characteristics of the four TRß1 mutants

 
Previously, to clearly demonstrate the formation of heterodimers by EMSA, it was necessary to use {Delta}TRß1 in which the A/B domain of wTRß1 has been deleted. Deletion of A/B domain has no effect on the T3 and DNA binding activity (13). Therefore, we used {Delta}TRß1 to demonstrate the formation of heterodimers of TRß1 with other mutants. Figure 1AGo shows that in the presence of equal amounts of mutant S and {Delta}TRß1 proteins, three well-resolved bands, representing F2-bound S/S, S/{Delta}TRß1 and {Delta}TRß1/{Delta}TRß1 dimers in an approximate ratios of 1:2:1, were seen (lanes 1–6). These results are similar to those seen for PV (13). Lanes 7 and 8 of Fig. 1AGo show the positions of {Delta}TRß1/{Delta}TRß1 and S/S homodimers, respectively. The intensities of these three bands increased in a F2-concentration dependent manner (lanes 1–6 of Fig. 1AGo). We determined the apparent affinity constants (Ka) in the binding of S/S, S/{Delta}TRß1 and {Delta}TRß1/{Delta}TRß1 dimers to F2. As shown in Table 2Go, these three dimers bound to F2 with very similar apparent binding affinity constants, indicating that the deletion of codon 337 (see Table 1Go) had no effect on the formation of S/S homodimers and S/{Delta}TRß1 heterodimers. We further carried out similar binding experiments for mutant ED (Fig. 1BGo) and mutant OK (Fig. 1CGo). Again, formation of three well separated mutant/mutant, mutant/{Delta}TRß1 and {Delta}TRß1/{Delta}TRß1 bands in a F2-concentration dependent manner was seen (lanes 1–6 of Fig. 1Go, B and C). We also determined their Ka to F2. As shown in Table 2Go, within experimental errors, there were no significant differences in Ka not only for binding to F2 for different dimers, but also for different mutants. The Ka for the binding of PV/PV, PV/{Delta}TRß1 and {Delta}TRß1/{Delta}TRß1 to F2 that were determined previously were also included in Table 2Go for comparison (13). These results indicate that in the absence of T3, the dimeric binding of mutant/mutant and mutant/{Delta}TRß1 to F2 was independent of mutation sites for these four mutants.



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  		Figure 1. Formation of dimers of {Delta}TRß1 with mutants S, OK, or ED. Each receptor was separately synthesized using TNT-coupled reticulocyte lysate as described in Materials and Methods. The amounts of synthesized receptors were determined by SDS-PAGE followed by quantification using PhosphoImager. Equal amounts of {Delta}TRß1 and mutants PV, OK, or ED proteins were incubated with increasing concentrations of 32P-labeled F2 (1.9, 3.8, 7.5, 5, 30, and 60 fmol). The reaction mixtures were analyzed by 5% nondenaturing gel electrophoresis. The dimers were visualized by autoradiography. M/M represents mutant/mutant homodimers; {Delta}TRß1/M represents {Delta}TRß1/mutant heterodimer; {Delta}TRß1/{Delta}TRß1 represents {Delta}TRß1 homodimers; Free = free probe.

 

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  		Table 2. Apparent association constants of the binding of F2 to {Delta}TRß1 and mTRß1 dimers

 
We further examined whether wTR{alpha}1 forms heterodimers with mutants. Lanes 9 and 8 of Fig. 2Go, A and B, respectively, show that different from the binding of {Delta}TRß1 to F2 (lane 7 of Fig. 1AGo), wTR{alpha}1 bound to F2 both as a homodimer and monomer (13, 16, 17). Lanes 8 and 9 of Fig. 2Go, A and B, show the binding of S and PV, respectively, as a homodimer to F2. In the presence of equal amounts of either mutant S (lanes 1–7 of Fig. 2AGo) or mutant PV (lanes 1–7 of Fig. 2BGo), in addition to the wTR{alpha}1 monomer, three discrete dimeric bands, representing mutant/mutant, mutant/wTR{alpha}1, and wTR{alpha}1/wTR{alpha}1 were seen, indicating that, similar to {Delta}TRß1, wTR{alpha}1 can also form heterodimers with mutants. As shown in Fig. 2Go, the binding of wTR{alpha}1 as monomer and three dimers to F2 was F2 concentration dependent. However, because of the complexity in the analysis of simultaneous multiple equilibria of the four binding species (three dimers and one monomer), we were unable to set up equations to determine accurately the Ka for each binding species. However, experiments shown in Fig. 2Go, A and B, were carried out under the same experimental conditions and the intensities of homodimeric and heterodimeric bands quantified by PhosphoImager were identical for mutant S and PV at each F2 concentration (e.g. the intensities of homodimeric and heterodimeric bands in lanes 7 of Fig. 2Go, A and B). Based on the identical intensities of the dimeric and monomeric bands, it is reasonable to conclude that there was no significant differences in the Ka between the binding of S/S and PV/PV, of S/wTR{alpha}1 and PV/wTR{alpha}1 dimers to F2. We also carried out the binding experiments for ED and OK similarly. We found that the intensities of F2-bound ED/ED and OK/OK homodimeric bands and F2-bound ED/wTR{alpha}1 and OK/wTR{alpha}1 heterodimeric bands, at each F2 concentration (data not shown), were similar to those of S/S and S/wTR{alpha}1, respectively. These results indicate that both TR isoforms could form heterodimers with many mutant TRs and that the binding of mutant/wild-type TR heterodimers to F2 is independent of the mutation sites in the four mutants examined.



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  		Figure 2. Formation of TR{alpha}1 and mutant PV or S dimers. The receptors were prepared and quantified as described in Fig. 1Go. Equal amounts of TR{alpha}1 and mutant S or PV were incubated in increasing concentrations of F2. EMSA was carried out similarly as described in Fig. 1Go. The F2-bound dimers and monomer were visualized by autoradiography. M/M represents mutant/mutant homodimers; wTR{alpha}1/M represents wTR{alpha}1/mutant heterodimer; wTR{alpha}1/wTR{alpha}1 represents wTR{alpha}1 homodimers; wTR{alpha}1 is the monomer; Free = free probe.

 
Differential sensitivity of F2-bound homodimer and heterodimer of TR isoforms to the T3-induced dissociation
In the presence of T3, however, a dramatically different picture emerged in the binding of homodimers and heterodimers of TR isoforms to F2. Figures 3DGo and 4IGo-A show that wTRß1/wTRß1, {Delta}TRß1/{Delta}TRß1 and wTRß1/{Delta}TRß1 dimers were similarly dissociated by T3 in a concentration-dependent manner, indicating that domain A/B has no effect on the T3-induced dissocation of TRß1 homodimers. Figure 3AGo shows that while {Delta}TRß1/{Delta}TRß1 was dissociated by increasing concentrations of T3, S/{Delta}TRß1 and S/S dimers were not affected by T3 up to 100 nM. These findings were similar to that found for PV/{Delta}TRß1 and PV/PV dimers (13). However, not all mutant/{Delta}TRß1 heterodimers and mutant/mutant homodimers were insensitive to the T3 effect. As shown in Fig. 3BGo, ED/ED and ED/{Delta}TRß1 were dissociated by T3 in a concentration-dependent manner. T3 also induced the dissociation of OK/OK and OK/{Delta}TRß1 dimers (Fig. 3CGo). For quantitative comparison, the intensities of the dimer bands at each concentration of T3 were quantified and plotted (Fig. 4Go). We have also included the previously reported dissociation curves of PV homodimers and heterodimers for comparison (13). Examination of the dissociation curves in Fig. 4IGo shows that the ED50 for T3-induced dissociation of {Delta}TRß1/{Delta}TRß1 was virtually identical within experimental error (3.1–4.1 nM), irrespective of the presence of the type of mutant (Table 3Go). The ED50s for T3-induced dissociation of ED/{Delta}TRß1 and OK/{Delta}TRß1 heterodimers were 48.5 ± 15 nM and 47.6 ± 12.9 nM, respectively; whereas no dissociation was detected for PV/{Delta}TRß1 and S/{Delta}TRß1 at the T3 concentrations examined (Table 3Go). The mutant homodimers were less affected by T3. However, at high T3 concentration (100 nM), approximately 10% and 50% of ED/ED and OK/OK homodimers were dissociated from F2 (Fig. 4IGo, B and C). But PV/PV and S/S homodimers were not dissociated by T3 as high as 100 nM. Thus, these results indicate that the sensitivity of T3-induced dissociation of F2-bound dimers correlates well with the T3 binding affinities of the receptor in that the ED50 is in the order of {Delta}TRß1<OK = ED<<S = PV (see Tables 1Go and 3Go).



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  		Figure 3. Effect of T3 on the formation of TR dimers. {Delta}TRß1 homodimers, {Delta}TRß1/S heterodimers, and S/S homodimers (A); {Delta}TRß1 homodimers, {Delta}TRß1/ED heterodimers, and ED/ED homodimers (B); {Delta}TRß1 homodimers, {Delta}TRß1/OK heterodimers, and OK/OK homodimers (C). {Delta}TRß1 homodimers, {Delta}TRß1/wTRß1 and wTRß1/wTRß1 homodimers (D). Equal amounts of {Delta}TRß1 and mutant (A–C) or wTRß1 (D) were incubated with F2 (60 fmol) in the absence or presence of T3 (1, 3, 6, 10, and 100 nM). EMSA was carried out similarly as described in Fig. 1Go.

 


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  		Figure 4. Quantative analysis of the F2-bound dimers. After the gel was dried, the intensities of the TRE-bund dimeric bands were quantified by PhosphoImager and plotted against the increasing concentrations of T3. In 4I-A, wTRß1/wTRß1 (•), {Delta}TRß1/{Delta}TRß1 ({circ}), {Delta}TRß1/wTRß1 ({blacksquare}); in 4I-A–E, {Delta}TRß1/{Delta}TRß1 ({circ}), {Delta}TRß1/mTR ({square}), mTR/mTR ({blacktriangleup}); in 4II A-D, wTR{alpha}1/wTR{alpha}1 ({circ}), wTR{alpha}1/mTR ({square}), mTR/mTR ({blacktriangleup}); data are expressed as mean ± SD (n = 3) with the SD in the range of 5–15%.

 

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  		Table 3. ED50 of T3-induced dissociation of F2-bound TR dimers

 
A more complex effect of T3 was seen for the binding of wTR{alpha}1 and mutants to F2. As shown in Fig. 5AGo, T3 induced not only the dissociation of wTR{alpha}1/wTR{alpha}1 homodimers, but also the dissociation of wTR{alpha}1/S heterodimers at the T3 concentrations that had no effect on the stability of F2-bound {Delta}TRß1/S dimers (Fig. 3AGo vs. Fig. 5AGo). Interestingly, binding of the wTR{alpha}1 monomers was increasing while the binding of wTR{alpha}1/wTR{alpha}1 and wTR{alpha}1/S were decreased by T3. The effects of T3 on the binding of wTR{alpha}1 with mutant PV (Fig. 5BGo), ED and OK (data not shown) were carried out similarly. The ED50s were calculated from the T3-induced dissociation curves shown in Fig. 4IGoI. The calculated ED50s shown in Table 3Go indicate that the ED50 for T3-induced dissociation of F2-bound wTR{alpha}1/wTR{alpha}1 homodimers was 1.2–1.5 nM that was 2- to 3-fold lower than that for {Delta}TRß1/{Delta}TRß1 homodimers. The ED50s were 20.7 ± 0.6, 33.1 ± 13.8, 11.4 ± 2.6 and 11.4 ± 2.4 nM for T3-induced dissociation of wTR{alpha}1/PV, wTR{alpha}1/S, wTR{alpha}1/ED and wTR{alpha}1/OK heterodimers, respectively. These values were clearly much lower than those for the corresponding {Delta}TRß1/M heterodimers. However, the ED50s for ED/ED and OK/OK were higher than 100 nM that were similar to those derived when {Delta}TRß1 was present. These results show that wTR{alpha}1, either as a homodimer or as a heterodimer with mutant TR, was more sensitive to T3-induced dissociation than {Delta}TRß1, indicating isoform-dependent stability of F2-bound TRs in the presence of T3. We have recently reported that wTR{alpha}1 binds to T3 with a 3-fold higher affinity than wTRß1(Zhu, X.-G., P. McPhie, K. H. Lin, and S. Y. Cheng, submitted for publication). Taken together, this differential sensitivity correlates with the T3-binding in that TR{alpha}1>TRß1>OK = ED>>S = PV.



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  		Figure 5. Effect of T3 on the formation of wTR{alpha}1 homodimers, wTR{alpha}1/S heterodimers and S homodimers (A) wTR{alpha}1 homodimers, wTR{alpha}1/PV heterodimers and PV homodimers (B). Equal amounts of wTR{alpha}1 and mutant S or PV were incubated with F2 in the absence or presence of T3 (1, 3, 6, 10, and 100 nM). EMSA was carried similarly as described in Fig. 1Go.

 
The potency of dominant negative action of PV on wTRß1 is higher than on wTR{alpha}1
Previously we have shown that the potency of dominant negative action of PV correlates well with the formation of TRß1/PV heterodimers (13). The isoform-dependent differential T3 sensitivity of the wild-type/mutant heterodimers provided us a powerful tool to further examine the role of wild-type/mutant heterodimers in the dominant negative action of mutants. The analyses from the above in vitro binding predicted that the dominant negative action of a mutant on wTRß1 would be stronger than on wTR{alpha}1. To see if this is the case, we carried out transient transfection assay in which we assessed the potency of dominant negative action of PV by comparing the transactivation activities of wTRß1 and wTR{alpha}1 in the presence of PV. Figure 6AGo shows that wTRß1 had a 2-fold higher transactivation activity than wTR{alpha}1 (bars 8 vs. 4) that is consistent with that reported previously (Zhu, X.-G., McPhie, P., Lin, K.-H., and Cheng, S.-Y., submitted for publication). When PV expression plasmid was cotransfected, the transactivation activity of wTRß1 and wTR{alpha}1 was both suppressed (bars 6 and 10). However, the transactivation activity of wTRß1 was suppressed nearly 2-fold more by PV than of wTR{alpha}1 (Fig. 6BGo). The similar extent of the dominant negative action of PV on {Delta}TRß1 mediated by F2 (Fig. 6BGo) further supported the observations from the in vitro F2 binding in that domain A/B has no effect on the activity of TRß1. Taken together, these results indicate that the dominant negative action of a mutant correlates with the stability of wild-type/mutant TR heterodimers.



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  		Figure 6. Comparison of the dominant negative potency of PV on the transactivation activity of wTRß1 or wTR{alpha}1 in CV1 cells. CV1 cells were transfected with either wTRß1, wTR{alpha}1 or {Delta}TRß1 expression vector (0.2 µg) together with ß-galactosidase expression plasmid pCH110 (0.2 µg) and pF2-TK-CAT (1 µg) in the absence or presence of mutant PV expression vector (0.2 µg). CAT activity was determined as described in Materials and Methods using equal amounts of lysate proteins, which were then corrected for transfection efficiency using ß-galactosidase activity. Data are mean ± SE of six independent determinations each with duplicates. The percent CAT activity (A); CAT activity is expressed as the percent inhibition of fold T3 induction of either the wTRß1 or wTR{alpha}1 (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have investigated the mechanism of the dominant negative effect of mutant TRs on the normal regulatory function of wild-type TRs, using the interaction of four representative mutant proteins (PV, S, ED, OK) with {Delta}TRß1 and wTR{alpha}1, in the presence of F2. Previously Yen et al. (18) used a similar approach, studying the interaction of another RTH mutant, Mf-1 (G345R), with wTRß1 and wTR{alpha}1, again in the presence of F2. Because of the limitations of the gel shift assay system, they were unable to resolve the various DNA/protein complexes in their reactions. Using super shifting by specific antibodies, they demonstrated the formation of both homo and heterodimers, in 1:1 mixtures of mutant and wTRs with F2. In such a mixture of Mf-1 and wTRß1, they observed that two-thirds of the DNA bound proteins were released in the presence of 100 nM T3. This is below the concentration range needed to release isolated Mf-1 homodimers from F2 (EC50 ~500 nM; see Ref.16) and Mf-1 itself has no detectable affinity for T3 (19, 20). They interpreted this observation to indicate the release of both TRß1/TRß1 and TRß1/Mf-1 dimers and concluded that only one of the TRs in a heterodimer needs to bind T3, for dissociation from DNA.

We took advantage of our recent observation that deletion of domain A/B from wTRß1 in no way changes its affinity for T3 or for TREs. However, the reduction in mol wt of {Delta}TRß1 makes it possible to resolve homo and heterodimers formed on DNA binding with mutant proteins by EMSA (13). Thus we can observe the specific behavior of each species present in a mixture, rather than inferring its properties from an average measurement. By studying mixtures of both wTR{alpha}1 and {Delta}TRß1, with four different naturally occurring mutant TRs, we have obtained a clearer picture of the interactions of wild-type and mutant TRs and their responses to increasing concentrations of T3.

The results summarized in Table 2Go show that, under the conditions of our experiments, all dimers derived from {Delta}TRß1, composed of wild-type and mutant proteins, have similar apparent affinities for F2 (Ka{cong} 1 x 109 M-1). The high concentrations of monomeric complexes, observed in experiments using wTR{alpha}1, precluded a similar simple analysis of that data. The constant ratios of homo and heterodimers (1:3.5:1, wTR{alpha}1/wTR{alpha}1: wTR{alpha}1/mutant: mutant/mutant) and the similar intensities of the bands in Figs. 1Go and 2Go, argue for a similar conclusion in this case. The intrinsic affinity of the dimers for F2 is independent of their composition.

However, the data shown in Fig. 4Go and summarized in Table 3Go demonstrate that, in marked contrast, the response of any particular F2 bound dimer to increasing concentrations of T3, is markedly influenced by its composition. Homodimers of wTR{alpha}1 are more easily dissociated by T3 than those of {Delta}TRß1. Heterodimers derived from mutants ED and OK with {Delta}TRß1 are more stable than those derived from wTR{alpha}1, whereas homodimers of these two mutants are only partially dissociated. Homodimers of mutants PV and S, which have no detectable affinity for T3, showed no dissociation over the range of T3 concentrations that we investigated (0–100 nM). The resistance of a dimer to dissociation from F2 by T3, as measured by its ED50s, parallels the reduction in the affinities for the hormone of both of its constituent proteins (9, 10). This correlation strongly suggests that both constituents of a TRE bound dimer must bind T3, before it can dissociate. Thus, under physiological conditions, heterodimers formed from wild-type and mutant TRs and wild-type homodimers will respond very differently to increases in T3 concentration. These heterodimers are therefore intimately involved in the dominant negative action of the mutant TRs.

One puzzling observation in our experiments, which seems to argue against this model, is the T3-induced dissociation of heterodimers of PV and S with wTR{alpha}1, because these two mutants have no detectable affinity for T3. However, a simple model predicts that the efficacy of T3 to dissociate any given dimer will depend on its affinity for both of the constituents of that dimer. Thus, the relative concentration of wTR{alpha}1 homodimers will fall rapidly with the second power of the hormone concentration. Because wTRß1 has a lower affinity for T3, higher concentrations of hormone will be required for full dissociation. Heterodimers of wTRs with mutants ED and OK will show higher ED50s for T3, reflecting very low affinities for hormone. In the cases of mutants PV and S, the concentration of heterodimers will decrease with only the first power of the hormone concentration, yielding very high values of ED50, as observed.

If one assumes a similar thermodynamic equilibrium is to occur in vivo, this would mean that at any given T3 concentration, there will be more wTRß1/mutant heterodimers bound to F2 than wTR{alpha}1/heterodimers. Therefore, wTRß1/mutant heterodimers can more effectively compete for binding to F2 than wTR{alpha}1/heterodimers. Based on these considerations, we predicted that the dominant negative action of a mutant on wTRß1 would be stronger than on wTR{alpha}1. We tested this hypothesis by comparing the dominant negative potency of PV on both TR isoforms. Indeed, we found that PV had a approximately 2-fold higher dominant negative action on wTRß1 than wTR{alpha}1. Thus, one of the factors that dictate the dominant negative potency of a mutant lies in the kind of the wild-type TR heterodimer partner.

The present studies clearly demonstrate that mutants not only formed heterodimers with TRß1, but with wTR{alpha}1. These heterodimers could account for the dominant negative effect of the mutant receptors. When equal amounts of wTRß1 and mutant receptors are present, the relative contribution of inactive forms will be enhanced due to the binding of a 2-fold higher amount of wTRß1/mutant heterodimers and of mutant/mutant homodimers to TREs, in competition with wTRß1 homodimers. It is reasonable to assume that a similar mechanism could operate in RTH patients as it has been shown that nearly equal amounts of normal and mutant TRß messenger RNA were found to be expressed in some tissues of the RTH patients (21, 22). In addition, mutants can also concurrently form inactive heterodimers with wTR{alpha}1 with binding of a approximately 3-fold higher amounts of wTR{alpha}1/mutant heterodimers to TRE, in competition with the wild-type homodimers. However, the differential stability of wTRß1/mutant and wTR{alpha}1/mutant heterodimers to T3 in the milieu added another additional layer of regulation on the potency of dominant negative action. However, it was reported that differential expression of normal and mutant TRß messenger RNA could occur in the kindred (23). This, together with the known differential expression of the wild-type isoform TRs in different tissues (24, 25) will result in different amounts of wild-type/mutant receptor heterodimers and mutant/mutant homodimers that in turn could fine tune the dominant negative action of the mutant receptors. Thus, the combinatorial diversities achieved via complexes of wild-type and mutant receptor heterodimers and mutant/mutant homodimers could contribute to the heterogeneity of tissue resistances observed in RTH patients.

Received October 4, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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