Dominant Negative Transcriptional Regulation by a Mutant Thyroid Hormone Receptor-/? in a Family with Generalized Resistance to Thyroid Hormone
Akihiro Sakurai, Takahide Miyamoto, Samuel Refetoff, and Leslie J. DeGroot Thyroid Study Unit University of Chicago Chicago, Illinois 60637
INTRODUCTION
An abnormal human thyroid hormone 0-receptor (hTR/3-Mf), which has a glycine to arginine substitution in the hormone-binding domain, has been identified in affected members of one family with generalized resistance to thyroid hormone. To better understand the mechanism by which this mutation produces the observed abnormality, expression vectors for the wild-type and mutant thyroid hormone receptors (TRs) were prepared to test hormone-binding activity and trans-activation function. Nuclear extracts of COS-7 cells transfected with wild-type TRs showed specific T3-binding activity, while mutant receptor-transfected COS-7 nuclear extract failed to bind T3. On the other hand, in a avidin-biotin complex DNA-binding assay, in vitro translated hTR/?-Mf showed high binding activity to the thyroid hormone response element, which was indistinguishable from that of wild-type TRs. In a transient expression study, only the wild-type TRs activated a rat GH gene promoter-chloramphenicol acetyltransferase fusion gene in a T3-dependent manner. Additionally, when wild-type TR and hTR0Mf were cotransfected, hTR/?-Mf inhibited gene activation regulated by wild-type TRs. From these results we conclude that 1) hTR0-Mf has no demonstrable T3 binding and appears to have minimal, if any, ability to activate a thyroid hormone-responsive gene in spite of its preserved ability to bind to a TRE in DNA; 2) hTR/7-Mf inhibits the transcriptional activation of a thyroid hormone-responsive gene by the wild-type TRs in a dominant manner; and 3) the dominant negative regulatory function of hTR/3-Mf appears to explain the clinical manifestations of thyroid hormone resistance produced by this mutation when present in the heterozygous state. (Molecular Endocrinology 4: 1988-1994, 1990)
The syndrome of generalized resistance to thyroid hormone (GRTH) is characterized by high serum levels of free thyroid hormone in the absence of increased basal metabolism and TSH suppression. Since its first recognition in 1967 (1), about 200 subjects with this syndrome have been identified, and both autosomal dominant and recessive modes of inheritance were found (2, 3). Although it had long been speculated that the syndrome could result from the existence of an abnormal thyroid hormone receptor (TR) (4), only recent progress in molecular biology enabled the identification of mutated human thyroid hormone 0-receptor (hTRjS) in affected subjects from two families (5, 6). To date there is agreement on the existence of two normal forms of /3-receptor encoded by the same gene. The hTR cDNA isolated by Weinberger et al. from placenta (7) is now referred to as 01, while the pituitary-specific form reported by Hodin et al. is referred to as /32 (8). TR/32 has been identified only in the rat to date. We, thus, refer to wild-type and mutant /3-receptor we identified as hTR/31-W and hTR/31-Mf, respectively, in the following text. These are identical to the previously designated hTRj8-W and hTR/3-Mf. The abnormal hTR/31 that we reported has a glycine (GGT) to arginine (CGT) substitution at amino acid codon 3451 in the ligand-binding domain, and as a result cannot bind thyroid hormone (5). Since affected members of this family are heterozygous and, thus, carry one normal allele, the question arises of how this mutation alters the function of normal receptors to cause the expression of an abnormal phenotype. Are their clinically evident abnormalities due to decreased expression of normal TR/3, or a negative function of the mutant recep1 This amino acid was previously numbered 340, but we recently found that hTRj81 has five additional amino acids at its amino-terminus, as deduced from the corrected sequence (9).
0888-8809/90/1988-1994$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
1988
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Function of Mutant Thyroid Hormone Receptor
1989
tor? In either case, the abnormality could be inherited in an autosomal dominant fashion. To answer these questions, we evaluated the function and interaction of the hTR/31-Mf with normal TRs.
50 -
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RESULTS a 20 -
We previously showed the lack of T3-binding activity by hTR/31-Mf using in vitro translation products (5). To confirm this result, we prepared vectors expressing various forms of hTRs (pCD series, see Materials and Methods) and transfected them into COS-7 cells. As anticipated, nuclear extracts of COS-7 cells transfected with pCDj81-W or pCD«1, which express the wild types of hTR/31 or hTRai, respectively, showed specific T3binding activity. On the other hand, nuclear extracts of COS-7 cells transfected with pCD/31-Mf or pCD«2, which express hTR/31 -Mf or non-T3-binding hTR variant «2 (hTRv«2), respectively, showed low T3 binding indistinguishable from mock (pCDM8)-transfected COS-7 cell extract (Fig. 1). We also examined the DNA-binding activity of the wild-type and mutant hTRs using the avidin-biotin complex DNA-binding (ABCD) assay. In vitro translated hTR/31-Mf showed high binding activity (mean ± SD, 35.9 ± 4.2%) to the thyroid hormone response element (TRE) sequence located in the rat GH gene promoter region. This binding activity was statistically not different from that Of hTR/31-W (39.7 ± 4.3%) or hTR«1 (40.5 ± 1.0%). However, binding of hTRv«2 to TRE was significantly {P < 0.01) lower than that of the three other forms (12.8 ± 2.8%; Fig. 2). This low binding of TRv«2 to the TRE sequence is in agreement with the data reported by Lazar et al. (10). Human TR binding was displaced by adding a 100-fold excess of nonbioti-
10 h
!• .
-pfii —rii 1 No Oligo
CONTROL
TRE
Fig. 2. Binding of in Vitro Synthesized Various hTRs to Biotinylated Oligonucleotides [35S]Methionine-labeled in vitro synthesized hTRs were allowed to bind to biotinylated oligonucleotides. CONTROL, A non-TRE-containing oligonucleotide sequence derived from a c/s-active element of adenovirus 5. TRE-containing oligonucleotide was derived from the rat GH gene 5' flanking region (-187/-158). For displacement, a 100-fold excess of nonbiotinylated TRE oligonucleotide was added to the reaction as a competitor (TRE + Comp.). Data are the mean ± SD of the percentage of total trichloroacetic acid-precipitable counts per min found in the streptoavidin-agarose precipitates. Experiments were performed in duplicate and were repeated three times.
* T3
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4
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|
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(-) cold T3
(+) cold T3
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2 2
TRE + Comp.
(-) (+) (-) (+) (-) (+) pCD(51-W pCDp"l-Mf pCDai
t
(-) (+) pCDa2
•
ft
(-) (+) pCDM8
Fig. 3. Transcriptional Regulation by Various hTRs Two micrograms of each expression vectors for various hTRs (hTR/31-W, /31-Mf, a 1 , and v«2) or a control plasmid (pCDM8) were transfected into COS-7 cells along with 4 ^g of the reporter plasmid pUrGH(S). After 48-h incubation in the absence (-) or presence (+) of 5 nw T3, cells were collected, and CAT activities were estimated.
1 -
pCDp1-W pCD|51-Mf pCDai
pCDa2
pCDM8
Fig. 1. T3-Binding Properties of Various hTRs Expression vectors for various hTRs were transfected into COS-7 cells. Nuclear extracts of transfected cells were prepared and assayed for binding to [125I]T3 in the absence or presence of a 1000-fold excess of unlabeled (cold) T3. Each bar indicates the mean value of two independent determinations.
nylated TRE oligonucleotide (TRE + Comp.), and no significant binding to control oligonucleotides was observed (CONTROL) (Fig. 2). These data demonstrate that the glycine to arginine substitution in hTR/31 -Mf abolishes or markedly reduces the hormone-binding activity of normal receptor, but does not affect the ability of the receptor to bind to TRE sequence in DNA. We, therefore, examined the frans-activating function of hTR/31-Mf using the TRE and a reporter gene. As shown in Fig. 3, hTR/31-W and
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MOL ENDO-1990 1990
VoUNo. 12
hTR«1 activated the rat GH gene promoter-thymidine kinase promoter-chloramphenicol acetyltransferase (CAT) fusion gene in a hormone-dependent manner, while hTR/31-Mf, hTRv«2, and pCDM8 showed no or very weak activation by T3. Figure 4 shows typical dose-response curves of CAT activation by the various hTRs and the variant hTR. In the absence of T3, all hTRs appeared to suppress the basal transcriptional activity [/31-W, 0.70 ± 0.37-fold; 01-Mf, 0.88 ± 0.26; «1, 0.68 ± 0.22; va2, 0.83 ± 0.41 (mean ± SD)]. This result is in agreement with the concept that in the absence of ligand, TRs function as TRE-specific repressors (11). Expression vectors for two functional TRs, pCD|81 -W and pCD«1, activated the CAT reporter gene in a T3 dose-dependent fashion. On the other hand, pCD«2 did not activate this reporter gene, and no significant activation was observed at T3 concentrations up to 500 nM. The pCD/31-Mf showed slight activation (mean ± SD, 1.37 ± 0.18-fold) at 50 nM T3. Thus, hTR/31-Mf might have a very low T3-binding activity which could not be detected by direct binding assay. Since affected subjects of this family are heterozygous and carry one normal allele, their clinical manifestation of thyroid hormone resistance may simply be the result of reduced expression of the normal hTR/31 or may be due to a dominant negative effect of the hTR/31 Mf. In fact, dominant inhibitory action of TRv«2 (12,13) and v-enbA (14,15) on the function of normal TRs has
been reported. We, therefore, evaluated the effect of coexpression of the hTR/31-Mf on the frans-activating function of hTR/31 -W and hTR«1. At 5 nM T3, pCD/31 W and pCD«1 activated the CAT gene by 7.3 ± 0.9fold (mean ± SD) and 9.9 ± 3.0-fold, respectively (Fig. 4). Cotransfected pCD/31-Mf inhibited CAT activation by pCD/31-W or pCD«1 by about 40% at a molar ratio of 1:1 (Fig. 5). When a 5-fold excess of pCD/31 -Mf was added, stronger inhibition was observed. The pCD/31Mf appeared to inhibit the action of pCD/31-W more effectively than that of pCD«1, although this difference was not statistically significant. This inhibitory effect is similar to that observed with TRv«2 (12, 13). We repeated the same experiments at T3 levels of 1 and 500 nM. Although CAT induction by hTR/31-W and hTR«1 varied at different T3 concentrations, the percent inhibition of the activation by hTRjSI -Mf did not significantly change at different T3 concentrations (data not shown).
DISCUSSION
Assuming that both the normal and mutant allele in affected subjects are equally expressed, a simple explanation for the dominant inheritance of thyroid hormone resistance would be the reduction of the wildtype hTR/3 to half the normal level. Although we have no direct information on the level of in vivo expression of the normal and mutant alleles, the presence of almost
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40
pCDa2 pCDM8
20
(-)
2
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pCD[5i-Mf 0.5
5
50
500
5000
T3 Concentration Fig. 4. T3 Dose Responsiveness of CAT Activation by Various hTRs The assay was performed as described in Fig. 3 in the presence of increasing concentrations of T3. CAT activation by pCDM8 in the absence of T3 was normalized to 1.0. Experiments were repeated four times. Maximum activation by hTRai and hTR/31-W varied in each experiment, but identical patterns of dose-response curve were obtained each time.
10
(H9)
pCDM8
Fig. 5. Negative Transcriptional Regulation by hTR/31-Mf Two micrograms of either pCD/31-W or pCDai were cotransfected into COS-7 cells along with 2 or 10 nQ pCD/31-Mf or 10 ng pCDM8 and 4 ng pUrGH(S). CAT activity in COS-7 cells transfected with either pCD/31 -W or pCDai and pllrGH(S) in the presence of 5 nM T3 was normalized to 100%. Data are the mean ± SD of four experiments. Each experiment was performed in duplicate. Statistical significance was examined for the effect of pCD#1-Mf on pCDj81 and pCD«1 by t test: *, P < 0.01; **, P < 0.05. n.s., Not significant.
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Function of Mutant Thyroid Hormone Receptor
equal number of wild-type and mutant hTR/31 cDNA clones obtained by amplification of mRNA from fibroblasts of affected member of the family (5) suggests that in this tissue both alleles are equally expressed. However, a recent finding in our laboratory indicates that reduced synthesis of the normal hTR/3 may not be sufficient to explain the clinical expression of the defect. We identified a large deletion encompassing the DNAand ligand-binding domains of the hTR/3 gene in another unrelated family with GRTH (16). In this family thyroid hormone resistance was present only in homozygous individuals with complete absence of the hTR/3 gene. Heterozygotes, possessing a single hTR/3 allele, showed no detectable abnormalities. This finding may indicate that the existence of only one allele is sufficient for normal hTR/3 function, although the possibility that mutant hTR/3 interferes with expression of the normal receptors cannot be ruled out. In rats, TR mRNA expression levels are regulated by thyroid hormone in a tissue-specific manner (17), implying that TR mRNA expression is autoregulated by TR per se. Thus, it is possible that a mutant TR affects the autoregulation feedback of the expression of normal TRs. Study of the regulation of TR expression will provide the answer to this question. A preferable explanation for the mechanism of thyroid hormone resistance in affected subjects is a dominant negative action by a mutant receptor (Fig. 5). Several mechanisms could account for this transcriptional regulation. The first possible mechanism is competition of normal and mutant receptors for binding to a TRE sequence in DNA. Since hTR/31-Mf has high binding activity to a TRE (Fig. 2), it could compete with other wild-type TRs for binding to a TRE, thus reducing their ability to activate thyroid hormone-responsive genes. TR-like molecules devoid of T3-binding activity (TRv«2 and v-enbA) function as dominant repressors of thyroid hormone-responsive genes (12-14). However, TRv«2 shows a lower DNA-binding activity than other TRs, although it has an identical DNA-binding domain (Fig. 2), and markedly reduced binding of v-enbA to TRE was demonstrated by gel retardation analysis (15). Therefore, this mechanism may not be enough to entirely explain dominant negative regulation by a mutant receptor. A second mechanism could be the formation of inactive receptor dimers. Although there are many studies concerning TRE sequences (18-24), a concensus sequence has not yet been confirmed, presumably because this sequence is poorly conserved from gene to gene. Brent et al. recently proposed a 6-base pair (bp) motif AGGT(C/A)A as a single receptor-binding site and demonstrated that at least two motifs are necessary for frans-activation (25). This supports the idea that TRs function as a dimer. In fact, as in the case of steroid hormone receptors (26-28), several studies support the concept of dimer formation by TRs. Leucine zipperlike motifs (heptad repeats) in the ligand-binding domain may be essential for dimer formation. Each of eight heptad repeats in hTR/3 contains hydrophobic amino
1991
acid residues at positions 1 and 8, and some of them also contain another hydrophobic residue at position 5 (29). Similar hydrophobic helices are found in several transcriptional factors, such as c-jun/c-fos (30-36), cmyc (37, 38), C/EBP (39), and GCN-4 (40), and are known to mediate dimerization of those protein molecules. In the case of TR, deletion of heptad repeats abolishes the frans-activation function of receptors (29). TRs are also able to form heterodimers with retinoic acid receptor, presumably through this motif, because when this region is deleted, the mutant cannot interact with retinoic acid receptor although intrinsic DNA-binding activity is preserved (41). Interestingly, an artificially prepared mutant TR/3, T/3I423437, which has two point mutations in its ligand-binding domain at amino acid codons 423 (leucine to isoleucine) and 437 (methionine to isoleucine),2 can form a dimer, but does not bind T3 (41). Human TR/31-Mf has one amino acid substitution in its ligand-binding domain, which is responsible for the loss of T3-binding activity (5). This mutation is located between the first and second heptads. However, a mutation in a heptad motif does not automatically indicate the loss of dimer-forming activity, since a mutation in T/3I423437 disrupts the heptad sequence without affecting its ability to form dimers. If hTR/31 -Mf preserved dimer-forming activity, as it probably does, it could form a mutant homodimer or a wild-mutant heterodimer. Formation of wild-mutant heterodimer would decrease the number of functional wild-type TR homodimers. The two mechanisms described above would simultaneously contribute to the dominant negative regulation by hTR/31 -Mf. Due to the formation of wild-mutant heterodimers, reduced amounts of wild-type homodimer will be formed, and this normal homodimer would then have to compete with heterodimer or mutant homodimer for binding to the TRE, further decreasing the number of functioning normal receptor. Another mechanism to be considered is competition between normal and mutant receptors for a limited amount of transcription factor. In a transient expression system, hTRv«2 inhibited the positive transcriptional regulation by hTR/31 or hTRai when high doses of expression plasmids were transfected into host cells, but this inhibition was not observed when low doses of plasmids were used (42). This result favors the hypothesis that hTRv«2 inhibits the function of hTR/31 and hTR«1 by depleting accessory factors that are essential for transcriptional activation by these receptors. Since we have not performed our experiment using small amounts of plasmid, we are unable to estimate this possibility or rule it out. Whatever the principal mechanism is, it is clear that hTR/31 -Mf interferes the transcriptional activity of normal receptors. Therefore, subjects expressing the mutant gene hTR/31-Mf would require supraphysiological doses of thyroid hormone to maintain appropriate 2
These numbers are according to the original report by Weinberger et al. (7). Corrected amino acid codon numbers (9), thus, should be 428 and 442, respectively.
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Vol4 No. 12
MOL ENDO-1990 1992
amounts of hormone-saturated wild-type TRs required for normal metabolism. We note that an attempt was made to normalize the blood thyroid hormone levels in one affected member of our sibship, and this resulted in aggravation of clinical symptoms (5). Although hTR/31-Mf slightly activated the TRE-CAT reporter gene at a high T3 concentration (Fig. 4), it is unlikely that this mutant protein is functional in vivo with such a low physiological activity. It is of interest that this weak activation by hTR/31-Mf was not enhanced by increasing T3 concentration (1.37 ± 0.18-fold at 50 nwi T3; 1.25 ± 0.22-fold at 500 nM T3). O'Donnell and Koenig (43) recently identified in rat TR/31 the region that is essential for transcriptional activation. Point mutations of amino acids 288, 290, and 300 of rat TR/31 made mutant receptors that have impaired transcriptional activity, but retain high T3-binding affinity. If weak transcriptional activity of hTR/31-Mf directly reflects its T3-binding activity, which is too low to detect by direct measurement, CAT activition at 500 nM T3 should have been higher than that at 50 nM. Therefore, it might be possible that a point mutation in hTR/31-Mf not only abolishes normal T3 binding of wild-type receptor, but also impairs the transcriptional activation. Little is known about the roles played by the various TR forms which are widely expressed in tissues (44, 45). It is not clear how they functionally interact with each other or whether they have distinct functions. Different TR defects are present in members of unrelated families with GRTH (5, 6,16,46). Their correlation with the observed genotypic and phenotypic abnormalities will facilitate understanding the pathophysiology of this syndrome and the mechanism of thyroid hormone action.
MATERIALS AND METHODS Plasmid Construction The wild-type /31 form of hTR (hTR/31) expression plasmid (pCD/31-W) was constructed from peA101, which contains the entire coding region of the hTR/?1 cDNA (7). The EcoRI insert of peA101 was ligated into the H/ndlll site of pCDM8 (47) using H/ndlll linkers. To prepare the expression plasmid for the mutant receptor hTR/31-Mf (pCD/31 -Mf), the EcoRI insert of phTR/3-Mf (5) was subcloned into pCDM8, as done for pCD]81-W. A human thyroid hormone «1 -receptor (hTRai) expression plasmid (pCD«1) was constructed by the same strategy using the EcoRI insert of pMe21 (48) and H/ndlll linkers. To make the hTR variant a2 (hTRv«2) expression plasmid (pCDa2), pKe711, which contains the entire hTRv«2 cDNA (49), was cut with Xba\, and the 3' «2-specific Xba\ fragment was ligated into Xbal-digested pCDai, which contained the 5' Xba\ fragment of hTR«1 cDNA. The reporter plasmid pUrGH(S) contained the BglU (-236)-Tnal (-147) fragment of the rat GH gene 5' flanking region. This fragment was ligated into the SamHI site of pUTKATI (50). This vector contains the herpes simplex virus thymidine kinase promoter, which directs expression of the CAT gene. Cell Culture and T3 Binding of hTRs Expressed by Transfected Cells COS-7 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Grand Island, NY) containing 10% (vol/
vol) fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 ng/m\) in 10-cm petri dishes at a density of 1 x 106 cells/ dish. Twenty hours before transfection, medium was changed to DMEM containing 10% hormone-depleted fetal bovine serum (51). Twenty micrograms of expression vector or pCDM8 were transfected into COS-7 cells using the calcium phosphate coprecipitate method (52). Twenty hours after transfection, the medium was changed, and incubation was continued for 48 h before harvest. Preparation of nuclear extracts from the transfected cells and T3 binding assay were performed as previously described (53). The protein concentration of nuclear extract was determined by the Coomassie blue method, using BSA as a standard (54). ABCD Assay The ABCD assay procedure used was as described by Glass et al. (55). The TRE oligomer (44 bp) contained the sequence -187 through -158 of the rat GH 5' flanking region and was designed to allow incorporation of eight biotin-11 -UMP residues into its 3' termini. After annealing the two complimentary oligonucleotides, the 5' overhangs at each end were filled in using Klenow fragment, 200 nM each of dATP, dCTP, and dGTP, and 200 M M biotin-11-dUTP. Unincorporated nucleotides were removed by a single passage through a Sephadex G-50 column, and the eluent was ethanol precipitated. The control oligomer (44 bp) contained the sequence from a cisactive element of adenovirus 5 (56). This oligomer contained six biotin-11 -UMP residues after Klenow fragment fill in. Highly efficient binding of both biotinylated oligomers to streptoavidinagarose was confirmed using 32P end-labeled biotin-DNAs. A nonbiotinylated TRE oligomer was also prepared using dTTP instead of biotin-11-dUTP. In vitro transcription and translation of hTR/31-W and hTR/31 -Mf were performed as previously described (5). [35S] Methionine-labeled hTR«1 and hTRva2 were synthesized as previously described (48, 49). Reticulocyte lysates programmed with each hTR were centrifuged through Sephadex G-50 columns equilibrated with ABCD buffer [50 mwi KCI, 20 mM KPO4 (pH 7.4), 1 mM MgCI2, 20% glycerol, and 1 HIM 0mercaptoethanol] to remove unincorporated [35S]methionine and change buffer. For the binding assay, 2-5 x 104 cpm trichloroacetic acid-precipitable [35S]methionine-labeled proteins were incubated with 1 pmol biotinylated oligomer and 0.2 n$ln\ poly(dl-dC) in 20 n\ ABCD buffer for 40 min at 22 C. Twenty microliters of streptoavidin-agarose suspension were added and incubated on ice for 10 min, and 1 ml ice-cold ABCD buffer was added before centrifugation. Streptoavidinagarose beads were washed with ABCD buffer, and radioactivity in the final pellets was estimated by scintillation counting. CAT Assay COS-7 cells were grown as described above. Cells were transfected with 20 ^g plasmids, including 4 ng reporter plasmid pUrGH(S). COS-7 cells express the simian virus 40 (SV40) T-antigen, which can cause high level replication of transfected CD expression plasmids containing the SV40 origin of replication (47). To maintain the amount of CD plasmid constant, pCDM8 was added when necessary. Twenty hours after transfection by the calcium phosphate method, the medium was changed to DMEM supplemented with 10% hormone-depleted fetal bovine serum with or without the addition of various amounts of T3. Cells were harvested after 48-h incubation, and CAT activity in cell extracts was measured using [14C] chloramphenicol (57). We found that the intraassay variability due to transfection efficiency was minimal, and standardization using a cotransfected GH expression plasmid did not significantly reduce this variability. Therefore, we did not include another expression plasmid for normalization, but all experiments in each figure were performed in duplicate on the same day, and experiments were repeated four times.
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1993
Function of Mutant Thyroid Hormone Receptor
Acknowledgments The authors wish to thank Drs. C. Weinberger and R. M. Evans for providing peA101, Drs. A. Aruffo and B. Seed for providing pCDM8, and Dr. D. D. Moore for providing pUTKATI.
15.
16. Received July 25, 1990. Revision received September 18, 1990. Accepted September 26, 1990. Address requests for reprints to: Dr. Leslie J. DeGroot, Box 138, Thyroid Study Unit, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637. This work was supported in part by USPHS Grants DK13377 and DK-15070, the March of Dimes Birth Defects Foundation, and the David Wiener Research Fund.
17.
18. 19.
REFERENCES 20. 1. Refetoff S, DeWind LT, DeGroot LJ 1967 Familial syndrome combining deaf-mutism, stippled epiphyses, goiter, and abnormally high PBI: possible target organ refractoriness to thyroid hormone. J Clin Endocrinol Metab 27:279-294 2. Refetoff S1982 Syndrome of thyroid hormone resistance. Am J Physiol 243:E88-98 3. Refetoff S 1989 The syndrome of generalized resistance to thyroid hormone (GRTH). Endocr Res 15:717-743 4. Refetoff S, DeGroot U , Benard B, DeWind LT 1972 Studies of a sibship with apparent hereditary resistance to the intracellular action of thyroid hormone. Metabolism 21:723-756 5. Sakurai A, Takeda K, Ain K, Ceccarelli P, Nakai A, Seino S, Bell Gl, Refetoff S, DeGroot LJ 1989 Generalized resistance to thyroid hormone associated with a mutation in the ligand-binding domain of the human thyroid hormone receptor 0. Proc Natl Acad Sci USA 86:8977-8981 6. Usala SJ, Tennyson GE, Bale AE, Lash RW, Gesundheit N, Wondisford FE, Accili D, Hauser P, Weintraub BD1990 A base mutation of the c-eroA/3 thyroid hormone receptor in a kindred with thyroid hormone resistance. Molecular heterogeneity in two other kindreds. J Clin Invest 85:93100 7. Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erb-A gene encodes a thyroid hormone receptor. Nature 324:641-6 8. Hodin RA, Lazar MA, Wintman Bl, Darling DS, Koenig RJ, Larsen PR, Moore DD, Chin WW 1989 Identification of a thyroid hormCne receptor that is pituitary-specific. Science 244:76-79 9. Sakurai A, Nakai A, DeGroot LJ 1990 Structural analysis of human thyroid hormone receptor /3 gene. Mol Cell Endocrinol 71:83-91 10. Lazar MA, Hodin RA, Darling DS, Chin WW 1988 Identification of a rat c-erbA a-related protein which binds deoxyribonucleic acid but does not bind thyroid hormone. Mol Endocrinol 2:893-901 11. Graupner G, Wills KN, Tzukerman M, Zhang X-k, Pfahl M 1989 Dual regulatory role for thyroid-hormone receptors allows control of retinoic-acid receptor activity. Nature 340:653-656 12. Koenig RJ, Lazar MA, Hodin RA, Brent GA, Larsen PR, Chin WW, Moore DD 1989 Inhibition of thyroid hormone action by a non-hormone binding c-ertoA protein generated by alternative mRNA splicing. Nature 337:659-661 13. Lazar MA, Hodin RA, Chin WW 1989 Human carboxylterminal variant of a-type c-erbA inhibits trans-activation by thyroid hormone receptors without binding thyroid hormone. Proc Natl Acad Sci USA 86:7771-7774 14. Damm K, Thompson CC, Evans RM 1989 Protein en-
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24. 25.
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33.
34.
coded by v-eroA functions as a thyroid-hormone receptor antagonist. Nature 339:593-597 Sap J, Munoz A, Schmitt J, Stunnenberg H, Vennstrom B 1989 Repression of transcription mediated at a thyroid hormone response element by the v-erb-A oncogene product. Nature 340:242-244 Takeda K, Balzano S, Sakurai A, DeGroot LJ, Refetoff S, Screening of 19 unrelated families with generalized resistance to thyroid hormone (GRTH) for known point mutations in the thyroid receptor (TR) /3 and the detection of a new mutation. 72nd Annual Meeting of The Endocrine Society, Atlanta GA, 1990, p 25 (Abstract) Hodin RA, Lazar MA, Chin WW 1990 Differential and tissue-specific regulation of the multiple rat c-erbA messenger RNA species by thyroid hormone. J Clin Invest 85:101-105 Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889-895 Izumo S, Mahdavi V 1988 Thyroid hormone receptor isoforms generated by alternative splicing differentially activate myosin HC gene transcription. Nature 334:539542 Norman MF, Lavin TN, Baxter JD, West BL 1989 The rat growth hormone gene contains multiple thyroid response elements. J Biol Chem 264:12063-12073 Burnside J, Darling DS, Carr FE, Chin WW 1989 Thyroid hormone regulation of the rat glycoprotein hormone asubunit gene promoter activity. J Biol Chem 264:68866891 Darling DS, Burnside J, Chin WW 1989 Binding of thyroid hormone receptors to the rat thyrotropin-/3 gene. Mol Endocrinol 3:1359-1368 Wondisford FE, Farr EA, Radovic S, Steinfelder HJ, Moates JM, McClaskey JH, Weintraub BD 1989 Thyroid hormone inhibition of the thyrotropin /3-subunit gene expression is mediated by a c/s-acting element located in the first exon. J Biol Chem 264:14601-14604 Petty KJ, Desvergne B, Mitsuhashi T, Nikodem VM 1990 Identification of a thyroid hormone response element in the malic enzyme gene. J Biol Chem 265:7395-7400 Brent GA, Harney JW, Warne RL, Moore DD, Larsen PR 1989 Mutations of the rat growth hormone promoter which increase and decrease response to thyroid hormone define a consensus thyroid hormone response element. Mol Endocrinol 3:1996-2004 Kumar V, Chambon P 1988 The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145-156 Tsai SY, Carlstedt-Duke J, Weigel NL, Dahlman K, Gustafsson J-A, Tsai M-J, O'Malley BW 1988 Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation. Cell 55:697-708 Wrange 6, Erikson P, Perlmann T 1989 The purified activated glucocorticoid receptor is a homodimer. J Biol Chem 264:5253-5259 Forman BM, Yang C-r, Au M, Casanova J, Ghysdeal J, Samuels HH 1989 A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors. Mol Endocrinol 3:1610-1626 Kouzarides T, Ziff E 1988 The role of the leucine zipper in the fos-jun interaction. Nature 336:646-651 Sassone-Corsi P, Ransone LJ, Lamph WW, Verma IM 1988 Direct interaction between fos and jun nuclear oncoproteins: role of the leucine zipper domain. Nature 336:692-695 Turner R, Tjian R 1989 Leucine repeats and an adjacent DNA binding domain mediate the formation of functional cFos-cJun heterodimers. Science 243:1689-1694 Gentz R, Rauscher III FJ, Abate C, Curran T1989 Parallel association of fos and jun leucine zippers juxtaposes DNA binding domains. Science 243:1695-1699 Neuberg M, Schuermann M, Hunter JB, Muller R 1989
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MOL ENDO-1990 1994
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Two functionally different regions in Fos are required for the sequence-specific DNA interaction of the Fos/Jun protein complex. Nature 338:589-590 Schuermann M, Neuberg M, Hunter JB, Jenuwein T, Ryseck RP, Bravo R, Muller R 1989 The leucine repeat motif in fos protein mediates complex formation with JUN/ AP-1 and is required for transformation. Cell 56:507-516 Ransone LJ, Visvader J, Sassone-Corsi P, Verma IM1989 Fos-Jun interaction: mutational analysis of the leucine zipper domain of both proteins. Genes Dev 3:770-781 Murre C, Schonleber-McCaw P, Baltimore D 1989 A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56:777-783 Dang CV, McGuire M, Buckmire M, Lee WMF 1989 Involvement of the leucine zipper region in the oligomerization and transforming activity of human c-myc protein. Nature 337:664-666 Landchultz WH, Johnson PF, McKnight SL1989 The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite. Science 243:1681-1688 Hope IA, Struhl K1987 GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J 6:2781-2784 Glass CK, Lipkin SM, Devary OV, Rosenfeld MG 1989 Positive and negative regulation of gene transcription by a retinoic acid-thyroid hormone receptor heterodimer. Cell 59:697-708 Rentoumis A, Chatterjee VKK, Madison LD, Datta S, Gallagher GD, DeGroot LJ, Jameson JL 1990 Negative and positive transcriptional regulation by thyroid hormone receptor isoforms. Mol Endocrinol 4:1522-1531 O'Donnell AL, Koenig RJ 1990 Mutational analysis identifies a new functional domain of the thyroid hormone receptor. Mol Endocrinol 4:715-720 Sakurai A, Nakai A, DeGroot LJ 1989 Expression of three forms of thyroid hormone receptor in human tissues. Mol Endocrinol 3:392-399 Macchia E, Nakai A, Janiga A, Sakurai A, Fisfalen ME, Gardner P, Soltani K, DeGroot LJ 1990 Characterization of site-specific polyclonal antibodies to c-erbA peptides recognizing human thyroid hormone receptors «1, a2 and 0 and native 3,5,3'-triiodothyronine receptor, and study of tissue distribution of the antigen. Endocrinology 126:3232-3239
Vol4No. 12
46. Usala SJ, Wondisford FE, Watson TL, Menke JB, Mueller OT, Ono S, Bercu BB, Weintraub BD, Generalized thyroid hormone resistance: clinical and molecular studies. 72nd Annual Meeting of The Endocrine Society, Atlanta GA, 1990, p 18 (Abstract) 47. Aruffo A, Seed B 1987 Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc Natl Acad Sci USA 84:8573-8577 48. Nakai A, Sakurai A, Bell Gl, DeGroot LJ 1988 Characterization of a third human thyroid hormone receptor coexpressed with other thyroid hormone receptors in several tissues. Mol Endocrinol 2:1087-1092 49. Nakai A, Seino S, Sakurai A, Szilak I, Bell Gl, DeGroot LJ 1988 Characterization of a thyroid hormone receptor expressed in human kidney and other tissues. Proc Natl Acad Sci USA 85:2781-2785 50. Prost E, Moore DD 1986 CAT vectors for analysis of eukaryotic promoters and enhancers. Gene 45:107-111 51. Flug F, Copp RP, Casanova J, Horowitz ZD, Janocko L, Plotnick M, Samuels HH 1987 C/s-acting elements of the rat growth hormone gene which mediate basal and regulated expression by thyroid hormone. J Biol Chem 262:6373-6382 52. Graham FL, Van der Eb AJ 1973 A new technique for the assay of infectivity of human adenovirus DNA. Virology 52:456-467 53. Ichikawa K, Hughes IA, Horwitz AL, DeGroot LJ 1987 Characterization of nuclear thyroid hormone receptors of cultured skin fibroblasts from patients with resistance to thyroid hormone. Metabolism 36:392-399 54. Bradford M 1976 A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248254 55. Glass CK, Holloway JM, DeVary OV, Rosenfeld MG 1988 The thyroid hormone receptor binds with opposite transcriptional effects to a common sequence motif in thyroid hormone and estrogen response elements. Cell 54:313323 56. Hearing P, Shenk T 1983 The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element. Cell 33:695-703 57. Gorman CM, Moffat LF, Howard BH 1982 Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:1044-1051
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Akihiro Sakurai, Takahide Miyamoto, Samuel Refetoff, and Leslie J. DeGroot Thyroid Study Unit University of Chicago Chicago, Illinois 60637
INTRODUCTION
An abnormal human thyroid hormone 0-receptor (hTR/3-Mf), which has a glycine to arginine substitution in the hormone-binding domain, has been identified in affected members of one family with generalized resistance to thyroid hormone. To better understand the mechanism by which this mutation produces the observed abnormality, expression vectors for the wild-type and mutant thyroid hormone receptors (TRs) were prepared to test hormone-binding activity and trans-activation function. Nuclear extracts of COS-7 cells transfected with wild-type TRs showed specific T3-binding activity, while mutant receptor-transfected COS-7 nuclear extract failed to bind T3. On the other hand, in a avidin-biotin complex DNA-binding assay, in vitro translated hTR/?-Mf showed high binding activity to the thyroid hormone response element, which was indistinguishable from that of wild-type TRs. In a transient expression study, only the wild-type TRs activated a rat GH gene promoter-chloramphenicol acetyltransferase fusion gene in a T3-dependent manner. Additionally, when wild-type TR and hTR0Mf were cotransfected, hTR/?-Mf inhibited gene activation regulated by wild-type TRs. From these results we conclude that 1) hTR0-Mf has no demonstrable T3 binding and appears to have minimal, if any, ability to activate a thyroid hormone-responsive gene in spite of its preserved ability to bind to a TRE in DNA; 2) hTR/7-Mf inhibits the transcriptional activation of a thyroid hormone-responsive gene by the wild-type TRs in a dominant manner; and 3) the dominant negative regulatory function of hTR/3-Mf appears to explain the clinical manifestations of thyroid hormone resistance produced by this mutation when present in the heterozygous state. (Molecular Endocrinology 4: 1988-1994, 1990)
The syndrome of generalized resistance to thyroid hormone (GRTH) is characterized by high serum levels of free thyroid hormone in the absence of increased basal metabolism and TSH suppression. Since its first recognition in 1967 (1), about 200 subjects with this syndrome have been identified, and both autosomal dominant and recessive modes of inheritance were found (2, 3). Although it had long been speculated that the syndrome could result from the existence of an abnormal thyroid hormone receptor (TR) (4), only recent progress in molecular biology enabled the identification of mutated human thyroid hormone 0-receptor (hTRjS) in affected subjects from two families (5, 6). To date there is agreement on the existence of two normal forms of /3-receptor encoded by the same gene. The hTR cDNA isolated by Weinberger et al. from placenta (7) is now referred to as 01, while the pituitary-specific form reported by Hodin et al. is referred to as /32 (8). TR/32 has been identified only in the rat to date. We, thus, refer to wild-type and mutant /3-receptor we identified as hTR/31-W and hTR/31-Mf, respectively, in the following text. These are identical to the previously designated hTRj8-W and hTR/3-Mf. The abnormal hTR/31 that we reported has a glycine (GGT) to arginine (CGT) substitution at amino acid codon 3451 in the ligand-binding domain, and as a result cannot bind thyroid hormone (5). Since affected members of this family are heterozygous and, thus, carry one normal allele, the question arises of how this mutation alters the function of normal receptors to cause the expression of an abnormal phenotype. Are their clinically evident abnormalities due to decreased expression of normal TR/3, or a negative function of the mutant recep1 This amino acid was previously numbered 340, but we recently found that hTRj81 has five additional amino acids at its amino-terminus, as deduced from the corrected sequence (9).
0888-8809/90/1988-1994$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
1988
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Function of Mutant Thyroid Hormone Receptor
1989
tor? In either case, the abnormality could be inherited in an autosomal dominant fashion. To answer these questions, we evaluated the function and interaction of the hTR/31-Mf with normal TRs.
50 -
40 -
• hTRpi-W • hTRpi-Mf a hTRai • hTRva2
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30 -
RESULTS a 20 -
We previously showed the lack of T3-binding activity by hTR/31-Mf using in vitro translation products (5). To confirm this result, we prepared vectors expressing various forms of hTRs (pCD series, see Materials and Methods) and transfected them into COS-7 cells. As anticipated, nuclear extracts of COS-7 cells transfected with pCDj81-W or pCD«1, which express the wild types of hTR/31 or hTRai, respectively, showed specific T3binding activity. On the other hand, nuclear extracts of COS-7 cells transfected with pCD/31-Mf or pCD«2, which express hTR/31 -Mf or non-T3-binding hTR variant «2 (hTRv«2), respectively, showed low T3 binding indistinguishable from mock (pCDM8)-transfected COS-7 cell extract (Fig. 1). We also examined the DNA-binding activity of the wild-type and mutant hTRs using the avidin-biotin complex DNA-binding (ABCD) assay. In vitro translated hTR/31-Mf showed high binding activity (mean ± SD, 35.9 ± 4.2%) to the thyroid hormone response element (TRE) sequence located in the rat GH gene promoter region. This binding activity was statistically not different from that Of hTR/31-W (39.7 ± 4.3%) or hTR«1 (40.5 ± 1.0%). However, binding of hTRv«2 to TRE was significantly {P < 0.01) lower than that of the three other forms (12.8 ± 2.8%; Fig. 2). This low binding of TRv«2 to the TRE sequence is in agreement with the data reported by Lazar et al. (10). Human TR binding was displaced by adding a 100-fold excess of nonbioti-
10 h
!• .
-pfii —rii 1 No Oligo
CONTROL
TRE
Fig. 2. Binding of in Vitro Synthesized Various hTRs to Biotinylated Oligonucleotides [35S]Methionine-labeled in vitro synthesized hTRs were allowed to bind to biotinylated oligonucleotides. CONTROL, A non-TRE-containing oligonucleotide sequence derived from a c/s-active element of adenovirus 5. TRE-containing oligonucleotide was derived from the rat GH gene 5' flanking region (-187/-158). For displacement, a 100-fold excess of nonbiotinylated TRE oligonucleotide was added to the reaction as a competitor (TRE + Comp.). Data are the mean ± SD of the percentage of total trichloroacetic acid-precipitable counts per min found in the streptoavidin-agarose precipitates. Experiments were performed in duplicate and were repeated three times.
* T3
•r
4
"
|
|
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(-) cold T3
(+) cold T3
C 3
2 2
TRE + Comp.
(-) (+) (-) (+) (-) (+) pCD(51-W pCDp"l-Mf pCDai
t
(-) (+) pCDa2
•
ft
(-) (+) pCDM8
Fig. 3. Transcriptional Regulation by Various hTRs Two micrograms of each expression vectors for various hTRs (hTR/31-W, /31-Mf, a 1 , and v«2) or a control plasmid (pCDM8) were transfected into COS-7 cells along with 4 ^g of the reporter plasmid pUrGH(S). After 48-h incubation in the absence (-) or presence (+) of 5 nw T3, cells were collected, and CAT activities were estimated.
1 -
pCDp1-W pCD|51-Mf pCDai
pCDa2
pCDM8
Fig. 1. T3-Binding Properties of Various hTRs Expression vectors for various hTRs were transfected into COS-7 cells. Nuclear extracts of transfected cells were prepared and assayed for binding to [125I]T3 in the absence or presence of a 1000-fold excess of unlabeled (cold) T3. Each bar indicates the mean value of two independent determinations.
nylated TRE oligonucleotide (TRE + Comp.), and no significant binding to control oligonucleotides was observed (CONTROL) (Fig. 2). These data demonstrate that the glycine to arginine substitution in hTR/31 -Mf abolishes or markedly reduces the hormone-binding activity of normal receptor, but does not affect the ability of the receptor to bind to TRE sequence in DNA. We, therefore, examined the frans-activating function of hTR/31-Mf using the TRE and a reporter gene. As shown in Fig. 3, hTR/31-W and
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MOL ENDO-1990 1990
VoUNo. 12
hTR«1 activated the rat GH gene promoter-thymidine kinase promoter-chloramphenicol acetyltransferase (CAT) fusion gene in a hormone-dependent manner, while hTR/31-Mf, hTRv«2, and pCDM8 showed no or very weak activation by T3. Figure 4 shows typical dose-response curves of CAT activation by the various hTRs and the variant hTR. In the absence of T3, all hTRs appeared to suppress the basal transcriptional activity [/31-W, 0.70 ± 0.37-fold; 01-Mf, 0.88 ± 0.26; «1, 0.68 ± 0.22; va2, 0.83 ± 0.41 (mean ± SD)]. This result is in agreement with the concept that in the absence of ligand, TRs function as TRE-specific repressors (11). Expression vectors for two functional TRs, pCD|81 -W and pCD«1, activated the CAT reporter gene in a T3 dose-dependent fashion. On the other hand, pCD«2 did not activate this reporter gene, and no significant activation was observed at T3 concentrations up to 500 nM. The pCD/31-Mf showed slight activation (mean ± SD, 1.37 ± 0.18-fold) at 50 nM T3. Thus, hTR/31-Mf might have a very low T3-binding activity which could not be detected by direct binding assay. Since affected subjects of this family are heterozygous and carry one normal allele, their clinical manifestation of thyroid hormone resistance may simply be the result of reduced expression of the normal hTR/31 or may be due to a dominant negative effect of the hTR/31 Mf. In fact, dominant inhibitory action of TRv«2 (12,13) and v-enbA (14,15) on the function of normal TRs has
been reported. We, therefore, evaluated the effect of coexpression of the hTR/31-Mf on the frans-activating function of hTR/31 -W and hTR«1. At 5 nM T3, pCD/31 W and pCD«1 activated the CAT gene by 7.3 ± 0.9fold (mean ± SD) and 9.9 ± 3.0-fold, respectively (Fig. 4). Cotransfected pCD/31-Mf inhibited CAT activation by pCD/31-W or pCD«1 by about 40% at a molar ratio of 1:1 (Fig. 5). When a 5-fold excess of pCD/31 -Mf was added, stronger inhibition was observed. The pCD/31Mf appeared to inhibit the action of pCD/31-W more effectively than that of pCD«1, although this difference was not statistically significant. This inhibitory effect is similar to that observed with TRv«2 (12, 13). We repeated the same experiments at T3 levels of 1 and 500 nM. Although CAT induction by hTR/31-W and hTR«1 varied at different T3 concentrations, the percent inhibition of the activation by hTRjSI -Mf did not significantly change at different T3 concentrations (data not shown).
DISCUSSION
Assuming that both the normal and mutant allele in affected subjects are equally expressed, a simple explanation for the dominant inheritance of thyroid hormone resistance would be the reduction of the wildtype hTR/3 to half the normal level. Although we have no direct information on the level of in vivo expression of the normal and mutant alleles, the presence of almost
n,s,(ct,P)
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o
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40
pCDa2 pCDM8
20
(-)
2
10
pCD[5i-Mf 0.5
5
50
500
5000
T3 Concentration Fig. 4. T3 Dose Responsiveness of CAT Activation by Various hTRs The assay was performed as described in Fig. 3 in the presence of increasing concentrations of T3. CAT activation by pCDM8 in the absence of T3 was normalized to 1.0. Experiments were repeated four times. Maximum activation by hTRai and hTR/31-W varied in each experiment, but identical patterns of dose-response curve were obtained each time.
10
(H9)
pCDM8
Fig. 5. Negative Transcriptional Regulation by hTR/31-Mf Two micrograms of either pCD/31-W or pCDai were cotransfected into COS-7 cells along with 2 or 10 nQ pCD/31-Mf or 10 ng pCDM8 and 4 ng pUrGH(S). CAT activity in COS-7 cells transfected with either pCD/31 -W or pCDai and pllrGH(S) in the presence of 5 nM T3 was normalized to 100%. Data are the mean ± SD of four experiments. Each experiment was performed in duplicate. Statistical significance was examined for the effect of pCD#1-Mf on pCDj81 and pCD«1 by t test: *, P < 0.01; **, P < 0.05. n.s., Not significant.
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Function of Mutant Thyroid Hormone Receptor
equal number of wild-type and mutant hTR/31 cDNA clones obtained by amplification of mRNA from fibroblasts of affected member of the family (5) suggests that in this tissue both alleles are equally expressed. However, a recent finding in our laboratory indicates that reduced synthesis of the normal hTR/3 may not be sufficient to explain the clinical expression of the defect. We identified a large deletion encompassing the DNAand ligand-binding domains of the hTR/3 gene in another unrelated family with GRTH (16). In this family thyroid hormone resistance was present only in homozygous individuals with complete absence of the hTR/3 gene. Heterozygotes, possessing a single hTR/3 allele, showed no detectable abnormalities. This finding may indicate that the existence of only one allele is sufficient for normal hTR/3 function, although the possibility that mutant hTR/3 interferes with expression of the normal receptors cannot be ruled out. In rats, TR mRNA expression levels are regulated by thyroid hormone in a tissue-specific manner (17), implying that TR mRNA expression is autoregulated by TR per se. Thus, it is possible that a mutant TR affects the autoregulation feedback of the expression of normal TRs. Study of the regulation of TR expression will provide the answer to this question. A preferable explanation for the mechanism of thyroid hormone resistance in affected subjects is a dominant negative action by a mutant receptor (Fig. 5). Several mechanisms could account for this transcriptional regulation. The first possible mechanism is competition of normal and mutant receptors for binding to a TRE sequence in DNA. Since hTR/31-Mf has high binding activity to a TRE (Fig. 2), it could compete with other wild-type TRs for binding to a TRE, thus reducing their ability to activate thyroid hormone-responsive genes. TR-like molecules devoid of T3-binding activity (TRv«2 and v-enbA) function as dominant repressors of thyroid hormone-responsive genes (12-14). However, TRv«2 shows a lower DNA-binding activity than other TRs, although it has an identical DNA-binding domain (Fig. 2), and markedly reduced binding of v-enbA to TRE was demonstrated by gel retardation analysis (15). Therefore, this mechanism may not be enough to entirely explain dominant negative regulation by a mutant receptor. A second mechanism could be the formation of inactive receptor dimers. Although there are many studies concerning TRE sequences (18-24), a concensus sequence has not yet been confirmed, presumably because this sequence is poorly conserved from gene to gene. Brent et al. recently proposed a 6-base pair (bp) motif AGGT(C/A)A as a single receptor-binding site and demonstrated that at least two motifs are necessary for frans-activation (25). This supports the idea that TRs function as a dimer. In fact, as in the case of steroid hormone receptors (26-28), several studies support the concept of dimer formation by TRs. Leucine zipperlike motifs (heptad repeats) in the ligand-binding domain may be essential for dimer formation. Each of eight heptad repeats in hTR/3 contains hydrophobic amino
1991
acid residues at positions 1 and 8, and some of them also contain another hydrophobic residue at position 5 (29). Similar hydrophobic helices are found in several transcriptional factors, such as c-jun/c-fos (30-36), cmyc (37, 38), C/EBP (39), and GCN-4 (40), and are known to mediate dimerization of those protein molecules. In the case of TR, deletion of heptad repeats abolishes the frans-activation function of receptors (29). TRs are also able to form heterodimers with retinoic acid receptor, presumably through this motif, because when this region is deleted, the mutant cannot interact with retinoic acid receptor although intrinsic DNA-binding activity is preserved (41). Interestingly, an artificially prepared mutant TR/3, T/3I423437, which has two point mutations in its ligand-binding domain at amino acid codons 423 (leucine to isoleucine) and 437 (methionine to isoleucine),2 can form a dimer, but does not bind T3 (41). Human TR/31-Mf has one amino acid substitution in its ligand-binding domain, which is responsible for the loss of T3-binding activity (5). This mutation is located between the first and second heptads. However, a mutation in a heptad motif does not automatically indicate the loss of dimer-forming activity, since a mutation in T/3I423437 disrupts the heptad sequence without affecting its ability to form dimers. If hTR/31 -Mf preserved dimer-forming activity, as it probably does, it could form a mutant homodimer or a wild-mutant heterodimer. Formation of wild-mutant heterodimer would decrease the number of functional wild-type TR homodimers. The two mechanisms described above would simultaneously contribute to the dominant negative regulation by hTR/31 -Mf. Due to the formation of wild-mutant heterodimers, reduced amounts of wild-type homodimer will be formed, and this normal homodimer would then have to compete with heterodimer or mutant homodimer for binding to the TRE, further decreasing the number of functioning normal receptor. Another mechanism to be considered is competition between normal and mutant receptors for a limited amount of transcription factor. In a transient expression system, hTRv«2 inhibited the positive transcriptional regulation by hTR/31 or hTRai when high doses of expression plasmids were transfected into host cells, but this inhibition was not observed when low doses of plasmids were used (42). This result favors the hypothesis that hTRv«2 inhibits the function of hTR/31 and hTR«1 by depleting accessory factors that are essential for transcriptional activation by these receptors. Since we have not performed our experiment using small amounts of plasmid, we are unable to estimate this possibility or rule it out. Whatever the principal mechanism is, it is clear that hTR/31 -Mf interferes the transcriptional activity of normal receptors. Therefore, subjects expressing the mutant gene hTR/31-Mf would require supraphysiological doses of thyroid hormone to maintain appropriate 2
These numbers are according to the original report by Weinberger et al. (7). Corrected amino acid codon numbers (9), thus, should be 428 and 442, respectively.
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Vol4 No. 12
MOL ENDO-1990 1992
amounts of hormone-saturated wild-type TRs required for normal metabolism. We note that an attempt was made to normalize the blood thyroid hormone levels in one affected member of our sibship, and this resulted in aggravation of clinical symptoms (5). Although hTR/31-Mf slightly activated the TRE-CAT reporter gene at a high T3 concentration (Fig. 4), it is unlikely that this mutant protein is functional in vivo with such a low physiological activity. It is of interest that this weak activation by hTR/31-Mf was not enhanced by increasing T3 concentration (1.37 ± 0.18-fold at 50 nwi T3; 1.25 ± 0.22-fold at 500 nM T3). O'Donnell and Koenig (43) recently identified in rat TR/31 the region that is essential for transcriptional activation. Point mutations of amino acids 288, 290, and 300 of rat TR/31 made mutant receptors that have impaired transcriptional activity, but retain high T3-binding affinity. If weak transcriptional activity of hTR/31-Mf directly reflects its T3-binding activity, which is too low to detect by direct measurement, CAT activition at 500 nM T3 should have been higher than that at 50 nM. Therefore, it might be possible that a point mutation in hTR/31-Mf not only abolishes normal T3 binding of wild-type receptor, but also impairs the transcriptional activation. Little is known about the roles played by the various TR forms which are widely expressed in tissues (44, 45). It is not clear how they functionally interact with each other or whether they have distinct functions. Different TR defects are present in members of unrelated families with GRTH (5, 6,16,46). Their correlation with the observed genotypic and phenotypic abnormalities will facilitate understanding the pathophysiology of this syndrome and the mechanism of thyroid hormone action.
MATERIALS AND METHODS Plasmid Construction The wild-type /31 form of hTR (hTR/31) expression plasmid (pCD/31-W) was constructed from peA101, which contains the entire coding region of the hTR/?1 cDNA (7). The EcoRI insert of peA101 was ligated into the H/ndlll site of pCDM8 (47) using H/ndlll linkers. To prepare the expression plasmid for the mutant receptor hTR/31-Mf (pCD/31 -Mf), the EcoRI insert of phTR/3-Mf (5) was subcloned into pCDM8, as done for pCD]81-W. A human thyroid hormone «1 -receptor (hTRai) expression plasmid (pCD«1) was constructed by the same strategy using the EcoRI insert of pMe21 (48) and H/ndlll linkers. To make the hTR variant a2 (hTRv«2) expression plasmid (pCDa2), pKe711, which contains the entire hTRv«2 cDNA (49), was cut with Xba\, and the 3' «2-specific Xba\ fragment was ligated into Xbal-digested pCDai, which contained the 5' Xba\ fragment of hTR«1 cDNA. The reporter plasmid pUrGH(S) contained the BglU (-236)-Tnal (-147) fragment of the rat GH gene 5' flanking region. This fragment was ligated into the SamHI site of pUTKATI (50). This vector contains the herpes simplex virus thymidine kinase promoter, which directs expression of the CAT gene. Cell Culture and T3 Binding of hTRs Expressed by Transfected Cells COS-7 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Grand Island, NY) containing 10% (vol/
vol) fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 ng/m\) in 10-cm petri dishes at a density of 1 x 106 cells/ dish. Twenty hours before transfection, medium was changed to DMEM containing 10% hormone-depleted fetal bovine serum (51). Twenty micrograms of expression vector or pCDM8 were transfected into COS-7 cells using the calcium phosphate coprecipitate method (52). Twenty hours after transfection, the medium was changed, and incubation was continued for 48 h before harvest. Preparation of nuclear extracts from the transfected cells and T3 binding assay were performed as previously described (53). The protein concentration of nuclear extract was determined by the Coomassie blue method, using BSA as a standard (54). ABCD Assay The ABCD assay procedure used was as described by Glass et al. (55). The TRE oligomer (44 bp) contained the sequence -187 through -158 of the rat GH 5' flanking region and was designed to allow incorporation of eight biotin-11 -UMP residues into its 3' termini. After annealing the two complimentary oligonucleotides, the 5' overhangs at each end were filled in using Klenow fragment, 200 nM each of dATP, dCTP, and dGTP, and 200 M M biotin-11-dUTP. Unincorporated nucleotides were removed by a single passage through a Sephadex G-50 column, and the eluent was ethanol precipitated. The control oligomer (44 bp) contained the sequence from a cisactive element of adenovirus 5 (56). This oligomer contained six biotin-11 -UMP residues after Klenow fragment fill in. Highly efficient binding of both biotinylated oligomers to streptoavidinagarose was confirmed using 32P end-labeled biotin-DNAs. A nonbiotinylated TRE oligomer was also prepared using dTTP instead of biotin-11-dUTP. In vitro transcription and translation of hTR/31-W and hTR/31 -Mf were performed as previously described (5). [35S] Methionine-labeled hTR«1 and hTRva2 were synthesized as previously described (48, 49). Reticulocyte lysates programmed with each hTR were centrifuged through Sephadex G-50 columns equilibrated with ABCD buffer [50 mwi KCI, 20 mM KPO4 (pH 7.4), 1 mM MgCI2, 20% glycerol, and 1 HIM 0mercaptoethanol] to remove unincorporated [35S]methionine and change buffer. For the binding assay, 2-5 x 104 cpm trichloroacetic acid-precipitable [35S]methionine-labeled proteins were incubated with 1 pmol biotinylated oligomer and 0.2 n$ln\ poly(dl-dC) in 20 n\ ABCD buffer for 40 min at 22 C. Twenty microliters of streptoavidin-agarose suspension were added and incubated on ice for 10 min, and 1 ml ice-cold ABCD buffer was added before centrifugation. Streptoavidinagarose beads were washed with ABCD buffer, and radioactivity in the final pellets was estimated by scintillation counting. CAT Assay COS-7 cells were grown as described above. Cells were transfected with 20 ^g plasmids, including 4 ng reporter plasmid pUrGH(S). COS-7 cells express the simian virus 40 (SV40) T-antigen, which can cause high level replication of transfected CD expression plasmids containing the SV40 origin of replication (47). To maintain the amount of CD plasmid constant, pCDM8 was added when necessary. Twenty hours after transfection by the calcium phosphate method, the medium was changed to DMEM supplemented with 10% hormone-depleted fetal bovine serum with or without the addition of various amounts of T3. Cells were harvested after 48-h incubation, and CAT activity in cell extracts was measured using [14C] chloramphenicol (57). We found that the intraassay variability due to transfection efficiency was minimal, and standardization using a cotransfected GH expression plasmid did not significantly reduce this variability. Therefore, we did not include another expression plasmid for normalization, but all experiments in each figure were performed in duplicate on the same day, and experiments were repeated four times.
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1993
Function of Mutant Thyroid Hormone Receptor
Acknowledgments The authors wish to thank Drs. C. Weinberger and R. M. Evans for providing peA101, Drs. A. Aruffo and B. Seed for providing pCDM8, and Dr. D. D. Moore for providing pUTKATI.
15.
16. Received July 25, 1990. Revision received September 18, 1990. Accepted September 26, 1990. Address requests for reprints to: Dr. Leslie J. DeGroot, Box 138, Thyroid Study Unit, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637. This work was supported in part by USPHS Grants DK13377 and DK-15070, the March of Dimes Birth Defects Foundation, and the David Wiener Research Fund.
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