T3 Binding Affinity Research Articles

Substrate-Induced Formation of Ribosomal Decoding Center for Accurate and Rapid Genetic Code Translation.

Accurate translation of genetic information is crucial for synthesis of functional proteins in all organisms. We use recent experimental data to discuss how induced fit affects accuracy of initial codon selection on the ribosome by aminoacyl transfer RNA in ternary complex ( T3) with elongation factor Tu (EF-Tu) and guanosine-5'-triphosphate (GTP). We define actual accuracy ([Formula: see text]) of a particular protein synthesis system as its current accuracy and the effective selectivity ([Formula: see text]) as [Formula: see text] in the limit of zero ribosomal binding affinity for T3. Intrinsic selectivity ([Formula: see text]), defined as the upper thermodynamic limit of [Formula: see text], is determined by the free energy difference between near-cognate and cognate T3 in the pre-GTP hydrolysis state on the ribosome. [Formula: see text] is much larger than [Formula: see text], suggesting the possibility of a considerable increase in [Formula: see text] and [Formula: see text] at negligible kinetic cost. Induced fit increases [Formula: see text] and [Formula: see text] without affecting [Formula: see text], and aminoglycoside antibiotics reduce [Formula: see text] and [Formula: see text] at unaltered [Formula: see text].

Clinical, Genetic, and Protein Structural Aspects of Familial Dysalbuminemic Hyperthyroxinemia and Hypertriiodothyroninemia.

Familial dysalbuminemic hyperthyroxinemia (FDH-T4) and hypertriiodothyroninemia (FDH-T3) are dominantly inherited syndromes characterized by a high concentration of thyroid hormone in the blood stream. The syndromes do not cause disease, because the concentration of free hormone is normal, but affected individuals are at risk of erroneous treatment. FDH-T4 is the most common cause of euthyroid hyperthyroxinemia in Caucasian populations in which its prevalence is about 1 in 10,000 individuals, but the prevalence can be much higher in some ethnic groups. The condition is caused by a genetic variant of human serum albumin (HSA); Arg218 is mutated to histidine, proline, or serine or Arg222 is changed to isoleucine. The disorder is characterized by greater elevation in serum l-thyroxine (T4) than in serum triiodothyronine (T3); T4 can be increased by a factor 8–15. The high serum concentration of T4 is due to modification of a binding site located in the N-terminal half of HSA (in subdomain IIA). Thus, mutating Arg218 or Arg222 for a smaller amino acid reduces the steric restrictions in the site and creates a high-affinity binding site. The mutations can also affect binding of other ligands and can perhaps cause modified pharmacokinetics of albumin-binding drugs. In normal HSA, the high-affinity site has another location (in subdomain IIIB). Different locations of these sites imply that persons with and without FDH-T4 can have different types of interactions, and thereby complications, when given albumin-binding drugs. FDH-T3 is caused by a leucine to proline mutation in position 66 of HSA, which results in a large increment of the binding affinity for T3 but not for T4. For avoiding unwanted treatment of euthyroid persons with hyperthyroxinemia or hypertriiodothyroninemia, protein sequencing and/or sequencing of the albumin gene should be performed.

Src-dependent phosphorylation at Y406 on the thyroid hormone receptor β confers the tumor suppressor activity

Association studies suggest that the thyroid hormone receptor β1 (TRβ1) could function as a tumor suppressor in cancer cells. However, the underlying molecular mechanisms remain to be elucidated. We explored how TRβ1 acted as a tumor suppressor in breast cancer MDA cells. Proliferation and invasiveness were markedly inhibited in cells stably expressing TRβ1 (MDA-TRβ1 cells). cSrc-phosphorylated TRβ1 at Y406 signaled T3-induced degradation. Mutation of Y406 to Phe (TRβ1Y406F) did not affect T3 binding affinity, but blocked T3-induced degradation in cells. Importantly, cell-based studies showed that TRβ1Y406F lost the inhibitory effects by TRβ1 on cell proliferation and invasion. Consistently, in xenograft models, MDA-TRβ1 cells exhibited significantly slower tumor growth rates than those of Neo control cells. In contrast, the tumor growth rates of MDA-TRβ1Y406F cells were indistinguishable from those of Neo control cells. We further showed that markedly more TRβ1Y406F than TRβ1 was physically associated with cSrc in cells, leading to constitutive activation of cSrc-FAK-ERK signaling. In contrast, degradation of T3-bound TRβ1 complexed with cSrc attenuated signaling to decrease cell proliferation and invasiveness, thus confirming TRβ1 as a tumor suppressor. Thus, the present studies suggested that TRβ1 could be tested as a novel potential therapeutic target.

Dysprealbuminemic Hyperthyroxinemia in a Patient with Hyperthyroid Graves Disease

Rare mutant forms of circulating albumin and prealbumin [transthyretin (TTR)] have increased binding affinity for thyroxine (T4). Patients with these variant plasma proteins, as a result of inherited mutations or as a paraneoplastic phenomenon, typically present with increased serum total T4 and, by some assay methodologies, an increased free T4 as well. Although these individuals are, in fact, euthyroid, nonspecific symptoms may lead to inappropriate treatment for hyperthyroidism. We present a 34-year-old woman in whom a mutant form of TTR with increased T4 binding affinity and coexisting Graves disease was present. Subsequent 131I therapy led to development of postablative hypothyroidism, which was obscured by her higher serum free T4 concentration. Circulating thyroid-binding globulin (TBG), albumin, and TTR concentrations were all within their respective reference limits. A T4-binding protein panel confirmed that TTR-bound T4 was significantly increased, whereas TBG- and albumin-bound T4 was normal, indicating that this patient had euthyroid dysprealbuminemic hyperthyroxinemia, which had been masked by the initial presentation of hyperthyroidism. These findings indicate that hypothyroidism can be masked by coexisting euthyroid dysprealbuminemic hyperthyroxinemia.

The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue.

Type 2 iodothyronine deiodinase (D2) is a selenoenzyme, the product of the recently cloned cAMP-dependent Dio2 gene, which increases 10- to 50-fold during cold stress only in brown adipose tissue (BAT). Here we report that despite a normal plasma 3,5,3'-triiodothyronine (T3) concentration, cold-exposed mice with targeted disruption of the Dio2 gene (Dio2(-/-)) become hypothermic due to impaired BAT thermogenesis and survive by compensatory shivering with consequent acute weight loss. This occurs despite normal basal mitochondrial uncoupling protein 1 (UCP1) concentration. In Dio2(-/-) brown adipocytes, the acute norepinephrine-, CL316,243-, or forskolin-induced increases in lipolysis, UCP1 mRNA, and O(2) consumption are all reduced due to impaired cAMP generation. These hypothyroid-like abnormalities are completely reversed by a single injection of T3 14 hours earlier. Recent studies suggest that UCP1 is primarily dependent on thyroid hormone receptor beta (TR beta) while the normal sympathetic response of brown adipocytes requires TR alpha. Intracellularly generated T3 may be required to saturate the TR alpha, which has an approximately fourfold lower T3-binding affinity than does TR beta. Thus, D2 is an essential component in the thyroid-sympathetic synergism required for thermal homeostasis in small mammals.

HYPOTHYROIDISM AND NUTRITIONAL STATUS ALTER 3,5,3′-TRIIODOTHYRONINE (T3) RECEPTORS IN ISOLATED LIVER NUCLEI OF ANABAS TESTUDINEUS (BLOCH)

T3 receptors are ligand-dependent transcriptional activators that can stimulate gene expression resulting in growth and development. In this study T3 binding sites on isolated liver nuclei are demonstrated in the teleost Anabas testudineus. The effect of hypothyroidism and feeding of polyunsaturated fatty acids (PUFA) on T3 receptor number and binding affinity was also studied. Hypothyroidism caused an increase in T3 receptor number and a decrease in receptor affinity. PUFA feeding also increased the T3 receptor number without any significant change in its receptor affinity.

Comparative Studies of Two Transthyretin Variants with Protective Effects on Familial Amyloidotic Polyneuropathy: TTR R104H and TTR T119M

Recently, a new nonpathogenic transthyretin (TTR) variant—TTR R104H (TTR H104)—has been described in heterozygotic and compound heterozygotic individuals from a Japanese family with familial amyloidotic polyneuropathy (FAP). The compound heterozygotic individual, a carrier of TTR V30M (TTR M30) and TTR R104H (TTR M30/H104) presented a very mild form of FAP with slow progression of the disease. TTR and retinol binding protein (RBP) levels were found to be increased in serum from TTR H104 carriers. These characteristics are very similar to those found in compound heterozygotic carriers of TTR V30M-T119M (TTR M30/M119). To structurally compare these variants, we performed stability and thyroxine (T4) binding studies. TTR M30/H104 showed an increased resistance to dissociation into monomers similar to TTR M30/M119. This suggests that the His104 substitution has the same stabilizing effect on tetrameric TTR as the Met119 substitution. Concerning T4 binding, TTR H104 presents a T4 binding affinity lower than that of TTR M119, but still higher than normal TTR. However, TTR from the compound heterozygotic carrier of TTR M30/H104 presented a T4 binding affinity lower than normal. The results indicate that the His 104 substitution induces structural alterations that increase the stability of the tetramer in compound heterozygotes for TTR M30 despite a lower affinity for T4 binding. Thus, stability of TTR and binding affinity for T4 may not be related. More detailed characterization of these variants is needed to clarify the structural alterations responsible for their increased stability.

DNA binding affinity of hTRβ1 mutants as heterodimers with traps from different tissues

Patients with generalized resistance to thyroid hormone (GRTH) show various organ-specific features, for example mental retardation, growth abnormalities, liver damage, delayed bone age or cardiac disorders. Could this reflect aberrant mutant thyroid hormone receptor β1 (TRβ1) heterodimerization with specific TR auxiliary proteins (TRAPs) from different tissues, altering the mutant's ability to transactivate tissue-specific genes? To answer this question, we examined the heterodimerization of TRβ1 mutants and TRAPs of several rat tissues (cerebrum, cerebellum, liver, heart, lung, spleen, and kidney), and in vitro translated RXRα, β and γ by electrophoretic gel mobility shift assay (EMSA). Mutant TRβ1 proteins, synthesized in reticulocyte lysate, were incubated with 32P rat malic enzyme (rME) thyroid hormone response elements (TRE) and nuclear extracts of rat tissues. The TRβ1 mutants used were Mf (G345R), and GH (R316H). Both have non-detectable T3 binding affinity. GH has weak dominant negative effect and Mf has strong dominant negative effect. Two major bands were observed in EMSA. Cerebrum, cerebellum, lung and liver extracts formed a slower migrating band than a TR homodimer, while kidney extracts formed a faster migrating band, and heart and spleen extracts had both bands. There were no qualitative differences in heterodimerization between TRβ1wt, and TRβ1 mutants, when using tissue extracts and DNA in excess ratio to TR. We found that RXRα, β, and γ were differentially expressed in each rat tissue and formed heterodimer complexes with wild type (WT) TRβ1. Scatchard analysis of affinity and capacity of the binding of TR-TRAP heterodimers to response elements was performed by competing with 2.5-, 5-, 10-, 25-, and 250-fold excess non-radiolabeled rME-TRE. When using kidney extract, the DNA binding affinity of heterodimers was significantly decreased both in wild type and mutant TRs, suggesting that the DNA binding affinity of the faster migrating band was lower than that of the slower migrating band. Mutant GH, which causes `pituitary RTH' and shows weak dominant negative effect, tended to form heterodimers with lower DNA binding affinity than TRβ1wt with all extracts. Mutant Mf, which has strong dominant negative effect, tended to show higher DNA binding affinity than TRβ1WT. When the data were pooled for all tissues, GH and Mf were found to form heterodimers with significantly lower, or higher, affinity for TREs than TRβ1wt. These results indicate that: 1) differences of DNA binding affinity of mutant TR-TRAP heterodimers to response elements in DNA play a part in its reduced or strong dominant negative effect; and 2) differences in formation of heterodimers with TRAPs present in tissues do not appear to explain the apparent tissue-specific and mutant-specific variations seen in RTH.

Lack of Coactivator Interaction Can Be a Mechanism for Dominant Negative Activity by Mutant Thyroid Hormone Receptors

We studied the interactions of two natural thyroid hormone receptor (TR) mutants from patients with resistance to thyroid hormone (RTH) and an artificial TR mutant with a nuclear receptor corepressor, N-CoR, and a steroid receptor coactivator, SRC-1. In electrophoretic mobility shift assays, wild-type TRbeta-1 interacted with N-CoR in the absence of ligand, whereas T3 caused dissociation of the TRbeta-1/N-CoR complex and formation of TRbeta-1/SRC-1 complex. In contrast, a natural mutant (G345R) with poor T3-binding affinity formed TRbeta-1/N-CoR complex, both in the absence and presence of T3, but could not form TRbeta-1/SRC-1 complex. Another TR mutant, which bound T3 with normal affinity and containing a mutation in the AF-2 region (E457D), had normal interactions with N-CoR but could not bind SRC-1. Both these mutants had strong dominant negative activity on wild-type TR transactivation. Studies with a TR mutant that had slightly decreased T3-binding affinity (R320H) showed a T3-dependent decrease in binding to N-CoR and increase in binding to SRC-1 that reflected its decreased ligand binding affinity. Additionally, when N-CoR and SRC-1 were added to these receptors at various T3 concentrations in electrophoretic mobility shift assays, TR/N-CoR and TR/SRC-1 complexes, but not intermediate complexes were observed, suggesting that N-CoR release is necessary before SRC-1 binding to TR. Our data provide new insight on the molecular mechanisms of dominant negative activity in RTH and suggest that the inability of mutant TRs to interact with coactivators such as SRC-1, which results from reduced T3-binding affinity, is a determinant of dominant negative activity.

Ligand- and nuclear factor-dependent change in hydrophobicity of thyroid hormone beta1 receptor.

An aqueous two-phase partitioning assay was performed using in vitro translated human thyroid hormone beta1 receptor (TRbeta1). Wild-type TRbeta1 was less hydrophobic in the presence of both triiodothyronine (T3) and nuclear extract. This reflects a conformational change, or change in electrostatic properties, of the TRbeta1-nuclear factor complex as a result of T3 binding. Mutant TRbeta1s with reduced T3 binding affinity required a higher concentration of T3 for the shift of hydrophobicity, and a mutant without T3 binding activity did not show any shift, even in the presence of 1 mM T3. The unique mutant receptor, R243Q, has impaired transcriptional function despite virtually normal binding affinity for T3. When this mutant was examined in this assay, the shift of hydrophobicity was significantly impaired even in the presence of both nuclear extract and a high concentration of T3. Nuclear extract of COS1 cells did not affect the T3-binding affinity of R243Q. These results indicate that the R243Q mutant has impaired a ligand-dependent conformational change and interaction with nuclear factor(s). Inability of R243Q to interact normally with nuclear factor(s) may explain, in part, the molecular mechanism of discordance between ligand binding and transactivation function of this mutant.

Determinants of chromatin disruption and transcriptional regulation instigated by the thyroid hormone receptor: hormone-regulated chromatin disruption is not sufficient for transcriptional activation.

Chromatin disruption and transcriptional activation are both thyroid hormone-dependent processes regulated by the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). In the absence of hormone, TR-RXR binds to nucleosomal DNA, locally disrupts histone-DNA contacts and generates a DNase I-hypersensitive site. Chromatin-bound unliganded TR-RXR silences transcription of the Xenopus TRbetaA gene within a canonical nucleosomal array. On addition of hormone, the receptor directs the extensive further disruption of chromatin structure over several hundred base pairs of DNA and activates transcription. We define a domain of the TR protein necessary for directing this extensive hormone-dependent chromatin disruption. Particular TR-RXR heterodimers containing mutations in this domain are able to bind both hormone and their thyroid hormone receptor recognition element (TRE) within chromatin, yet are unable to direct the extensive hormone-dependent disruption of chromatin or to activate transcription. We distinguish the hormone-dependent disruption of chromatin and transcriptional activation as independently regulated events through the mutagenesis of basal promoter elements and by altering the position and number of TREs within the TRbetaA promoter. Chromatin disruption alone on a minichromosome is shown to be insufficient for transcriptional activation of the TRbetaA gene.

Resistance to thyroid hormone caused by two mutant thyroid hormone receptors beta, R243Q and R243W, with marked impairment of function that cannot be explained by altered in vitro 3,5,3'-triiodothyroinine binding affinity.

Resistance to thyroid hormone (RTH) is a syndrome of reduced responsiveness to thyroid hormone caused by mutations in the thyroid hormone receptor beta (TRbeta) gene. Mutant TRbetas exhibit variable degrees of impaired T3 binding resulting in reduced T3-mediated function. The dominant mode of inheritance is attributed to the ability of mutant TRbetas to interfere with the function of the wild-type (WT) TR, a phenomenon known as dominant negative effect (DNE). We recently identified two families with RTH having mutations in amino acid 243 (R243Q and R243W) in whom the mechanism of RTH appears to be distinct from that of other natural TRbeta mutations. These mutations, which are located in the hinge domain of the TRbeta, do not significantly alter the binding affinity for T3, measured in vitro. The present study was undertaken to characterize the properties of these mutant TRbetas to understand the molecular basis of the RTH phenotype. Two other mutant TRbeta producing RTH with mild (320H) and severe (345R) impairment of T3 binding were studied in parallel. The results demonstrate that TRbetas 243Q and 243W could be translocated into the nucleus where they exerted normal ligand-independent repression of positively regulated thyroid hormone response elements. Yet, the addition of 10 nmol/L T3 failed to normalize the transactivation (16-13% of WT) and revert the DNE exerted by the two TRbeta mutants. In contrast, at this T3 concentration, the transactivation function of 320H was significantly higher (50% of WT), and the DNE was completely abolished, in keeping with the mild clinical form of RTH. Formation of 243Q and 243W homodimers on thyroid hormone response elements could not be as readily prevented by T3 as those formed by the WT and 320H TRbetas. These results suggest that the substitution of R243 in TRbeta produces RTH by increasing the propensity for the formation of tightly bound homodimers or by reduction of the receptor affinity for T3 only after it binds to DNA.

The function of retinoid X receptors on negative thyroid hormone response elements

Retinoid X receptors (RXRs) form heterodimers with thyroid hormone receptors (TRs). RXRs increase DNA binding affinity of TRs and T3-mediated transactivation on positive T3 response elements (TREs). However, the role of RXRs on negative TREs, and the relation of RXRs to the dominant negative effect of mutant TRs, are not defined. To clarify the function of RXRs on negative TREs, we performed transient cotransfection studies using the rat glycoprotein hormone α promoter fused to luciferase gene ( αLuc), and human TRH promoter fused to luciferase gene (TRH-Luc) as reporters. We found that the JEG-3 cell- αLuc system was very sensitive to TR regulation. Using TR β1 wild-type (WT) expression vector, 6.2 ng/well (170 ng/10 cm dish), and 0.2 ng/well (11 ng/10 cm dish) caused maximal, and half maximal, inhibition of Luc activities in the presence of 1 nM T3. A T3 dose dependent inhibition study was also performed. From these studies, we determined that the appropriate conditions in which to study αLuc transactivation, in a linear portion of the dose response curve, was using 0.8 ng/well TR β1 expression vector and 0.1 nM T3. Under these conditions, TR β1 mutant R316H (GH), but not G345R (Mf), showed a weak dominant negative effect at a 1:1 ratio in the presence of 0.1 nM T3 although neither mutant had detectable T3 binding affinity. Moreover this dominant negative effect of R316H on the αLuc reporter was enhanced in the presence of RXR γ. Mutant G345R showed a stronger dominant negative effect than did R316H when using a double palindromic TRE fused to herpes simplex thymidine kinase-Luc reporter as a positive TRE. These results conform to the clinical features of R316H which is associated with apparent pituitary resistance of thyroid hormone (PRTH). Mutant R316H also showed a weak dominant negative effect with TRH-Luc at a 1:1 ratio in the absence or presence of RXR γ. However RXR γ did not enhance the dominant negative effect as it did using αLuc reporter gene. Electrophoretic gel mobility shift assay (EMSA) showed that RXR α augmented the DNA binding affinity of wild type and R316H TRs as heterodimers on the previously reported negative TREs of glycoprotein hormone α promoter, suggesting that RXR does not produce its response by removing TRs from these TREs. RXR α augmented DNA binding affinity of TR β1WT, and R316H showed a weaker heterodimer band than did the wild type in EMSA. Using the TRH-Luc reporter, basal activity was increased by wild type TR β1. However a TR β1 DNA binding domain mutant, (C127S) which can not bind to DNA, did not increase the basal activity. This indicates that DNA binding of the TR is required for increasing basal activity of TRH promoter. These results indicate that (1) RXR-TR heterodimers play a role in basal transactivation and T3 suppression of negatively regulated genes, and (2) RXRs increase the dominant negative effect of some mutant TRs on specific negative TREs. (3) This effect occurs without removing TRs from the TRE. (4) The differential dominant negative effect of mutant R316H (negative TRE>positive TRE) may explain, at least in part, the presentation of R316H as PRTH. (5) Augmentation of basal activity by wild type TRs on a negative TRE requires DNA binding.

Amino acid substitutions of thyroid hormone receptor-beta at codon 435 with resistance to thyroid hormone selectively alter homodimer formation.

Thyroid hormone action is mediated through its nuclear receptors (TRs), which bind to target DNA sequences [thyroid hormone response element (TRE)] as a homodimer or a heterodimer with 9-cis-retinoic acid receptors. Mutations of TR beta identified in patients with resistance to thyroid hormone (RTH) cluster primarily at two areas separated by the putative dimerization region. Two TR beta mutations were newly found in patients with RTH at codon 435 histidine (H435L and H435Q) close to the dimerization region. Recent crystallographic study suggested that H435 is critical for direct contact with T3. To study how the side-chain charge of amino acids at this position affects receptor characteristics, T3-binding activity, receptor dimerization, transcriptional activity, and dominant negative action were analyzed in two RTH mutants and two additional artificial mutants (H435R and H435E). The T3 binding affinities of all four mutants were below detection. In electrophoretic mobility shift assay using TRE-DR4 or the inverted palindrome (Lap), heterodimer formation of mutant receptors with 9-cis-retinoic acid receptor was similar to that of wild type receptors. However, homodimer formation varied among mutant receptors, especially using TRE-DR4, with a rank order of wild type = H435R > H435Q > H435L > > H435E. In the presence of a basic amino acid at codon 435, homodimer formation was preserved, whereas substitution to neutral or acidic amino acids resulted in decreased homodimer formation. In transient transfection assays using reporter genes under the control of 2xPal-thymidine kinase (TK), DR4-TK, Lap-TK, or TSH alpha promoter, these four mutants were inactive in T3-dependent transcriptional activation. Dominant negative inhibition was similar for all four mutants. These results indicate that 1) newly found TR beta mutations at codon 435 are responsible for RTH; and 2) codon 435 in TR beta is located at a position that can predominantly alter homodimer formation on certain TREs, such as DR4.

Understanding the molecular mechanism of dominant negative action of mutant thyroid hormone beta 1-receptors: the important role of the wild-type/mutant receptor heterodimer.

The clinical manifestations of patients with resistance to thyroid hormone result from inhibition of the functions of wild-type thyroid hormone receptors (wTRs) by the dominant negative effect of mutant TR beta 1 receptors (mTR beta 1). One of the proposed mechanisms by which mTR beta 1 exerts its dominant negative action is via formation of the putative inactive wTR beta 1/mTR beta 1 heterodimer. However, the nature of the wTR beta 1/mTR beta 1 heterodimer is poorly understood. The present study characterizes the wTR beta 1/mTR beta 1 heterodimer by electrophoretic mobility shift assay. The mutant TR beta 1 used was PV, which contains a frame shift mutation in the C-terminal part of TR beta 1 and has less than 1% of the T3 binding affinity of the wTR beta 1. Because of the difficulty in resolving wTR beta 1 and mutant PV dimers, we used a truncated wTR beta 1 in which the A/B domain was deleted (delta TR beta 1) to demonstrate the formation of the heterodimer on thyroid hormone response elements (TREs) in which the half-site binding motifs are oriented in an inverted repeat (F2), a direct repeat separated by four nucleotides (DR4), or an inverted repeat (Pal). Deletion of the A/B domain had no effect on the binding of T3 and TREs to wTR beta 1. In the presence of equal amounts of delta TR beta 1 and PV, three types of molecular complexes. delta TR beta 1 homodimer, delta TR beta 1/PV heterodimer, and PV homodimer bound to each TRE in a ratio of approximately 1:2:1. The identities of these complexes were confirmed by their ability to be supershifted by anti-TR beta 1 and/or anti-PV antibodies. delta TR beta 1/PV heterodimer formation varied with different TREs. The ratio of apparent affinity constant (Ka) in the binding of delta TR beta 1/PV to TREs was F2:DR4:Pal = approximately 6:2:1. The effect of T3 on delta TR beta 1/PV heterodimer formation was TRE dependent. No T3-induced dissociation was observed for the delta TR beta 1/PV heterodimer when bound to F2 and Pal. In contrast, the delta TR beta 1/PV heterodimer bound to DR4 was dissociated by T3 with an ED50 of 3.9 +/- 0.9 nM. The T3-induced dissociation of delta TR beta 1 homodimer bound to F2, DR4, and Pal had ED50 values of 4.1 +/- 1.2, 1.3 +/- 0.3, and more than 100 nM, respectively. By transfection assays, the dominant negative action of PV was found to be TRE dependent with the rank order of F2 >> Pal > ME (a DR4-like TRE in the rat malic enzyme gene). Taken together, these results indicate a strong correlation between wTR beta 1/mTR beta 1 heterodimer formation and the dominant negative potency of PV. These results suggest that the wTR beta 1/mTR beta 1 heterodimer could play an important role in the dominant negative action of mTR beta 1.

Understanding the molecular mechanism of dominant negative action of mutant thyroid hormone beta 1-receptors: the important role of the wild- type/mutant receptor heterodimer

The clinical manifestations of patients with resistance to thyroid hormone result from inhibition of the functions of wild-type thyroid hormone receptors (wTRs) by the dominant negative effect of mutant TR beta 1 receptors (mTR beta 1). One of the proposed mechanisms by which mTR beta 1 exerts its dominant negative action is via formation of the putative inactive wTR beta 1/mTR beta 1 heterodimer. However, the nature of the wTR beta 1/mTR beta 1 heterodimer is poorly understood. The present study characterizes the wTR beta 1/mTR beta 1 heterodimer by electrophoretic mobility shift assay. The mutant TR beta 1 used was PV, which contains a frame shift mutation in the C-terminal part of TR beta 1 and has less than 1% of the T3 binding affinity of the wTR beta 1. Because of the difficulty in resolving wTR beta 1 and mutant PV dimers, we used a truncated wTR beta 1 in which the A/B domain was deleted (delta TR beta 1) to demonstrate the formation of the heterodimer on thyroid hormone response elements (TREs) in which the half-site binding motifs are oriented in an inverted repeat (F2), a direct repeat separated by four nucleotides (DR4), or an inverted repeat (Pal). Deletion of the A/B domain had no effect on the binding of T3 and TREs to wTR beta 1. In the presence of equal amounts of delta TR beta 1 and PV, three types of molecular complexes. delta TR beta 1 homodimer, delta TR beta 1/PV heterodimer, and PV homodimer bound to each TRE in a ratio of approximately 1:2:1. The identities of these complexes were confirmed by their ability to be supershifted by anti-TR beta 1 and/or anti-PV antibodies. delta TR beta 1/PV heterodimer formation varied with different TREs. The ratio of apparent affinity constant (Ka) in the binding of delta TR beta 1/PV to TREs was F2:DR4:Pal = approximately 6:2:1. The effect of T3 on delta TR beta 1/PV heterodimer formation was TRE dependent. No T3-induced dissociation was observed for the delta TR beta 1/PV heterodimer when bound to F2 and Pal. In contrast, the delta TR beta 1/PV heterodimer bound to DR4 was dissociated by T3 with an ED50 of 3.9 +/- 0.9 nM. The T3-induced dissociation of delta TR beta 1 homodimer bound to F2, DR4, and Pal had ED50 values of 4.1 +/- 1.2, 1.3 +/- 0.3, and more than 100 nM, respectively. By transfection assays, the dominant negative action of PV was found to be TRE dependent with the rank order of F2 >> Pal > ME (a DR4-like TRE in the rat malic enzyme gene). Taken together, these results indicate a strong correlation between wTR beta 1/mTR beta 1 heterodimer formation and the dominant negative potency of PV. These results suggest that the wTR beta 1/mTR beta 1 heterodimer could play an important role in the dominant negative action of mTR beta 1.

Studies on the repression of basal transcription (silencing) by artificial and natural human thyroid hormone receptor-beta mutants.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. Interestingly, in the absence of ligand, TRs also can repress basal transcription of positively regulated target genes, suggesting that unliganded TR may have a distinct role in gene regulation. In this paper, DNA binding, truncation, and natural human TR beta mutants were used in cotransfection and electrophoretic mobility shift assays to study various aspects of TR-mediated basal repression. Presently, little is known about the role(s) of natural human TR beta mutants on basal repression. These results show that: 1) TR binding to DNA likely is required for basal repression; 2) the amino-terminal region of TR is not required for basal repression; 3) TR homodimer binding is not absolutely required for basal repression, as TR mutants that selectively form TR-retinoid X receptor heterodimers can mediate basal repression; and 4) TR mutants with poor T3-binding affinity likely have constitutive basal repression, even in the presence of ligand. These findings provide new insight on the mechanism of basal repression by unliganded TRs.

Studies on the repression of basal transcription (silencing) by artificial and natural human thyroid hormone receptor-beta mutants

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. Interestingly, in the absence of ligand, TRs also can repress basal transcription of positively regulated target genes, suggesting that unliganded TR may have a distinct role in gene regulation. In this paper, DNA binding, truncation, and natural human TR beta mutants were used in cotransfection and electrophoretic mobility shift assays to study various aspects of TR-mediated basal repression. Presently, little is known about the role(s) of natural human TR beta mutants on basal repression. These results show that: 1) TR binding to DNA likely is required for basal repression; 2) the amino-terminal region of TR is not required for basal repression; 3) TR homodimer binding is not absolutely required for basal repression, as TR mutants that selectively form TR-retinoid X receptor heterodimers can mediate basal repression; and 4) TR mutants with poor T3-binding affinity likely have constitutive basal repression, even in the presence of ligand. These findings provide new insight on the mechanism of basal repression by unliganded TRs.

Thyroxine binding properties of glycosylated human serum albumin as measured by fluorescence.

Thyroid hormone, thyroxine (T4) binding to glycosylated human serum albumin (G-HSA), and native human serum albumin (HSA) were studied as a function of pH using the fluorescence method. T4 binding affinity for G-HSA was remarkably reduced in an alkaline pH as compared with the native HSA. The thermodynamic parameters for binding are estimated at pH 7.5: (a) for G-HSA, delta G = -8.50 +/- 0.04 kcal mol-1 (30 degrees C), delta H = -5.2 kcal mol-1, delta S = +11 e.u.; (b) for HSA, delta G = -8.89 +/- 0.04 kcal mol-1 (30 degrees C), delta H = -3.5 kcal mol-1, delta S = +18 e.u. These results suggest that the glycosylation of HSA causes a variation in the electrostatic interaction between T4 and HSA.

Mutations of CpG dinucleotides located in the triiodothyronine (T3)-binding domain of the thyroid hormone receptor (TR) beta gene that appears to be devoid of natural mutations may not be detected because they are unlikely to produce the clinical phenotype of resistance to thyroid hormone.

Thyroid hormone receptor (TR) beta gene mutations identified in patients with resistance to thyroid hormone (RTH) revealed two clusters ("hot" areas) of mutations (RTHmut) in the triiodothyronine (T3)-binding domain. Furthermore, 45% of RTHmuts and 90% of recurring mutations are located in CpG dinucleotides ("hot spots"). To investigate why the region between the two hot areas lacks RTHmuts, we produced 10 artificial mutant TR beta s (ARTmut) in this "cold" region according to the hot spot rule (C-->T or G-->A substitutions in CpGs). The properties of ARTmuts were compared with those of six RTHmuts. Among all RTHmuts, R320H manifesting a mild form of RTH showed the least impairment of T3-binding affinity (Ka). In contrast, Ka was normal in six ARTmuts (group A), reduced to a lesser extent than R320H in three (group B), and one that was truncated (R410X) did not bind T3. All RTHmuts had impaired ability to transactivate T3-responsive elements and exhibited a strong dominant negative effect on cotransfected wild-type TR beta. Group B and A ARTmuts had minimally impaired or normal transactivation and weak or no dominant negative effect, respectively. R410X showed neither transactivation nor dominant negative effect. Natural mutations expected to occur in the cold region of TR beta should fail to manifest as RTH (group A) or should escape detection (group B) since the serum thyroid hormone levels required to compensate for the reduced binding affinity should be inferior to those found in subjects with R320H. R410X would manifest RTH only in the homozygote state. The cold region of the putative T3-binding domain is relatively insensitive to amino acid changes and, thus, may not be involved in a direct interaction with T3.