Biomedical subjects

J W Harney

Publications and source records attributed to J W Harney.

At least 55 records · Page 3Linked to original sources

PubMed1995

Cloning and functional characterization of human selenophosphate synthetase, an essential component of selenoprotein synthesis.

Selenocysteine is co-translationally incorporated into prokaryotic and eukaryotic selenoproteins at in-frame UGA codons. However, the only component of the eukaryotic selenocysteine incorporation machinery identified to date is the selenocysteine-specific tRNA(Sec). In prokaryotes, selenocysteine is synthesized from seryl-tRNA(Sec) and the active selenium donor, selenophosphate. Selenophosphate is synthesized from selenide and ATP by the selD gene product, selenophosphate synthetase, and is required for selenocysteine synthesis and incorporation into bacterial selenoproteins. We have now cloned human selD and shown that transfection of the human selD cDNA into mammalian cells results in increased selenium labeling of a mammalian selenoprotein, type 1 iodothyronine deiodinase. Despite significant differences between the mechanisms of selenoprotein synthesis in prokaryotes and eukaryotes, human selD weakly complements a bacterial selD mutation, partially restoring selenium incorporation into bacterial selenoproteins. Human selenophosphate synthetase has only 32% homology with the bacterial protein, although a highly homologous region that has similarity to a consensus ATP/GTP binding domain has been identified. Point mutations within this region result in decreased incorporation of selenium into type 1 iodothyronine deiodinase in all but one case. Further analysis revealed that reduced selenium labeling was due to altered ATP binding properties of the mutant selenophosphate synthetases.

Adenosine Triphosphate↗

PubMed1995

Topological analysis of the integral membrane protein, type 1 iodothyronine deiodinase (D1).

Type 1 iodothyronine deiodinase (D1) is a microsomal selenoenzyme which catalyzes deiodination of thyroxine to 3,5,3'-triiodothyronine. Immunoblotting showed that endogenous hepatic, renal, and transiently expressed D1 remains in microsomes after pH 11.5 treatment. In vitro translation studies using pancreatic microsomes identified a single transmembrane domain with a cytosolic carboxyl-terminal catalytic portion. The transmembrane domain is located between conserved basic amino acids at positions 11 and 12 and a group of charged residues at positions 34-39. A transiently expressed D1 protein in which residues 2-25 were deleted was inactive and not integrated into membranes. Activity was not restored by replacing these residues with transmembrane domains from a cytochrome P450 or type 3 deiodinase enzyme despite their incorporation into membranes. Elimination of the positive charges at positions 11 and 12 reduced the amount of transiently expressed protein by 70%, but the enzyme formed was catalytically normal. Similar results were found after conversion of the Lys-27 in the transmembrane domain to Met or Glu. We conclude that the amino terminus of D1 contains uncleaved signal and stop transfer sequence properties. In addition, positively charged residues at positions 11, 12, and 27 are required for optimal formation of the protein but not for catalysis.

Amino Acid Sequence↗

PubMed1995

A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene.

We identified two thyroid hormone response elements (TREs) in the 2.5-kb, 5'-flanking region of the human gene encoding type 1 iodothyronine deiodinase (hdio1), an enzyme which catalyses the activation of thyroxine to 3,5,3'-triiodothyronine (T3). Both TREs contribute equally to T3 induction of the homologous promoter in transient expression assays. The proximal TRE (TRE1), which is located at bp -100, has an unusual structure, a direct repeat of the octamer YYRGGTCA hexamer that is spaced by 10 bp. The pyrimidines in the -2 position relative to the core hexamer are both essential to function. In vitro binding studies of TRE1 showed no heterodimer formation with retinoid X receptor (RXR) beta or JEG nuclear extracts (containing RXR alpha) and bacterially expressed chicken T3 receptor alpha 1 (TR alpha) can occupy both half-sites although the 3' half-site is dominant. T3 causes dissociation of TR alpha from the 5' half-site but increases binding to the 3' half-site. Binding of a second TR to TRE1 is minimally cooperative; however, no cooperativity was noted for a functional mutant in which the half-sites are separated by 15 bp, implying that TRs bind as independent monomers. Nonetheless, T3 still causes TR dissociation from the DR+15, indicating that dissociation occurs independently of TR-TR contact and that rebinding of a T3-TR complex to the 3' half-site occurs because of its slightly higher affinity. A distal TRE (TRE2) is found at bp -700 and is a direct repeat of a PuGGTCA hexamer spaced by 4 bp. It has typical TR homodimer and TR-RXR heterodimer binding properties. The TRE1 of hdio1 is the first example of a naturally occurring TRE consisting of two relatively independent octamer sequences which do not require the RXR family of proteins for function.

Base Sequence↗

PubMed1995

Type 3 lodothyronine deiodinase: cloning, in vitro expression, and functional analysis of the placental selenoenzyme.

Type 3 iodothyronine deiodinase (D3) catalyzes the conversion of T4 and T3 to inactive metabolites. It is highly expressed in placenta and thus can regulate circulating fetal thyroid hormone concentrations throughout gestation. We have cloned and expressed a 2.1-kb human placental D3 cDNA which encodes a 32-kD protein with a Km of 1.2 nM for 5 deiodination of T3 and 340 nM for 5' deiodination of reverse T3. The reaction requires DTT and is not inhibited by 6n-propylthiouracil. We quantitated transiently expressed D3 by specifically labeling the protein with bromoacetyl [125I]T3. The Kcat/Km ratio for 5 deiodination of T3 was over 1,000-fold that for 5' deiodination of reverse T3. Human D3 is a selenoenzyme as evidenced by (a) the presence of an in frame UGA codon at position 144, (b) the synthesis of a 32-kD 75Se-labeled protein in D3 cDNA transfected cells, and (c) the presence of a selenocysteine insertion sequence element in the 3' untranslated region of the mRNA which is required for its expression. The D3 selenocysteine insertion sequence element is more potent than that in the type 1 deiodinase or glutathione peroxidase gene, suggesting a high priority for selenocysteine incorporation into this enzyme. The conservation of this enzyme from Xenopus laevis tadpoles to humans implies an essential role for regulation of thyroid hormone inactivation during embryological development.

Amino Acid Sequence↗

PubMed1995

Effect of 3,5,3'-Triiodothyronine (T3) administration on dio1 gene expression and T3 metabolism in normal and type 1 deiodinase-deficient mice.

The type 1 deiodinase (D1) catalyzes the monodeiodination of T4 to produce T3, the active thyroid hormone. In the C3H mouse, hepatic D1 and the dio1 messenger RNA (mRNA) are only 10% that in the C57 strain, the common phenotype. Low activity cosegregated with a series of five GCT repeats located in the 5'-flanking region of the C3H dio1 gene that impaired C3H promoter potency and provided a partial explanation for the lower D1. The present studies were performed to search for additional explanations for low D1 activity in C3H mice. Previous studies have shown that T3 up-regulates the dio1 gene. Therefore, loss of the capacity to respond to endogenous T3 is a possible additional cause of the lower D1 levels in the C3H mice. The hepatic C3H dio1 mRNA increases 10- to 20 fold after T3 administration. The t3 effect occurs at a transplantation level and T3 does not alter the dio1 mRNA half-life. Despite the transcriptional response to T3, no functional thyroid response elements were identified in the 1.5-kilobase 5'-flanking region of either the C57 or C3H dio1 gene. After the same dose of exogenous T3, both dio1 mRNA and D1 of the C3H mouse respond to a greater extent than those of the C57 strain. This can be explained in part by the reduction in T3 clearance due to the lower D1 levels in C3H mice in which higher concentrations of circulating T3 are maintained. The decrease in serum T3 levels and T3 production observed in fasting and systemic illness in both human and experimental animals has been attributed in part to a decrease in hepatic D1. In contrast, despite markedly lower hepatic and renal D1 levels, serum T3 concentrations remain normal in C3H mice. The present studies suggest that the absence of stress-induced hypothalamic-pituitary suppression that allows T4 production to be maintained together with the reduced clearance of T3 and T4 via inner ring deiodination compensate for the D1 deficiency.

Animals↗

PubMed1995

Retinoid-X receptor (RXR) differentially augments thyroid hormone response in cell lines as a function of the response element and endogenous RXR content.

Retinoid-X receptor (RXR) forms heterodimers with thyroid hormone receptor (TR) and significantly enhances binding to thyroid hormone response elements (TREs). Expression of RXR in a transient transfection assay augments the T3 response, but the influences of the specific cell line and TRE used have not been systematically studied. We determined RXR alpha and -beta augmentation of the TR alpha-mediated T3 response in transient transfection assays of COS, JEG, and mouse embryonic stem (ES) cell lines for a series of eight wild-type thyroid hormone (T3) and retinoic acid response elements (previously shown to bind TR). RXR augmented T3-induced expression in COS and ES cells (1.5- to 4-fold greater expression with added RXR compared to TR alone), but had minimal effect on augmentation of response in JEG cells. For most elements studied there was a proportional augmentation of basal and T3-stimulated expression. TREs from rat GH and laminin-B1, however, had relatively higher levels of T3-induced expression as a result of RXR cotransfection (T3 induction ratios increased 2-fold or greater). Previous characterization of these elements demonstrates that they contain more than two hexameric binding domains, all of which can simultaneously bind TR. The influence of endogenous RXR expression in a cell line on RXR augmentation of the T3 response was determined. RXR alpha and -beta messenger RNA (mRNA) expression was quantitated by Northern blot in each cell line. COS and JEG cells expressed almost exclusively RXR alpha mRNA, although expression was almost 2-fold higher in JEG compared to COS cells (12 +/- 2.5 vs. 6.8 +/- 0.5 density units relative to actin; mean +/- SE; P < 0.05). ES cells expressed only RXR beta mRNA, but at a very low level (0.4 +/- 0.1). Nuclear extracts prepared from JEG and COS cells augmented TR binding proportional to the endogenous RXR mRNA expression, and the heterodimer band was supershifted by the addition of antibody to RXR alpha. Nuclear extracts from ES cells had no detectable TR heterodimer binding to a range of response elements. RXR augmentation of the T3 response differs among cell lines and is greater in those with reduced endogenous RXR. Furthermore, the functional augmentation of the T3 response ratio by RXR is likely to require additional sequences contained in only a subset of elements in which RXR augments TR binding.

Animals↗

PubMed1995

Pituitary cells respond to thyroid hormone by discrete, gene-specific pathways.

Type 1 iodothyronine deiodinase (D1) converts T4 to T3, the active thyroid hormone, by removal of the outer ring iodine. Previous studies in liver and thyroid cells have shown that T3 regulates Type 1 deiodinase (dio1) gene expression by a mechanism not requiring ongoing protein synthesis. For certain T3-regulated genes, such as rat GH, T3-induced transcription is blocked by protein synthesis inhibitors. Because the somatotrope tumor cell lines express both dio1 and GH, we compared these two positively T3-regulated genes to establish whether cycloheximide blockade of T3 effects is cell-type or gene specific. In these cells, the T3 stimulation of dio1 messenger RNA (mRNA) is not blocked by cycloheximide, whereas the T3 effect on GH mRNA synthesis is eliminated. Other differences between these two genes were also noted. Retinoic acid does not alter dio1 gene expression or the response to T3 but increases GH and synergizes with T3. Dexamethasone alone had no effect on dio1 mRNA but did enhance the effect of T3 on both dio1 and GH. These results point to distinct pathways for T3 induction of mRNA synthesis from different genes within the same cell.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Structural and functional differences in the dio1 gene in mice with inherited type 1 deiodinase deficiency.

The type 1 deiodinase (D1) provides the major portion of the circulating T3 in vertebrates. In C3H and certain other inbred mice, liver and kidney D1 activity is 5- to 10-fold lower than in the common phenotype, C57. The lower D1 levels are paralleled by a decreased normal-sized dio1 mRNA and hyperthyroxinemia. Low activity cosegregates with a restriction fragment length variant (RFLV) in both inbred and recombinant strains, indicating it is due to differences in the dio1 gene. The exonic structure and the deduced amino acid sequences are identical for both strains and highly homologous to that of the rat. The RFLV is due to an approximately 150-base pair expansion of repetitive sequences in the second intron of the C3H gene, but this segment does not differentially affect the transient expression of a human GH gene. The promoter and 5'-flanking regions of the C3H and C57 dio1 genes are very similar and are GC rich without TATA or CCAAT boxes. However, functional assays of 1.5-kilobase 5'-flanking dio1-CAT constructs showed 2- to 3-fold higher activity of the C57-CAT constructs. Deletion mutants showed that sequences between -705 and -162 were the cause of this. In this region, the only major difference between the two genes is a 21-base pair insert containing five CTG repeats in the C3H promoter. This difference also cosegregates with low D1 activity and the intron RFLV in four other mouse strains. The correlation of the CTG repeat insert with both in vitro and in vivo expression and the absence of other significant sequence differences in the 5'-flanking region argue that this is the major explanation for the impaired expression of the dio1 gene and the resulting hyperthyroxinemia of the C3H mouse.

Amino Acid Sequence↗

PubMed1994

Selenocysteine insertion or termination: factors affecting UGA codon fate and complementary anticodon:codon mutations.

Translation of UGA as selenocysteine instead of termination occurs in numerous proteins, and the process of recording UGA requires specific signals in the corresponding mRNAs. In eukaryotes, stem-loops in the 3' untranslated region of the mRNAs confer this function. Despite the presence of these signals, selenocysteine incorporation is inefficient. To investigate the reason for this, we examined the effects of the amount of deiodinase cDNA on UGA readthrough in transfected cells, quantitating the full-length and UGA terminated products by Western blotting. The gene for the selenocysteine-specific tRNA was also cotransfected to determine if it was limiting. We find that the concentrations of both the selenoprotein DNA and the tRNA affect the ratio of selenocysteine incorporation to termination. Selenium depletion was also found to decrease readthrough. The fact that the truncated peptide is synthesized intracellularly demonstrates unequivocally that UGA can serve as both a stop and a selenocysteine codon in a single mRNA. Mutation of UGA to UAA (stop) or UUA (leucine) in the deiodinase mRNA abolishes deiodinase activity; but activity is partially restored when selenocysteine tRNAs containing complementary mutations are contransfected. Thus, UGA is not essential for selenocysteine incorporation in mammalian cells, provided that codon:anticodon complementarity is maintained.

Anticodon↗

PubMed1994

Identification of critical amino acids for 3,5,3'-triiodothyronine deiodination by human type 1 deiodinase based on comparative functional-structural analyses of the human, dog, and rat enzymes.

The selenoenzyme, type 1 iodothyronine deiodinase (type 1 DI), catalyzes the activation of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) but 3,3',5'-triiodothyronine (rT3) is the preferred substrate for the human enzyme. Since the dog type 1 DI has a significantly lower affinity for rT3, we cloned the dog type 1 DI to identify amino acids critical for rT3 binding. The Km of the transiently expressed dog enzyme for rT3 5'-deiodination is 25-fold higher than that of the human enzyme. However, the Ki of T4 for rT3 deiodination by dog type 1 DI is only 3-fold higher than that for the human, suggesting that the differences between the two proteins affect binding of rT3 more than that of T4. Comparative competition studies in which rT3 or T4 is used to block covalent bromoacetyl T3 binding to the two proteins support this. Mutational studies showed that the critical differences between the dog (D) and human (H) enzymes are Asn (D) 45 Gly (H), Gly (D) 46 Glu (H), and Leu (D) 60:Phe (H) 65. Substitution of the human residues for those of the dog at these positions causes the predicted changes in the Km (rT3) and vice versa. A Phe65 to Leu mutation alone in the human enzyme increases the Km (rT3) 10-fold. We speculate that Phe65 is especially important for rT3 binding due to an interaction between the tyrosyl ring of rT3 and the aromatic ring of Phe65.

Amino Acid Sequence↗

PubMed1994

Activation and inactivation of thyroid hormone by type I iodothyronine deiodinase.

The prohormone thyroxine (T4) is activated by outer ring deiodination (ORD) to 3,3',5-triiodothyronine (T3) and both hormones are degraded by inner ring deiodination (IRD) to 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine, respectively. Indirect evidence suggests that the type I iodothyronine deiodinase (ID-I) in liver has both ORD and IRD activities, with preference for rT3 and sulfated iodothyronines as substrates. To establish this, we have compared the ORD of rT3 and IRD of T3 and T3 sulfate by homogenates of cells transfected with rat ID-I cDNA and by rat liver microsomes. In both preparations rT3 is the preferred substrate, while deiodination of T3 is markedly accelerated by its sulfation. Kinetic analysis provided similar Km and Vmax values in cell homogenates and liver microsomes. These data demonstrate unequivocally that ID-I is capable of both activating and inactivating thyroid hormone by ORD and IRD, respectively.

Animals↗

PubMed1994

Thyroid hormone receptor binds with unique properties to response elements that contain hexamer domains in an inverted palindrome arrangement.

Thyroid hormone receptor (T3R)-accessory protein heterodimers preferentially bind to thyroid hormone response elements (TREs), which contain hexamer domains arranged as a direct repeat separated by a 4-basepair gap (DR + 4). T3R homodimers, however, preferentially bind to elements that consist of an inverted palindrome. We now report on unique T3R binding patterns and functional characteristics of two such elements that mediate T3 regulation. We performed mutational analysis of the chicken lysozyme silencer F2 (LysF2) TRE and demonstrated that the two functional binding domains are arranged as an inverted palindrome separated by 6 basepairs. Both the LysF2 TRE and the similarly arranged myelin basic protein TRE bind T3R dimers at very low T3R concentrations. Despite the high relative affinity for T3R dimer binding, the T3 induction conferred by these elements is low compared to that of previously characterized TREs with a DR + 4 arrangement. The laminin-B1 gene element, previously shown to bind retinoic acid receptor, contains at least four hexameric binding domains. All of the domains can bind T3R simultaneously and are involved in conferring T3 induction, but bind with a different pattern than that reported for retinoic acid receptor. T3R homodimer binding to a series of mutant laminin-B1 elements and T3 induction were significantly correlated (r = 0.82; P < 0.05). T3R homodimer binding to LysF2 element mutants was not correlated with T3 induction (r = 0.32; P > 0.05); however, T3R-nuclear protein heterodimer binding was significantly correlated (r = 0.67; P < 0.05). T3R-nuclear protein heterodimers, but not homodimers, bound consistently to mutations of the LysF2 element that altered the gap between hexamers. The overall discordance between strong T3R binding to these elements and weak T3 induction indicates that the unusual hexamer arrangement places the T3R complex in an unfavorable configuration for maximal T3-dependent transactivation. The differential T3 sensitivity of generalized resistance to thyroid hormone-associated T3R mutants to the LysF2 element compared with the DR + 4 arrangement suggests that these unique features may have physiological significance.

Animals↗

PubMed1993

Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons.

We investigated the requirements for selenocysteine insertion at single or multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements were identified in the 3' untranslated region of the rat selenoprotein P mRNA, with predicted stem-loops and critical nucleotides similar to those in the SECIS elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS elements confirmed that conserved nucleotides in the loop and in unpaired regions of the stem are critical for activity. This indicates that multiple contact sites are required for SECIS function. Stop codon function at any of five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in UGA codon position.

Animals↗

PubMed1993

Dominant negative inhibition by mutant thyroid hormone receptors is thyroid hormone response element and receptor isoform specific.

The heterogeneity of tissue-specific manifestations of generalized resistance to thyroid hormone (GRTH) could result from differential interactions between the mutant thyroid hormone (T3) receptor-beta (TR beta) on T3 response elements (TREs) in different T3-responsive genes. To explore this hypothesis, the mutant TR beta associated with kindred A, P448H; a TR beta mutant, P448L; and a comparable TR alpha mutant (P398H) were tested for intrinsic function and for inhibition of wild-type TR alpha- and -beta-induced expression from four structurally distinct TREs, the rGH ABC*, the rGH palindrome (PAL), the rat malic enzyme (ME), and the chicken lysozyme silencer F2 (F2). The relative function of the mutants was similarly reduced on the four TREs studied and was T3 concentration dependent. The TR alpha mutant retained the intrinsically greater potency characteristic of this isoform, but remained impaired with respect to wild-type TR alpha even at 500 nM T3. In general, dominant negative inhibition of wild-type TR alpha and -beta function was dependent upon the T3 concentration, as expected from the decreased affinity for ligand conferred by this mutation. A T3 concentration sufficient to relieve the inhibition of wild-type TR function on the ABC*, PAL, and ME TREs (50 nM) had no effect on inhibition of the F2 TRE by the mutant TRs. Receptor isoform preferential inhibition was observed on the ABC*, PAL, and ME TREs by the mutant TRs. Thus, both TRE structure and the isoform of endogenously active receptor could determine the degree of inhibition of a specific gene in GRTH individuals. Further, the lack of dominant negative potentials does not explain the absence of TR alpha mutations in GRTH kindreds.

Animals↗

PubMed1992

Substitution of cysteine for selenocysteine in type I iodothyronine deiodinase reduces the catalytic efficiency of the protein but enhances its translation.

Type I iodothyronine 5' deiodinase (5'DI) contains selenocysteine, encoded by a UGA codon, and this amino acid is essential for maximum catalytic efficiency in this enzyme. We recently showed that translation of UGA as selenocysteine in this protein requires a specific sequence of about 250 nucleotides in the 3' untranslated region of the messenger RNA. Translation of a 5'DI cysteine mutant does not require the 3' untranslated region. To examine both the efficiency of UGA codon recognition and the relative catalytic efficiency of selenocysteine vs. cysteine in 5'DI, we used bromoacetyl 125I-T3 labeling to quantitate transiently expressed selenocysteine (wild type) and cysteine containing type I iodothyronine deiodinases in transfected COS-7 and JEG-3 cell lines. Kinetic analyses of the same cell sonicates were performed to determine the apparent maximum velocity and Michaelis-Menten constant values for reverse T3 5' deiodination. COS-7 cells express the cysteine mutant protein at about 20-fold and JEG-3 cells about 400-fold higher levels than the selenoenzyme. However, in both cell types, the apparent catalytic constant values were at least 100-fold higher for the wild-type enzyme, compared with the cysteine mutant. These results indicate that cell lines differ markedly in their capacity to translate UGA-containing messenger RNAs. The much higher catalytic constant values for the selenium-containing enzyme illustrate the biochemical advantage of this element as compared with sulfur in the catalysis of iodothyronine deiodination.

Catalysis↗

PubMed1992

Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.

The type I 5' iodothyronine deiodinase (5' DI) catalyzes the deiodination of T4 to the biologically active hormone T3 and accounts for a significant fraction of its production. We have recently cloned the complementary DNA (cDNA) for the rat 5' DI, which contains the rare amino acid selenocysteine, and used this to screen human liver and kidney cDNA libraries to identify a human 5' DI cDNA clone. From these, we constructed a cDNA encoding a functional 5' DI. The 2222 base pair human 5' DI cDNA is approximately 200 nucleotides shorter than the 2.4-kilobase hybridizing band in Northern blots of human liver, kidney, and thyroid, because of missing 5' untranslated sequence and the poly A tail. The deduced amino acid sequence codes for a protein of 28.7 kilodaltons assuming the UGA codon at position 382 encodes selenocysteine, and is highly homologous (88% similarity) to the rat. We transiently expressed the 5' DI in COS-7 cells to establish that it encodes a functional enzyme and to study its kinetics. These show saturable deiodination of rT3 (Ka 0.52 +/- 0.04 mumol/L and Vmax 63.2 +/- 16.4 pmol min-1 mg-1). T4 and gold thioglucose are competitive inhibitors of rT3 deiodination. 6-n-Propylthiouracil (PTU) is an uncompetitive inhibitor (with rT3) and competitive inhibitor (with dithiothreitol) of rT3 deiodination. 6-n-Propylthiouracil inhibits T4 to T3 conversion. Labeling of COS-7 cells transiently transfected with the human 5' DI cDNA with bromoacetyl-125I-T3 demonstrates a 28-kilodalton protein. This indicates that in the human, as well as in the rat messenger RNA, the UGA encodes selenocysteine and translation terminates at the UAA codon at nucleotides 754 to 756. Reverse T3 and gold thioglucose (100 nmol/L) block bromoacetyl-125I-T3 labeling of the transiently expressed human and rat 5' DI proteins. These results demonstrate that the human 5' DI is a selenoprotein, analogous to the rat enzyme. Given the previously demonstrated critical role of the selenium atom in catalyzing deiodination by this protein, we conclude that this trace element is essential for normal thyroid hormone action in man.

Affinity Labels↗

PubMed1992

Differential capacity of wild type promoter elements for binding and trans-activation by retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR) and thyroid hormone receptor (T3R) are structurally similar and can bind as homodimers or T3R-RAR heterodimers to a single synthetic DNA response element. The interaction of these two types of receptors with wild type elements, however, has not been systematically investigated. Promoter elements from genes regulated by retinoic acid (RA) or thyroid hormone (T3) were tested for response to T3 and RA in transient transfections in both JEG and COS cells. The elements were classified as primarily responsive to RA or to T3 or responsive to both ligands. Binding of highly purified RAR alpha and T3R alpha to the various elements was assessed using the gel shift assay. Those elements predominantly responsive to one ligand showed preferential binding to the appropriate receptor. A series of point mutations were introduced into the rat GH T3 response element to further define sequence requirements for response to both RA and T3. Down-mutations in any of the three hexamers (previously demonstrated to be required for full response to T3 and full binding of T3R) also decreased RA induction and RAR binding. However, only one of two sets of up-mutations for T3 response also increased RA induction, demonstrating differences in hexamer preference between RAR and T3R. Variation in spacing of the three hexamers did not influence RA vs. T3 induction or RAR vs. T3R binding according to the predictions of a simple hexamer spacing model. There was a strong correlation between the extent of T3R dimer binding and strength of T3 induction for a subset of elements studied in JEG cells (r = 0.97, P < 0.01) and a weaker but significant correlation in COS cells (r = 0.65, P < 0.05)). In contrast, RAR dimer binding by the wild type elements did not quantitatively correlate with RA induction in either JEG (r = 0.13, P > 0.05) or COS cells (r = 0.21, P > 0.05). These results suggests that RAR interacts with a heterodimer partner(s) which influences binding site specificity, whereas T3R heterodimer partner(s) is less likely to alter binding site recognition. The observed difference in COS and JEG cells as well as the weak T3R binding-function relationship of the malic enzyme element, however, suggest that the influence of T3R heterodimer partner(s) on binding site specificity is likely to vary with cell type and the specific element tested.

Animals↗

PubMed1992

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.

Animals↗