Cytosolic Thyroid Hormone Binding Protein Research Articles

A review of the peripheral levels of regulation by thyroid hormone.

Thyroid hormone (TH) regulates many physiological processes that differ between tissues, developmental stages and in response to specific environmental cues. It can therefore play very different signaling roles depending on specific physiological contexts. Much progress has been made in resolving mechanisms for TH signaling over the past 2 decades, and there has been increasing emphasis on the role of peripheral levels of regulation in determining ultimate TH action. This progress has revealed a complex regulatory network, where TH bioavailability and bioactivity are peripherally regulated by sometimes subtle mechanisms at various levels of organization, including membrane receptors and transporters on the cell surface, intracellular deiodinase enzymes, thyroid receptor isoforms and cytosolic thyroid hormone binding proteins, and via accessibility and subtypes of thyroid hormone response elements in the promoters of target genes. The majority of this research comes from disease models, and so the biological relevance of each of these regulatory levels has not been comprehensively explored. This review synthesizes what is known of these local levels of TH regulation, with particular focus on their functional roles in regulating animal response to environmental cues. While thorough analysis for all of these regulatory levels in any one study is currently unrealistic, an appreciation for their collective importance is necessary to frame comparative analyses in a relevant context. This is important because common biomarkers for TH action can have very different meanings, not only for different tissues, but also for individuals, populations and species from different developmental or environmental backgrounds.

Regulation of thyroid hormone-induced development in vivo by thyroid hormone transporters and cytosolic binding proteins

Differential tissue sensitivity/responsivity to hormones can explain developmental asynchrony among hormone-dependent events despite equivalent exposure of each tissue to circulating hormone levels. A dramatic vertebrate example is during frog metamorphosis, where transformation of the hind limb, brain, intestine, liver, and tail are completely dependent on thyroid hormone (TH) but occurs asynchronously during development. TH transporters (THTs) and cytosolic TH binding proteins (CTHBPs) have been proposed to affect the timing of tissue transformation based on expression profiles and in vitro studies, but they have not been previously tested in vivo. We used a combination of expression pattern, relative expression level, and in vivo functional analysis to evaluate the potential for THTs (LAT1, OATP1c1, and MCT8) and CTHBPs (PKM2, CRYM, and ALDH1) to control the timing of TH-dependent development. Quantitative PCR analysis revealed complex expression profiles of THTs and CTHBPs with respect to developmental stage, tissue, and TH receptor β (TRβ) expression. For some tissues, the timing of tissue transformation was associated with a peak in the expression of some THTs or CTHBPs. An in vivo overexpression assay by tail muscle injection showed LAT1, PKM2, and CRYM increased TH-dependent tail muscle cell disappearance. Co-overexpression of MCT8 and CRYM had a synergistic effect on cell disappearance. Our data show that each tissue examined has a unique developmental expression profile of THTs and CTHBPs and provide direct in vivo evidence that the ones tested are capable of affecting the timing of developmental responses to TH.

Expression levels of TRα, LAT1, and PKM2 alter the rate of metamorphic change

Event Abstract Back to Event Expression levels of TRα, LAT1, and PKM2 alter the rate of metamorphic change Jinyoung Choi1* and Daniel R. Buchholz1 1 University of Cincinnati, Department of Biological Science, United States Thyroid hormone (TH) plays a key role for most physiological and developmental changes during metamorphosis. TH exerts its function by binding thyroid hormone receptors (TRs) in the nucleus to initiate TH-dependent gene expression. Intracellular TH levels are regulated by thyroid hormone transporters (THTs) and cytosolic thyroid hormone binding proteins (CTHBPs). Intracellular TH levels and TR expression levels likely regulate the timing of transformation of different tissues during metamorphosis. We hypothesized that altered TR, LAT1 (a THT), and PKM2 (a CTHBP) expression affects the rate of metamorphosis when overexpressed. In this study, we overexpressed TR α, LAT1, and PKM2 in tail muscle cells by intramuscular plasmid DNA injection and quantified the disappearance of tail muscle cells during T3-induced metamorphosis. In cells overexpressing TR, the rate of cell death was significantly faster compared to controls.. Moreover, cell death was accelerated or delayed in muscle cells overexpressing LAT1 or PKM2, respectively, compared to controls. These results show that the expression levels of TR and proteins affecting intracellular TH levels can affect the rate of metamorphic changes. References Shi, Y., Wong, J., Puzianowska-Kuznicka, M., and Stolow, M.A. (1996). Tadpole competence and tissue-specific temporal regulation of amphibian metamorphosis: roles of thyroid hormone and its receptors. BioEssay 18(5), 391-399. Buchholz, D.R., Moskalik, C.L., Kulkarni, S.S., Hollar, A.R.,and Ng, Allison (2011).Hormone regulation and the evolution of frog metamorphic diversity. Mechanisms of Life History Evolution. Oxford publishers (in press). Hollar, A.R., Choi, J., Grimm, A.T., and Buchholz, D.R. (2011). Higher thyroid hormone receptor expression correlates with short 4 larval periods in spadefoot toads and increases metamorphic rate. Submitted to Gen. Comp. Endocrinol Friesema, E.C., Ganguly S., Abdalla, A., Fox, J.E.M., Halestrap, A.P.H. and Visser, T.J. (2003). Identification of monocarboxlyate transporter 8 as a specific thyroid hormone transporter. J. Bio. Chem. 278 (41), 40128-40315. Luze, A.D., Sachs, B., and Demeneix, B.A. (1993). Thyroid hormone-dependent transcriptional regulation of exogenous genes transfected into Xenopus tadpole muscle in vivo. Pro. Natl. Acad. USA 90 (August) 7322-7326. Kinne, A., Kleinau, G., Hoefig, G.S., Gruters, A., Kohrle, J., Krause, G., and Schweizer, U. (2010). Essential molecular determinants for thyroid hormone transport and first structural implications for monocarboxlyate transporter 8. J. Bio. Chem. 285 (36) 28054-28063 Connors, K.A., Korte, J.J., Anderson, G.W., and Degitz, S.J. (2010). Characterization of thyroid hormone transporter expression during tissue-specific metamorphic events in Xenopus tropicalis. Gen. Comp. Endocrinol. 168, 149-159. Keywords: cytosolic thyroid hormone binding protein, LAT1, metamorphosis, PKM2, Thyroid hormone, Thyroid hormone transporter, tissue asynchrony, TRα Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Thyroid Citation: Choi J and Buchholz DR (2011). Expression levels of TRα, LAT1, and PKM2 alter the rate of metamorphic change. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00116 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Miss. Jinyoung Choi, University of Cincinnati, Department of Biological Science, Cincinnati, Ohio, 45221, United States, jinyoung.angela@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jinyoung Choi Daniel R Buchholz Google Jinyoung Choi Daniel R Buchholz Google Scholar Jinyoung Choi Daniel R Buchholz PubMed Jinyoung Choi Daniel R Buchholz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Effective Cellular Uptake and Efflux of Thyroid Hormone by Human Monocarboxylate Transporter 10

Cellular entry of thyroid hormone is mediated by plasma membrane transporters, among others a T-type (aromatic) amino acid transporter. Monocarboxylate transporter 10 (MCT10) has been reported to transport aromatic amino acids but not iodothyronines. Within the MCT family, MCT10 is most homologous to MCT8, which is a very important iodothyronine transporter but does not transport amino acids. In view of this paradox, we decided to reinvestigate the possible transport of thyroid hormone by human (h) MCT10 in comparison with hMCT8. Transfection of COS1 cells with hMCT10 cDNA resulted in 1) the production of an approximately 55 kDa protein located to the plasma membrane as shown by immunoblotting and confocal microscopy, 2) a strong increase in the affinity labeling of intracellular type I deiodinase by N-bromoacetyl-[(125)I]T(3), 3) a marked stimulation of cellular T(4) and, particularly, T(3) uptake, 4) a significant inhibition of T(3) uptake by phenylalanine, tyrosine, and tryptophan of 12.5%, 22.2%, and 51.4%, respectively, and 5) a marked increase in the intracellular deiodination of T(4) and T(3) by different deiodinases. Cotransfection studies using the cytosolic thyroid hormone-binding protein micro-crystallin (CRYM) indicated that hMCT10 facilitates both cellular uptake and efflux of T(4) and T(3). In the absence of CRYM, hMCT10 and hMCT8 increased T(3) uptake after 5 min incubation up to 4.0- and 1.9-fold, and in the presence of CRYM up to 6.9- and 5.8-fold, respectively. hMCT10 was less active toward T(4) than hMCT8. These findings establish that hMCT10 is at least as active a thyroid hormone transporter as hMCT8, and that both transporters facilitate iodothyronine uptake as well as efflux.

Thyroid hormone receptor expression in the obligatory paedomorphic salamander Necturus maculosus

Amphibian metamorphosis is under the strict control of thyroid hormones (TH). These hormones induce metamorphosis by controlling gene expression through binding to thyroid hormone receptors (TRs). Necturus maculosus is considered to be an obligatory paedomorphic Amphibian since metamorphosis never occurs spontaneously and cannot be induced by pharmacological means. Since metamorphosis depends on the acquisition of response of tadpole tissues to thyroid hormone, we aimed to determine TR gene expression patterns in Necturus maculosus as well as the expression of two TH-related genes: Cytosolic Thyroid Hormone-Binding Protein (CTHBP)-M2-pyruvate kinase, a gene encoding a cytosolic TH binding protein and stromelysin 3, a direct TH target gene in Xenopus laevis. Tissue samples were obtained from specimens of Necturus maculosus. We performed in situ hybridization using non-cross-hybridizing RNA probes obtained from the cloned Necturus TRalpha and TRbeta genes. We found clear expression of Necturus TRalpha gene in several tissues including the central nervous system, epithelial cells of digestive and urinary organs, as well as myocardium and skeletal muscle. TRbeta was also expressed in the brain. In other tissues, hybridization signals were too low to draw reliable conclusions about their precise distribution. In addition, we observed that the expression of CTHBP and ST3 is largely distinct from that of TRs. The fact that we observed a clear expression of TRalpha and TRbeta which are evolutionary conserved, suggests that Necturus tissues express TRs. Our results thus indicate that, in contrast to previously held hypotheses, Necturus tissues are TH responsive.

Characterization of Xenopus cytosolic thyroid-hormone-binding protein (xCTBP) with aldehyde dehydrogenase activity

Multiple cytosolic thyroid-hormone-binding proteins (CTBPs) with varying characteristics, depending on the species and tissue, have been reported. We first purified a 59-kDa CTBP from Xenopus liver (xCTBP), and found that it is responsible for major [ 125I]T 3-binding activity in Xenopus liver cytosol. Amino acid sequencing of internal peptide fragments derived from xCTBP demonstrated high identity to the corresponding sequence of mammalian aldehyde dehydrogenases 1 (ALDH1). To confirm whether or not xCTBP is identical to xALDH1, we isolated cDNAs encoding xALDH1 from an adult Xenopus hepatic cDNA library. The amino acid sequences deduced from the two isolated xALDH1 cDNAs were very similar to those of mammalian ALDH1 enzymes. The recombinant xALDH1 protein exhibited both T 3-binding activity and ALDH activity converting retinal to retinoic acid (RA), which were similar to those of xCTBP purified from liver cytosol. The T 3-binding activity was inhibited by NAD, while the ALDH activity was inhibited by thyroid hormones. Our results demonstrate that xCTBP is identical to ALDH1 and suggest that this protein might modulate RA synthesis and intracellular concentration of free T 3. Communications between thyroid hormone and retinoid pathways are discussed.

Xenopus Cytosolic Thyroid Hormone-binding Protein (xCTBP) Is Aldehyde Dehydrogenase Catalyzing the Formation of Retinoic Acid

Amino acid sequencing of an internal peptide fragment derived from purified Xenopus cytosolic thyroid hormone-binding protein (xCTBP) demonstrates high similarity to the corresponding sequence of mammalian aldehyde dehydrogenase 1 (ALDH1) (Yamauchi, K., and Tata, J. R. (1994) Eur. J. Biochem. 225, 1105-1112). Here we show that xCTBP was co-purified with ALDH and 3,3',5-triiodo-L-thyronine (T3) binding activities. By photoaffinity labeling with [125I]T3, a T3-binding site in the xCTBP was estimated to reside in amino acid residues 93-114, which is distinct from the active site of the enzyme but present in the NAD+ binding domain. The amino acid sequences deduced from the two isolated xALDH1 cDNAs (xALDH1-I and xALDH1-II) were 94.6% identical to each other and very similar to those of mammalian ALDH1 enzymes. The two recombinant xALDH1 proteins exhibit both T3 binding activity and ALDH activity converting retinal to retinoic acid (RA), which are similar to those of xCTBP. The mRNAs were present abundantly in kidney and intestine of adult female Xenopus. Interestingly, their T3 binding activities were inhibited by NAD+ and NADH but not by NADP+ and NADPH, whereas NAD+ was required for their ALDH activities. Our results demonstrate that xCTBP is identical to ALDH1 and suggest that this protein might modulate RA synthesis and intracellular level of free T3.

Tissue-Dependent and Developmentally Regulated Cytosolic Thyroid-Hormone-Binding Proteins (CTBPs) in Xenopus

Xenopus cytosolic thyroid-hormone-binding proteins (CTBPs) were examined by a photoaffinity labeling and [ 125I]T 3-binding assay. An affinity-labeled protein of 59 kDa, which was responsible for the major T 3-binding activity in adult tissues, was predominant in liver. The 59-kDa CTBP first appeared in significant amounts at the metamorphic climax stage in liver cytosol and continued to be expressed after metamorphosis. Another affinity-labeled CTBP of 38 kDa appeared at the metamorphic climax stage in cytosol from head region, but disappeared after this stage. T 3-binding assay using whole cytosol showed that a distinct CTBP, although not photoaffinity-labeled, was present in cytosol from hindlimb bud and gradually disappeared as the hindlimb grew. The cytosol from liver, head region, and hindlimb bud contained high affinity binding sites for T 3 with K d values ranging from 10 −9 to 10 −8 M. These results suggest that there are at least three distinct CTBPs in Xenopus cytosol, which are expressed in a tissue-dependent and developmentally regulated manner.

Tadpole competence and tissue‐specific temporal regulation of amphibian metamorphosis: Roles of thyroid hormone and its receptors

Amphibian metamorphosis is a post-embryonic process that systematically transforms different tissues in a tadpole. Thyroid hormone plays a causative role in this complex process by inducing a cascade of gene regulation. While natural metamorphosis does not occur until endogenous thyroid hormone has been synthesized, tadpoles are competent to respond to exogenous thyroid hormone shortly after hatching. In addition, even though the metamorphic transitions of individual organs are all controlled by thyroid hormone, each occurs at distinct developmental stages. Recent molecular studies suggest that this competence of premetamorphic tadpoles to respond to the hormone and the developmental stage-dependent regulation of tissue-specific transformations are determined in part by the levels of thyroid hormone receptors and the concentrations of cellular free thyroid hormone. In addition, at least two genes, encoding a cytosolic thyroid hormone binding protein and a 5-deiodinase, respectively, are likely to be critical players in regulating cellular free thyroid hormone concentrations. This review discusses how all of these molecular components coordinate to induce amphibian metamorphosis in a correct spatial and temporal manner. These studies provide us with general clues as to how and why tissues become competent to respond to hormonal signals.

Tissue-dependent developmental expression of a cytosolic thyroid hormone protein gene in Xenopus: Its role in the regulation of amphibian metamorphosis

We have cloned the cDNA encoding the Xenopus laevis homolog of mammalian cytosolic thyroid hormone binding protein (CTHBP). We found that while its mRNA level varies little in whole animals during development, the expression of CTHBP is inversely correlated with tissue-specific transformations during metamorphosis. A high level of its mRNA was observed in the tail of premetamorphic tadpoles. However, the expression is dramatically repressed with the onset of rapid tail resorption. In the hindlimb, the expression of CTHBP is very low during morphogenesis. Subsequently, its expression continuously increases during the period of limb growth. In contrast, a low level of CTHBP expression was detected in the intestine throughout metamorphosis. These results suggest that CTHBP could function to modulate the metamorphic process by regulating the level of intracellular thyroid hormones.

Regulation by thyroid hormone of the synthesis of a cytosolic thyroid hormone binding protein during liver regeneration

To understand the regulation by thyroid hormone, 3,3′,5-triiodo-L-thyronine (T 3), of the synthesis of a cytosolic thyroid hormone binding protein (p58-M 2) during liver regeneration, the synthesis of p58-M 2 was evaluated. The synthesis of p58-M 2 was measured by metabolic labeling of primary cultures derived from the regenerating liver of euthyroid, hypo- or hyperthyroid rats. During regeneration, the increase in the liver/body weight ratio is ∼25% higher in hyper- than in hypothyroid rats. However, T 3 has no effect on the rate of overall liver regeneration observed in four days. In mature liver, T 3 increased the synthesis of p58-M 2 by ∼2.5-fold. During regeneration, however, the change in the synthesis of p58-M 2 varied with the thyroid status. In euthyroid rats, the synthesis of p58-M 2 continued to increase up to 2-fold during liver regeneration. In hyperthyroid rats, after an initial increase by 1.5-fold on day 1, the synthesis of p58-M 2 subsequently declined during regeneration. In hypothyroid rats, the synthesis of p58-M 2 remained virtually unchanged during regeneration. These results indicate that T 3 regulates the synthesis of p58-M 2 in mature and regenerating liver.

Regulation of thyroid hormone receptor-mediated transcription by a cytosol protein.

Thyroid hormone receptors (TRs) are members of the steroid hormone/retinoic acid receptor superfamily, which regulate homeostasis, development, and differentiation. Their transcriptional activity is modulated by the thyroid hormone 3,3',5-triiodo-L-thyronine (T3). The present study evaluated the effect of the availability of cytoplasmic T3 on the modulation of transcriptional responses of the TRs. In human choriocarcinoma JEG-3 and monkey COS-1 cells, the cytosolic thyroid hormone binding protein is a monomer of the tetrameric pyruvate kinase, subtype M2, which does not bind T3. The in vivo monomer-tetramer interconversion is regulated by glucose via fructose 1,6-bisphosphate. At the physiological T3 concentration, lowering the glucose concentration led to an increase in the cellular concentration of the cytosolic thyroid hormone binding protein. By using a transient transfection system, a concomitant reduction in the transcriptional activity of the human beta 1 thyroid hormone receptor was detected in both cell lines. In the absence of glucose, the transcriptional activity of the human beta 1 thyroid hormone receptor in JEG-3 and COS-1 cells was reduced by 65-75% and 90-95%, respectively. However, glucose had no effect on the basal transcriptional activity. These findings demonstrate an important prenuclear step in the modulation of the gene regulating activity of the TRs.

Transcriptional stimulation by thyroid hormone of a cytosolic thyroid hormone binding protein which is homologous to a subunit of pyruvate kinase M1.

We have recently shown that the monomer of rat pituitary pyruvate kinase subtype M1 (p58-M1) is a cytosolic binding protein for 3,3',5-triiodo-L-thyronine (T3). To understand the role p58-M1 plays in thyroid hormone action, we examined the regulation of p58-M1 by T3 in GH3 cells. Expression of p58-M1 was evaluated by metabolically labeling GH3 cells cultured in regular medium, thyroid hormone depleted medium (Td medium), or Td medium supplemented with T3 (Td + T3 medium) followed by immunoprecipitation. T3 stimulates the expression of p58-M1 by 2-fold. Analysis by pulse-chase experiments indicates that the increased expression is not due to the increase of stability of p58-M1. Northern analysis of mRNA prepared from cells cultured in regular, Td, or Td + T3 medium demonstrates that T3 increases the accumulation of cytoplasmic mRNA by 2-fold. Nuclei from cells cultured in the three conditions were prepared, and the rates of synthesis of nascent nuclear RNA were compared by an in vitro transcription assay. Addition of T3 stimulates the rate of transcription by 2-fold. The parallel and identical magnitude in the increase of transcription rate and the accumulation of mRNA indicates that T3 stimulates the synthesis of p58-M1 by increasing the transcriptional activity of its gene.

The monomer of pyruvate kinase, subtype M 1, is both a kinase and a cytosolic thyroid hormone binding protein

Using a T7 expression system, the monomer of rat pituitary pyruvate kinase, subtype M 1 (PKM 1), was overexpressed in Escherichia coli and purified to homogeneity. The monomeric p58-M 1 has intrinsic enzymatic activity with a Vmax of 79 ± 20 units/mg and Km's for ADP and PEP of 1.43 ± 0.76 and 0.14 ± 0.07 mM, respectively. The monomer binds 3,3′,5-triiodo-L-thyronine (T 3) with Ka = 1.5 × 10 7 M −1. The order of analog specificity is L-T 3 > L-thyroxine > D-T 3 > 3′-isopropyl-3,5-diiodo-L-thyronine ≥ 3′,5′,3-triiodo-L-thyronine. In contrast, tetrameric PKM 1 lacks T 3 binding activity. The kinase activity of p58-M 1 is inhibited by T 3 and its analogs in a concentration-dependent manner with the order of inhibitory activity similar to that of binding activity. This inhibition, however, is reversed by the addition of fructose 1,6-bisphosphate. p58-M 1 is the second PK isoenzyme monomer to be identified as having thyroid hormone binding activity.

An in vitro novel mechanism of regulating the activity of pyruvate kinase M2 by thyroid hormone and fructose 1,6-bisphosphate

We have recently shown that the cytosolic thyroid hormone binding protein (p58-M2) in human epidermoid carcinoma A431 cells is a monomer of pyruvate kinase, subtype M2 (PKM2). To characterize further the molecular properties of p58-M2, we overexpressed p58-M2 in Escherichia coli and purified it to homogeneity. At 22 degrees C, the monomeric p58-M2, exhibited kinase activity with an apparent Vmax of 22 +/- 9 units/mg. The Km for adenosine diphosphate (ADP) and phosphoenolpyruvate (PEP) are 3.85 +/- 2.4 and 1.55 +/- 0.73 mM, respectively. Upon activation by fructose 1,6-bisphosphate (Fru-1,6-P2), Vmax and Km for ADP and PEP were changed to 490 +/- 27 units/mg and 0.63 +/- 0.09 and 0.13 +/- 0.01 mM, respectively. These results indicated that p58-M2 has intrinsic kinase activity. Analysis of the molecular size indicated that the activation of p58-M2, by Fru-1,6-P2 resulted in the association of the monomeric p58-M2 to the tetrameric PKM2. p58-M2 bound to 3,3',5-triiodo-L-thyronine (T3) (Ka = 1.7 x 10(7) M-1) and exhibited analogue specificity, whereas PKM2 did not bind thyroid hormone. The order of binding affinity was L-T3 greater than L-thyroxine greater than 3,3',5-triiodothyropropionic acid greater than 3'-isopropyl-3,5-triiodo-L-thyronine greater than 3'5',3-triiodo-L-thyronine. Binding of T3 and its analogues resulted in the inhibition of the kinase activity of p58-M2. The order of kinase inhibitory activity and preventing its association to tetrameric PKM2 was parallel to that of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular binding proteins of thyroid hormones

Cellular binding proteins of thyroid hormones are present in the cell nucleus, cytosol, cell membrane, and mitochondria. While nuclear binding is proven to mediate hormone action, the exact roles of the other binding sites remain to be established. Nuclear receptor associates with DNA, core histone, and nuclear matrix and preferentially distributes in transcriptionally active chromatin due to interaction with H1 histone. Of particular importance is the binding of nuclear receptor to specific DNA sequences of target genes, termed thyroid-responsive elements. The binding is stabilized by non-receptor nuclear protein. Upon binding thyroid hormone, nuclear receptor is activated through alterations in the steric configuration, leading to changes in the rate of transcription of the target genes. Multiple nuclear receptor forms exist with likely distinct functional roles. Cytosolic thyroid hormone binding proteins are also heterogeneous. One form is under the control of cell metabolism (NADP and NADPH) and it may have a role in transport of the hormone to mitochondria and nucleus. Membrane-linked thyroid hormone binding proteins may have dual functional roles: one is to mediate hormone action and the other is to support active uptake of hormones by cells. Mitochondrial function may be regulated by thyroid hormone through mitochondrial binding sites in cooperation with nuclear receptor-mediated pathway. Further studies are required to elucidate the exact functional roles of non nuclear thyroid hormone binding proteins.

Chromosomal localization of the gene for a human cytosolic thyroid hormone binding protein homologous to the subunit of pyruvate kinase, subtype M2

A cDNA for the gene that encodes a human cytosolic thyroid hormone binding protein (p58) recently has been isolated and sequenced. Analysis of the p58 sequence indicates that it is identical to the subunit of pyruvate kinase, subtype M2. By in situ hybridization, the gene for p58 was mapped to 15q24-25. This localization shows that the p58 gene is not linked to the L-type of pyruvate kinase, which is located on chromosome 1. The p58 gene was found to be activated in several forms of cancer. Current localization will permit us to assess the effect of alterations involving chromosome 15 on the structure and activity of the p58 gene in neoplasms or chromosome syndromes.

Regulation of thyroid hormone binding to its cytosolic binding protein by L-α-alanine

The human cytosolic thyroid hormone binding protein (p58) was recently shown to be a monomer of pyruvate kinase, subtype PKM 2, and have intrinsic pyruvate kinase activity. The present study evaluated the effect of L-α-alanine on the binding of 3,3′,5-triiodo-L-thyronine (T 3) and enzymatic activity of p58. Analysis of the competitive binding data indicated that alanine, at the physiological concentration, is a non-competitive inhibitor of T 3 binding to p58. Furthermore, alanine was found to be a “mixed” inhibitor of the substrate phosphoenol pyruvate. However, binding of alanine to p58 did not block the association of p58 to form the tetrameric pyruvate kinase.

Cytosolic thyroid hormone-binding protein is a monomer of pyruvate kinase

Cytosolic thyroid hormone-binding protein is a monomer of pyruvate kinase

Cytosolic thyroid hormone-binding protein is a monomer of pyruvate kinase.

A cDNA clone encoding a human cytosolic thyroid hormone-binding protein (p58) has been isolated. The human sequence was found to be homologous to that of rat pyruvate kinase (EC 2.7.1.40) subtype M2. p58 is a monomer that has approximately 5% the enzymatic activity of the tetrameric pyruvate kinase M2. The tetrameric M2 does not bind 3,3',5-triiodo-L-thyronine (T3). Binding of p58 to T3 and its analogs resulted in the inhibition of its pyruvate kinase activity. The apparent Ki values of T3, L-thyroxine, and D-T3 are 30 nM, 100 nM, and 2 mM, respectively. L-Thyronine and 3,3',5'-triiodo-L-thyronine had no effect. This order of activity correlates with the thermogenic effects reported for T3 and its analogs. Conversion of p58 to the tetramer is reversible and is under the control of fructose 1,6-bisphosphate. The conversion is inhibited by T3 in a dose-dependent manner. Since pyruvate kinase is a key enzyme in regulating cellular ADP, ATP, and pyruvate, our findings suggest that p58 may be involved in mediating some of the cellular metabolic effects induced by thyroid hormones.