Thyroid hormone binding to the rat heart cytosol proteins

The properties of the thyroid hormone binding to rat heart cytosol were studied. Cytosol proteins were found to bind specifically T4 with high affinity (Ka approximately equal to 10(8)M-1) and rT3 with lower affinity (Ka approximately equal to 10(7)M-1), but they do not bind T3. The binding of both T4 and rT3 was pH dependent, however, while T4 binding had the highest values between pH 7.0 and 10, rT3 binding increased from pH 6.0 to 10.7. Divalent ions also stimulated T4 and rT3 binding. Sulfhydryl groups blocking agents such as N-ethylmaleimide (NEM) and iodoacetamide significantly decreased rT3 binding and had less profound effect on binding of T4 to cytosol proteins. The importance of free -SH groups remains unclear as dithiothreitol was found to diminish the binding of T4 and rT3.     

Vitamin A and thyroxine carrier proteins in chicken plasma: steady-state control of the plasma level of free retinol-binding protein and free thyroxine.

1. The binding parameters of prealbumin-2 with retinol-binding protein and thyroxine (T4) revealed the existence of distinct and multiple sites for both retinol-binding protein and T4. 2. From the analysis of binding parameters of retinol-binding protein with prealbumin-2 it is clear that under steady-state conditions about 99% of the holo-retinol-binding protein remains bound to prealbumin-2. 3. Equilibrium dialysis studies on binding properties of thyroid hormones with prealbumin-2 revealed that it has a single high affinity site and three low affinity sites. 4. The occurrence of three carrier proteins for thyroid hormones, thyroxine-binding globulin, prealbumin-2 and albumin has been demonstrated. However, the chicken thyroxine-binding globulin differs from human thyroxine-binding globulin by being relatively less acidic and occurring at a two-fold lower concentration. But the thyroid hormone binding parameters are comparable. 5. Highly sensitive methods were developed for determination of T4 binding capacities of the various proteins and plasma level of total T4 by fractionation of carrier proteins and further quantitatively employing in electrophoresis and equilibrium dialysis. 6. The thyroxine-binding proteins were found to be of two types, one (viz., thyroxine-binding globulin) of great affinity but of low binding capacity, which mainly acts as reservoir of T4, and another (viz., prealbumin-2) of low affinity but of high binding capacity, which can participate predominantly in the control of the free T4 pool.     

Specific binding of iodothyronines with apolipoprotein A-1 in high density lipoprotein particle isolated from human serum by affinity chromatography on thyroxine-sepharose

The kinetic and equilibrium characteristics of interaction of thyroxine (T4) and its structural analogs with a high density lipoprotein (HDL) fraction isolated from human serum by T4-Sepharose affinity chromatography and containing apolipoprotein A-I (apo A-I) as a sole protein component, were studied. The binding of [125I]T4 to apo A-I-HDL reached a maximum after 40 min and did not change during the next 80 min of incubation at 0 degrees--22 degrees C. Dissociation of [125I]T4 induced by the addition of excess unlabeled T4 to the complex solution proceeded more intensely on a time scale at 0--2 degrees C than at 22 degrees C. Incubation of apo A-I-HDL with increasing concentrations of T4 showed that the binding is saturable. The data analysis using different computer programs revealed the presence in apo A-I-HDL of a single class of binding sites with K alpha = (4.0 +/- 2.1).10(-7) M- and Bmax = 1.7 +/- 0.8 nmol T4/mg of protein. Naturally occurring iodothyronines, their analogs and D-isomers of thyroid hormones competed with [125I]T4 for the binding sites on apo A-I-HDL with the following inhibitory potencies: L-T4 = D-T4 greater than or equal to 3,3',5-triiodo-L-thyronine = 3,3',5-triiodo-D-thyronine greater than 3,5-diiodo-L-thyronine = 3,3',5- triiodothyroacetic acid greater than 3,3',5-triiodothyropropionic acid greater than or equal to 3,5-diiodo-L-thyrosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation in vitro of rabbit erythrocyte cytosol phospholipid-dependent protein kinase activity. A novel action of thyroid hormone

L-Thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) at 10(-10) M stimulated phospholipid- and Ca2+-dependent protein kinase activity in rabbit red cell cytosol in vitro by 151 and 176%, respectively. Kinase of 30-fold greater specific activity, developed with 0.4 mM NaCl from cytosol applied to DEAE-cellulose, was also stimulated up to 2-fold by thyroid hormone. Hormone enhancement of kinase activity occurred after 60 min of incubation at 37 degrees C prior to enzyme assay. Thyroid hormone analogues triiodothyroacetic acid, 3,5-dimethyl-3'-isopropyl-L-thyronine, D-T3, D-T4, and 3,3',5'-L-triiodothyronine (reverse T3) were inactive. These results support a role for thyroid hormone endogenously in regulation of phospholipid-dependent protein kinase activity.     

A cytoplasmic thyroid hormone binding protein: characterization using monoclonal antibodies

We have previously purified a cellular thyroid hormone binding protein (p58) from a human carcinoma cell line [Kitagawa, S., Obata, T., Hasumura, S., Pastan, I., & Cheng, S.-y. (1987) J. Biol. Chem. 262, 3903-3908]. In the present study, the binding characteristics, the molecular properties, and subcellular localization of p58 were further characterized. Binding of the purified p58 to thyroid hormones was examined. Analysis of binding data indicates that p58 binds to 3,3',5-triiodo-L-thyronine (T3) with a Kd of 24.3 +/- 0.3 nM and n = 0.71. p58 binds to L-thyroxine similarly as to T3. However, D-T3 and reverse-T3 bind to p58 with an affinity 4- and 20-fold less than that of T3, respectively. By use of the purified p58 as an immunogen, two hybridomas, J11 and J12, secreting monoclonal antibodies to p58 were isolated; both antibodies belong to the IgG1K subclass. J12 recognizes p58 from human, monkey, dog, hamster, and rat, but not mouse. J11 exhibits a similar species specificity except that it does not react with p58 from hamster. With these antibodies, p58 was found to be not posttranslationally modified by glycosylation, sulfation, or phosphorylation. It has a cellular degradation rate t1/2 congruent to 2.1 h. Immunocytochemical studies indicate that p58 is located in the nonmembranous cytoplasm (cytosol). These results are consistent with subcellular fractionation studies which show that greater than 95% of J11 and J12 reactivity and T3 binding activity can be found in the 110,000g supernatant.     

Identification of thyroid hormone receptors in rat liver nuclei by photoaffinity labeling with L-thyroxine and triiodo-L-thyronine

Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of binding of thyroid hormone analogues to hepatic organic anion binding proteins

Dv protein and ligandin are two hepatic cytosolic proteins which bind organic anions, including endogenous thyroid hormones. Binding studies were performed using the ANS displacement technique to compare the binding of a variety of thyroid hormone analogues to purified organic anion binder and ligandin. Inhibition of ANS binding by these compounds was competitive. Both proteins bound L- and D-thyroxine with comparable affinity (Kd 30-45 microM), whereas ligandin bound 3',3',5-triiodo-L-thyronine, 3',3',5-triiodo-L-thyronine and most analogues with greater affinity. Nevertheless, the order of ligand affinities for both binders was highly correlated, suggesting that the nature of the binding site on both proteins is similar. The binding affinities of these organic anion binders are 2-3 orders of magnitude lower than an hepatic cytosolic thyroid binder reported by others, suggesting that ligandin and organic anion binder may not be important in intracellular thyroid hormone transfer.     

Glucocorticoids,thyroid hormones,and iodothyronine deiodinases in embryonic saltwater crocodiles

We investigated the relationship between glucocorticoids, thyroid hormones, and outer ring and inner ring deiodinases (ORD and IRD) during embryonic development in the saltwater crocodile (Crocodylus porosus). We treated the embryos with the synthetic glucocorticoid dexamethasone (Dex), 3,3',5-triiodothyronine (T(3)), and a combination of these two hormones (Dex + T(3)). The effects of these treatments were specific in different tissues and at different stages of development and also brought about changes in plasma concentrations of free thyroid hormones and corticosterone. Administration of Dex to crocodile eggs resulted in a decrease in 3,3',5,5'-tetraiodothyronine (T(4)) ORD activities in liver and kidney microsomes, and a decrease in the high-K(m) rT(3) ORD activity in kidney microsomes, on day 60 of incubation. Dex treatment increased the T(4) ORD activity in liver microsomes, but not kidney microsomes, on day 75 of incubation. Dex administration decreased T(3) IRD activity in liver microsomes. However, this decrease did not change plasma-free T(3) concentrations, which suggests that free thyroid hormone levels are likely to be tightly regulated during development.     

Proton nuclear magnetic resonance assignments of thyroid hormone and its analogues

1H NMR data of a series of thyroid hormone analogues, e.g., thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), 3-monoidothyronine (3-T1), 3'-monoiodothyronine (3'-T1), and thyronine (TO) in dimethylsulfoxide (DMSO) have been obtained on a 300 MHz spectrometer. The chemical shift and coupling constant are determined and tabulated for each aromatic proton. The inner tyrosyl ring protons in T4, T3, and 3,5-T2 have downfield chemical shifts with respect to those of the outer phenolic ring protons. Four-bond cross-ring coupling has been observed in all the monoiodinated rings. However, this long-range coupling does not exist in T4, diiodinated on both rings, and T0, containing no iodines on the rings. There is no evidence that at 30 degrees C these iodothyronines have any motional constraint in DMSO solution. In addition to identification of the hormones, the potential use of some characteristic peaks as probes in binding studies is discussed.     

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.     

Binding of thyroid hormones and their analogues to thyroxine-binding globulin in human serum.

The present study was undertaken to study the binding of several thyroid hormones and structurally related compounds to human serum thyroxine-binding alpha-globulin (TBG). The source of TBG was normal human serum diluted 1:100 in 0.035 M barbital buffer, pH 7.4. In the binding assays, 125I-thyroxine, unlabeled thyroxine, and diluted serum were incubated for 20 h at 37 degrees in Plexiglas equilibrium dialysis units. Two orders of binding sites were discerned: a high affinity, low capacity binding site with an affinity constant of approximately 2.5 X 10(9) M-1, and a low affinity, very high capacity binding site with an affinity constant of less than 10(6) M-1. Studies with purified TBG, serum deficient in TBG, and purified human serum albumin indicated that the high affinity site represented binding to TBG and the low affinity site represented binging to albumin. The ability of several groups of thyroid hormone analogues to bind to TBG was then investigated. As a result of these studies, the following structural features of thyroid hormones were found to be important for optimal binding activity: (a) the L-alanine side chain conformation, (b) the presence of a 4'-hydroxyl group, (c) the presence of two substituents in the inner and outer rings (positions 3, 5, 3', and 5'), and (d) the presence of either bromines or iodines in the inner ring and iodines in the outer ring. Of lesser importance was the presence of an oxygen atom in the ether position.     

Nongenomic effects of thyroid hormones on the immune system cells: New targets, old players

It is now widely accepted that thyroid hormones, l-thyroxine (T(4)) and 3,3',5-triiodo-l-thyronine (T(3)), act as modulators of the immune response. Immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen species, and cytokine synthesis and release, are altered in hypo- and hyper-thyroid conditions, even though for many immune cells no clear correlation has been found between altered levels of T(3) or T(4) and effects on the immune responses. Integrins are extracellular matrix proteins that are important modulators of many cellular responses, and the integrin αvβ3 has been identified as a cell surface receptor for thyroid hormones. Rapid signaling via this plasma membrane binding site appears to be responsible for many nongenomic effects of thyroid hormones, independent of the classic nuclear receptors. Through the integrin αvβ3 receptor the hormone can activate both the ERK1/2 and phosphatidylinositol 3-kinase pathways, with downstream effects including intracellular protein trafficking, angiogenesis and tumor cell proliferation. It has recently become clear that an important downstream target of the thyroid hormone nongenomic pathway may be the mammalian target of rapamycin, mTOR. New results demonstrate the capability of T(3) or T(4) to induce in the short time range important responses related to the immune function, such as reactive oxygen species production and cell migration in THP-1 monocytes. Thus thyroid hormones seem to be able to modulate the immune system by a combination of rapid nongenomic responses interacting with the classical nuclear response.     

Rat liver iodothyronine monodeiodinase. Evaluation of the iodothyronine ligand-binding site

Ligand binding characteristics of rat liver microsomal type I iodothyronine deiodinase were evaluated by measuring dose-response inhibition and apparent Michaelis-Menten or inhibitor constants of iodothyronine analogues to compete as substrates or inhibitors for the natural substrate L-thyroxine. These data show strong correlations with the binding requirements of hormone analogues to serum thyroxine-binding prealbumin since iodothyronine analogues with a negatively charged side chain, a negative charge or hydrogen bonding function in the 4'-position, tetraiodo ring substitution, and a skewed hormone conformation are structural features shared in common which markedly affect enzyme activity and protein binding affinity. 3,3',5'-Triiodo-L-thyronine is the most potent natural substrate (IC50 = 0.3 microM) and tetraiodothyroacetic acid is the most potent inhibitor (IC50 = 0.2 microM). Both thyroxine (T4)-5'- and T4-5-deiodination pathways are inhibited by these potent analogues, providing further evidence for a single enzyme catalyzing the rat liver microsomal deiodination reactions. These data also show that L-hormone analogues are preferentially deiodinated via the T4-5'-deiodination pathway, whereas D-analogues produce products via the T4-5-deiodination pathway. The thyroxine-binding prealbumin complex was used to model the interaction of thyroid hormones with the deiodinase active site. Computer graphic modeling of the prealbumin complex showed that only those analogues which are potent deiodinase inhibitors or substrates can be accommodated in the hormone binding site. This model suggests the design of functionally specific ligands which can modulate peripheral thyroid hormone metabolism and act as antithyroidal drugs.     

Linkage between the hormone binding site and the reactive center loop of thyroxine binding globulin

Thyroxine binding globulin (TBG) is the major carrier of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) in plasma. TBG is member of the serpin family of proteins although it has no proteinase inhibitory activity. In this study we show that TBG has properties typical of a metastable serpin and provide evidence that occupancy of the hormone binding site alters the conformation of the reactive center loop. After reactive center loop cleavage by endoproteinase Asp-N or neutrophil elastase the protein became more stable to guanidine hydrochloride denaturation compared to the native protein, as a result of loop insertion. In addition, incubation of the native protein with a reactive center loop peptide, caused a change in mobility on a native gel. This is consistent with the idea that thyroxine binding globulin is able to form a binary complex with the peptide as a result of beta-sheet A expansion. To assess the effect of cleavage and loop insertion on the hormone binding site we used the specific binding of a fluorophore, 1,8-anilinonaphthalene sulfonic acid (ANS). Loop insertion itself had no effect on ANS affinity, but cleavage with elastase at the P4'-P5' bond caused a reduction in affinity, presumably because this cleavage site is located within the hormone binding site. These data support the concept that cleavage of TBG by proteinases released in inflammation is a mechanism to deliver thyroid hormones to target tissues. A linkage between the occupancy state of the hormone binding site and the conformation of the reactive center loop was indicated by the observation that binding of T3 to native TBG reduced proteolytic susceptibility by both endoproteinase Asp-N and elastase.     

Hormone binding by protein disulfide isomerase, a high capacity hormone reservoir of the endoplasmic reticulum

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3',5-triiodo-l-thyronine (T(3)). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 +/- 0.5 microm). There are two binding sites with comparable affinity for T(3) (4.3 +/- 1.4 microm). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T(3) inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T(3) is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 microm) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.     

Goitres in rats fed polychlorinated biphenyls.

Rats fed a polychlorinated biphenyl (PCB) mixture in a high- or low-iodine diet (HID or LID respectively) for 15 days had thyroid enlargement, low serum thyroxine (T4), and high serum thyrotropin concentrations. Although binding of thyroid hormones to serum proteins was reduced in PCB-fed animals, the free T4 index (reflecting free T4 in serum) was less in these rats. Both serum triiodothyronine (T3) and the free T3 index were elevated in rats fed PCB in HID. LID-maintained rats elevated serum T3 concentrations but the free T3 index was similar to that in HID-fed rats, owing to enhanced binding of thyroid hormone to serum proteins. Addition of PCB to LID reduced serum T3 levels but did not alter the free T3 index because binding was less. In rats fed HID containing PCB, thyroid 131I uptake was increased.     

Effect of thyroid hormones and their analogues on the mitochondrial calcium transport activity.

In this paper the authors studied the effects of thyroid hormones and their structural analogues on the mitochondrial calcium transport activities. The thyroid hormones, 3,5,3' L-triiodothyronine (LT3) and 3,5,3'5' L-tetraiodothyronine (LT4) at physiological intracellular concentrations between 7.2 and 9 nM, decouple total Ca++ transport, as well as inhibit the passive transport of Ca++, either due to oxidation of pyruvate, malate or succinate or after inhibition with rotenone. The optical isomers 3,5,3' D-triiodothyronine (DT3) and 3,5,3',5' D-tetraiodothyronine (DT4) are less effective at all the used concentrations. Furthermore the structural analogues 3,3',5' L-triiodothyronine (LrT3), 3,5-dicloro, 3',5' L-diiodothyronine (LDiClT2) and 3,5 L-diiodothyronine (LT2) furnished even less effects on the same activities. The effect of the thyroid hormones and of their structural analogues has revealed that the mitochondrial calcium transport may be influenced both by a stereospecific interaction between hormones and protein ligands and by a lipophilic chaotropic action on the mitochondrial membranes lipids. In this context it is interesting to consider that both thyroid hormones and Ca++ transport activity are interacting with the energetic metabolism by means of phosphorylation and substrate oxidation mechanism.     

Specific 3,3′,5′-Triiodothyronine (Reverse T3) Binding Sites on Rat Liver Plasma Membranes: Comparison with Thyroxine (T4) Binding Sites

Abstract

The binding characteristics of thyroxine (T4), triiodothyronine (T3), and reverse T3 (rT3) to rat liver plasma membranes (RLPM) were examined to explore the interactions of thyroid hormones with cell surface receptors. Scatchard analysis suggested that all three ligands bound to two classes of binding sites. The high affinity rT3 binding sites appeared to be distinct from the high affinity T4 sites, on the basis of differing optimum physicochemical conditions for binding, and analog displacement studies. The higher affinity constant for T4 was 1.7 ± 0.2 × 10⁹ M^-1 (mean ± SEM) and binding capacity was 3.1 ± 0.3 pmol mg ^-1 protein whereas for rT3 binding the Ka was 2.5 ± 0.4 × 10⁸ M^-1 and capacity was 6.2 ± 0.9 pmol mg ^-1. (¹²⁵ I) T3 bound with lower affinity and T3 tracer was readily displaced by T4. Moreover, comparatively higher concentrations of T3 were needed to displace either radiolabeled T4 or rT3, suggesting that T3 was binding to both the T4 and rT3 sites with lower affinity. Marker enzyme studies on RLPM, of varying purity prepared by different methods, showed a positive correlation between the activity of the plasma membrane enzyme magnesium-stimulated ATPase and high affinity rT3 and T4 binding. Column chromatography of the radioligands, after dissociation from membrane binding sites, confirmed that the integrity of the hormones was not altered during association or dissociation. Our results raise the possibility that the high affinity T4 and rT3 binding sites on RLPM may be hormone receptors mediating biological actions at the membrane level.     

Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in Escherichia coli

Using a T7 expression system, large amounts of the human placental c-erbA protein (h-TR beta 1) were expressed. From 1 liter of Escherichia coli culture, approximately 50-100 micrograms of purified h-TR beta 1 were obtained. Analysis of the binding data indicated that the purified h-TR beta 1 binds to 3,3',5-triiodo-L-thyronine (T3) with a Ka = 2.8 x 10(9) M-1. It binds to 3,3',5-triiodo-L-thyropropionic acid, 3,3',5-triiodo-L-thyroacetic acid, D-T3, L-thyroxine (T4), and 3',5',3-triiodo-L-thyronine with 475, 120, 39, 7, and 0.1%, respectively, of the activity of L-T3. This order of binding activity to T3 analogs is similar to that reported for the T3 nuclear receptor identified in tissues or cultured cells. Furthermore, the purified h-TR beta 1 binds to the T3 response element of the rat growth hormone gene. Thus, the purified h-TR beta 1 is active. To identify the hormone binding domain, the purified h-TR beta 1 was affinity labeled with underivatized [3',5'-125I]T4. A partial digestion by trypsin yielded a 125I-labeled 25-kDa fragment which was identified to be the domain Phe240-Asp456 by amino acid sequencing. Thus, the purified h-TR beta 1 appears suitable for other structural and functional studies.