Subcellular localization and binding of 125I-triiodothyronine in calf thyroid

The subcellular distribution of 125I-T3 was studied in calf thyroid slices, under the same experimental conditions where T3 inhibits protein and RNA synthesis, labelled hormone was found mainly in the 20,000 X g supernatant. The specificity of each subcellular localization was determined by incubating the slices with 10(-5)M T3. Only in the purified nuclei a significant decrease was found, indicating a specific localization of the labelled hormone. When slices were incubated with 125I both labelled T3 and T4 were found in purified nuclei, indicating that endogenously synthesized hormones can reach thyroid nuclei. Purified thyroid nuclei were incubated with labelled T3 and increasing amounts of cold hormone. Specific binding reached a plateau after 90 min of incubation at 20 degrees C. When the displacement curves were analysed by a Scatchard plot a binding site with a Ka of 5.2 X 10(7) M-1 and a capacity of 3.0 X 10(-15) moles/microgram DNA was observed. Digestion of nuclei with trypsin and protease abolished completely the binding of 125I-T3 thus indicating the protein nature of the receptor. The hormone-receptor complex could be extracted with 0.4M KCI and eluted in the void volume after Sephadex G-25 column chromatography, similar to peripheral tissues nuclear T3 receptors. The present studies provide the first evidence for the existence of nuclear receptors for T3 in the thyroid, an event probably related to the autoregulatory mechanism.     

High-affinity binding of thyroid hormones to neuroblastoma plasma membranes

The binding of thyroid hormones to isolated plasma membranes was studied in NB41A3 neuroblasts. Saturable binding of L-T3, D-T3 and L-T4 was observed. Binding was time-dependent, with equilibrium reached in less than 60 min and maximal binding occurring between pH 7.4 and 7. Saturation experiments demonstrated two classes of sites for L-T3: a high-affinity site with Ka 8.4 X 10(9) M-1 and a low-affinity site with Ka 7.3 X 10(6) M-1.L-T3 and D-T3 inhibited each other's binding, L-T3 being several-times more potent. Affinity labeling of isolated membranes with bromoacetylated thyroid hormones disclosed stereospecific binding to SDS-PAGE bands with approximate molecular masses of 27 kDa (preferentially labeled by BrAc-L-T3), 32 kDa (preferentially labeled by BrAc-D-T3), and 48 and 87 kDa (preferentially labeled by BrAc-L-T4). Binding of BrAc-L-T3 to the 27 kDa band accounted for 3.4% of total binding, was selectively inhibited by excess L-T3, and may be involved in intracellular transport of L-T3.     

Triiodothyronine receptor sites in serum-free cultured hepatocytes from adult rat liver

Nuclear T3 specific binding sites were characterized by Scatchard analyses of L-125I-T3 binding to nuclei extracted from freshly isolated and 1, 2 and 6 day-cultured hepatocytes. The results demonstrate a marked decrease in T3 binding capacity of nuclei extracted from 1 day-cultured cells followed by an almost complete recovery within 6 days. The affinity constant value of nuclear receptor sites is significantly decreased in 1 day-cultured cells with a subsequent partial recovery. The affinity and capacity pattern of nuclear T3 binding sites appears to be in line with the delayed responses of hepatocyte primary cultures to T3.     

Decrease in triiodothyronine binding sites in chick embryo erythrocytes during early development

Specific thyroid hormone (TH) binding sites have been detected in nuclei of erythrocytes obtained from developing chick embryos. The binding characteristics and relative affinities for TH analogs were those expected of TH receptors. Nuclear triiodothyronine (T3) saturation analysis was carried out in vitro by incubating intact erythrocytes in M199 medium with 3-200 pM [125I]T3 for 1 hr at 37 degrees C or 20-24 hr at 21 degrees C. Nuclei were obtained by centrifugation after lysing the erythrocytes in a stabilizing buffer containing 0.3% saponin, followed by addition of Triton X-100 (final concentration 0.2%) to minimize the nonspecific binding. Scatchard analysis of equilibrium binding data suggested that the nuclei possess a single class of binding sites. The binding is reversible and the rate of dissociation is temperature dependent. T3 and T4 appear to bind to the same sites, but the affinity of T3 was 16 times greater. Among TH analogs tested, Triac had the highest affinity followed by L-T3, D-T3, Tetrac, L-T4, D-T4, T2, and rT3. Serial studies performed on different days of chick embryogenesis demonstrated a rapid and significant decrease of the erythrocyte nuclear T3 receptor. On Day 5, the number of T3 binding sites was maximal at 1600 +/- 100 per nucleus. The number declined steadily until, by Day 20, it had reached about 60 +/- 10 sites/nucleus. RBC from adult and baby chickens had less than 1% as many binding sites as those from Day 5 embryos. There was no significant change in the affinity of the sites (Kd approximately equal to 20 pM at 37 degrees C). The reason for the loss of T3 binding sites during embryogenesis is not known. Since the plasma level of the TH increases during embryogenesis, this may reflect down regulation. Another possibility is that the change in erythrocyte population which occurs during this period involves production of erythrocytes which contain fewer T3 binding sites.     

Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.     

Studies on the nuclear 3, 5, 3'-triiodo-L-thyronine binding sites in cytotrophoblast

Human placental trophoblasts contain 3, 5, 3'-triiodo-L-thyronine (T3) nuclear receptors not only at term but all throughout pregnancy. We determined which of the trophoblast, cytotrophoblast (C-cell) or syncytiotrophoblasts (S-cell) respond to thyroid hormone, and whether the T3 binding capacity changes with placental aging. Nuclear protein of mononuclear cells purified from term chorionic tissue by enzymatic digestion and Percoll gradient centrifugation had an apparent association constant (Ka) of 5.2 x 10(9)M-1 and a binding capacity of 445 fmol T3/mg DNA, 8 times greater than that of term trophoblasts. Primary harvested mononuclear cells reacted against neither anti-hCG-beta nor anti-hPL antibodies, although some of them reacted immunocytochemically against anti-hCG-alpha antibody. These cells aggregated with each other and transformed into multinuclear cells in culture after 96 hrs of incubation, showing that these primary harvested cells were C cells that had morphologically transformed into S cells. The transformed cells secreted hCG and hPL and immunocytochemically stained for these markers, suggesting that the C cells had functionally transformed into S cells. Nuclear binding of T3 in trophoblastic tissue is present not only at term but throughout pregnancy. Although each nuclei had a similar Ka value, the binding capacity decreased towards term. These findings suggest that nuclear T3 receptors of placental trophoblast change with placental aging and this change is mainly due to the change in the C/S cell ratio. We concluded that the cytotrophoblast is an active target cell of thyroid hormone.     

Thyroid hormone receptors in human cultured fibroblasts: evidence for cellular T4 transport and nuclear T3 binding

In cultured normal human skin fibroblasts specific and saturable binding sites for triiodothyronine (T3) have been revealed. In fact radiolabelled T3 binds rapidly to intact cells with maximum uptake after 1 hour, while nuclear binding is delayed, the equilibrium being reached after 2 hours. In intact cells it is possible to identify a single binding site for 125I-T3, with a Ka = 1.8 X 10(10)M-1 and Ro = 1.25 X 10(-11)M, similarly in nuclei it was possible to identify a single binding site of Ka = 8.8 X 10(9)M-1 and Ro = 2.3 X 10(-11)M. Intact human fibroblasts take up thyroxine (T4) even more rapidly than T3, with maximum after 5 min, showing a lower affinity for T4 than for T3 and a negligible specific and saturable binding sites for T4, the presence of a cellular transport system for T4 may be hypothesized, considering that iodothyronine cellular binding is increased by preincubation with low doses of T4.     

An essential role of domain D in the hormone-binding activity of human beta 1 thyroid hormone nuclear receptor

By analogy with steroid receptors, human placental thyroid hormone nuclear receptor (hTR beta 1) could be divided into four functional domains: A/B (Met1-Leu101), C (Cys102-Ala170), D (Thr171-Lys237), and E (Arg238-Asp456). The E domain was thought to bind thyroid hormone. To evaluate whether domain E alone is sufficient to bind T3 or requires the presence of other domains for functional T3-binding activity, a series of deletion mutants was constructed. The mutants were expressed in Escherichia coli, and the expressed proteins were purified. Analysis of the T3-binding affinity and analog specificity of the purified truncated hTR beta 1 indicated that domain E alone did not have T3-binding activity. Extension of the amino-terminal sequence of domain E to include part of domain D yielded a mutant (Lys201-Asp456) with a Ka for T3 of 0.5 +/- 0.2 x 10(9) M-1. Further extension to include the entire domain D (Met169-Asp456) yielded a mutant with T3-binding activity with a Ka of 0.8 +/- 0.1 x 10(9) M-1. Further extension of the amino-terminal sequence to include domain C increased the affinity for T3 by nearly 2-fold (Ka = 1.5 +/- 0.4 x 10(9) M-1). The Ka for the wild-type hTR beta 1 is 1.5 +/- 0.2 x 10(9) M-1. Furthermore, mutant (Met169-Asp456) binds to 3',5',3-triiodo-L-thyropropionic acid, D-T3, L-T4, and L-T3 with 307%, 37%, 7%, and 0.1%, respectively, of the activity of L-T3. This order of analog affinity is similar to that of the wild-type hTR beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific receptors for triiodothyronine in nuclei isolated from normal human polynuclear neutrophils

In normal subjects, nuclei isolated and purified from circulating granulocytes bound 125I-T3. Binding was reversible and inhibited by unlabelled hormone. Scatchard plots showed a single class of high affinity sites (Kd: approximately 1,5 nM) with a high maximal binding capacity (MBC: approximately 400 fmol of T3 bound/100 micrograms of DNA). Structural analogs partially competed with 125I-T3 binding. These data suggest that human normal polymorphonuclear neutrophils possess specific nuclear receptors for triiodothyronine.     

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)     

High blood triiodothyronine and the euthyroid state

A high excess of circulating T3 was observed in an euthyroid woman. Agarose gel electrophoresis of serum preincubated with 125I-T3 revealed an abnormal T3-binding in gamma-globulin zone. This binding interfered with the hormone radioimmunoassay. Immunological characterization identified this protein as an IgG-K and IgG-lambda polyclonal antibody that bound T3 but not T4. Scatchard analysis of 125I-T3 binding to the gamma-globulin fraction isolated showed a single class of binding sites with a high affinity Ka = 0.4 X 10(9) L/M and maximal binding capacity of 5.2 X 10(-9) M.     

Saturable binding of thyroid hormone to isolated rat hepatocytes

The binding of [125I]triiodothyronine (T3) to freshly prepared rat hepatocytes was studied at 0 degrees C. The abundant non-saturable binding could be suppressed by washing the cells with alkaline buffer, pH 10.5 at 0 degrees C, without loss of cell viability, thus allowing detection of saturable binding. Three classes of binding sites were identified from analysis of the saturable T3 binding in the presence and absence of bromosulfophthalein (BSP). One of these classes was inhibited by BSP. The T3 dissociation constants were 3.5, 35 and 115 nM and the number of sites was respectively 0.9, 20 and 36 X 10(6) sites/cell. L-T3 had a 10-times higher affinity than D-T3 and a 50-times higher affinity than triiodothyroacetic acid. Saturable T3 binding was associated with plasma membrane-containing subcellular fractions. These binding sites may be related to those previously described in isolated plasma membranes from rat liver and could be involved in the entry of T3 into the hepatocyte.     

Purification and characterization of NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine binding protein in rat kidney

The NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine(T3)-binding protein (CTBP) has been purified over 30,000-fold from rat kidney by using charcoal extraction, Mono Q-Sepharose, Blue Sepharose CL-6B, and Sephacryl S-200 column chromatography. Purified CTBP had a sedimentation coefficient of 4.7 S, Stokes radius of 32.5A, and calculated molecular weight of 58,000. The apparently homogeneous protein consisted of a single polypeptide chain with Mr of 58,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Scatchard analysis of T3 binding showed that NADPH increases maximal binding capacity without changes in the affinity constant (Ka = 2.43 X 10(9) M-1). Double reciprocal analysis of NADPH and binding capacity gave maximal binding capacity of 16,400 pmol/mg of CTBP, Mr = 58,000. The order of affinity of iodothyronine analogues to purified CTBP was as follows: L-T3 = D-T3 greater than triiodothyroacetic acid greater than L-thyroxine. [125I]T3 bound to purified CTBP spontaneously dissociated from CTBP at 20 degrees C (t 1/2 = 22 min) in the absence of NADPH, whereas the dissociation was not observed in the presence of NADPH. The optimal pH for T3 binding was 7.2-7.5 Na+, K+, Ca2+, and Mg2+ (0-200 mM) did not influence T3 binding to CTBP. The purified CTBP did not bind to DNA and was not adsorbed to concanavalin A-Sepharose.     

Phaseolus vulgaris isolectin binding to human erythrocytes.

The Phaseolus vulgaris isolectins L4,L3E1, L2E2, L1E3, and E4 were isolated by affinity and ion exchange chromatography. Pure isolectins were radiolabeled by the chloramine-T method with Na125IO4 and their binding to human erythrocytes was studied. A normal erythrocyte has approximately 8 times 10(5) receptor sites for each isolectin; however, the association constants (Ka) of binding increased from 1.1 times 10(7) M-1 to 3.8 times 10(8) M-1, with increasing number of E subunits per tetrameric isolectin molecule. Isolectin to erythrocyte binding reached equilibrium rapidly and was reversed by fetuin. All isolectins competed with 125I-E4 for erythrocyte binding sites, with a constant (KI) similar to the Ka calculated for each respective radiolabeled isolectin. When isolectin binding at 0 degrees C, 4 degrees C, or 8 degrees C was compared to that at 25 degrees C, there was no reduction in the number of binding sites per cell, but the Ka of E4 was reduced to 3 times 10(7) M-1. Fixed erythrocytes displayed similar isolectin binding characteristics.     

Thioredoxin and glutaredoxin enhance the binding of L-triiodothyronine to its hepatic nuclear receptors

The influence of thioredoxin and glutaredoxin on binding of L-triiodothyronine (T3) to the rat hepatic nuclear T3 receptor was compared with that of the exogenous activator dithiothreitol. Specific [125I]T3 binding, the affinity constant, Ka, and the maximal binding capacity, MBC, were measured using whole nuclei, solubilized preparations of receptor, and chromatographed nuclear receptor. Both the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) and the glutaredoxin system (glutaredoxin, glutathione reductase, glutathione, and NADPH) increased specific binding of T3 to nuclei, solubilized receptor, and chromatographed receptor significantly. Compared with the values obtained in the absence of added thiol (Ka = 1.6 +/- 0.1 x 10(9) M-1, MBC = 1.7 +/- 0.06 pM), the thioredoxin and glutaredoxin systems increased Ka by 147 and 112%, respectively, while decreasing MBC by 51 and 45%, respectively, when chromatographed receptor was used. The same tendency was observed with solubilized receptor. However, dithiothreitol increased Ka without affecting MBC when solubilized receptor was used. These results, the first demonstration of endogenous disulphide reductant systems enhancing binding of T3 to its receptor, suggest that the thioredoxin and (or) glutaredoxin systems may modulate the physiological effects of thyroid hormone.     

Multiple thyroid hormone binding sites on rat liver nuclear envelopes

Nuclear envelopes relatively free of plasma membrane contamination were isolated from the male rat liver. Equilibrium binding of T3 to nuclear envelopes occurred after incubation for 3 h at 20 degrees C. Scatchard analysis revealed two classes of binding sites; a high affinity site having a KD of 1.8 nM with a maximum binding capacity of 14.5 pmol/mg protein and a low affinity site having a KD of 152.1 nM with a maximum binding capacity of 346.8 pmol/mg protein. No degradation of the radioligand occurred during incubation with the nuclear envelope. T4, rT3 and Triac competed effectively for the binding of T3 to the high affinity site whereas only T4 competed well for binding to the lower affinity site. The binding site was protease sensitive but not salt extractable. Multiple T3 binding sites having similar affinities have been reported on plasma membranes. An intriguing possibility is that membrane binding sites may be involved in translocation of thyroid hormone across membrane barriers.     

Multiple thyroid hormone binding sites on male rat liver nuclear matrices

Equilibrium binding of T3 to nuclear matrices isolated from male rat liver occurred after incubation for 3h at 20 degrees C. Two binding sites, having KD's of 6 and 95 nM, were revealed by Scatchard analysis. T3 and Triac competed for the binding of [125I]T3 to the high affinity site whereas only T3 competed for binding to the lower affinity site. Reverse T3 (rT3) did not compete for the binding of T3 to either class of binding sites. The binding sites were highly DNAse-sensitive, and less sensitive to protease treatment. The effect of binding of T3 to nuclear matrices by ATP, DTT and EDTA indicated that the sites are dissimilar to previously identified cytosolic binding sites. The higher affinity site resembles the T3 receptor in affinity and thyroid hormone specificity. The second site represents a new class of thyroid hormone binding sites. Its role in the regulation of thyroid hormone action warrants further investigation.     

Effects of D- and L-thyroxine on the glucocorticoid binding capacity of adult rat liver

Administration of either D- or L-thyroxine (T4) significantly increased the glucocorticoid binding capacity of cytosol of the livers of adrenalectomized adult rats. Administration of up to 0.5 mg/100 g body wt. of L-T4 was more effective than that of D-T4, but higher doses (0.8-3 mg/100 g body wt.) of D-T4 increased the binding capacity markedly to more than that with L-T4. T4- administration did not alter the apparent dissociation constant of glucocorticoid binding proteins for glucocorticoid binding, or their behavior on DEAE-cellulose chromatography either before or after thermal activation (23 degrees C for 40 min). Thus the increased binding capacity seemed to be due to increase in the level of glucocorticoid receptor in rat liver.     

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.     

An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit.

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).