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Endocrinology Vol. 144, No. 6 2297-2303
Copyright © 2003 by The Endocrine Society

Identification of 3,5-Diiodo-L-Thyronine-Binding Proteins in Rat Liver Cytosol by Photoaffinity Labeling

Maria Moreno, Elena Silvestri, Assunta Lombardi, Theo J. Visser, Fernando Goglia and Antonia Lanni

Dipartimento di Scienze Biologiche ed Ambientali (M.M., E.S., F.G.), Università degli Studi del Sannio, I-82100 Benevento, Italy; Dipartimento di Fisiologia Generale ed Ambientale (A.Lo.), Università degli Studi di Napoli "Federico II," I-80134 Naples, Italy; Dipartimento di Scienze della Vita (A.La.), Seconda Università degli Studi di Napoli, I-81100 Caserta, Italy; and Department of Internal Medicine (T.J.V.), Erasmus MC, 3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Professor Antonia Lanni, Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Via Vivaldi 43, 81100 Caserta, Italy. E-mail: antonia.lanni{at}unina2.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we obtained evidence for the presence of cytosolic-binding proteins for 3,5-diiodo-L-thyronine (3,5-T2). UV irradiation of rat liver cytosol with [125I]3,5-T2 resulted in specific covalent attachment of 125I to three polypeptides with apparent molecular masses of 86, 66, and 38 kDa. The photoaffinity labeling of all three proteins was strongly inhibited (by about 90%) when the reaction was carried out in the presence of a 10-fold excess of unlabeled 3,5-T2 or T3. However, whereas inhibition by 3,5-T2 was nicotinamide adenine dinucleotide phosphate reduced (NADPH) independent, T3 inhibited only in the presence of NADPH. The 38-kDa protein, which showed the greatest affinity for 3,5-T2, was partially purified by preparative fast-performance liquid chromatography. Its binding activity was optimal at pH 7.4, stable between 0 and 37 C, and already maximal after 5–10 min of incubation. The finding that a 38-kDa cytosolic-binding protein binds 3,5-T2 in the absence of NADPH, but T3 only in a NADPH-dependent manner, suggests that it may serve to regulate intracellular T3/3,5-T2 translocation in a way that depends on the nicotinamide adenine dinucleotide phosphate/NADPH ratio.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A MAJOR CHARACTERISTIC of the thyroid hormone T3 is the multiplicity of physiological processes it regulates at the cellular level. These include such diverse functions as fetal and neonatal development, growth and cell differentiation in many organisms, and energy meta-bolism in homeotherms. No single mechanism has been found to account for all of these diverse actions (for review, see Ref. 1).

Most of the effects exerted by thyroid hormone are nuclear mediated, being initiated through the interaction of T3 with highly specific nuclear thyroid hormone receptors, serving to regulate the expression of several genes (2, 3). However, some of the effects of T3 do not involve protein synthesis and are termed nonnuclear mediated. These have to be accounted for by alternative mechanisms. Extranuclear T3-binding sites might mediate such actions, and indeed they have been found in several compartments of the cell, including mitochondria, the plasma membrane, the cytoskeleton, and the cytoplasm (for review, see Ref. 4). These nonnuclear-mediated effects, unlike the nuclear-mediated ones, can be induced by iodothyronine analogs that show low or no activity toward nuclear thyroid hormone receptors; these include T4, rT3, 3,3'-diiodo-L-thyronine (3,3'-T2), and 3,5-diiodo-L-thyronine (3,5-T2). In the last few years, evidence has accumulated to suggest that among these iodothyronines 3,5-T2 mediates effects on energy metabolism by acting as a biologically active analog of T3 (5, 6, 7, 8). Laboratories other than ours have demonstrated metabolic effects of this diiodothyronine at the mitochondrial level in rats (9, 10), human cells (11), and goldfish (12). These effects are independent of protein synthesis and mediated by a rapid and direct interaction of 3,5-T2 with mitochondria (13, 14). In addition, we recently reported a direct effect of 3,5-T2 on rat liver cytosol in which it induced a rapid cycloheximide-independent stimulation of glucose-6-phosphate dehydrogenase activity (15). Even more recently we showed that when T3 is injected into euthyroid rats, not all the observed effects on resting metabolic rate are attributable to T3 itself because its in vivo deiodination to 3,5-T2 gives rise to nonnuclear-mediated (actinomycin D-independent) metabolic effects (16).

Because the effects of 3,5-T2 can be clearly demonstrated in vivo, even though experiments performed in vitro on isolated mitochondria have so far failed to show an effect, we postulated the presence of cytosolic binding sites for 3,5-T2. Much of the previous work has been performed on T3-binding activity, but in the present study, we sought evidence of the presence of 3,5-T2-binding proteins in rat liver cytosol as a prerequisite for any hypothesis invoking their involvement in mediating the effects of 3,5-T2 at the cellular level. Efforts to identify and characterize 3,5-T2-binding sites by conventional ligand-binding assays were hampered by the low specific radioactivity of the [125I]3,5-T2 and the presence apparently of multiple 3,5-T2 binding sites. We therefore performed direct photoaffinity-labeling of rat liver cytosol using underivatized [125I]3,5-T2. This covalently modified the binding sites within the proteins and provided direct evidence for 3,5-T2 binding. This method has proved useful before for identifying thyroid hormone-binding proteins (17), and it allowed us to examine the specificity of 3,5-T2 binding and its dependence on nicotinamide adenine dinucleotide phosphate reduced (NADPH).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
L-T3, L-3,5-T2, L-3,3'-T2, 3,5-diiodothyroacetic acid, L-3',5'-T2, and D-3,5-T2 were from MMDRI Henning (Berlin, Germany) and were more than 99.5% pure. [125I]3,5-T2 (specific radioactivity 50–80 mCi/mg) was synthesized by thermal iodine exchange at 120 C using N-acetyl-3,5-diiodo-4'-O-methyl-thyronine ethyl ester followed by acid hydrolysis and HPLC purification (Formula GmbH, Berlin, Germany). SDS-PAGE reagents were from Bio-Rad Laboratories, Inc. (Segrate-Milano, Italy). Molecular weight markers and Coomassie Brilliant Blue R-250 were from Sigma (St. Louis, MO).

Animals
Male Wistar rats (250–300 g) living in a temperature-controlled room at 28 C were kept one per cage under an artificial lighting regime of 12-h light/12-h darkness. A commercial mash (Charles Rivers, Lecco, Italy) was available ad libitum, and the animals also had free access to water. All experiments were performed in accordance with local and national guidelines on the care and use of laboratory animals.

Preparation of cytosol
Livers were homogenized on ice in four volumes (wt/vol) of ice-cold 20 mM HEPES buffer, pH 7.4, containing 0.32 M sucrose and 1 mM dithiothreitol [DTT (buffer A)] using a glass-Teflon Potter-Elvehjem homogenizer. The homogenate was centrifuged at 25,000 x g for 10 min at 4 C, and the resulting supernatant was centrifuged for 60 min at 100,000 x g at the same temperature. Aliquots of cytosol were stored at -80 C until further analysis. Protein levels were determined by the method of Hartree (18) with BSA as a standard.

Binding assays
Individual cytosol samples were analyzed separately. Aliquots of 20 µg protein were incubated for 60 min at 0–4 C with 10-7 M [125I]3,5-T2 in the absence or presence of a 1000-fold excess of unlabeled 3,5-T2 in a final volume of 0.1 ml of 0.32 M sucrose, 20 mM HEPES, and 1 mM MgCl2 (pH 7.4) containing 1 mM DTT (buffer B). To test the effect of NADPH, parallel incubations were carried out in the presence of NADPH (3 x 10-7 M). Protein-bound [125I]3,5-T2 was separated from free hormone by the Dowex method. Thus, the reaction was stopped by the addition of 0.15 ml of 10% AG-X8-Cl resin in buffer B, and the tubes were then vortexed (5 sec) and centrifuged (12,000 x g for 5 min at 4 C). The radioactivity in the supernatant represents protein-bound [125I]3,5-T2, whereas that present in the Dowex pellet represents the free hormone. The total binding of [125I]3,5-T2 was evaluated from the incubation with labeled 3,5-T2 only, whereas the nonspecific binding was determined from the incubation with [125I]3,5-T2 in the presence of excess unlabeled iodothyronine. The specific binding was derived by subtracting the nonspecific binding from the total 3,5-T2 binding. The radioactivity was counted in a 4000 {gamma} counter (Beckman Coulter, Fullerton, CA). The association constant (Ka) and maximum binding capacity (MBC) were calculated using the method of Scatchard (19).

Photoaffinity labeling
Aliquots of 1 mg cytosolic proteins were incubated for 30 min at 4 C in 0.2 ml buffer A, containing 1 x 10-8 M [125I]3,5-T2, in the absence or presence of a 10- to 1000-fold excess of unlabeled 3,5-T2 or T3, with or without NADPH. DTT (1 mM) was included as a free-radical scavenger. Samples were exposed to UV light from a 150-W mercury arc lamp for 5 min at 0 C at a distance of 4.5 cm form the center of the lamp. The irradiated proteins were precipitated with ice-cold ethanol, then resolved by SDS-PAGE (20), and visualized using Coomassie Brilliant Blue R250 staining. The apparent molecular weight of the 125I-labeled protein was calculated from the migration of standard proteins (high-molecular-weight markers, Sigma-Aldrich Corp., St. Louis, MO). The labeled proteins were detected by autoradiography after exposure to x-ray film (Fuji Photo Film Co., Ltd. Medical, Tokyo, Japan) at -80 C for 2–3 d. X-ray films were scanned, and the band intensities were quantified using NIH Image, version 1.60 (http://rsb.info.nih.gov/nih-image/).

Purification and characterization of cytosolic binding proteins by fast-performance liquid chromatography (FPLC)
Cytosol (1 mg/ml, final concentration) was applied to a Superdex 75 HR 10/30 column (Pharmacia, LKB Biotechnology, Uppsala, Sweden) equilibrated with buffer B, and the proteins were eluted with the same buffer using a flow rate of 1 ml/min. The column was calibrated using gel-filtration calibration kits to allow the estimation of protein molecular weights. The eluted fractions were tested for specific 3,5-T2-binding activity with [125I]3,5-T2 in the presence or absence of NADPH (3 x 10-7 M). Nonspecific binding was determined in the presence of 10-4 M 3,5-T2. The peak fractions were further analyzed for specific [125I]3,5-T2 binding as a function of the protein concentration, incubation time, incubation temperature, and pH. Ka and MBC values were determined by Scatchard analysis. For competition experiments, incubations were carried out using 10-8 M [125I]3,5-T2 and increasing concentrations of unlabeled analogs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
3,5-T2-binding characteristics of rat liver cytosol
During incubation, rat liver cytosol bound a significant proportion of the [125I]3,5-T2 (10-7 M) added to the incubation medium (50% on average). The addition of a 1000-fold excess of unlabeled 3,5-T2 caused a 50–60% displacement of bound [125I]3,5-T2. When NADPH (3 x 10-7 M) was present in the incubation mixture, the results were not different. Scatchard analysis of the binding of 10-7 to 10-6 M [125I]3,5-T2 to rat liver cytosol provided apparent Ka and MBC values of 0.8 x 106 M-1 and 360 pmol/mg cytosolic protein, respectively. However, the limited range of 3,5-T2 concentrations used (due to the low specific activity of [125I]3,5-T2) did not allow the detection of possible higher-affinity 3,5-T2-binding sites. Furthermore, the Scatchard plot was clearly nonlinear, suggesting the contribution of multiple 3,5-T2-binding sites. Therefore, we attempted to characterize these 3,5-T2-binding proteins by photoaffinity labeling of rat liver cytosol with underivatized [125I]3,5-T2.

Photolabeling of rat liver cytosol with [125I]3,5-T2
UV cross-linking of [125I]3,5-T2 to rat liver cytosol resulted in the predominant labeling of three protein bands with apparent molecular of 86 kDa (p86), 66 kDa (p66), and 38 kDa (p38), as determined by SDS-PAGE (Fig. 1Go). The covalent incorporation of 125I in p66 was higher than in the other two proteins. This could be due to differences in protein concentration, which were indeed revealed by Coomassie blue staining of cytosolic proteins (Fig. 2Go). Photoaffinity labeling by [125I]3,5-T2 was performed in the presence of 10–1000 times higher concentrations of unlabeled 3,5-T2 or T3. In the absence of NADPH, the photoaffinity labeling of the three bands already showed strong inhibition when the reaction was carried out in the presence of a 10-fold excess of unlabeled 3,5-T2 (Fig. 3AGo). Quantification of the data indicated that the inhibition of the labeling of the different proteins by 10-fold excess of unlabeled 3,5-T2 decreased in the following order: p38 (90%), greater than p86 (56%), p66 or more (53%). Increasing the excess of unlabeled 3,5-T2 to 100- or 1000-fold did not significantly further decrease the photoaffinity labeling of the different proteins. In the absence of added NADPH, the addition of unlabeled T3 had no significant effect at any concentration tested on the photoaffinity labeling of the different proteins by [125I]3,5-T2 (Fig. 3BGo).



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  		Figure 1. Identification by photoaffinity labeling of rat liver cytosolic 3,5-T2-binding proteins. Aliquots of 1 mg cytosolic proteins were incubated for 30 min at 0–4 C in 0.2 ml buffer A, containing 1 x 10-8 M [125I]3,5-T2, in the absence (lane 1) or presence of unlabeled 3,5-T2 at a concentration 10 (lane 2), 100 (lane 3), or 1000 (lane 4) times higher than that of [125I]3,5-T2. Aliquots were irradiated for 5 min at 0 C. The irradiated cytosolic proteins were precipitated with ice-cold ethanol, collected by centrifugation, electrophoresed on a sodium dodecyl sulfate-15% polyacrylamide gel, and visualized by Coomassie Brilliant Blue R250 staining. The gel was dried and exposed to x-ray film. A representative autoradiograph is shown.

 


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  		Figure 2. Coomassie staining of a representative gel containing the cytosolic proteins from the following conditions: lane 1, 1 x 10-8 M [125I]3,5-T2; lane 2, 1 x 10-8 M [125I]3,5-T2 in the presence of 3 x 10-7 M NADPH; lane 3, 1 x 10-8 M [125I]T3; lane 4, 1 x 10-8 M [125I]T3 in the presence of 3 x 10-7 M NADPH.

 


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  		Figure 3. Photoaffinity labeling of p86, p66, and p38 with [125I]3,5-T2 (1 x 10-8 M) in the absence (lane 1) and presence of unlabeled 3,5-T2 (A) or T3 (B) at a concentration 10, 100, or 1000 times higher than that of [125I]3,5-T2 (lanes 2–4). Incubations were done at 0–4 C. Representative autoradiographs are shown. The lower panels in A and B show quantification of the data, expressed as a percentage of the relative density obtained in the absence of unlabeled 3,5-T2 or T3 and presented as the mean ± SEM of triplicate determinations in each of three different cytosolic preparations.

 
To test for a possible influence of NADPH on 3,5-T2 binding and the competitive effect (if any) of T3, incubations were performed in the presence of 3 x 10-7 M NADPH, a concentration that has been reported to be associated with a maximal activation of T3 binding to cytosolic proteins (21). Comparison of Fig. 4AGo with Fig. 3AGo indicates that the binding of 3,5-T2 to the three proteins was not significantly influenced by the presence of NADPH. In contrast, our data suggest that NADPH is involved in T3 binding, as evidenced by the inhibitory effect of the unlabeled T3 on the photo labeling of [125I]3,5-T2 in the presence of NADPH (Fig. 4BGo). Quantification of these data indicated that the labeling of p38 was almost completely inhibited by T3, even at a concentration only 10 times higher than that of [125I]3,5-T2. At the same concentration, T3 inhibited the labeling of p86 and p66 by 52% and 76%, respectively. There was no further dose-dependent inhibition when 100- or 1000-fold excess of unlabeled T3 was used (Fig. 4BGo). The NADPH dependence of the T3 binding has also been shown in incubations in which rat liver cytosolic proteins were photoaffinity labeled by [125I]T3 (Fig. 5Go).



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  		Figure 4. Effect of NADPH on the photoaffinity labeling of p86, p66, and p38. Cytosol was incubated with [125I]3,5-T2 (1 x 10-8 M) without (lane 1) or with unlabeled 3,5-T2 (A) or T3 (B) at a concentration 10, 100, or 1000 times higher than that of [125I]3,5-T2 (lanes 2–4) in the presence of NADPH (3 x 10-7 M). Representative autoradiographs are shown. The lower panels in A and B show quantification of the data, expressed as a percentage of the relative density obtained in the absence of unlabeled 3,5-T2 or T3, and presented as the mean ± SEM of triplicate determinations in each of three different cytosolic preparations.

 


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  		Figure 5. Photoaffinity labeling of rat liver cytosol with [125I]T3 (1 x 10-8 M) in the absence (lane 1) and presence (lane 2) of NADPH (3 x 10-7 M). Representative autoradiographs are shown.

 
The NADPH dependence of the inhibition of 3,5-T2 binding by T3 was further evidenced by the results of incubations performed in the presence of increasing concentrations of NADPH. The inhibition of 3,5-T2 binding by T3 was found to be dependent on the concentration of NADPH (10-10 to 10-7 M), with apparently maximum inhibition at 10-7 M NADPH (data not shown).

Purification and characterization of the cytosolic binding proteins
The FPLC elution profile shown in Fig. 6Go contains three peaks of [125I]3,5-T2-specific binding activity corresponding to proteins with apparent molecular masses of 86, 66, and 38 kDa. Among these three partially purified proteins, p38 showed by far the highest specific binding. Determination of the [125I]T3-binding activity in each fraction confirmed that T3 is able to abolish 3,5-T2 binding only if NADPH is present in the incubation medium (data not shown). Figure 7Go shows the binding kinetics of the three partially purified proteins. Specific 3,5-T2 binding increased in a linear fashion with the amount of protein up to approximately 10 µg. However, there was no further increase at higher protein concentrations (Fig. 7AGo). We therefore chose to evaluate the other characteristics of this binding using a protein concentration of 10 µg. Figure 7BGo shows that maximum binding was reached within 5–10 min of the start of the incubation and it decreased strongly from 10–20 min. No significant differences in 3,5-T2 binding were detected over a range of incubation temperatures from 0 to 37 C, whereas at 60 C specific binding was effectively abolished (Fig. 7CGo). Specific [125I]3,5-T2 binding was maximal at pH 7.4, with decreases being evident at more acidic or basic values (Fig. 7DGo). Because the highest specific binding activity was shown by p38 (see Fig. 1Go), we chose it for further evaluation of specificity, capacity, and affinity values. The specificity of 3,5-T2 binding was evaluated by competition studies using 3,5-T2 analogs. Figure 8Go shows that the binding is specific for 3,5-T2 because none of the analogs tested (3,5-Diac, 3,5-T2, D-3,5-T2, 3,3'-T2, or T3) proved able to inhibit it in the presence of NADPH.



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  		Figure 6. Purification of cytosolic 3,5-T2-specific binding proteins from rat liver. The chromatography of the cytosol was performed using a Superdex 75 HR 10/30 column. One-milliliter fractions were collected, and the eluant was monitored for protein concentration. The molecular mass standards used were transferrin (81 kDa), BSA (66 kDa), ovalbumin (43 kDa), amylase c (29 kDa), and cytochrome c (12.4 kDa). The void volume (Vo) was determined using Dextran Blue 2000. The distribution coefficient (Kav) for a given protein was calculated from the elution volume (Ve) as follows: Kav = (Ve - Vo)/(Vt - Vo), where Vt is the total column volume. The [125I]3,5-T2-specific binding activity of the aliquots was measured as described in Materials and Methods. The figure shows a representative FPLC elution profile of rat liver cytosol (1 mg/ml, final concentration) containing three peaks of [125I]3,5-T2-specific binding activity. These correspond to protein fractions with estimated molecular masses of 86, 66, and 38 kDa.

 


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  		Figure 7. [125I]3,5-T2 binding to the partially purified proteins p86, p66, and p38. [125I]3,5-T2 (1 x 10-7 M) binding was measured in the presence of 10-4 M unlabeled 3,5-T2. The temperature of incubation was 0–4 C unless otherwise indicated. A, Binding to various protein concentrations during a 30-min incubation. B, Time course of [125I]3,5-T2 binding to 10 µg protein. C and D, Dependence on temperature (C) and pH (D) shown by [125I]3,5-T2 binding to 10 µg protein during a 10-min incubation. Specific binding of [125I]3,5-T2 is presented as mean ± SEM of triplicate determinations in each of three different cytosolic preparations.

 


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  		Figure 8. Effects of iodothyronine analogs on [125I]3,5-T2 binding to partially purified p38. The p38 (10 µg) was incubated with [125I]3,5-T2 10-8 M in the presence of various concentrations of unlabeled analogs, in each case for 10 min at 0–4 C. Each value indicates the mean of triplicate determinations in each of three independent experiments.

 
Notwithstanding the low specific activity of the [125I]3,5-T2, attempts were made to carry out saturation-binding analysis using partially purified p38. The standard assay conditions chosen for the saturation-binding experiments were 10 min of incubation at 0 C with 10 µg protein at pH 7.4. The apparent Ka value for 3,5-T2 binding (1 x 106 M-1) was only slightly higher than that obtained using whole cytosol. In contrast, MBC increased markedly to a value of 7.2 nmol/mg protein vs. 0.36 nmol/mg protein for whole cytosol. A representative Scatchard plot is shown in Fig. 9Go.



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  		Figure 9. Representative Scatchard plot of 3,5-T2 binding to p38. Incubations were performed with [125I]3,5-T2 concentrations within the range 10-8 to 10-7 M and 10 µg p38. Bound labeled 3,5-T2 was determined as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In view of the clear effects exerted by 3,5-T2 on energy metabolism when injected in vivo and the difficulty experienced in demonstrating such effects in vitro using isolated mitochondria, we felt it important to look for evidence both of the presence of 3,5-T2-binding proteins in the cytosol and a possible interaction between T3 and 3,5-T2 affecting such binding. This work reports the identification by photoaffinity labeling of three cytosolic proteins able specifically to bind 3,5-T2.

Cytosolic T3-binding proteins have been described in tissues obtained from various animal species: a 58-kDa monomeric protein was purified from rat kidney cytosol (22), and a protein with similar binding properties and molecular mass was purified from rat brain cytosol by Lennon (23). In addition, a T3-binding protein has been purified from rat liver cytosol as a dimer consisting of 38-kDa subunits (21), whereas a 36-kDa protein has been cloned from human kidney, which revealed identity to the amino acid sequence deduced from a human cDNA homologous to kangaroo µ-crystallin (24). Although they differ in tissue distribution and molecular mass, all of these proteins exhibit regulation of their T3-binding activity by NADPH in vitro. Other proteins with T3-binding activity have been identified. These are a 58-kDa protein in human cells (a subunit of pyruvate kinase subtype M2) whose activity is regulated by fructose 1,6-bisphosphate (25) and a 59-kDa protein in Xenopus liver cytosol (an aldehyde dehydrogenase) whose activity is dependent on -SH groups (26, 27).

Several functions have been suggested for these proteins: 1) a cytosolic reservoir of thyroid hormone, 2) a carrier involved in T3 translocation from cytoplasm to nucleus and/or mitochondrion, and 3) a modulator of nuclear receptor-mediated transcription (28, 29, 30). However, direct evidence in support of such roles is still lacking, and the physiological function of cytosolic T3-binding proteins (CTBPs) remains unclear. Cytosolic proteins with molecular masses from 26 to 70 kDa have been found to bind photoactivated T3 (17). In rat kidney, a 58-kDa protein with a very high affinity (Ka 2.4 x 109 M-1) for T3 has been characterized by Hashizume et al. (28). It can be activated by NADPH (22) or nicotinamide adenine dinucleotide phosphate (NADP) plus DTT (31). This NADPH-dependent CTBP stimulates T3 binding to mitochondria but inhibits T3 binding to isolated nuclei in vitro, suggesting that it may be involved in transferring T3 to mitochondria (32) but not to the nucleus (33). The same CTBP, when activated by NADP in the presence of DTT, accelerates the transport of T3 from cytosol to nuclear T3 receptors in vitro (31). These findings suggest that T3-CTBP complexes may regulate the transport of T3 to its nuclear receptor in a way that depends on the NADP/NADPH concentration ratio within the cell. Another NADPH-dependent CTBP, whose function is unknown, has been identified in rat liver (21). It differs from the 58-kDa CTBP in that it is not activated by NADP plus DTT and it displays different analog-binding specificity. Using CTBP-expressing Chinese hamster ovary or GH3 cells, Mori et al. (34) reported recently that CTBP regulates the T3-induced gene expression by increasing nuclear T3 content.

Unlike the cytosolic T3-binding proteins, the cytosolic 3,5-T2-binding proteins identified in this study each had an activity that was independent of NADPH. However, T3 proved able to inhibit the 3,5-T2 binding, provided NADPH was present. This indicates that the same protein may bind either T3 or 3,5-T2, depending on the redox state of the cell, and it suggests an important role for this protein in the in vivo regulation of the intracellular T3 and 3,5-T2 translocation. By photoaffinity labeling with [125I]3,5-T2, three putative binding proteins of about 86, 66, and 38 kDa were identified. Binding studies performed on proteins obtained by preparative FPLC showed that among these, p38 bound 3,5-T2 with by far the highest affinity. Although the Scatchard analysis performed on the purified protein failed to show an improvement in apparent Ka values, the MBC value (7.2 nmol/mg protein) is much higher than that estimated in whole cytosol (0.36 nmol/mg protein). Because rat liver cytosol contains multiple CTPBs, the ratio of these MBCs provides only a minimum estimate of the purification of p38, i.e. 20-fold. Assuming that one p38 molecule binds one iodothyronine molecule, the MBC value of 7.2 nmol/mg protein indicates that the p38 fraction is 28% pure. These results indicate that p38 represents a low-affinity, high-capacity 3,5-T2/T3-binding protein.

The finding that p38 is able to bind T3 in the presence of NADPH, but not in its absence, may suggest that depending on the energy status of the cell, this protein binds either T3 or 3,5-T2, and may then transport the bound iodothyronine to its destination, T3 into the nucleus and/or mitochondria and 3,5-T2 into the mitochondria. Once in the nucleus, T3 will activate the transcription of responsive genes, whereas in the mitochondria 3,5-T2 will increase respiratory chain activity. In support of the former is the evidence that 3,5-T2-specific binding sites have been demonstrated in isolated mitochondria (13) and suggested to be involved in the regulation of energy metabolism by 3,5-T2. Another possibility is that the low-affinity, high-capacity CTBPs could serve to sequester hormone in a NADPH/NADP-dependent manner, limiting its access to target sites such as the mitochondria and degrading enzymes.

As mentioned above, the effects of 3,5-T2 can be clearly demonstrated in vivo, but experiments using isolated mitochondria fail to show an effect. Indeed, the absence of CTBPs would prevent a 3,5-T2 effect if they are required to facilitate binding of this iodothyronine to mitochondrial sites or prevent it from degradation.

A function for these cytosolic molecules as a cytosolic reservoir cannot be excluded, and future studies should reveal whether 3,5-T2 mitochondrial signaling operates through these cytosolic-binding proteins.


   Footnotes
 
This work was supported by the Ministero dell’Università e delle Ricerche Scientifiche e Tecnologiche (MURST-COFIN 2000 Protocol MM05C48114).

Abbreviations: CTBP, Cytosolic T3-binding protein; 3,3'-T2, 3,3'-diiodo-L-thyronine; 3,5-T2, 3,5-diiodo-L-thyronine; DTT, dithiothreitol; FPLC, fast-performance liquid chromatography; Ka, association constant; MBC, maximum binding capacity; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate reduced.

Received December 19, 2002.

Accepted for publication February 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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