Biochem. J. (1984) 219, 1001-1007 1001Printed in Great Britain

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

Yvonne A. LEFEBVRE and Jaya T. VENKATRAMANDepartment of Medical Biochemistry, Faculty of Medicine, University of Calgary, 3330 Hospital Drive N. W.,

Calgary, Alberta, Canada T2N 4N1

(Received 18 October 1983/Accepted 30 January 1984)

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incuba-tion of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fractionresulted in specific binding of T3 to the membranes. Maximum specific bindingoccurred at 30°C after 2h incubation. Storage for 1 week at -80°C resulted in no lossof binding. Scatchard analysis revealed a class of binding sites with KD 86nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclearenvelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L-and D-thyronine did not compete for [1 251]T3 binding. Incubation of nuclearenvelopes with 0.6M-NaCl before addition of T3 resulted in the complete loss ofspecific binding sites, whereas exposure of the membranes to 2.0M-NaCl after incu-bation with T3 did not extract binding sites. Nuclear matrices, after incubation with[1 251]T3 under the same conditions, were shown to possess a class of binding sites witha similar KD but with approx. 30% of the maximum binding capacity. Nuclearenvelopes from hypothyroid animals may possess slightly lower numbers of bindingsites compared with nuclear envelopes from the intact animal, whereas nuclearmatrices from hypothyroid animals have the same number of binding sites as donuclear envelopes from the intact animal. In conclusion, nuclear envelopes andnuclear matrices have a class of binding sites with relatively high affinity for T3. It isdistinct from nuclear and cytosolic binding sites.

Much evidence suggests that thyroid hormones(Oppenheimer, 1979) must enter the nucleus beforeexerting their action. Clearly the hormones musttraverse the nuclear-envelope barrier duringtranslocation from the cytoplasm into the nucleus.It is possible that modulation of thyroid-hormoneaction occurs via interaction of the hormones withnuclear-envelope components during their passageacross the nuclear envelope. To investigate this, wehave characterized an L-T3 binding site on a male-rat liver nuclear-envelope preparation. In addi-tion, as some components of the nuclear envelopeare also present in nuclear matrices, we haveinvestigated whether the T3-binding site is alsopresent on nuclear matrices. We report here that aT3-binding site having similar affinity is presenton both nuclear envelopes and nuclear matrices.

Abbreviations used: T3, 3,3',5-tri-iodothyronine; rT3,3,3',5'-tri-iodothyronine; T4, thyroxine.

Materials and methods

MaterialsL-[1 251]T3 (3800MuCi/Mg) was purchased from

New England Nuclear (Montreal, Canada). T3,3,3',5-tri-iodothyroacetic acid, deoxyribonucleaseI, Triton X-100 and protease Type VI were pur-chased from Sigma Chemical Co. (St. Louis, MO,U.S.A.). rT3 was purchased from Henning-Berlin(Berlin, Germany). L-T4 and D-T4 were generouslygiven by Dr. N. Henderson (Department ofBiology, University of Calgary).

AnimalsMale Sprague-Dawley rats, weighing 200-300g

(average wt. 340g), were obtained from CharlesRiver Canada Inc. (Montreal, Canada) and main-tained on a diet of Wayne Lab Blox (Allied Mills,Chicago, IL, U.S.A.) and tap water ad libitum. Therats were killed by decapitation. The livers were

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Y. A. Lefebvre and J. T. Venkatraman

quickly removed, rinsed in 0.32M-sucrose contain-ing 3mM-MgCl2, stripped of connective tissue,weighed, and placed on ice.

In two experiments, athyreotic animals (maleSprague-Dawley rats, average wt. 310g, 16-18weeks old) were used. Surgical thyroidectomy wasperformed by the supplier. On receipt in thelaboratory, thyroidectomized animals were fed ona diet supplemented with calcium lactate (2%, w/v)for 2 weeks and then injected intraperitoneallywith lOOpCi of Na13 1. The animals were killed 6weeks after injection. The serum T3 concentrationof these rats was undetectable (less than2ng/100ml) as measured by radioimmunoassay.

Preparation of nuclear envelopesAll procedures were carried out at 4°C. Nuclei

were isolated by modifications of the procedure ofWidnell & Tata (1964). Tissue (50-60g) in 250mlof 0.32M-sucrose containing 3mM-MgCl2 washomogenized with a Polytron (Brinkman Instru-ments, Rexdale, Ont., Canada) at setting 4 for1 min. The homogenate was passed through fourlayers of cheesecloth. The filtrate was then dilutedto 1 litre and centrifuged at 4000g for 20min. Thepellet was resuspended in 2.4M-sucrose containing1mM-MgCl2 (adjusted to pH7.4 with NaHCO3),and this suspension was centrifuged at 50000g for60min. The resulting pellet of purified nuclei wasthen used to prepare nuclear envelopes by modi-fications of the procedure of Kay et al. (1972). Thenuclei were resuspended in 0.25M-sucrose contain-ing 1 mM-MgCl2 (adjusted to pH 7.4 withNaHCO3) and centrifuged at 750g for 5min. Thepellet was resuspended in digestion buffer (10mM-Tris/HCl, 0.30M-sucrose, 0.1 mM-MgCl2, 5mM-2-mercaptoethanol, pH8.5) to which freshly dis-solved deoxyribonuclease I was added to a concen-tration of 10jug/ml. The suspension was incubatedat room temperature for 20min. After centrifuga-tion at 12000g for 5min, the crude nuclearenvelopes were resuspended in 10mM-Tris/HCl,pH7.4, and 2ml was layered on a discontinuoussucrose gradient of 5ml of 0.25M-sucrose and 10mleach of 1.5M-, 1.8M- and 2.0M-sucrose (all sucrosesolutions made up in O0mM-Tris/HCl, pH 7.4). Thegradients were centrifuged at 100000g for 90min.The major band of purified nuclear envelope wasremoved from the gradients with a Pasteur pipetteand washed twice in 10mM-Tris/HCl, pH7.4, bycentrifugation at 33000g for 5min.

Preparation of nuclear matricesNuclear matrices were prepared by the method

of Barrack & Coffey (1980). Nuclei were sequen-tially extracted with Triton X-100 (1%, w/v),deoxyribonuclease (200pg/2mg of protein), low-Mg2+ buffer (0.25mM-MgCl2) and 2M-NaCl. The

insoluble residue remaining after these extractionswas the nuclear matrix.

Protein determinationsProtein content of subcellular fractions was

determined by the method of Lowry et al. (1951),with bovine serum albumin as standard.

Polyacrylamide-slab-gel electrophoresisNuclear-envelope and nuclear-matrix proteins

were analysed by sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis in 12%-acrylamidegels (Laemmli, 1970).

Assay of T3 binding to nuclear envelopes and nuclearmatrices

Stock solutions of T3 and analogues were madein 0.1 M-NaOH. Working solutions were made bydiluting 0.02ml of the stock solution in 9.98ml of20mM-Tris/HCl, pH7.4. NaOH was added to eachassay tube to a concentration of 40pM. [125I]T3(0.lml) was diluted to l0.0ml in 0.1M-NaOH andthen further diluted 3.33-fold in 20mM-Tris/HCl,pH7.4.

Nuclear envelopes or nuclear matrices isolatedfrom male-rat liver (5-20ug of protein/assay tube)were incubated in a final volume of 0.25ml of20mM-Tris/HCl, pH 7.4, containing [1251]T3(46500d.p.m./assay tube) for the times and at thetemperatures specified. Each sample was assayedin triplicate. To identical tubes 1O0M-T3 was alsoadded to determine non-specific binding. Thereaction was terminated by centrifugation in aBeckman Microfuge B and removal of the super-natant with a Pasteur pipette. The pellet waswashed twice by addition of 0.25ml of 20mM-Tris/HCl, pH 7.4, centrifuged again and the super-natant was removed. Radioactivity was assayed ina Tracor Analytic model 1185 Gamma System(counting efficiency 86%). Specific binding wasdefined as that binding obtained by subtracting theradioactivity in the tube containing excess un-labelled T3 (non-specific binding) from that tubecontaining only labelled T3 (total binding).

Studies of the effects of NaCl on T3 bindingwere done in two ways. In one series of experi-ments, nuclear envelopes (20ug of protein/assaytube) were preincubated with 0.3ml of 0.6M-NaClin 20mM-Tris/HCl, pH 7.4, for 30min at 20°C. Thetubes were then centrifuged and the resulting pelletwas washed once in 20mM-Tris/HCl, pH7.4. Asdescribed above, the pellet was then incubated inthe assay mixture to determine T3 binding to theextracted pellet. Control nuclear envelopes werepreincubated with 20mM-Tris/HCl, pH7.4, in-stead of NaCl. In another series of experiments,nuclear envelopes were incubated with T3 and thenthe nuclear envelopes obtained by centrifugation

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were washed. Then the pellet was incubated with0.3ml of NaCl at concentrations of 0.6M, 1.0M and2.OM in 20mM-Tris/HCl, pH7.4, for 30min at20°C. The nuclear envelopes were then pelletedand washed once again before being assayed forradioactivity. Control nuclear envelopes wereincubated with 20mM-Tris/HCl, pH 7.4, instead ofNaCl.

Results

Characterization of T3 binding to male-rat livernuclear envelopeThe yield of nuclear-envelope material was

1.90mg of protein/rat or 1.16mg of protein/g ofstarting tissue. The major band of purified nuclearenvelopes usually formed at the 1.5M-/1 .8M-sucrose interface, but occasionally was positioned atthe 1.8M-/2.0M-sucrose interface. Agutter & Gleed(1980) have reported that the density of sheep livernuclear-envelope preparations varies according tothe concentrations of nuclei during the lysis stage.This preparation of rat liver nuclear envelopes con-sists of relatively intact nuclear envelopes, whichretain the inner and outer membranes withmorphologically well-preserved nuclear-pore com-plexes (Kay et al., 1972). We obtained intact ghostsand some torn membrane sheets, as monitoredwith phase-contrast microscopy.We studied conditions for optimal binding of T3

to nuclear envelopes. Fig. 1 shows that this bindingwas temperature-dependent. Little or no specificbinding occurred at 4°C. At 20°C and 30°C very

1000

_ 800

F c; 600

c) E 400

c 0c.) o

co "- 200

0 1 5 30 60 75 90 105 120Time (min)

Fig. 1. Effect oftime and temperature on the binding of T3to male-rat liver nuclear envelopes

Nuclear envelopes (20ug of protein/assay tube)were incubated in triplicate with [1225I]T3 (0.07nM)and 0.5 nM-T3 in the presence and absence of 10#M-T3 at 4°C (0), 20°C (A), 30°C (-) and 37°C (0) for0-2h. Specific binding was calculated from thedifference in binding in the presence and absence oflOM-T3.

little specific binding occurred for the first 15 minof incubation. With increasing lengths of incuba-tion, increasing binding occurred until 1 h, atwhich a plateau was reached. At 30°C there was aslight decrease in binding thereafter to 2 h, whereasbinding remained constant at 20°C. Longer incu-bations, for 4h, 8 h and 24 h, at 30°C resulted in de-creasing binding of 20%, 35% and 43% respectively(results not shown). T3 binding at 37°C reached amaximum at 15 min and remained at that value to30min. Thereafter a rapid decrease in bindingoccurred, so that at 60min no specific binding wasdetectable. As the highest extents of T3 binding tonuclear envelopes occurred at 30°C after 2h incu-bation, these conditions were chosen for furtherexperiments. At each temperature, specific bind-ing represented approx. 25% of the total binding.

Fig. 2 demonstrates that, after 2h incubation at30°C, a linear relationship exists between T3 bind-

00.s._

0 10 20 30 40

Protein (,ug/assay tube)

Fig. 2. Effect of increasing concentrations of nuclear-envelope protein on the binding of T3 to male-rat liver

nuclear envelopesNuclear envelopes (5-40ug of protein/assay tube)were incubated in triplicate with [125I]T3 (0.07nM)and 0.5 nM-T3 in the presence (-) and absence (0)of IOM-T3 for 2h at 30°C. Specific binding (A) wascalculated from the difference in binding of [I 25I]T3in the presence and absence of unlabelled T3.

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Y. A. Lefebvre and J. T. Venkatraman

ing and membrane protein concentrations to40pg/assay tube. Fig. 2 also shows that there isconsiderable non-specific binding of T3 to themembrane preparation.The effect of storage of nuclear envelopes at

-80°C on the binding ofT3 was investigated. Stor-age at -80°C for 7 days did not result in decreasedT3 binding. After 2 weeks storage, however, T3binding to two nuclear-envelope preparations de-creased to 39.8% and 55.0% of the binding ob-served with the fresh preparation. For routinework, nuclear-envelope preparations were stored inliquid N2 and used within 1 week of preparation.

Fig. 3(a) shows the saturation plot of T3 bindingto nuclear envelopes from male-rat liver. Scatchard(1949) analysis of these data (Fig. 3b) showed thatthe KD for specific binding could be calculated as86 x 10-9M, with an apparent number of bindingsites of 18 x 10-9M or 223 pmol/mg of protein. Wehave not yet ruled out the existence of more thanone class of binding sites, especially in view of thefact that there is considerable non-specific bind-ing.

Specificity of T3 binding to the male-rat livernuclear-envelope preparation was examined bycompetition experiments in which analogues of T3were included in incubation mixtures. Fig. 4 showsthat L-T4 and D-T4 are not effective in inhibitingbinding of [1 251]T3. The concentration of un-labelled L-T3 required to inhibit the binding of[1 251]T3 by 50% was 72.5% the concentration of un-labelled tri-iodothyroacetic acid required to inhibitthe binding of [125I]T3 by 50%. Therefore un-labelled tri-iodothyroacetic acid was 72.5% aspotent as L-T3. On the other hand, rT3 was as effec-tive in inhibiting the binding of [125I]T3 as un-labelled L-T3. [1251]T3 binding at 0% inhibitionwas 2200fmol/mg of protein, and binding at 100%inhibition was 650fmol/mg of protein.Two kinds of experiments were performed to

investigate whether salt extraction of nuclearenvelopes caused any change in binding of T3 tonuclear envelopes. When nuclear envelopes werepreincubated with 0.6M-NaCl, 73% of the specificT3 binding to nuclear envelopes preincubated in20mM-Tris/HCl, pH7.4, was lost. On the otherhand, nuclear envelopes extracted with 0.6M-,1.0M- and 2.0M-NaCl after incubation with T3retained 98.5%, 97.9% and 100% respectively ofthe T3-binding sites.We have also characterized T3 binding to

nuclear matrices prepared from male-rat liver. Ratliver nuclear matrices were verified to be matricesby phase contrast microscopy. Fig. 5 shows peptideprofiles of nuclear envelopes and nuclear matricesprepared from male-rat liver. Although the matrixand the nuclear envelope consist of many proteins,and many dissimilarities exist between the two

60 f (a)

50

40

30-

H 0

20

10

0 100 200 300 400Free T3 (nM)

0.24 (b)

0.20

0.16

0Mr 0.12 0

0.08

0

0.04

0 4 8 12 16 20

T3 bound (nM)

Fig. 3. Saturation analysis of T3 binding to male-ratliver nuclear envelopes

Nuclear envelopes (20ug of protein/assay tube)were incubated in triplicate for 2h at 30°C with in-creasing amounts of T3 (0.1-300.OnM) and [125I]T3(0.07nM) with (-; to measure non-specific binding)and without (A; to measure total binding) 1OpM-un-labelled T3. Specific binding (0) was calculatedfrom the difference in retained radioactivity in theabsence and presence of unlabelled T3 (a) andplotted according to Scatchard (b). The line of bestfit was determined by linear-regression analyses,and the correlation coefficient was 0.86 (P> 0.005).

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peptide profiles, both preparations are primarilycomposed of three major polypeptides, ranging inMr from 60000 to 68000. Table 1 shows thatScatchard analysis of binding to nuclear matricesreveals a class of binding sites having a KD of118 x 10-9M and a capacity of 5 x 10-9M or65 pmol/mg of protein. The affinity of T3 fornuclear matrices is similar to the affinity of T3 fornuclear envelopes. However, the number of bind-ing sites is approx. 70% less.We also investigated binding of T3 to nuclear

envelopes and nuclear matrices from thyroid-ectomized animals. Scatchard analysis (Table 1)revealed that the affinity of T3 for these prepara-

tions was similar to affinities of fractions preparedfrom intact animals. The number of binding siteson nuclear envelopes, however, may be slightly de-creased in the thyroidectomized animal comparedwith the intact animal.

68 kDa-

0

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30

g 40

50Q 60

70

80

90

100

45kDa-

24kDa-

18.4kDa -

14.3kDa-

0 0.5 5.0 50.0 500 5000 10000Hormone concn. (nM)

Fig. 4. Effect ofunlabelled L-T3, L-T4, D-T4, tri-iodothyro-acetic acid and rT3 on the specific binding of L-[1251]T3Nuclear envelopes (20ug of protein/assay tube)were incubated for 2h at 300C with 0.5nM-T3 and[125I]T3 (0.07nM) with unlabelled L-T3 (O), L-T4(L), D-T4 (0), tri-iodothyroacetic acid (0) or rT3(A) at concentrations of 0.5, 5.0, 50.0, 500, 5000 andlOOOOnM. Two triplicate experiments were per-formed. The values represent the means.

Fig. 5. Electrophoretic analysis of nuclear-envelope andnuclear-matrix peptides

A sample (200pug of protein) of each preparationwas applied to the polyacrylamide gel and, afterseparation of peptides, gels were stained with Coo-massie Blue. Lanes: a, M, markers; b, nuclear-envelope protein; c, nuclear-matrix protein.

Table 1. Scatchard analyses ofT3 binding to nuclear-envelope and nuclear-matrix preparations obtainedfrom intact male ratsand thyroidectomized rats

Scatchard analyses were performed as described in the legend to Fig. 3. Values are the means of triplicates from twopreparations. The values in parentheses are the results obtained in each experiment.

PreparationNuclear envelopes from

intact malethyroidectomized male

Nuclear matrices fromintact malethyroidectomized male

KD(nM)

86 (73,99)90 (105, 75)

118 (99, 137)140 (100, 180)

No. of binding sites(pmol/mg of protein)

223 (225, 221)133 (96, 170)

65 (85, 45)72 (60, 82)

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Discussion

Present evidence strongly suggests that thyroidhormone action is initiated by the binding of L-T3to non-histone chromatin receptors (Oppen-heimer, 1979; Schwartz & Oppenheimer, 1978)within the nucleus. To date, no one has investi-gated the mechanism by which the hormones tra-verse the two membranes comprising the nuclearenvelope on their way to the chromatin receptors.It is possible that T3, a small and relatively lipo-philic molecule, enters the nucleus via passivediffusion, perhaps via the nuclear-pore complexes.It is equally possible that T3 entry into the nucleusis by a controlled passage across the nuclearenvelope. Thyroid hormone action may be modu-lated by such a transport process. The first step to-ward identifying such a process is identification ofa binding component for T3 on nuclear envelopesfrom a T3-target tissue. We have therefore isolatedintact nuclear envelopes from male-rat liver andhave characterized T3 binding to the membranes.The binding experiments involved incubation in

vitro of the isolated nuclear-envelope fraction withlabelled hormone and were not designed todistinguish between binding of the labelled T3 tomembrane sites previously occupied by endo-genous hormone and binding to unoccupied sites.However, at 0°C we were unable to measure anyspecific binding, which, we conclude, shows eitherthat the binding process requires energy or thatdissociation of endogenous ligand is required.Silva et al. (1977) have shown that 40% of themeasured binding sites in extracts from rat livernuclei are occupied. Since the KD of the T3-bindingsite from the unthyroidectomized animals was notdifferent, however, the binding process probablyrequires energy and is not a reflection of endo-genously bound hormone.

Scatchard analysis of the binding of T3 tonuclear envelopes from livers of intact ratsrevealed a class of binding sites having a KD of86 x 10-9M. The dissociation constant of T3 bind-ing to isolated nuclei as well as to chromatin recep-tors is reported to be 0.1 X 10-10M-2x 10-9M(Schwartz & Oppenheimer, 1978; DeGroot &Torresani, 1975; Surks et al., 1975). A cytoplasmicbinding component has also been identified whichin the rat liver has a KD of approx. 10- 7M (Dillmanet al., 1974; Davis et al., 1974; Visser et al., 1976).We conclude therefore that the nuclear-envelopebinding site characterized in the present studiesdiffers in its affinity from both the nuclear and thecytosolic binding sites.The maximum binding capacity of the nuclear

envelope for T3 is 223 pmol/mg of protein, or 24ngof T3 for nuclear envelopes from 1g of liver.Reported values for the maximum binding capa-city of nuclei are approx. 10-fold less (DeGroot &

Torresani, 1975). Although previous estimates ofnuclear binding capacities have been obtainedwith nuclei which have been treated with non-ionic detergents, and such treatment removes theouter nuclear membrane (Aaronson & Blobel,1975), perhaps a rich source of T3-binding sites,the nuclear-envelope site is probably a higher-capacity lower-affinity site than are previouslyreported nuclear sites.

Specificity studies showed that the most effec-tive competitors for binding of [125I]T3 to thenuclear envelopes were T3 itself and rT3. Tri-iodo-thyroacetic acid was a less effective competitor,and L-T4 and D-T4 did not compete at all. Theorder of potency of the T3 analogues correlateswith that demonstrated for the binding of T3 tonuclei and chromatin receptors, except that rT3 is aless effective competitor in nuclei and chromatinthan is tri-iodothyroacetic acid (Schwartz &Oppenheimer, 1978; Smith et al., 1980). Tri-iodo-thyroacetic acid is a more effective competitor ofT3 binding to nuclear envelopes than it is in thecytosol binding system (Smith et al., 1980).We have also identified a binding site with a KD

similar to that of nuclear envelopes on nuclearmatrices, but with approx. 30% of the binding sitescompared with the nuclear-envelope site. Thesematrices were prepared after high-salt extraction.Our studies of NaCl extraction of nuclear enve-lopes showed that NaCl extracted most of thebinding sites, and perhaps this explains the de-creased number of sites in the matrix. However,once T3 has bound to the sites, they are protectedfrom salt extraction and then even high concentra-tions of salt cannot remove the occupied site.Wilson et al. (1982) have described specific T3-binding sites on rat liver nuclear matrices with aKD of 1.3nm. The T3-binding site reported here isof lower affinity and appears to be present on bothnuclear envelopes and nuclear matrices.Oppenheimer et al. (1975) reported that the T3-

binding capacity of hepatic nuclei is the same ineuthyroid and thyroidectomized animals. Ourstudies suggest that, although the affinity of thenuclear-envelope T3-binding site in the thyroid-ectomized animal is similar, the number of bindingsites is slightly decreased. On the other hand,nuclear matrices prepared from thyroidectomizedanimals did not display a lower binding capacity.

In summary, we have identified in the rat liver anuclear-envelope binding site which differs frompreviously described nuclear and cytosolic bindingsites in its affinity, binding capacity and specifi-city. The site is also present on nuclear matrices.The physiological role of this binding site remainsto be elucidated.

This work was supported by the Medical ResearchCouncil of Canada and by the National Cancer Institute

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Tri-iodothyronine, nuclear envelopes and nuclear matrices 1007

of Canada. Y. A. L. is a Scholar of the Alberta HeritageFoundation for Medical Research. J. T. V. is a Post-Doc-toral Fellow of the A.H.F.M.R. We gratefully acknowl-edge the advice of Dr. N. E. Henderson, Dr. H. J. Gorenand Mr. Tim Magnus and the help of Dr. L. J. Haydenwith the thyroidectomized animals. We thank Ms. J.Gayford for typing the manuscript.

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