Nontransformed rabbit liver glucocorticoid receptor: purification, characterization and transformation

BIOCHIMIE, 1985, 67, 1267-1278

Nontransformed rabbit liver glucocorticoid receptor" purification, characterization and transformation. Patrick L U S T E N B E R G E R °, Philippe B L A N C H A R D I E , Marc D E N I S , Pierre F O R M S T E C H E R * , Jean-Luc O R S O N N E A U and Serge B E R N A R D .

Laboratoire de Biochimie Mddicale, UER de Mddechle, 1, rue G. Veil, 44035 Nantes Cedex. * Laboratoire de Biochimie Structurale, UER de Mddech+e, 1 place de Verdun, 59045 Lille Cedex. (Regu le 8-7-1985, accept~ le 6-9-1985).

R~sum~ - - La fonne non transform~e du rdcepteur h gh+cocortico?des d,t foie de laphl a dtd purifide environ 8 O00 fois grtice ~ u n protocole en trois drapes. La premiOre dtape a dtd rdalisde par prdcipitation au sulfate de protamhle permettant une purification de 5-6 fois avec un rendement de 85 %. La seconde dtape par chromatographie d'affinitd sur N-(12-dodecyl-amhlo) 9 a-fluoro-16 a-mdthyl-ll fl, 17 ct-dihydroxy-3-oxo-1,4-androsta-diOne-17 fl-carboxamide Sepharose a apportd un facteur de purification de i'ordre de 1 500 gt 2 O00fois. L'dtape finale mettant en oeuvre la chromatographie d'exclusion haute performance aboutit h u n facteur de purification voisin de 8 O00fois. La fraction obtenue s'est rdvdlde pure h 60 %. Le rdcepteur purifid sous forme n o n transformde possdde un coefficient de sddimentation de 9 S en gradient de sucrose 5-20 % prdpard en tampon phosphate O,16 M, un rayon de Stokes de 6,1-6,3 nm en gel filtration conventionnelle et haute performance. La spdcificitd de liaison du rdcepteur purifid est identique celle ddtermhlde sur des prdparations cytosoliques. La chromatographie sur dchangeurs d'ions et l'isofocalisation ont montrd que la purification affecte la charge globale de la protdine. Ce phdnom~ne pourrait dtre d:t h des interactions entre le rdcepteur et des facteurs cytosoliques. L'dlectrophor~se sur gel de polyacrylamide, en conditions ddnaturantes, a montrd une bande majeure M~ = 94 000 en parfait accord avec les valeurs prdcddemment ddcrites pour d'autres rdcepteurs h glucocorticoMes. AprOs dlimination du molybdate puis exposition h 25°C en prdsence de 0,4 M KCI, le rdcepteur purifid reste transformable. La caractdrisation des formes moldculaires transformde et n o n transformde a dtd rdalisde gr6ce gtla mesure de la liaison :t des noyaux isolds, gt l'affinitd vis-gt-vis des dchangeurs anioniques et par HPLC. Les rdsultats montrent que, clans ces conditions, 40 % du rdcepteur purifid est transformd. Mots-cl~s : r~cepteur/! glucocorficoides / purification / transformation.

Summary - - The molybdate-stabilized nontransformed form of the glucocorticoid receptor from rabbit liver has been purified approximately 8,000-foM by a three-step procedure. The f r s t step involved protamine sulfate precipitation which allowed a 5-6-fold purification with 85 % yield. The second step, affinity chromatography ushlg a N-(12-dodecyl-amh~o) 9 ct-fluoro-16 a-methyl-11 fl, 0 To whom all correspondance should be addressed Abbreviations : Dexamethasone : 9 a-fluoro-16 a-methyl-ll ft. 17 a, 21-trihydroxy-l,4-pregnadiene-3,20 dione;

BSA : bovine sen+m albumin: SDS : sodium dodecyl sulfate; CBG : corticosteroid binding globulin.

P. Lustenberger and coll.

1268

17 ct-dihydro:~T-3-oxo-l,4-androstadiene-17 fl-carboxamide substituted Sepharose gel, purified the receptor 1,500-2,000-fold as calculated by specific radioactivity. The third step involved high performance liquid chromatography resulthlg hi overall purification near 8,000-fold. The final glucocorticoM receptor appeared about 60 % pure. The purified nontransformed glucocorticoid receptor had a sedimentation coefficient of 9 S in 0.16 M phosphate containing 5-20 % sucrose gradients and the Stokes radius was 6.1-6.3 nm as determhled by low pressure gel filtration and HPLC. Bhlding specificity of the purified receptor was identical to that previously reported in crude rabbit liver cytosoL lsoelectricfocushTg and ion-exchange chromatography showed that the purification procedure affected the net charge of the receptor protein. This phenomenon could be related to hlteractions between the glucocorticoid receptor and cytosolic factors. SDS polyacrylamide gel electrophoresis showed a major Mr = 94.000 protein band which is in good agreement with previously reported values for ghtcocorticoid receptors. Transformation of the purified receptor was achieved after removal of molybdate by exposure at 25°C to 0.4 M KCI. Characterization of the molecular forms was performed by means of incorporation hlto isolated mtclei, affinity towards polyanionic exchangers and high pressure size exchtsion chromatography. Results show that about 40 % of the receptor is ht the transformed stiTte. Key-words : glucocorticoid receptor / purification / transformation.

Introduction

Materials and methods

Tremendous progress has been made in the past years in the understanding of glucocorticoid hormone action. Exhaustive investigation needs highly purified nontransformed receptor preparation in order to study the physico-chemical properties, functions and transformation process of receptor. Only few reports have appeared which are related to this purification [I-6]. In these studies, rat liver cytosol or HTC cells were used as biological material. Using a home made steroid affinity resin in molybdate containing buffer, we have developed a simple two-step procedure for the purification and characterization of rat liver glucocorticoid receptor [7]. Preliminary investigation with rabbit liver receptor [8] showed us that it seemed to be a more suitable material for purification, providing a more important source of receptor than the rat liver. In the present paper, we describe a three-step procedure including affinity chromatography to purify the nontransformed molybdate stabilized form of glucocorticoid receptor from rabbit liver. Properties of purified material are similar to those of crude cytosolic receptor. After removal o f molybdate, purified receptor complexes can undergo heat and salt-dependent transformation similar to that observed for cytosolic receptor.

Chemicals [l, 2, 4(n)-3H] dexamethasone (40 Ci/mmol) was obtained from the Radiochemical Centre (Amersham, U.K.); radioinert steroids were from Sigma. DEAE Trisacryl, Ultrogei AcA 34, Trisacryl GF 05 and PhosphoUitrogel were purchased from IBF (Villeneuve la Garenne, France); hydroxylapatite (DNA grade) was from BioRad (Richmond, U.S.A.). Sepharose CL 4B, gel filtration calibration kits, and SDS-polyacrylamide gel electrophoresis calibration kits were obtained from Pharmacia (Uppsala, Sweden); electrophoresis and isoelectrofocusing reagents and calibration kits were from Serva (Heidelberg, F.R.G.). All other reagents were of analytical grade from Merck (Darmstadt, F.R.G.).

Buffers Buffers used were buffer A: 20 mM potassium phosphate, 10mM sodium molybdat.e, 20mM 2-mercaptoethanol and 20 % (v/v) glycerol, pH 7.4 at 0-4"C; buffer B: 160mM potassium phosphate, 10mM sodium molybdate, 20 mM 2-mercaptoethanol and 20 % (v/v) glycerol, pH7.4 at 0-4°C; buffer C" 10mM tris-HCl, 10 mM sodium molybdate, 20 mM 2-mercaptoethanol, and 20 % (v/v) glycerol, pH 7.4 at 0-4°C.

Animals and preparation of cytosol Male rabbits (Fauve de Bourgogne) weighing 2,000g were adrenalectomized under Flunitrazepam

Purification of rabbit liver ghtcocorticoid receptor and Thiopental anesthesia and received 0.9 % NaCI to drink. Animals were killed 2-3 days after surgery. Livers were perfused with cold 0.9 % NaCI and then with buffer A. All subsequent procedures were carried out at 0-4°C. The excised and minced livers were homogenized in 2 volumes of buffer A with a Teflon-glass Potter homogenizer. The homogenate was centrifuged at 20,000 x g for 10 min. The upper layer of floating lipids was discarded and the supernatant fraction centrifuged at 105,000 x g for 120 min to obtain the cytosol, pH was adjusted to 7.4 with K:HPO4 1 M and the resulting cytosol was stored at -70"C until further use, without loss of binding activity up to 3 months.

Assay of steroid receptor bhlding Duplicate samples of cytosol were incubated with 2.10-SM [~H] dexamethasone in the presence or absence of a 1000-fold excess of non radioactive dexamethasone at 0-4"C for 16 h. Charcoal adsorption assays [9] were performed in duplicate to measure the extent of steroid binding. Specific binding in cytosol and purified samples was also determined using a hydroxylapatite technique [10]. Non specific binding was estimated after heat denaturation of the steroid receptor complexes at 50°C for 30 min. When protein content was lower than 1 mg/ml samples were supplemented with BSA up to 2 mg/ml. The binding activity of purified receptor after gel filtration and ion-exchange chromatographies was determined directly by counting aliquots of each fraction.

Purification procedure The affinity gel was prepared by coupling a dexamethasone derivative to a 12-carbon spacer arm attached to the polymer support (Sepharose CL4 B). The details of its preparation are described elsewhere [3]. Estimation of bound ligand by incorporation of a tracer dose of ['H] dexamethasone allow to calculate a capacity of 10-' M/ml gel. The affinity matrix was equilibrated in buffer B before use. Fractionation of cytosol was performed with 0.5 % (w/v) protamine sulfate solution [31. This was added dropwise to the cytosol to give a 0.075 % final concentration of protamine sulfate. Material precipitated was recovered by centrifugation at 20,000 x g for 10 min and dissolved in buffer B in a volume equivalent to one-fifth the volume of cytosol. The redissolved protamine sulfate fraction, usually 40 to 50 ml (corresponding to one rabbit liver) was incubated with I ml of affinity gel. Batchwise adsorption was performed at 0-4°C in a rotating shaker for 20 h. The suspension was centrifuged at 750 x g for 2 rain and the supernatant was removed and assayed for specific binding. The resin was transferred in a column ( 0 2.5 cm) and washed at a flow rate of 10 ml/min

1269

with the followings : buffer B 40 ml; buffer B supplemented with 0.4M KCI 30ml; buffer A 3 0 m l and buffer B warmed at 20"C 30ml. The gel was then suspended in 4 ml of buffer B containing 5.10 -~ M ['H] dexamethasone (13 Ci/mmol). Elution was performed batchwise at 20"C for 16 h. The slurry was filtered and the gel rinsed with I ml buffer B. The combined eluate was assayed for specific binding and protein content. Affinity chromatography eluates were stored at - 70"C. The final purification step included first a concentration of the affinity eluate by DEAE chromatography followed by size exclusion HPLC. Conditions were as follows: column (1.5 × O 1.I cm) filled with DEAE Trisacryl; flow rate 36 m l / h ; washing with 20 ml buffer A and elution with 2 ml buffer B; the samples were diluted five-fold with buffer A before passing through the column. Size exclusion HPLC was performed as mentioned below, with repeated injections of 0.5 ml sample. Pooled fractions of receptor were supplemented with 10 % glycerol, 10 mM 2-mercaptoethanol and 10-~M [3HI dexamethasone to prevent dissociation. Eluates were stored at - 7 0 ° C .

Gel fihration Analytical AcA 34 gel filtration. A column (90 x O 1.6 cm) of Ultrogel AcA 34 was equilibrated in buffer B at 0-4"C. 0.5 ml samples were run on to the column at a flow rate of 7.5 ml/h. Calibration was carried out with the following proteins : chymotrypsinogen A (Rs 2.2 nm), ovalbumin (Rs 2.9 nm), bovine serum albumin (Rs 3.6 nm), catalase (Rs 5.2 nm) and ferritin (Rs 6.1 nm). Blue dextran 2000 was used to estimate the void volume (V,) and total volume (V,) was determined by potassium bichromate filtration. The standard curve was plotted according to the method of Porath [11]. The Stokes radii of receptor preparations were estimated using linear regression analysis of KI~~ vs Rs of the standards. Under our conditions, most of the ferritin tented to aggregate and did not fit the standard curve, the Rs of the receptor was thus estimated by linear extrapolation of the calibration plot [12].

Size exclusion HPLC. Samples were applied on a 60 x O 0.75 cm LKB Ultropae TSK G-3000 column refrigerated at 0-4°C, connected to a WATERS 440 HPLC apparatus. Elution was performed at a flow rate of 4 8 m l / h with 20mM Tris-HCi, 10mM sodium molybdate, 400 mM KCI, 1 mM EDTA buffer pH 7.0 at 0-4"C. Calibration and exploitation of results were performed as for conventional gel filtration. Ion-exchange chromatography DEAE Trisacryl, PhosphoUltrogei and hydroxylalYatite were prepared as suggested by the manufacturers. Gels were packed into 1.5 ml columns ( 0 1.1 cm) and equilibrated in buffer A except for PhosphoUl-

1270

P. Lustenberger and coll.

trogel which was equilibrated in buffer C. Samples of purified receptor were diluted ten-fold with equilibrating buffer and loaded on to the columns. Free steroid and unbound material were removed from the column with a 10 ml wash with starting buffer. Elutions were carried out with linear salt gradients (20 mi), 0.02 M to 0.4 M phosphate in buffer A and 0 to 0.4 M NaC! in buffer C for PhosphoUltrogel. Two min fractions were collected at a flow rate of 36 ml/h and assayed for radioactivity.

Sucrose density gradients Linear 5-20 % (w/v) sucrose gradients were prepared in buffer A. Samples (0.2 ml) of purified receptor were layered on the top of the gradients. Centrifugation was carried out for 24 h at 280,000 x g in a Beckman SW50 rotor. Standard proteins, catalase (11.2 S), aldolase (7.35 S), alburhin (4.6 S) and myoglobin (2.0 S) were run separately. Fractions of 7 drops were collected by piercing the bottom of the tubes and assayed for r~dioactivity. Sedimentation coefficients were determined according to Martin et Ames [13].

Receptor transformation Samples o f purified receptor were cleared of molybdate by chromatography on Trisacryl G F 0 5 . Eluting buffer (buffer B) contained 2 mg/ml BSA. The excluded volume was pooled and supplemented with 0.4 M KCI. A control aliquot was withdrawn in which 10 mM molybdate was added to block transformation. After 30rain at 25°C the extent of transformation was measured by PhosphoUItrogel chromatography, size-exclusion HPLC and nuclear uptake. Molybdate (10 mM) was added in test tube and included in all buffers to prevent further transformation during the prolonged contact with chromatographic supports or isolated nuclei. Ion exchange chromatography and size exclusion HPLC were performed as described above. Incorporation of [3H] dexamethasone bound complexes into nuclei was carried out as follows. A 50 lai aliquot of purified receptor was mixed with the suspension of nuclei (200 lag of DNA) and 500 ttl of 5 mM MgCI2, 2.5 mM KCI, 10 mM Na,MoO~, 5 mM Tris/HCl buffer pH 7.50 (TKM buffer) and incubated at 0-4°C with gentle shaking. After 30 min the pellets were washed 4 times with 1 ml TKM buffer and assayed for radioactivity.

Isoelectricfocusing One millimeter thick agarose gels (245×110 mm) were prepared between 1 and 3 days before use and Were stored at 0-4°C. The gels were supported on a thin hydrophilic polyester sheet (Gel Bond) and contained 1% (w/v) agarose, 12 % (w/v) sorbitol and 0.6 % (v/v) ampholines (pH range 3-10). Samples (0.02 ml) were applied near the cathode using paper sample applicator. Running conditions were as follows: 15 W and 1,500 volts maximum, current unlimited, 2,000 volts/h. A coolant temperature of 0-4°C was maintained all over the experiment. After migration, gels containing labelled samples were cut into 2 mm slices for counting. Strips with standard proteins were stained with Coomassie brilliant blue G 250. pI of the receptor was estimated using a calibration plot established from standard proteins.

Miscellaneous Proteins were measured by the Coomassie blue adsorption.method [17] using bovine serum albumin as standard. Adaptation to the Cobas analyzer (Hoffman-La Roche, Switzerland) allows the determination up to 5 lag/ml. Radioactivity was determined in a Beckman LS 2800 liquid scintillation spectrometer. The samples (0.15-0.20 ml) were counted in 2.5 ml Beckman Ready Solv HP ~ scintillation fluid with a tritium efficiency of 45 %. Nuclei were isolated as described by Tata [18]. DNA concentration was estimated according to Kapuscin.ski and Skoczylas [19].

Results

Polyacrylamide gel electrophoresis Aliquots to be analysed under denaturing conditions were concentrated by precipitation with trichloracetic acid (10 % final concentration) in the presence of SDS (0.1%). The pellets were rinsed with ether/ethanol (1/1) and treated with 1% SDS and 1% mercaptoethanol. After 3 min boiling, 50 I11 samples were layered on 5-20 % linear acrylamide slab gels and electrophoresis was carried out according to the procedure of Laemmli [14] at room temperature for 4 h at 45 mA. After electrophoresis, the gels were stained with silver nitrate [15]. The standard curve between relative mobilities (distance of migration/length of the gel) of the standard proteins and their log molecular weight was plotted according to the method of Weber and Osborn [16].

Glucocorticoid receptor purification Previously d e s c r i b e d p u r i f i c a t i o n p r o c e d u r e [3] was used with m i n o r m o d i f i c a t i o n s . T h e p r e l i m i n a r y step, p r o t a m i n e sulfate p r e c i p i t a t i o n , allow e d a p a r t i a l p u r i f i c a t i o n o f the r e c e p t o r ( u p to

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Sepharose C L 4 B

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Structure of the affinity matrix.

Purification of rabbit liver glucocorticoid receptor 5-6-fold) with good yield and induced an important reduction of volume. Affinity chromatography was performed with a dexamethasone derived matrix (Fig. 1). A 12-carbon aliphatic spacer arm was used because it appeared more efficient than the previous 9-carbon spacer arm, especially for the adsorption of the receptor. Extensive washing with several buffers at different ionic strenghts and temperatures was necessary in order to remove non specific proteins. Biospecific elution in the presence of isotopically diluted [3HI dexamethasone was performed for 16 h at 20°C. At 0°C, the rate of dissociation of glucocorticoid receptor complex from rabbit liver cytosol was too slow to allow the exchange of the steroid [8]. Purification after affinity chromatography was 1,500-2,000-fold; elution and total yields varied between 30-40% and 15-25%, respectively. The affinity eluate was further purified by size exclusion H P L C on a TSK 3000 column. This step resulted in a 5-6-fold additional purification with 80 % yield. In Table I, the results of a typical experiment are shown. The overall purification was about 8,000-fold as calculated by specific radioactivity. The final yield was about 15 %.

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Molecular parameters Gelfihration chromatography. Stokes radii were determined by gel filtration on Ultrogel AcA 34 column and size exclusion HPLC. In high ionic strength and in the presence of molybdate, the purified receptor eluted as a single symmetrical peak (Fig. 2). Rs of the glucocorticoid receptor calculated from calibration plots, was found to be 6.1 nm in conventional low pressure gel filtration and 6.3 nm in HPLC. Sucrose gradient centr~tgation. On 5-20 % linear sucrose gradients, the purified receptor sedimented at 9.0S in the presence of 10mM molybdate and high ionic strength (0.16 M phosphate). A typical sedimentation profile is shown on Figure 3. From the Stokes radius and the sedimentation coefficient values, an apparent M~=240,000248,000 can be calculated [20]. In crude cytosol, the molecular parameters were estimated using similar experimental conditions [8]. Reported values of Rs (5.6nm) and molecular weight (280,000) were calculated from the standard plots KD VS log Rs and KD VS log M~. According to Sherman [27], it is better to determine Rs with the

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FIG. 2. - - Pilrified ghwocorticoid receptor : measmz'ment o f Stokes radius. A) Gel filtration on Ultrogel AeA 34. 0.5 ml affinity eluate was run on an AcA 34 column as well as standard proteins as described under Materials and Methods. Elution was achieved with buffer B. 1.43 ml fractions were collected and 0.2 ml aliquots were assayed for radioactivity. Kgj3 was plotted as a function of Rs (Inset). The standard proteins were : a, catalase: b, aldolase; c, BSA: d, ovalbumin: e, chymotrypsinogen A. B) Size exclusion HPLC. After removal of excess [SH] dexamethasone by DEAE chromatography a 0.2 ml sample was applied on a TSK G 3000 column and eluted as described under experimental section. I rain fractions were collected and assayed for radioactivity. Distribution coefficients and Stokes radii were calculated as described in Materials and Methods. Reference proteins were : a, thyroglobulin" b, ferritin: c, BSA: d, ovalbumin: e, Soja trypsic inhibitor.

1272

P. Lustenberger and coll. TABLE I Nontransformed rabbit liver glucocorticoid receptor : three-step purification.

Volume ml

Total protein mg

Cytosol Protamine ext. Affinity XTO eluate

220 45

3256 522

45.19 38.20

0.37

HPLC

9.5

0.057

Fraction

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[3H] Dexamethasone bound specific act. cpm.10 -6 cpm/mg.10 -3

at

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Fractions Sucrose gradient ultracentrifugation of pmified untransformed ghzcocorticoid receptor. FIG. 3.

-

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0.2 ml samples of purified eluate desalted on DEAE Trisacry[ were layered on 5-20 % (w/v) sucrose density gradients prepared in buffer B. Ultracentrifugation was performed at 280,000g for 24 h. The standard proteins were: Mb, myoglobin, BSA, albumin; Aid, aldolase and Cat, catalase.

linear correlation of K1f3vs Rs. Using this relation, a Rs value of 6.2nm was calculated for the cytosolic glucocorticoid receptor. This information when combined with the value of the sedimentation coefficient, allowed to calculate an apparent Mr = 244,000.

Ionic properties Ion-exchange chromatography analysis. The purified molybdate stabilized glucocorticoid re-

Purification fold

Yield %

13.88 73.17

1 5.3

100 84.5

8.66

23 400

1687

19.2

6.84

120 025

8647

15.1

ceptor was analysed with three different ionexchange resins. When applied to hydroxylapatite, a single peak of radioactivity eluted at 0.125M phosphate (Figure 4A). On DEAE Trisacryl, the receptor was completely adsorbed. Elution with a linear 0.02-0.4 M phosphate gradient yielded a single peak of radioactivity at 0.125M phosphate (Fig. 4B). When purified material was applied on a PhosphoUltrogel packed column (Fig. 4C) all protein bound radioactivity was eluted in the void volume. In each case, the peak of specifically bound [3H] dexamethasone was deleted when heat inactivated eluate was chromatographed (data not shown).

Isoelectricfocusing. Analysis in agarose slab gels with 3-10 pH range ampholines showed that the purified glucocorticoid receptor focused at pH 5.9 (Fig. 5) Good results were only obtained with samples containing no salts. Before electrofocusing the eluate was concentrated and dialyzed against buffer A under vacuum. After staining with Coomassie brilliant blue, no proteins were detectable. When compared with results obtained with [3H] dexamethasone receptor in cytosol, it appeared that the purified receptor behaved slightly differently. The average isoelectric point was 5.3 for receptor from crude cytosol, but 5.9 for purified eluates. This dissimilarity was also found w i t h ion-exchange analysis on cationic resins. The purified receptor was eluted with a lower salt concentration (0.125 vs 0.160 M phosphate) than the receptor in cytosol. Reincubation of purified samples with heat inactivated cytosol prior to focusing led to a 0.4 pH acidic shift of the peak of bound radioactivity.

Purification of rabbit liver glucocorticoid receptor /

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FIG. 4. -- Ion-exchange chromatograph.r of the purified ghtcocorticoid receptor. A) Hydroxylapatite (HA).B) DEAE Trisacryl (DEAE). C) PhosphoUltrogel (PU). Ten fold diluted aliquots (3 ml) of purified glucocorticoid receptor were loaded on 1.5 ml columns filled with the different gels, Chromatography was performed at a flow rate of 0.6 ml/min. After a preliminary washing with 10 ml of buffer A (HA and DEAE) or 10 ml of buffer C (PU), elution was carried out with linear gradients (20 ml total volume) 0.02 M-0.4 M phosphate in buffer A (HA and DEAE) or 0-0.4 M NaCI in buffer C (PU). 1.2 ml fractions were collected and assayed for radioactivity (o) and salts (A).

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FIG. 5. -- lsoelectricfocusing of cytosolic [ JH ] de.vamethasone receptor and purified ghwocorticoid receptor. Crude cytosol was incubated overnight at 0-4°C with 2.10-SM [3H] dexamethasone. Purified glucocorticoid receptor preparation was concentrated and dialysed under vacuum against buffer A. Isoelectricfocusing on 1% agarose and 0.6 % ampholines (pH range 3-10) slab gels was performed as described in Materials and Methods. For each fraction triplicate samples (0.02 ml) were run concomitantly. After focusing the strip of gel corresponding to three samples was sliced into 2 mm fractions and analysed for radioactivity. Strips with standard proteins were stained with Coomassie brilIiant blue G 250. A) Rabbit liver eytosolie glucocorticoid receptor. B) Purified glucocorticoid receptor, C) Purified glucocorticoid receptor reincubated with an equal volume of heat-inactivated cytosol.

Binding parameters Kinetic study. Purified receptor was obtained in its steroid bound form. In the absence of a convenient procedure able to strip the hormone from the binding site, it was not conceivable to study the association rate and the equilibrium o f binding. Only the dissociation process could be studied after addition of an excess of unlabelled dexamethasone. The dissociation followed a first

order kinetics (Fig. 6). At 0-4 °C, the rate constant was 6 . 3 x l 0 - S m i n -t. The rate of dissociation increased ten-fold at 20°C (7.8 x 10-4min-~). At the same time, the stability of the purified receptor was investigated. Samples of purified receptor kept in the presence of an excess of [3H] dexamethasone were left at 0-4 or 20°C. At regular time intervals, the residual binding activity was measured. The half-lives of purified receptor were 200 h at 0-4°C and 15 h at 20°C.

P. Lustenberger and coll.

1274

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1~o Time (h) [H3 Dexamethasone]

Dissociation kinetics o[the lmr~lied ghwocorticoid

Aliquots of affinity eluate were desahed on DEAE Trisacu(I. Afterwards dissociation was initiated by the addition of 5.!0-+M unlabelled dexamethasone. At the indicated times, duplicate samples were removed and assayed for macromolecular bound radioactivity by the hydroxylapatite batch assay. Kinetics of dissociation was followed at 0-4°C ( o ) and 20°C (o). Estimations of dissociation rate constants were achieved by using semi-log plot of residual binding (B/Bo) vs time. Initial binding value (B0) was about 1000 cpm/0.1 ml.

From these values of inactivation and dissociation, it appeared that exchange experiments must be carried out at 20°C.

Hormone bhTding specificity (Fig. 7). Eluate o f the affinity chromatography gel was cleared of excess of [3H] dexamethasone by D E A E chromatography. Samples of labelled pure receptor were incubated overnight at 20°C in the presence of 10 - a M [3HI dexamethasone and increasing amounts (3.10 -9 to 10 -5 M) o f competing steroids. The remaining bound [3HI dexamethasone was measured by the dextran coated charcoal assay. Dexamethasone, betamethasone and triamcinolone acetonide were the best competitors, followed by the natural glucocorticoids, cortisol and corticosterone. Progesterone and aldosterone caused only little displacement, while 5 ct-dihydrotestosterone and 17 13-estradiol did not display any competition. Electrophoretic analysis Receptor peak fractions eluted from size exclusion HPLC were pooled and analysed by SDS polyacrylamide gel electrophoresis. After staining with silver nitrate, the protein pattern shows a major M,=94,000 band (Fig. 8). Two other

FIG.

7.

--

Purified rabbit liver ghwocorticoid receptor:

hormone specificio'. Affinity eluate was cleared of excess of labelled dexamethasone by DEAE chromatography. Samples (0.5ml) of purified receptor were incubated overnight at 20°C in the presence of 10-SM [3H]dexamethasone and increasing concentrations of non radioactive competitors (3.10-+-10-5M). Residually bound radioactivity was assayed by the dextran coated charcoal assay. Ordinate: ratio of binding of [~H1dexamethasone in the presence of competitor (B) to binding in the absence of competitor (B0). Abcissa: concentration ratios. Maximal binding value was B0 = 3000 cpm/0.1 ml. Triamcinolone acetonide (A); Dexamethasone (o): Betamethasone(.); Progesterone (,,); AIdosterone (o): Cortisol (,,): Corticostetone (n); 17 ~-oestradiol (O); 5 tt-dihydrotestosterone (,).

protein bands corresponding to apparent M r = 70,000 and 50,000 were also detected. These lower molecular weight proteins may be either components of the receptor complex or may derived from the Mr=94,000 protein by proteolytic cleavage.

Receptor transformation Purified samples were first cleared of molybdate which is a potent inhibitor of transformation process [8]. Desalting was carried out in the presence of 2 m g / m l BSA in the eluting buffer in order to prevent receptor degradation. Conditions of in vitro transformation (0.4 M KCI and 25°C for 30 min) of purified receptor were established from previous results with cytosol [8]. Results of quantification o f the transformation process are depicted in Table II. Ion-exchange analysis with PhosphoUltrogel showed that a fraction of [3H] dexamethasone b o u n d was retained and eluted at a concentration o f 0.125 M NaCI.

Purification of rabbit liver glucocorticoid receptor ,q-

receptor acquired the capacity to enter into nuclei. The extent of transformation measured with PhosphoUltrogel chromatography and size exclusion HPLC was about 40 %.

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f .... i

u FIG. 8. -- Polvacrrlamide gel electrophoresis under denaturating condition's of purified ghwocorticoid receptor. Samples of pure glucocorticoid receptor eluted from size exclusion HPLC (0.5 ml) were treated as described in Materials and Methods. Bromophenol blue indicated the migration front. The molecular weight standards were : a) thyroglobulin, M,=330,000; b) ferritin (half-unit), M,=220,000; c) phosphorylase b, M,=97,000; d) albumin, Mr=67,000; e) ovalbumin, M,=43,000; f) lactate dehydrogenase, M, = 36,000. Plot of the log of M, vsthe migration of reference proteins allowed calculation of the M, of the purified glucocorticoid receptor.

Estimation of Stokes radii using exclusion size HPLC revealed that transformation is accompanied by a decrease in the Stokes radius of the predominant peak from 6.3 nm (untransformed) to 3.5 nm (transformed). Experiments performed with isolated nuclei showed that about 20-30 % of

Using a three-step procedure including affinity chromatography, the rabbit liver glucocorticoid receptor has been purified about 8,000-fold with 15 % yield. This is mainly due to the combination of affinity chromatography and HPLC size exclusion filtration. The first allows to purify several hundred times the receptor, with reasonable yield. Size exclusion HPLC was chosen amongst the current methodologies so far available due to the highly labile nature of steroid hormone receptors. The results reported here show that this procedure is very efficient and avoids the loss of receptor structure. Assuming a molecular weight of 94,000 and a single steroid binding site per molecule, the final preparation appears to be about 60 % pure. The results presented here are comparable to the purifications previously reported. Among the different methods described for the purification of the untransformed glucocorticold receptor, two kinds of affinity matrices were depicted. In one case [I, 4, 5] deoxycorticosterone was immobilized by an ether linkage. Using this affinity matrix and subsequent DEAE chromatography, rat liver glucocorticoid receptor has been purified approximatively 4,000-fold with 33 % yield by Grandics et al. [5]. It stands to reason that this ligand is not highly specific for glucocorticoid receptor. Both progesterone receptor and CBG are retained by this matrix. The second kind of biospecific adsorbant was synthetized from the 17 I~-carboxylic acid derived from dexamethasone [22] linked through an amide bond [2, 3, 6]. This latter appears highly specific towards the glucocorticoid receptor, since CBG from rat and rabbit serum does not bind fluorinated steroids such as dexamethasone (data not shown). Very recently, Govindan and Gronemeyer [6], using a two-step procedure, announced a 6,000-fold purification of rat liver receptor with 90% yield. Regarding overall yield, our results appear less efficient. However, elution of rat liver receptor was carried out at 0°C whereas rabbit liver receptor could only be exchanged at 20°C. All attempts of elution carried out at 0-4°C were unsuccessful. To improve the elution yield, it would be interesting to test the conditions used by Govindan and Gronemeyer [6], including

P. Lustenberger and coll.

1276

stabilized molecular form (Rs=6.3 nm and 9S). These values are in close agreement with the estimated values obtained in cytosol [8]. While the hydrodynamic characteristics seem unchanged, some minor differences pertaining to ionic properties can be noticed after purification. The behaviour o f purified receptor towards ionic exchangers ( D E A E Trisacryl and hydroxylapatite) is similar to that of crude cytosolic receptor, but purified receptor is eluted with a lower concentration o f salt. This result agrees well with the more basic pl of the purified receptor 5.9 vs 5.3 measured in cytosol. Such a result can be related to several explanations. It would be due to the transformation process induced by the ionic exchangers or the ampholines. However, analysis by gel filtration or sucrose density gradients after D E A E chromatography exhibit species having the characteristics of untransformed receptor ( R s = 6.3 nm and 9S). Moreover, purified receptor does not bind to PhosphoUltrogel (Fig. 4C) and to isolated nuclei (Table II). Based on physicochemical and functional properties, it seems that purified receptor is in its untransformed state. Further studies show that this receptor can be transformed in vitro after the elimination of molybdate and characterized in its transformed state.

5 0 m M N a S C N in eluting buffer. Nevertheless, thiocyanate proved to be a potent transforming factor with rabbit liver glucocorticoid receptor (data not shown). For the final step of purification, size exclusion HPLC appeared more suitable just as well in efficiency and yield than ion-exchange chromatography on D E A E Trisacryl or hydroxylapatite [23]. The chemical structure of the immobilized ligand, a dexamethasone derivative, ensure the high specificity o f the affinity matrix. In purified material, binding of [3H] dexamethasone is displaceable only by synthetic fluorinated steroids and to a lesser extent by natural glucocorticoids. Progesterone has only a weak affinity, thus proving the specific purification of glucocorticoid receptor and not C B G. ! Because chromatographic procedures expose t'he receptor to salt or dilution, it is important to use conditions which prevent transformation [24], ~illowing isolation o f a single molecular form o f the receptor. Transformation of the receptor was hindered by the use of the inhibitor sodium molybdate [25-27]. Physicochemical characterization of the purified material shows that the receptor still remains in its large molybdate

TABLE II Characteristics of[~H] dexamethasone-receptor complexes of purified extracts before and after heat and salt exposure.

Treatment

Purified glucocorticoid receptor in presence of 10 mM molybdate + heat/salt (0.4 M KCI; 25°C; 30 rain) Gel filtration in the presence of 2 mg/ml BSA + l0 mM molybdate + heat/salt (0.4 M KCI; 25°C; 30 rain) Gel filtration in the presence of 2 mg/ml BSA + heat/salt (0.4 M KCI; 25°C; 30 min)

Binding to isolated nuclei % (a)

Binding to PhosphoUltrogel % (b)

Size exclusion HPLC Rs = 6.3 nm Rs = 3.5 nm % (c) %

0

0

100

0

5-9

8-12

80

20

35-45

18-30

60

40

Chromatography on PhosphoUItrogeland binding to isolated nuclei were performed as described under Materials and Methods. Estimation of Stokes radii was obtained by reference to the standard curve. (a) values of [~H]dexamethasone-receptor complexr~ bound b.y isolated nuclei relating to the total receptor complexes added in the assay ( - 3,000 cpm). (b) values of [JH] dexamethasone-receptor complexes retained by PhosphoUltrogel relating to the total receptor complexes loaded on the columns ( - I0,000cpm). (c) distribution of bound [3HIdexamethasone among the different peaks obtained in size exclusion HPLC (= 10.000cpm total receptor). Estimation of [3HIdexamethasone-receptorcomplexes was performed with hydroxylapatite batch assay.

Purification o f rabbit liver ghtcocorticoid receptor

A second explanation might be a proteolytic cleavage of the receptor. This conclusion can be dealt with only if this cleavage affects a low molecular weight fragment, since no modifications of gel filtration and sedimentation profiles appear. Moreover, reincubation of purified material with inactivated cytosol would not restore the original pI if a proteolytic cleavage had occurred. Thus, it seems to be a more plausible explanation that there may be interactions of receptor with cytosolic factors which affect the net charge of the protein. This is consistent with the shift of the pl observed after reincubation of receptor with cytosol (Fig. 5). Such differential isoelectricfocusing properties of crude and purified forms of a protein were already described for human ct2 macroglobulin [281. SDS polyacrylamide gel electrophoresis shows a major 94,000 band. This correlates with the previously r'eported values of 89,000 [21, 90,000 [5, 71 and 94;000 [29] for the rat liver glucocorticoid receptor. The occurrence of minor bands, 70,000 and 50,000 can be related to the receptor degradation or to contaminant proteins. After ct chymotrypsifi digestion of the rat liver glucocorticold receptor, Wrange et al. [29] demonstrate the occurrence of fragments of Mr=72,000 and 50,000. In HTC cells, the molecular weight of the major proteolytic fragments obtained by Reichman et al. [30] are 30,400 and 28,400 for trypsin, 42,000; 32,100 and 30,700 for ~t chymotrypsin and 62,500; 51,I00 and 31,200 for Vs protease. In the absence of protease inhibitors in the cytosol, proteolytic cleavage cannot be avoided. Moreover, we cannot rule out the possibility that contaminants would be distributed into two species. But it seems more plausible that the distribution affects all the proteins and thus is not revealed by staining. Thus the appearance of smaller components from receptor degradation can be explained either by proteolytic cleavage [29] or by dissociatiofi of a polymeric structure [31]. A heterooligomeric structure of the glucocorticoid receptor, as suggested by Grandics et al. [5], seems to be a more plausible explanation for these observations. Further investigations are required to elucidate the exact structure of the protein and to identify the factors which interact with the receptor in cytosol. The most instructive findings are those that show the transformation of the purified receptor. As shown in Table II, after heat and salt exposure, a part of the purified steroid receptor complexes exhibit characteristics similar to those

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•of the transformed cytosolic receptor [8]. Highly purified receptor is transformed to the same extent as cytosolic receptor under the same conditions. Already, the mere fact to desalt the purified receptor by Trisacyl G F 05 chromatography induce a N 10 % transformation. These data contrast with those reported by Grandics et al. [5]. In the same way, we demontrate that addition of cytosolic factor(s) is not necessary.'From experiments performed with isolated nuclei, a ~ 20 % yield of transformation is obtained versus ~ 40 % with PhosphoUItrogel or size exclusion HPLC estimations. This difference would be explained by the occurrence of dysactivation process as proposed by Milgrom [24]. In the dysactivated state, the receptor retains the ability of binding hormone but has lost its capacity to bind to isolated nuclei. Thus, our results relating to transformation are consistent with the hypothesis of a heterooligomeric structure of the glucocorticoid receptor as suggested by previous studies [5, 31]. In conclusion, the rabbit liver glucocorticoid receptor has been isolated in the untransformed state and the purified protein still remains functional. It can now be subject to detailed studies, that are clearly essential for understanding the transformation process which is the key to elucidate the mechanism of glucocorticoid hormone action and regulation.

Acknowledgments This work was supported by grants from I N S E R M (contract CRL n ° 81.30.15), from the Association pour la Recherche contre le Cancer (ARC) and by UER o f Medicine. We are grateful to M.R. Sherman for insightful comments. We thank Institut National de la Santd et de la Recherche Mddicale (U 211)for its material assistance. The expert secretarial assistance o f A. Combalot is greatly appreciated.

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20. Siegel, L.M. & Monty, K.J. (1966) Biochim. Biophys. Acta, I12, 346-362. 21. Sherman, M.R. (1975) in : "Methods in Enzymolog),'" vol. XXXVI (O'Malley, B.W. & Hardman, J.G. Eds.) pp. 211-234, Academic Press, New York. 22. Formstecher, P., Lustenberger, P. & Dautrevaux, M. (1980) Steroids, 35, 265-272. 23. Lustenberger, P., Blancbardie, P., Orsonneau, J.L. & Bernard, S. (1985) in : "Protides of the Biological Fluids" 32th Colloquium (Peeters, H. Ed.), pp. 1121-1124, Pergamon Press, Oxford. 24. Milgrom, E. (1981) in : "'Biochemical Actions of Hormones'" vol. 8, (Litwack, G. Ed.), pp. 465-492, Academic Press, New York. 25. Barnett, C.A., Schmidt, T.J. & Litwack, G. (1980) Biochemistry, 19, 5446-5455. 26. Dahmer, M.K., Housley, P.R. & Pratt, W.B. (1984) Ann. Rev. PhysioL, 46, 67-81. 27. Housley, P.R., Grippo, J.F., Dahmer, M.K. & Pratt, W.B. (1984) in : "Biochemical Actions of Hornlones'" vol. 11 (Litwack, G. Ed.), pp. 347-376, Academic Press, New York. 28. Back, S.A. & Alhadeff, J.A. (1983) J. Chromatogr., 278, 43-51. 29. Wrange, O., Okret, S., Radojcic, M., CarlstedtDuke, J. & Gustafsson, J.A. (1984) J. BioL Chem., 259, 4534-4541. 30. Reichman, M.E., Foster, C.M., Eisen, L.P., Eisen, H.J., Torain, B.F. & Simons, S.S. (1984) Biochemistry, 23, 5376-5384. 31. Vedeckis, W.V. (1981) Biochemistry, 20, 7237-7245.