Resolution of the molecular forms of rat liver glucocorticoid receptor by affinity chromatography on immobilized heparin

BIOCHIMIE, 1984, 66, 505-511

Resolution of the molecular forms of rat liver glucocorticoid receptor by affinity chromatography on immobilized heparin. P. B L A N C H A R D I E ' : , P. L U S T E N B E R G E R , J.L. O R S O N N E A U

a n d S. B E R N A R D .

Laboratoire de Biochimie Mddicale -- U.E.R. de MEdecine 1, rue Gaston Veil -- 44035 Nantes Cedex, France. (Refue le 3-4-1984, acceptde le 3-6-1984).

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- - Le r~cepteur cytosolique ?t glucocorticoides du foie de rat, lid ?t ia dexamethasone et stabilisd en prdsence de molybdate, interagit avec l'hEparine-ultrogel. L'dlution en prdsence de NaCI ou d'hdparine met en dvidence une seule forme molEculaire. En l'absence de molybdate et apr~s exposition ~ la chaleur, l'analyse chromatographique du cytosol, dans les mEmes conditions, rdv~ie ia prdsence de deux formes distinctes. Ces formes moldculaires ont dtd caractdrisdes par mesure de la liaison aux noyaux isolds, par centrifugation en gradient de sucrose et par chromatographie d'exclusion liquide ~ haute performance. Nos rdsultats montrent que les deux formes sdpardes sur hdparine-ultrogei correspondent aux dtats non transformd et transformd du rdcepteur. L'utilisation de la chromatographie sur hdparine-ultrogel permet donc d'envisager l'dtude de la transformation du r~cepteur ~ glucocorticofdes et de quantifier le r~cepteur sous ses deux formes grace ?t l'utilisation d'un seul support chromatographique. Mots-cl6s : h6parine-agarose / r6cepteur ~ glucocorticoides / transformation.

Summary - -

Rat liver cytosolic glucocorticoid receptor labelled with [3HI dexamethasone and stabilized with molybdate was bound to heparin-ultrogel and eluted with NaCi or heparin as a single peak o f radioactivity. After heat exposure o f cytosol, two steroid receptor complexes could be separated by NaC! or heparin. Characterization o f the two forms was performed by means of affinity towards isolated nuclei, sucrose g-adient centrifugation and gel exclusion high performance liquid chromatography. 7he results presented here suggest that the two forms eluted from heparin-agarose correspond to the untransformed and transformed states o f the glucocorticoid receptor complex. Taken together, these observations argue in favor o f heparin-ultrogel as a suitable procedure to study the mechanism o f glucocorticoid-receptor transformation. Key-words : heparin-agarose / glucocorticoid-receptor / transformation.

O To whom all correspondence should be addressed Abbreviations : BSA : bovinesenml albumin DCC : dextran coated charcoal Dex : de.~'amethasone

HAP : h.vdroxylapatite HPLC • high perlbrmance liquid chromatography

506

P. Blanchardie and Coll.

Introduction In rat liver cytosol, numerous studies have demonstrated the presence of two forms of glucocorticoid receptor complexes [I]. The untransformed glucocorticoid receptor complex may be characterized in the presence of sodium molybdate. The second form appears after heat exposure o f the cytosol and acquires an increased affinity for isolated nuclei by a process defined as transformation [2]. Heparin-agarose has been shown to interact with the estrogen receptor [3,4], progesterone receptor [5] and androgen receptor [6]. Therefore, chromatography on immobilized heparin has been employed for purification [7,8] and for characterization of receptors [6]. This led us to investigate whether or not the same methodology might also be used with the glucocorticoid receptor. In this report we describe the separation and purification of the two forms of glucocorticoid receptor using a single chromatographic step on heparin linked to acrylamide/ agarose.

Materials and methods

Chemicals 1,2 (n) -- [3H] dexamethasone (spec. act. 40 Ci/ mmole) was provided by the Radiochemical Centre (Amersham, England). Heparin-ultrogel and heparin ........... ,j were supplied by IBF (Viiieneuve-iaGarenne, France). Hydroxylapatite (Bio-Gel HTP) was obtained from Bio-Rad Laboratories (Richmond, USA). All other reagents were of analytical grade.

Animals Male Wistar rats were purchased from C.E.R.J. (Le Genest, France). Animals were adrenalectomized, as described previously [9].

Preparation of subcellular fractions All experiments were performed at 0-4°C. Livers were homogenized in 2 vol. (w/v) of 20 mM 2-mercaptoethanol, 20% glycerol (v/v), 20 mM potassium phosphate buffer, pH 7.40 (PMG buffer). Cytosol was obtained by centrifugation of the homogenate for two hours at 105 000 g. Nuclei were isolated as described by Tata [10].

Assay of specific binding Samples were incubated at 0-4°C with 10-SM [3H]-dexamethasone. Macromolecular-bound radioacti-

vity was measured by the dextran-coated charcoal technique [Ill or by batch assay of hydroxylapatite according to Mc Carty and Schwartz [12]. Correction for non-specific binding was made in the presence of an ! 000-fold excess of unlabelled dexamethasone.

Receptor transformation Heat transformation was induced by incubation for 30minutes at 25°C. Afterwards molybdate (10mM) was included in the assay to prevent secondary transformation during the prolonged contact with nuclei or heparin-agarose. In all cases where untransformed receptor was used the transformation process was blocked by addition of 10 mM sodium molybdate in the homogeneization buffer. The extent of receptor transformation was measured by incubation of the [3H] Dex bound complexes with isolated nuclei. Briefly, a 50~! aliquot of cytosol containing [3H] dexamethasone-receptor complexes (350 l'mol) was mixed with the suspension of nuclei (20-3001ig of DNA) and 5001il of 5 mM MgCi,, 2.5raM KCI, 10mM Na2 MoOn, 5 mM Tris/HCl buffer, pH 7.50 (TKM buffer) and incubated at 0-4 °C with gentle shaking. After 30 minutes the pellets were washed 4 times with ! ml TKM buffer and assayed for radioactivity.

Heparin-ultrogel chromatography 5 ml heparin-ultrogel were packed in a IBF I I column (0 1.14 cm) and equilibrated in 20 mM 2-mercaptoethanol, 10 mM Na.~ MOO4, 20 % glycerol (v/v), 20 mM potassium phosphate buffer pH 7.40 (PMG Mo buffer). Cytosol was loaded and e!ution was performed at a flow rate of 4.1 ml/h/cm 2. Fractions were collected, and aliquots were assayed for proteins, radioactivity and salts.

Gel exclusion high performance liquid chromatography Samples (0.05 ml to 0.2 ml) were applied on a 7.5 x 600 mm LKB Ultropac TSK-G 3 000 SW column connected to a WATERS Model 440 HPLC apparatus. Elution was performed at a flow rate of 60 ml/h with 1 mM EDTA, 400 mM KCI, 20 mM Tris/HC! buffer, pH 7.0. 1 ml fractions were collected and assayed for radioactivity.

Other techniques Proteins were measured by the Coomassie blue adsorption method [13] using BSA as standard. Radioactivity was determined in a Beckman LS2800 liquid scintillation spectrometer. Samples were counted in 2.5 ml Beckman Ready-Solv TM HP/b scintillation fluid. DNA concentration was estimated according to Kapuscinski and Skoczylas [14].

Heparin agarose chromatography of glucocorticoid receptor

507

Results and discussion 100 A

Interaction of untransformed receptor with immobilized heparin

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Figure 1 shows the elution patterns on heparin acrylamide/agarose of labelled rat liver cytosol. Cytoplasmic receptor was retained by the matrix and eluted with both NaCI (0 to 1 M) or heparin (0 to 0.1 mg/ml) gradients. Two peaks of radioactivity were observed, one in the void volume, the other at 0.25 M NaC! or 0.08 mg/ml heparin. The first peak (I) represents the unbound [3H] dexamethasone and non specific binding. The second peak (II) refers to the [3HI dexamethasone-receptor complex : (i) a 1 000-fold excess of unlabelled dexamethasone during incubation abolished the peak, (ii) radioactivity is resistant to DCC treatment. Under these conditions, with NaCI gradients, the purification factor was about 4-5-fold with 90 % yield, with heparin gradients, the receptor was purified 20-fold with 75 % yield. The behaviour of glucocorticoid receptor towards immobilized heparin is quite similar to that of other steroid receptors [3, 6]). One major difference lies in the efficiency of the purification.

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FIG. 2. -- Effect of increasing concentrations of magnesium chloride on the interaction of [3HI dexamethasone-receptor complexes with heparin-ultrogei. Aliquots of heparin-ultrogel (0.5ml) were incubated batchwise with 1.5 ml labelled cytosol and increasing concentrations of MgCI_,. After 30 minutes at 4 "C, the gel was washed with 4.5 ml PMG Mo buffer. Elution was performed in the presence of PMG Mo buffer containing 0.8 M NaCI. Specifically bound ['H] Dex in the eluate was measured by the DCC assay. The amount of receptor bound to immobilized heparin is given as percent of total receptor present in the cytosol.

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FIG. 1. -- Chromatography of molybdate-stabilized O'to;,lasn#c glucocorticoid-receptor complex front rat liver on hepalqn-ultrogel. Rat liver cytosol prepared in PMG Mo buffer was incubated overnight with I0-" M [-'H] dexamethasone. 5 ml labelled cytosol were applied to a column filled with 5 ml heparin-uitrogel. The column was washed with 2 ml of PMG Mo buffer and eluted with " A a 10 ml linear 0-1 M NaCI gradient in PMG Mo buffer. B a 10 ml linear 0-0.1 mg/ml heparin gradient in PMG Mo buffer. Each 1.45 ml fraction was assayed for total radioactivity ( e - - e ) , proteins (A--A) and salts ( e - - e ) .

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P. Bianchardie and Coll.

30 minutes at 25 °C. In contrast with the results presented above, three peaks of radioactivity were obtained. The first peak (I) is eluted in the void volume and can be accounted for by free [31-t] dcxamethasone and non specific binding. The second peak (II) is eluted in the gradient with the same concentration of NaCI or heparin than the earlier obtained with untransformed receptor (0.25 M NaCI or 0.08 mg/ml heparin). The third peak (III) is eluted later in the gradient (0.34 M NaCI or 0.33 mg/ml heparin). This result is consistent with the presence of a molecular form which possesses an increased affinity towards immobilized polyanions. It is likely that the complex present in the third peak corresponds to the transformed species of [3H] dexamethasonereceptor complex, in agreement with earlier results obtained with glucocorticoid receptor and different polyanionic exchangers [16]. In order to confirm the nature of the molecular forms of [3H] dexamethasone-receptor complexes, we investigated the optimal conditions of separation of the different peaks. As seen in Figure 4, step by step elution with NaC! or heparin, allowed a complete resolution of the three peaks of radioactivity. Under these conditions, form II was purified ,-10-fold with NaCI and heparin, whereas form III was purified -.- 25-fold with NaCI and -,- 4t3-fold with heparin. The two peaks (II and III) corresponding to the [aH] dexamethasone-receptor

Purification factors up to 100-fold were obtained by Molinari et al. [4] for the [3H] estradiol receptor complex. However, the experiments were conducted in the presence of magnesium chloride which increases about I 0-fold the affinity of the receptor for heparin-agarose. Therefore a smaller amount of gel can be used (one tenth of cytosol volume) with a subsequent lower contamination and a smaller e!ution volume. In our experiments, the ratio gei/cytosol was about 1 which makes our purification less impressive than that previously described. As shown in Figure 2, the addition of 50mM MgCI2 increases about five fold the amount of bound [3H] Dex-receptor complex. Under these conditions the purification factor reached 70-fold with 80 % yield.

Interaction of transformed receptor with immobilised heparin In the absence of molybdate, heat,exposure of the cytosol brings about the apparition of a molecular form of [3H] Dex-receptor complex able to translocate into isolated nuclei [15] or to interact with polyanions [16]. Figure 3 illustrates the NaCI and heparin gradient elution profiles from heparin-acrylamide/agarose for glucocorticold-receptor complexes in cytosol pre-exposed

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FIG. 3. -- Chromatoglaphy on heparin-uhrogel of preheated rat liver cytosol prepared in the absence of molybdate. Cytosol prepared in PMG buffer was incubated overnight with 10 -" M [3H] dexamethasone. After a 30 minutes exposure at 25 "C, samples (2 mi) were loaded onto the column of heparin-ultrogel. Washing was achieved using 2 ml 0.17 M NaC! in PMG Mo buffer and elution was performed in the presence of PMG Mo buffer with : A a 35 ml linear (0.17-0.37 M) NaCI gradient. B a 30 mi linear (0-1 mg/ml) heparin gradient. Each 0.8 ml fraction was assayed for total radioactivity ( o - - o ) and salts ( , - - , ) .

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Heparin-uhrogel chromatography of O'tosolic [sH] Dex'-receptor complexes : Stepwise elution.

2 ml [3H] Dex labelled cytosol prepared in PMG buffer and treated as de~cri.bed in Fig. 3 were loaded on the heparin-ultrogei column. The column was washed with l0 ml 0.17 M NaCl in P M G Mo buffer. Stepwise elution was performed as follows : A : l0 mi 0.27 M NaCI and 5 ml 0.37 M NaCl in PMG Mo buffer. B : 5 ml 0 15 m g / m l heparin; 5 ml 0.17 M NaCl and 5 mi 0.5 mg/mi heparin in PMG Mo buffer. Each 0.8 ml fraction was assayed for total radioactivity ( e - - e ) , preteins ( a - - A ) and salts ( , - - , ) or ( , - - , ) .

complexes were pooled and supplemented with 10-SM [3H] Dex and 2 mg/ml BSA. Preliminary results have shown that excess of free hormone was necessary to prevent dissociation of [3H] Dex-receptor complex and that supplementation with BSA was needed for measurement of specific binding with HAP batch assays. Total bound radioactivity (peak II + peak IlI) corresponded closely to the [3HI Dex-receptor complex applied on the heparin column. The mean U~UWut,u, between the two peaks was 36•5 + 6.0% for peak II and 62.8 + 5.8 % for peak III (n = 6).

Characterization of the molecular forms of the glucocorticoid receptor

To correlate the two receptor forms seen on heparin chromatography with those appearing in cytosol, receptor complexes were analyzed by different methods (Table I). In a cell-free system, using isolated nuclei, [3H] Dex-receptor complex present in prewarmed cytosol displayed affinity towards nuclei and addition of l0 mM molybdate blocked almost completely the transformation process. Only glucocorticoid receptor present in peak III interacted significantly with nuclei• Estimation of Stokes radii using exclusion size HPLC revealed the presence of a major peak with a Stokes radius of 6.6 nm in the molybdate-

stabilized cytosol. Transformation is accompanied by a decrease in the Stokes radius of the predominant peak form 6.6 nm (untransformed) to 3.4nm (transformed). The analysis of [3HI Dex-receptor complex in heparin chromatography eluates showed that peak II contained only the untransformed species (Rs = 6.6 rim) whereas peak Ill was constituted by the 3.4rim form similar to the transformed receptor present in ,-~ ! r d. ,...L ~ ,,-~ J q , . , ~ L11..7;3 ~ . / | .

Sucrose gradient analysis of molybdate-stabilized cytosol revealed a single sharp peak which sedimented as a 8.5 S complex (Table I). In contrast, complexes from heated cytosol gave two separate peaks, one corresponding to the 8.5 S form and the other sedimenting at 4.5 S. The [3H] Dex-receptor complex in peak If contained only the faster sedimenting form (8.5 S). Unfortunately, it is likely that the receptor present in peak III is very unstable, which compromized the characterization by sucrose gradient analysis. The disappearance of specific binding in peak Ill after 16 hours at 0-4 °C confirmed this unstability of the transformed receptor. Taken together, these data, in agreement with earlier results [2, 17, 18, 19l, indicate that peak II contains only the untransformed form and peak III the transformed species. Based on the data derived from heparin chromatography we calculated a -- 60% level of transformation

P. Blanchardie and Coll.

510

TABLE I Characteristics of [3H] Dex-receptor complexes in cytosolic extracts and eluates from heparin-uitrogel

Properties Elution from Heparin ultroge! ~d,,_i (M) Hep mg/m! ~.:0(a) Binding to isolated nuclei

% (b)

Sedimentation in S.~0.~ Sucrose gradient % HPLC

Rs(nm) % (c)

Peaks eluted from heparin-ultrogel II II I

Labelled cytosol + Molybdate 0.25 0.08 100

-- Molybdate 0.25 0.34 0.08 0.33 36.5 __ 6 62.8 + 5.8

0.7 ___ 0.07

35 + 2.7

10.7 _ !.6

41.4 _+ 4.2

8.5 100

8.5 36

4.5 64

8.5 100

not detected

6.6 100

6.6 37.6 _ 3.1

3.4 62.4 + 3.1

6.6 100

3.4 100

Chromatography on heparin-ultrogel and bindmg to isolated nuclei were performed as described under Materials and Methods. Sedimentation coefficients were obtained from sucrose gradient analysis. 5 and 20 % ~w/v) sucrose were prepared in PMG Mo buffer. 0.2 ml samples were layered on each gradient and centrifugation was performed at 280 000 g for 16 hours at 4 "C. 7-drop fractions were collected after puncturing the bottom of the tubes. Markers were : myoglobin (2 S), albumin (4.6 S), aldolase (7.35 S) and catalase (!i.35 S). Estimation of Stokes radii was obtained by reference to a standard curve K~'3 vs Rs. Calibration was carried out with the following markers : thyroglobulin (8.6 nm), ferritin (6.1 nm), albumin (3.6 nm), ovalbumin (2.8 nm) and trypsic inhibitor (2.2 nm). (a) values are the mean _ S.D. of 6 separate experiments. (b) values are the mean + S.D, of 4 separate experiments. (c) values are the mean + S.D. of 6 separate determinations.

which c o r r e s p o n d s to values currently observed in rat liver cytosol [20]. F r o m e x p e r i m e n t s p e r f o r m e d with isolated nuclei, a -- 35 % yield o f transform a t i o n was o b t a i n e d ; this difference could be e x p l a i n e d by the occurrence o f the dysactivation process as p r o p o s e d by M i l g r o m [21]. In the d y s a c t i v a t e d state the receptor retained its property o f binding h o r m o n e but h a d lost its ability to b i n d to nuclei. F u r t h e r m o r e o u r results show that p e a k III, only constituted by the 3.4 nm form, did not interact completely with nuclei. A similar o b s e r v a t i o n was recently r e p o r t e d by M u n c k a n d H o l b r o o k [22] showing a g r e a t e r efficiency o f D N A - c e l l u l o s e versus nuclei to m e a s u r e the extent o f receptor t r a n s f o r m a t i o n . Q u a n t i f i c a t i o n of the t r a n s f o r m a t i o n process by h e p a r i n c h r o m a t o g r a p h y gives closely identical results to those obtained in size exclusion H P L C a n d sucrose gradient centrifugation. H e p a r i n c h r o m a t o g r a p h y a p p e a r s as a convenient procedure for qualitative a n d quantitative estimation o f the t r a n s f o r m a t i o n process. This p r o c e d u r e allows a g o o d s e p a r a t i o n of the two f o r m s of glucocortic o i d - r e c e p t o r complexes by m e a n s o f a single matrix a n d can be easily t r a n s p o s e d to microsampie assays. F u r t h e r m o r e , with larger a m o u n t s o f

h e p a r i n - a c r y l a m i d e / a g a r o s e , one m a y expect to purify the glucocorticoid receptor c o m p l e x e s in the u n t r a n s f o r m e d o r / a n d t r a n s f o r m e d states.

Acknowledgments This work was supported by grants from LN.S.E.R.M. (contract CRL n ° 813015) and by U.E.R. of Medicine. We are grateful to A. Combalot for helping to prepare the manuscript. We thank Institut National de la Santd et de la Recherche Mddicale. Unitd 211 (Dr. J. Aubry) for its material assistance.

REFERENCES 1. Cake, M.H. & Litwack, G. (1976) in Biochemical actions of hormones (Litwack, G. ed.), vol. 3, pp. 317-390, Academic Press, New-York and London. 2. Schmidt, T.J. & Litwack, G. (1982) Physiol. Rev., 62,

! 131-1192. 3. Secco, C., Redeuilh, G., Radanyi, C., Baulieu, E.E. et Richard-Foy, H. (1979) C.R. Acad. Sc. Paris, 289, 907-910.

Heparin agarose chromatography o f glucocorticoid receptor 4. Molinari, A.M., Medici, N., Moncharmont, B. & Puca, G.A. (1977) Proc. Natl. Acad. Sci. USA, 74, 4886-4890. 5. Yang, C.R., Mester, J., Wolfson, A., Renoir, J.M. & Baulieu, E.E. (1982) Biochem. J., 208, 399-406. 6. Mulder, E., Foekens, J.A., Peters, M.J. & Van Der Molen, H.J. (1979) FEBS Lett., 97, 260-264. 7. Ratajczak, T. & Hahnel, R. (1980) J. Steroid Biochem.. 13, 439-444. 8. Puca, G.A., Medici, N., Molinari, A.M., Moncharmont, B., Nola, E. & Sica, V. (1980) J. Steroid Biochem.. 12, 105-113. 9. Lustenberger, P., Formstecher, P. & Dautrevaux, M. (1981) J. Steroid Biochem., 14, 697-703. 10. Tata, J.R. (1974) Methods in Enzymology (Fleischer, S. & Packer, S. ed.), vol. 31 A, pp. 253-262, Academic Press, New-York. II. Rousseau, G.G., Baxter, J.D. & Tomkins, G.M. (1972) J. Mol. Biol., 67, 99-1 i 5. 12. McCarty, G.R. & Schwartz, B. (1982) Invest. Ophtalmol. Vis. Sci., 23, 525-528.

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13. Bradford, M.M. (1976) Analyt. Biochem. 72_, 248-254. 14. Kapuscinski, J. and Skoczylas, B. (1977) Analyt. Biochem., 83, 252-257. 15. Milgrom, E., Atger, M. & Baulieu, E.E. (1973) Biochemistry, 12, 5198-5205. 16. Atger, M. & Milgrom, E. (1976) Biochemistry. 15, 4298-4304. 17. Grody, W.W., Schrader, W.T. & O'Malley, B.W. (i982) Endocrine Rev., 3, 141 ~ . 18. Vedeckis, W.V. (1983) Biochemistry, 22, 1975-1983. 19. Vedeckis, W.V. (1983) Biochemistry, 22, 1983-1989. 20. Barnett, C.A., Speck, L. & Litwack, G. (1983) Eur. J. Biochem., 134, 231-235. 21. Milgrom, E. (1981) in : Biochemical Actions of Hormones (Litwack, G. ed.), vol. 8, pp. 465-492, Academic Press, New-York. 22. Munck, A. & Holbrook, N.J. (1984) J. Biol. Chem., 259, 820-831.