Physicochemical characterization and transformation of the cytosolic glucocorticoid receptor from rabbit liver

J. steroid Biochem. Vol. 18, No. 6, pp. 789-799, Printed in Great Britain. All rights reserved

0022-473 1/83/060789-l 1$03.00/O Copyright 0 1983 PergamonPressLtd

1983

PHYSICOCHEMICAL CHARACTERIZATION AND TRANSFORMATION OF THE CYTOSOLIC GLUCOCORTICOID RECEPTOR FROM RABBIT LIVER P. BLANCHARD]E*, P. LUSTENBERGER,J. L. ORKINNEAU and S. BERNARD Laboratoire de Biochimie Medicale, U.E.R. de Medecine. I, rue Gaston Veil,

44035 Nantes Cedex, France (Received 27 April 1982) SUMMARY

Characterization of the glucocorticoid receptor from rabbit liver cytosol was studied in vitro. Binding of [3H]-dexamethasone showed a high affinity (K, = 2.4. 10m9M) and a concentration of binding sites of 0.3. lo-l2 mol/mg proteins. Association and dissociation rate constants for [3H]-dexamethasone were and 2.5. lo-“ min-‘. Competition experiments showed that dexamethrespectively 1.6. lo5 M-“mine’ asone and triamcinolone acetonide were the most effective competitors while progesterone and aldosterone competed very poorly. The stabilities of bound and unbound forms were investigated with and without molybdate and other oxyanions. Calibrated gel filtration gave a stokes radius of 5.6 nm and an apparent mol. wt of 280,000. The untransformed complex sedimented at 9 s in 5-20% sucrose density gradients and the transformed species sedimented near 4 s. The behaviour of the three forms of receptor. untransformed, transformed and molybdate-stabilized was studied on ionic exchangers. Transformation of the [3H]-dexamethasone-receptor complex was shown to occur both with high ionic strength and elevated temperature. The transformation step was almost completely inhibited by tungstate and molybdate while vanadate revealed a very slight inhibitory effect.

MATERIALS AND METHODS

INTRODUCIION

Animals and preparation

Until now glucocorticoid receptors have been described from a wide variety of tissues [l-lo]. These proteins are located mainly in the cytosol at very low concentrations and interact very strongly with synthetic glucocorticoids such as dexamethasone and triamcinolone acetonide. Preliminary experiments with various rabbit tissues have demonstrated the occurrence of a cytoplasmic glucocorticoid receptor in liver [ll, 123, but the characterization of these receptors has not been extensive. Indeed, in the view of the purification of the glucocorticoid receptor, the rabbit liver might provide a more important source of receptor than the rat liver. The purpose of this study was to define the characteristics of the glucocorticoid receptor from rabbit liver in the untransformed and transformed states?. This was carried out using different oxyanions like molybdate, vanadate and tungstate which are known to affect the stability and transformation of steroid receptors [14, 151. This investigation was necessary before purification properties

and allowed

us to compare

of the rabbit glucocorticoid

receptor

of cytosol

Male rabbits (Fauve de Bourgogne or New Zealand strains) were adrenalectomized under Nembutal anesthesia (30 mg/kg) and received 9?/,,NaCl to drink. Animals were killed by decapitation 3 days after surgery. The livers were perfused with cold NaCl 150 mM. All subsequent procedures were carried out at 4°C. The livers were homogenized in two vol. (w/v) of buffer A or C using an Ultra Turrax for 10 s and a Teflon-glass potter homogenizer. The crude homogenate was centrifuged at 105,000 g for 90 min in a Beckman L-5-50 ultracentrifuge. The supernatant was removed by aspiration after discarding the upper layer of floating lipids. pH was adjusted to 7.4 with K,HPO, 0.8 M and the resulting cytosol fraction was used immediately or stored at -70°C until further use. The cytosol preparation contained lS20 mg/ml proteins. Reagents

the with

receptors described in other species.

* To whom reprint requests should be sent. t Transformation refers to the process by which steroidbound cytoplasmic receptor acquires the property to bind to nuclei and polyanions [13]. 789

[1,2 (n)-3H]-dexamethasone, 20 Ci mmol- ’ and [ 1,2,4 (n)-3H]-triamcinolone acetonide, 22 Ci mmol- ’ were obtained from the Radiochemical Centre (Amersham, U.K.). [ 1,2,6,7-3H]-progesterone, 57 Ci mmol- ’ and [ 1,2-3H]-aldosterone, 22 Ci mmol- ’ were from C.E.A. (Gif sur Yvette, France). Unlabelled triamcinolone acetonide, cortisol, progesterone, 17/Sestradiol. corticosterone, estrone, estriol, testosterone, tetrahydrocortisone, cortisone, predniso-

790

P.

BLANCHARDIE

lone, Sa-dihydrotestosterone and aldosterone were purchased from Sigma Chemical Co (St. Louis, MO). Triamcinolone was a gift from Specia (Paris, France). Desoxymetasone and dexamethasone were obtained from Roussel UCLAF (Romainville, France). All other reagents were of analytical grade. Buffers Buffer A: potassium phosphate 20 mM, Na,MoO, 10 mM, 2-mercaptoethanol 20 mM, glycerol 20% (v/v), pH 7.4. Buffer B: potassium phosphate 160 mM, Na,MoO, 10 mM, 2-mercaptoethanol 20 mM, glycerol 20% (v/v), pH 7.4. Buffer C: potassium phosphate 20 mM, 2-mercaptoethanol 20 mM, glycerol 20% (V/V), pH 7.4. Buffer D: Tris 10 mM, EDTA 1 mM, 2-mercaptoethanol 1 mM, pH 7.4. Assay of specific binding Duplicate samples were incubated at @4”C with lo-* M C3H]-dexamethasone. The non-specific binding was determined by incubating parallel samples in the presence of lOOO-fold excess of unlabelled dexamethasone. Charcoal adsorption assays [ 161 were performed in duplicate to measure the extent of steroid binding. Binding parameters were calculated according to Scatchard[ 171. Competitiae binding studies Competition assays were performed in duplicate with 10e8M [3H]-dexamethasone in the absence or presence of increasing concentrations of non radioactive steroids. Specific and nonspecific binding were measured in duplicate after a 16 h incubation time at @4”C as described above. Metabolism

of steroids in liver cytosol

0.5 ml Cytosol were incubated with lo-* M [3H]-aldosterone, C3H]- dexamethasone, [3H]-cortisol and [3H]-progesterone for 16 h at &4”C. Incubations were stopped by adding 1 ml water-satured ethylacetate containing 5 pg of unlabelled homologous steroid as described by Snochowski et ul. [18]. Steroids were extracted twice with 1 ml ethylacetate and the pooled extracts were evapored to dryness. Extracts were chromatographed on silicagel thin layer plates (0.25 mm silicagel 60 F 254, Merck, Darmstadt, GFR). The plates were developed in two standard systems: chloroform-ethylacetate (3 : 2, v/v) for proand dichloromethaneeacetone-methanol gesterone (7: 2: 1, v/v) for cortisol, dexamethasone and aldosterone. Control samples containing steroids were run in the same conditions. Evaluation of radioactivity was performed by scraping the areas corresponding to the standard steroids and those corresponding to metabolites. The radioactive products were eluted from silicage1 with 2 ml methanol directly into scintillation vials. After evaporation 2.5 ml scintillation liquid were added.

et al.

Sucrose density gradient analysis Cytosol preincubated 16 h at &4 C with 2. lo-’ M [3H]-dexamethasone was treated on a charcoal pellet as described by Thorsen[19]. Duplicate samples (0.2 ml) were layered on linear 5-2O”,b sucrose gradients (4.8 ml) prepared in the appropriate buffer. Centrifugation was carried out for 30 h at 48,000 rev.jmin using a swinging bucket rotor SW-50. Fractions of 7 drops were collected from the bottom of the tubes. Myoglobin (2s) bovine serum albumin (4.6 s), aldolase (7.35 s) and catalase (11.35 s) were run in separate tubes to determine sedimentation constants. Molecular

sieve chromatograph!

One hundred and eighty ml of AcA 34 from IBF (Villeneuve-la-Garenne, France) equilibrated in buffer B was packed into a K 16/100 column Pharmacia (Uppsala, Sweden). The void volume (V,) and total volume (V,) were determined with Dextran blue (Pharmacia) and [3H]-dexamethasone respectively. Preincubated cytosols (5 ml) were loaded onto the column and elution was performed at a flow rate of 4.35 ml/h/cm’. 1.45 ml fractions were collected and aliquots were assayed for proteins and radioactivity. The calibration proteins used were: ovalbumin. albumin, aldolase, catalase from Boehringer Mannheim. Ion exchange Chromatography DEAE-trisacryl, phospho-ultrogel and hydroxylapatite-ultrogel (HAP) were purchased from IBF and were used as suggested by the manufacturer. Routine chromatography was performed as follows: columns (volume: 30 ml; 4 2.5 cm) were poured and equilibrated in buffer A pH 7.2 (DEAE and HAP) or buffer D pH 7.4 (phospho-ultrogel). Preincubated cytosol (3.5 ml) was applied and the column washed until 28 ml or dropthrough was collected. Receptor was then eluted with a linear salt gradient (60 ml total volume) and columns were rinsed with 30 ml high salt buffer. 3.5 ml fractions were collected. Flow rates were 18 ml/h/cm2 for HAP and DEAE, and 4.8 ml/h/cm’ for phospho-ultrogel. Aliquots (0.25 ml) were analyzed by liquid scintillation counting and salt concentration was estimated by flame emission spectrophotometry (Klina flame photometer, Beckman). The linear salt gradients (in buffer A or D) utilized were as follows: DEAE-trisacryl and HAP-ultrogel (0.02 M-0.40 M potassium phosphate): phospho-ultrogel (up to 0.4 M NaCl). Receptor

transjtirmation

Two methods of receptor transformation were utilized: exposure to high salt concentration (0.4 M KCl) or to elevated temperature (23’C). The extent of receptor transformation was measured by DEAE-trisacryl, phospho-ultrogel chromatography. sucrose gradient centrifugation and

Rabbit

liver glucocorticoid

uptake [13]. Ion-exchange chromatography was performed as described above with mini-columns (1 ml packed gel). Sample volumes were 0.5 ml. Elution was carried out as follows: flow rate 36 ml/h, washing volume 9 ml, gradient 20ml total volume. 1.2 ml fractions were collected and analyzed as previously described. Kinetics and inhibition of receptor transformation were studied using a batch assay with phospho-ultrogel. An aliquot of 0.05 ml cytosol was transferred to a test-tube containing 0.2 ml phospho-ultrogel and 0.85 ml buffer D. Adsorption was performed by gentle shaking at O-4’C for 30 min. Subsequently, the tubes were centrifuged at 5008 for 5 min. The supernatant was discarded and the gel washed four times with 1 ml buffer D. After the last washing, the gel was transferred to a scintillation vial and counted. Radioactivity in the gel pellet corresponded to the transformed species of the glucocorticoid receptor complex. When high salt exposure had been performed, the samples were diluted prior to adsorption on ionexchangers.

791

receptor

nuclear

Protein and radioacticity

measurements

Proteins were measured by the Coomassie blue adsorption method [20] using bovine serum albumin as standard. Radioactivity was determined in an Intertechnique SL 4000 liquid scintillation spectrometer. The samples (0.15 ml) were counted in 2.5 ml Beckman ReadySolvTM HP scintillation fluid with a tritium efficiency of 4045%. RESULTS AND DISCUSSION

Binding parameters In order to determine the total amount of receptor bound, we have reinvestigated the differential dissociation with dextran-coated charcoal (Fig. 1). The dif-

i-A_

--.10 Time

20

30

(min)

Fig. 1. Effect of length of charcoal adsorption. Cytosol was incubated for 4 h at G4”C with lo-* M C3H]-dexamethasone. Non-specific binding was achieved in the presence of a 1000-fold excess of unlabelled dexamethasone. A half volume dextran-coated charcoal solution (3% Norit-A (w/v), 0.3% Dextran T-70 (w/v) in buffer A without glycerol) was added and the mixture was incubated for various lengths of time. (A---A) Total binding; (A--A) Non specific binding.

I 5

I IO

I 15

Free nM Fig. 2. Specific binding of [3H]-dexamethasone to rabbit liver cytosol. Aliquots of cytosol were incubated for 16 h at @4”C with increasing concentrations of C3H]-dexamethasone alone or supplemented with a lOOO-fold excess of unlabelled dexamethasone. The amount of specifically bound

labelled steroid was measured by the charcoal assay. (A) Binding curve. (B) Scatchard plot of the binding data.

ference between total and non-specific binding which represents specific binding remains constant from 2 min. But the ratio of non-specific binding to specific binding still decreases with longer incubation times. These observations led us to choose a 10 min adsorption time. This timepoint appeared to be optimal when several assays were performed simultaneously. Binding curoe and Scatchard analysis. The amount of specific binding was determined at several concentrations (2. lo- ‘O-3. 10m8 M) of labelled dexamethasone. Figure 2 shows on the reciprocal plot a single class of binding sites for the ligand with an apparent KD of 2.5. lo-’ M. Average values for equilibrium dissociation constant and concentration of receptor were respectively 2.4. lo- 9 M and 0.3 . lo- I2 mol/mg proteins. Previously reported values were in the range of 1.5.1O-‘M to 8.1O-‘M for K, and 0.12.10-12 mol receptor/mg proteins. But Giannopoulos et a[.[ 1 l] stressed on the underestimation of this number of binding sites. In rat liver cytosol the apparent dissociation constant is slightly higher (3.7. lo-’ M), [21] also and the number of binding sites: 0.6. lo- l2 mol/mg proteins [21,22]. Association and dissociation kinetics. The time course study of [3H]-dexamethasone (Fig. 3A) showed that: (i) equilibrium was reached after 5 h and (ii) a plot of binding of l/free steroid versus initial time is linear indicating a second order kinetics [23]. The mean association rate constant calculated was 1.6. lo5 Mm’.min-’ for C3H]-dexamethasone. The binding remained maximal up to 20 h. Therefore overnight incubations (1618 h) were performed as often as possible. Dissociation was studied after addition of an excess of unlabelled dexamethasone (Fig. 3B). C3H]-dexamethasone dissociates with a half-life of 48 h. The dis-

P. BLANCHARVIErt al

B

min

h Time

Time

(h)

Fig. 3. A-Association kinetics of [3H]-dexamethasone binding to rabbit liver cytosol. Cytosol prepared in buffer A was incubated at G4”C with lo-* M [3H]-dexamethasone alone or with a IOOO-fold excess of unlabelled dexamethasone. At the indicated times duplicate samples were removed and assayed for specifically bound radioactivity. Inset: Inverse of free [3H]-dexamethasone UStime. (B) Dissociation rate of [‘HI-dexamethasone. Cytosol was incubated for 16 h with 1Om8 M [3H]-dexamethasone. Dissociation was initiated by the addition of a IOOO-fold excess of unlabelled dexamethasone. At the indicated times triplicate aliquots of cytosol were removed and assayed for residual bound radioactivity using charcoal adsorption assay.

sociation follows a first order kinetics and the average value of the rate constant was 2.5. 10d4 min-‘. From the kinetic constants, the equilibrium dissociation constant was 1.6. 10m9 M which is in good agreement with the value obtained from the equilibrium data (2.4. 1O-9 M). In the case of [3H]-triamcinolone acetonide, the apparent rate constant of dissociation after isotopic dilution was 4.5. 10m4 min- ’ with a half-life of 28 h (data not shown). The association rate constant for C3H]-dexamethasone is in the same order of magnitude in rat liver [21]. The main difference appears with the dissociation rate. The dissociation rate constant of the glucocorticoid receptor [3H]-dexamethasone complex from rat liver is five times greater [21]. Moreover the faster dissociation rate of triamcinolone acetonide versus dexamethasone contrasts with the results obtained in other species [24-261. Specijicity of binding. The specificity of C3H]-dexamethasone binding to cytoplasmic glucocorticoid receptor was studied by a competitive binding assay at equilibrium. Non radioactive steroids were added at concentrations up to 10m6 M. Results are expressed as “competition factors” i.e. ratio of the concentrations of the competitor sample to the reference substance required for 50% inhibition of specific binding (Table 1). The synthetic glucocorticoids are the most effective competitors. Dihydrotestosterone, testosterone, 17@estradiol, estrone and estriol are ineffective to displace the tritiated ligand. In rabbit lung [4, 1l] the binding of triamcinolone acetonide, cortisol, corticosterone, desoxycorticosterone and progesterone is in the same order of potency. All the 17a-hydroxylated steroids have a relatively higher affinity towards the receptor. This result agrees

with the fact that the rabbit is a cortisol secreting species. A marked change in the order of potency of the different steroids for binding to the receptor is observed in the rat, a corticosterone secreting species [4]. The most noticeable differences are a reversal in the order of affinities of cortisol and corticosterone, dexamethasone and desoxymetasone, and in contrast Table 1. Specificity

of [3H]-dexamethasone to rabbit liver cytosol

Unlabelled competitor Triamcinolone acetonide Desoxymetasone Triamcinolone Prednisolone Cortisol Promegestone (R 5020) Cortisone Corticosterone Aldosterone Desoxycorticosterone (DOC) Progesterone Tetrahydrocortisone Dihydrotestosterone Testosterone 17/Gestradiol Estrone Estriol

binding

Competition factor 4 9 10 10 19 160 200 250 250 605 800 > 1000 > 1000 > 1000 > 1000 >lOOo >lOOO

Aliquots of cytosol were incubated for 16 h at with 1Om8 M [3H]-dexamethasone alone or with varying concentrations of unlabelled competitor. Non-specific binding was determined in the presence of a lOOO-fold excess of unlabelled dexamethasone. Results are expressed as “competition factor”, i.e. ratio of the concentrations of the competitor steroid to the reference substance required for 50:~ inhibition of specific binding O-4°C

Rabbit

liver glucocorticoid

793

receptor

Table 2. Half-lives of glucocorticoid receptor inactivation: Effect of temperature and molybdate Half-life (h)

[Competitor]

/ [‘H-Dexamethasone]

Fig. 4. Effect of competitors on the binding of [3H]-dexamethasone to rabbit liver cytosol. After 16 h at G4”C, specific binding was determined as described in Materials

and Methods. Ordinate: ratio of binding of C3H]-dexamethasone in the presence of competitior (B) to binding in the absence of competitor (E,,,). Abcissa: concentration ratios. t-0 Dexamethasone. x~ x Corticosterone. APA Desoxymetasone. Lm Aldosterone. 0-O Cortisol. A-A Progesterone.

with results obtained with rat liver cytosol[27] progesterone and aldosterone are only poor competitors (Fig. 4). Under the conditions used to assay the steroid binding, the metabolism of dexamethasone, cortisol and aldosterone was low: 0, 11 and 25% respectively. The metabolism of progesterone was very greater (88%) and may account for the low binding affinity of progesterone in rabbit liver. This observation corroborates the finding of Giannopoulos et a/.[4]. Stability of the receptor We first compared the stability of the bound and unbound forms of the receptor and the effects of temperature, ionic strength and pH. The stability of the unbound form was measured by incubating cytosol prepared in buffer C for various lengths of time at 0, 20 and 30°C. Binding activity was

100

200

KCL

400

(mM)

0°C

20°C

Unbound form - MO + MO IO-‘M +MolO-‘M

I1 20 23

2 4f 5:

Bound form - MO + MO IO-’ M

128 235

+ MO lo-‘M

570

30°C

’

;L : 37

12 16 44

I_) 5 9

Inactivation of unbound glucocorticoid receptor was assayed by incubating aliquots of cytosol at c-4. 20 or 30°C for various times with or without molybdate followed by duplicate incubation with lO-‘H [3M]-dexamethasone for 2 h at &4”C. A control sample was treated in the same conditions (2 h at O-4°C with IO-* M [3H]-dexamethasone). Inactivation of the bound form was performed in

the same way except that preincubated cytosol was used. Estimations of half-lives of inactivation were achieved by using semi-log plot of residual binding

t’s time.

then assayed after a 2 h incubation time with lo-’ M [3H]-dexamethasone. The inactivation followed a first order kinetics. Results presented in Table 2 show that 50% of binding activity is lost after 11 h at 0°C. The half-lives of receptor inactivation at 20 and 30°C are 2 h and 30 min respectively. The binding activity is stable for 3 h at 0°C in KC1 0.15 M up to 0.30M but 50% of binding activity is lost in 0.4 M KCI (Fig. 5A). As shown in Fig. SB, maximal binding activity occurs between pH 6.4-6.7, but 90% binding activity still remains at pH 7.4. The inactivation of the unbound form of the glucocorticoid receptor varies greatly according to the tissue. At 0°C the half-lives of

6

7

PH

Fig. 5. Stability of the unbound form. (A) Effect of salts. Cytosol prepared in buffer A (A-A) or C (A---A) was supplemented with varying concentrations of KCl. Duplicate samples were incubated at &4”C for 3 h and the residual binding activity was determined by incubation 2 h at G4”C with 10-s M [3H]-dexamethasone. Residual binding is expressed in relation to maximal binding (B/B,,,). (B) Effect of pH. Cytosol was prepared in buffer A (A--A) or C (A-A). pH was adjusted by adding 0.25 M phosphate buffer. Aliquots were incubated 3 h at &4”C and then 2 h at (t4”C with 10-s M C3H]-dexamethasone to measure residual binding. All data arc expressed in relation to maximal binding (B/B,,,).

794

P. BLANCHARDIE et al.

inactivation of the rat spleen glucocorticoid receptor is 15 h [28], 4 h for the mammary cytoplasmic receptor [29] and 4 h in rat thymocytes [30]. In rat liver the inactivation rate is much slower (15-25 h) [31]. The discrepancies among the values obtained for the inactivation rates would be influenced by cytosolic factors not identified [28] or by conditions of preparation and recuperation of cytosol which are not identical [32]. As compared to the rat liver [33] the unbound rabbit receptor is more stable under high ionic strength up to 0.3 M KU. When the glucocorticoid receptor is complexed with [3H]-dexamethasone 50”,, of binding activity is lost after 128 h at 0 C. Higher incubation temperatures also show a stabilizing effect of the steroid (Table 2). lZj%ct of‘ m/_hhte orz binding stability. Over the past few years numerous reports have shown the stabilizing effect of molybdate on steroid hormone receptors [14, 15,341. To determine if this was true for this tissue, we examined the effects of sodium molybdate on the stability of the unbound form of the receptor in the same conditions of temperature. ionic strength and pH. Cytosol was prepared in buffer C containing varying amounts of molybdate up to 0.1 M. The presence of molybdate enhances significantly the half-lives of inactivations at all temperatures tested (Table 2). The effect was concentration dependent, it appeared at a concentration of 5. lo- 3 M molybdate and increased slightly while increasing molybdate (Fig. 6). Dexamethasone binding remains maximal in high ionic strength in presence of lo-’ M molybdate (Fig. 5A). This protective effect of molybdate was already demonstrated by Miras et al.[l], McBlain ef a!.[291 and Nielsen et al.[31,32]. With a similar concen-

4

0

I

I

I

IO Anions

k-b--

5

E d Li

100 Fractions

Fig. 7. Ultrogel ACA 34 chromatography of the glucocorticoid receptor. Chromatography was performed as described in Materials and Methods. Cytosol was labelled with 2’ IO-* M [3H]-dexamethasone. Elution was achieved with buffer B. Standard proteins were I. catalase: 2. aldolase; 3, BSA; 4, ovalbumin.

tration of molybdate the area of optimal binding occurs in a narrow range of pH (Fig. 5B). The addition of both dexamethasone and molybdate revealed a high stabilizing effect (Table 2). &%ct of various compounds on binding stability. Fluorure, tungstate and vanadate were tested in order to determine their effectiveness to stabilize the steroid binding activity. These compounds were added to cytosol preparations at concentrations varying from 10m3 M to 0.1 M. Samples were incubated at 20°C for 3 h and residual binding activity was tested with lo-’ M [3H]-dexamethasone for 2 h (Fig. 6). Fluorure gives no significant effect on the binding activity in the concentration range tested. Vanadate gives a maximal stabilizing effect at 25. 10e3 M and appears to inhibit binding activity above 50, 10e3 M. Tungstate stabilizes the binding activity at a concentration of 5’ 10m3 M and has an inhibitory effect above 50. 10m3 M. Molybdate, in the same experiment, has a protecting effect at the concentration of 5. lo-” M without inhibitory effect for highest concentrations. The most protective effect appears with tungstate and molybdate. In rat liver and IM-9 cell cytosols, inhibition of inactivation of unoccupied receptor by molybdate and vanadate is similar [35].

100

(mM)

Fig. 6. Dose-response curve for the effect of molybdate, vanadate, tungstate and fluorure on binding stability. Cytosol prepared in buffer C with increasing concentrations of molybdate (A---A), vanadate (+o), tungstate (t-m) and fluorure (x __ x ) up to 0.1 M, was kept at 20°C for 3 h. Afterwards triplicate samples were incubated with lO-s M [3H]-dexamethasone for 2 h at G4”C. Residual specific binding was determined by the charcoal assay. Results are expressed as the ratio of residual binding to initial binding activity. Initial binding was determined using an aliquot incubated 2 h with the radiolabelled Itgand.

Molecular

parameters

Geljikration

chromatography.

and in the presence

of molybdate

In high ionic strength the C3H]-dexameth-

complex eluted as a single symmetrical peak (Fig. 7). The Stokes radius was found to be 5.6 nm. The apparent molecular weight of the molybdate stabilized complex calculated from the calibration plot was 280,000. Similar sizes have been found several steroid hormones receptors of for [23,34,36,37]. In the same conditions, with rat liver cvtosol. we found a Stokes radius of 5.0-5.1 nm. asone

receptor

Rabbit

liver glucocorticoid

4

3 ’

1

..

.I\.. ! \

I/

I

~~

,<*

IO

T

20

B

Fractions Fig. 8. Sucrose density gradient pattern of the rabbit liver cytosol. Cytosol was incubated overnight with 2. IO-* M [3H]-dexamethasone and treated as described in Materials and Methods before layering on 5-20% sucrose gradient. Centrifugation was performed at 2OO.OOOg for 30 h. A---A Cytosol incubated in buffer A. x-x Cytosol incubated in buffer B. C-W Cytosol incubated in buffer C. t-0 Salt transformed receptor in buffer A (20°C; 0.4M KCI; 30min). O----O Cytosol incubated with a IOOO-fold excess of unlabelled dexamethasone in buffer A. Standard proteins were 1, myoglobin; 2, BSA: 3, aldolase; 4, catalase.

Sucrose grudient centrijirgation. On 5520% linear

su-

glucocorticoid receptor complex was found to sediment at 9 s under low salt conditions. The C3H]-dexamethasone-receptor complex was detected as a single peak and a lOOO-fold excess of unlabelled dexamethasone completely abolished the radioactive peak (Fig. 8). Similar results were obtained with [3H]-triamcinolone acetonide (data not shown). Under high salt-conditions, the presence of 10. 10e3 M molybdate did not alter the sedimentation pattern of the complex [37]. Heating at crose

gradients,

the

untransformed

receptor

795

23°C or exposure to 0.4 M KC1 caused the occurrence of a second peak of radioactivity at 4 s corresponding to the transformed state of the receptor [38]. Recently, Murakami et a/.[381 showed that addition of molybdate did not cause aggregates of larger size. Ionic properties. When cytosol was applied to hydroxylapatite in presence of 10’ 10m3M molybdate a single peak of radioactivity eluted at 0.16 M phosphate (Fig. 9A). The first peak corresponded to nonspecific binding (data not shown). On DEAE-trisacryl, untransformed C3H]-dexamethasone receptor was completely adsorbed. Elution with a linear 0.02-0.4 M phosphate gradient yielded a single peak of radioactivity at 0.16 M phosphate (Fig. 9B). The same result was obtained with molybdate stabilized receptor. After transformation of the receptor, 2 peaks of radioactivity were obtained: (i) non-retained radioactivity corresponds to the transformed species; (ii) adsorbed radioactivity eluted at 0.16 M phosphate refers to the untransformed state (Fig. 9B). When untransformed cytosol was applied on a phospho-ultrogel packed column, all protein bound radioactivity was eluted in the void volume. Here again, the same result was obtained with molybdate stabilized receptor. On the same matrice transformed receptor eluted at 0.18 M NaCl (Fig. 9C). For each kind of ion exchange chromatography, the peak of radioactivity corresponded to specifically bound [3H]-dexamethasone: (i) lOOO-fold excess of unlabelled dexamethasone during the incubation abolished the peak; (ii) the radioactivity is resistant to dextran-charcoal treatment. Similar results were obtained recently by Vedeckis[39] for the AtT-20 mouse pituitary tumor cell line glucocorticoid receptor. With the same chromatographic procedures the glucocorticoid receptor from rat liver cytosol showed similar patterns of elution (data not shown).

Fractions Fig. 9. Ion exchange chromatography of the rabbit liver cytosol. Cytosol was labelled with 2’ lo-* M [3H]-dexamethasone in buffer A or C. After 5 h aliquots of untransformed (-0) and transformed cytosol (M) were chromatographed as described under Materials and Methods. Cytosol was transformed by 30 min exposure to 0.4 M KC1 and chromatographed on the same column after l/3 dilution. All samples were treated by dextran-charcoal prior to chromatography. (A) hydroxylapatite. (B) DEAEtrisacryl. (C) phospho-ultrogel.

796

P. BLANCHARIJIE et (11

The differential behaviour of the two forms of glucocorticoid receptor complexes on cationic and anionic exchangers allowed us to evaluate quantitatively the proportions of transformed and untransformed states. Transformation

of the steroid-receptor

complex

It is commonly

accepted that receptors may be transformed after binding with horthe mone [34,38,40]. This step would be necessary for the transfer of the hormone to the nucleus of the cell. Different factors have been reported to promote steroid-receptor transformation in citro: elevated temperature, ionic strength. ATP, dilution or alkaline pH [41]. It was therefore interesting to check whether the rabbit liver glucocorticoid receptor could also be transformed by one or several of these treatments. The transformation step was measured using three different procedures: chromatography on DEAE-trisacryl and phospho-ultrogel, and centrifugation on sucrose density gradients. The transformed complex exhibited an increased affinity towards phosphoultrogel, a decreased affinity for DEAE-trisacryl and a 4s sedimentation constant. In each assay an aliquot of cytosol was incubated in presence of isolated nuclei. Nuclear uptake by isolated nuclei only showed a qualitative response. Milgrom, in a recent report, stressed the difficulty to obtain good quantitative results with isolated nuclei [41]. Temperature and salt mediated tran.sfiwmation. Cytosol prepared in buffer C was labelled “overnight” with 10m8 M C3H]-dexamethasone. Aliquots were either left at O-4 ‘C or exposed at 23 C or exposed to elevated ionic strength (0.4 M KCI). Warming at 23’C for 120 min gave IO?,> transformation. When the cytosol was brought to 0.4 M KC1 during the same time, Table 3 shows that 35”;, transformation occurred. Treatment of cytosol with 10e2 M ATP at 0 C gave no significant transformation. This result differs from the progesteroneereceptor complex transformation

Table virro

3. ERect of different factors on the ill transformation of the glucocorticoid receptor

Treatment

Transfol(mation

30 min 0 C 30 min 23’ C 120 min 120min 60 min 30 min 120 min

0 5 IO 0 IO <5 15520 35

0 C 23 C IO mM ATP 0 C 0.4 M KC1 O‘C 0.4 M KCI OC

Cytosol prepared in buffer C was incubated with 10-s M [3H]-dexamethasone at O-4°C. Samples were treated as indicated. In each case a control sample was treated in the same conditions except the factor inducing transformation. Estimation of transformation was performed by three different methods: DEAE-trisacryl and phospho-ultrogel chromatographies and sucrose density gradient centrifugation. The three methods gave comparable rest&s. The percentages given on the table are the average values of the results obtained.

which has been reported to occur with 10m2 M ATP at 0 C [42]. In order to determine the optimal conditions of transformation of the rabbit liver glucocorticoid receptor a phospho-ultrogel batch assay was performed. This assay was quicker and more convenient than chromatographic procedures. Figure 10 shows the kinetics of transformation and the effect of ionic strength. KC1 concentrations varying from 0.1 to 0.4 M showed that 307; transformation occurred with 0.4M KC1 at 0 C and 50”,, at 20’C with an incubation time of 120min. The percentages of transformation appeared to be highly dependent upon the conditions used. 0.4 M KC1 at 20-C was the most effective factor of transformation

IOOC B

A

KC1

(MI

Time

(h)

Fig. 10. Influence of temperature, ionic strength and incubation time on the transformation. Cytosol prepared in buffer C was labelled with 10-s M [3H]-dexamethasone for 16 h at O-4°C. (A) Labelled cytosol was incubated in presence of varying concentrations of KC1 at 0°C (A--A) and 20°C (A---A) for 2 h. (B) Incubations were performed with (--) or without (. .) 0.4 M KC1 at O’C (A---A) and 20°C (A-A) during various lengths of time. Specific binding to phospho-ultrogel batch assav was determined in duplicate at each concentration as described under Materials and Methods. Each point represents an average of two separate experiments

liver glucocorticoid receptor

Rabbit

797

Pp G

05 MoOa

I7

mM

Oxyanions

(mM)

Fig. Il. Dose-response curve for the effects of oxyanions on the transformation. Labelled cytosol prepared in buffer C was incubated at 20°C for 2 h in the presence of 0.4 M KC1 and various concentrations of molvbdate (A---A), vanadate (t--O) and tungstate (6W). (A) Residual specific binding of [3H]-dexamethasone to receptor was determined in duplicate for each concentration. An aliquot was left 2 h at 0°C without treatment with 0.4 M KC1 to evaluate initial binding (En). (B) Transformation of [3H]-dexamethasone receptor was measured in duplicate for each concknration by phospho-ultrogel batch assay (see Materials and Methods). CSO refers to the concentration of oxyanions required to reduce 50% inactivation and transformation.

for the rabbit liver glucocorticoid receptor. With the usual conditions of transformation (i.e. 30 min at 23°C or 0.4 M KC1 at 0°C for 60 min) we could not obtain more than 25’7: transformation. McBlain et a/.[431 obtained 60% transformation using mammary tissues of Balb/c Mice with 0.4 M KC1 at @4”C. With liver or kidney cytosol, heat treatment gave 4@45% transformation [44]. The transformed receptor of rabbit liver appears to be similar to that described in other species. But the conditions of transformation seem to be different, optima1 results being obtained only in conjunction with ionic strength and temperature. Effects of oxJlanions on the transformation. McBlain et a!.[431 suggested recently that inactivation and transformation could be two related processes. According to this hypothesis inhibitors of transformation like vanadate, molybdate, and tungstate would have similar effects on the two mechanisms. In order to test this eventuality, inactivation and transformation were assayed in presence of various concentrations of oxyanions. When preincubated cytosol was submitted to transformation, there is a loss of receptor due to inactivation. Results presented in Fig. 11A show that molybdate and tungstate appeared highly effective against inactivation in a dose-dependent manner. In the same concentration range, vanadate was ineffective to protect the glucocorticoid receptor against inactivation. These results are in good agreement with those obtained with unoccupied receptor. As shown in Fig. llB, the transformation step was also inhibited by tungstate and molybdate; vanadate appeared to be less effective. The effects of molybdate and tungstate are dose-dependent. The effective dose of transformation inhibition is very similar to the concentration necessary to protect occupied or unoccupied receptor against inactivation. The inhibitory effect of these compounds seems to be related to their common chemical structure rather

than by inhibition of phosphatases [39] but their mechanism of action is still unclear. It has been proposed that tungstate would bind directly to the glucocorticoid receptor complex [38], however, an other explanation would be that oxyanions bind with an other factor which could interact with the glucocorticoid receptor complex [45]. The results presented here, particularly the dosedependent effect of oxyanions on the prevention of both receptor inactivation and transformation, reinforce the hypothesis of McBlain et al. that the processes of in vitro transformation of glucocorticoid receptor complex and the in vitro inactivation of receptor are related. CONCLUSION

The glucocorticoid receptor from rabbit liver appears to be closely related to receptors from other species. Analysis of binding studies show that like glucocorticoid receptor from chicken [28], the rabbit receptor seems to be more specific for glucocorticoids. The molecular parameters of molybdate stabilized, transformed and untransformed receptor complexes presented are similar to those from other species. In vitro functional analysis reveals that transformation occurs in a similar manner. The differences observed, namely in the extent of transformation, may not be taken as proper molecular characteristics without further investigation, because of possible cytosol factors may greatly influence the parameters studied. The effects of molybdate and other oxyanions are consistent with the hypothesis of a relationship between in vitro transformation and in vitro inactivation of the glucocorticoid receptor complex. Further studies are now in progress to explain the mechanism of action of these compounds on the transformation and inactivation steps. Another way of

P. BLANCHARDIE rt

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research is to apply to the rabbit liver glucocorticoid receptor the purification procedure that we have already described for the receptor from rat liver [46,47]. This would allow us to investigate the precise mechanism on a better defined fraction of untransformed glucocorticoid receptor. Ackno~~lrdgements-This work was supported by grants from INSERM (contract CRL No. 8130151.and bv U.E.R. of Medicine. We thank U 211 INSERM (Dr J. Aubry) for putting ultracentrifuge our disposal.

and

scintillation

spectrometer

17.

18.

19.

at 20.

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