Microheterogeneity of agonist and antagonist glucocorticoid receptor complexes detected by isoelectric focusing and modifications induced by receptor activation

Biochimica et Biophysica Acta 927 (1987) 129-138 Elsevier

129

BBA 11764

M i c r o h e t e r o g e n e i t y of a g o n i s t and a n t a g o n i s t glucocorticoid r e c e p t o r c o m p l e x e s d e t e c t e d by isoelectric f o c u s i n g a n d m o d i f i c a t i o n s i n d u c e d by r e c e p t o r a c t i v a t i o n P i e r r e - M a r i e D a n z e , Pierre F o r m s t e c h e r , C l a u d e R i c h a r d and Michel Dautrevaux Laboratoire de Biochirnie Structurale, Facult~ de M~decine, Lille (France (Received 17 July 1986)

Key words: Glucocorticoid receptor; Antiglucocorticoid: Isoelectrofocusing

Rat-liver glucocorticoid receptor was incubated with either [3H]triamcinolone acetonide or [3HIRU 486, a well known antiglucocorticoid. Once formed, the steroid-receptor complexes were analyzed by isoelectric focusing in agarose gel slabs. A careful slicing of the receptor tracks revealed the presence of three distinct radioactive peaks focused at the following pl values: 5.3 5=0.2 (n = 17), 4.83 5=0.04 (n = 17) and 4.4 5=0.1 (n = 17). All these peaks correspond with receptor isoforms as suggested by control experiments. The receptor state was analyzed after focusing by a chromatographic assay on DNA-cellulose, DEAE-trisacrylT M and hydroxyapatite minicolumns. The peak of pl 4.4 apparently corresponded to the non-transformed receptor and was greatly stabilized in the presence of RU 486, whereas the peaks of pl 4.8 and 5.3 were probably made of transformed receptor and meroreceptor. These results were confirmed by autoradiographic studies after isoelectric focusing of receptor molecules covalently labelled with [3H]dexamethasone mesylate. Thus, the rat-liver glucocorticoid receptor appeared to be a rather acidic protein which became less acidic after transformation by heat, displaying a pl shift which was strongly reduced in case of steroid-receptor complexes formed with the antiglucocorticoid RU 486.

Introduction

Glucocorticoid effects are mediated in target cells by specific receptor proteins which play an essential role in the triggering of the steroidal response. Glucocorticoid receptor complexes, once formed, undergo an 'activation' step by which they acquire affinity for nuclei and DNA [1,2]. Despite intensive research over the past decade, our understanding of the molecular events involved in activation has remained limited. One of its most clearly established features is the Correspondence: Dr. P.M. Formstecher, Laboratoire de Biochimie Structurale, Facult6 de M6dicine, 1, Place de Verdun, 59045 Lille-Cedex, France.

acidophilic change suffered by the receptor molecule, which became able to bind to a variety of natural or synthetic polyanions [3]. However, contrasting with the numerous data accumulated about the easy resolution of both activated and non-activated forms of the glucocorticoid receptor complex by DNA-cellulose, phosphocellulose, heparin agarose and DEAE-cellulose chromatography, very few reports dealt with the use of isoelectric focusing to this purpose. Moreover, according to the technique used or to the authors, several pI values ranging from 5.1 to 7.5 have already been published for the rat-liver glucocorticoid receptor, by far the most studied [4-12]. However the lack of a reliable assay of the receptor, by far the most studied [4-12]. However the

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

130 lack of a reliable assay of the receptor state after the focusing step precluded any clear assignment of each p l isoform to its corresponding receptor species (i.e., unactivated, activated or meroreceptor). The recent description by Ben Or and Chrambach [13] of the possibilities afforded by isolectric focusing in a Sephadex G-75 matrix rather than in a polyacrylamide gel for the quantitative recovery and subsequent analysis of the focused binding activity yielded an attractive solution to this problem. In this paper we resorted to a similar agarose isoelectric focusing technique with subsequent assay of the focused receptor and we applied it to the analysis of rat-liver glucocorticoid receptor samples preincubated with either triamcinolone acetonide or RU 486 and submitted or not to heat-activation prior to focusing. The study of RU 486 glucocorticoid receptor complexes could seem of critical interest because this recently described potent antiglucocorticoid [14,15] appeared to bind the glucocorticoid receptor with a very high affinity, yielding a steroid hormone complex able to undergo only partial activation [15-18] and stabilized in a large size 8 S form, as we have recently shown [18[. Thus, this steroid was expected to allow the clear identification of a non-activated form of the receptor after focusing. Moreover, dexamethasone-21 mesylate was used to label the glucocorticoid receptor covalently [19] and to confirm the result obtained with triamcinolone acetonide and RU 486. Material and Methods

Chemicals [1,24(n)-3H]Triamcinolone acetonide (28 Ci. mmol 1) and [6,7(n)-3H]dexamethasone mesylate (49 Ci. mmol l) were purchased from (Amersham International, U.K.) and New England Nuclear Research Products (Dreieich, F.R.G.), respectively. RU 486 (ll/3-(4-Dimethylaminophenyl)17]3-hydroxy-17a-prop-l-ynyl)estra-4,9-dien-3-one) and its labelled derivative ([6,7-3H]RU 486; 50 • 6 Ci- mmol 1) came from Roussel-Uclaf (Romainville, France). Unlabelled triamcinolone acetonide was from Serva (Heidelberg, F.R.G.). Ampholines (3.5-9.5) and Isogel T M agarose-EF came from LKB (Bromma, Sweden) whereas the calibration

kit for isoelectric point determination was purchased from Pharmacia (Uppsala, Sweden). DNA-cellulose was from Sigma Chemicals (St Louis, MO, USA), DEAE-trisacryl from Industrie Biologique Fran~aise (Villeneuve-la-Garenne, France) and Biogel H T P T M from Bio-Rad (Richmond CA, U.S.A.). Seraclear T M tubes (Technicon Instruments Corporation, Tarrytown, NY, U.S.A.) were used as minicolumns for DNA cellulose, DEAE-trisacryl and hydroxyapatite chromatography.

Buffers and stock solutions Anode and cathode electrolytes used for isoelectric focusing were 0.5 M acetic acid and 0.5 M sodium hydroxide solution, respectively. The buffer used was 50 mM Tris-HCl, 1 mM EDTA, 10% glycerol (pH 7.4) supplemented with 10 mM sodium molybdate when specified. The buffer used for cytosol preparation was supplemented with 1 mM phenylmethylsulfonyl fluoride.

Cytosol preparation Male Wistar rats (200 g) were adrenalectomized 2-3 days before killing by cervical dislocation. After removal, livers were perfused with 10 ml 0.9% NaC1 and 10 ml ice-cold buffer. All the subsequent operations were performed at 4 ° C unless otherwise mentioned. After weighing, the livers were homogenized in the Tris-buffer with or without 10 mM sodium molybdate (1.5 ml buffer per g of liver) using a Teflon-glass Potter homogenizer and centrifuged at 300000 × g for 40 min. The supernatant was then removed, and after adjusting pH at 7.4 with 1 M Tris, it was immediately used for steroid binding. The cytosol samples were incubated with 30 nM [3H]steroid at 4°C for 4-16 h by 0.5 ml fractions. Some aliquots were stored at - 2 0 ° C for 1 or 2 weeks before use.

Isoelectrofocusing 0.5 mm thick agarose gel slabs (245 × 110 mm) were prepared 1 or 2 days before use according to the LKB instruction manual 1818A. The gel composition was 0.8% (w/v) isogel agarose EF, 0.5% (v/v) glycerol and 0.6% (v/v) ampholines (pH range 3.5 9.5). The slab was placed in a LKB multiphor 2117 apparatus connected to a LKB 2103 power supply. Samples (0.02 ml) were applied 1.5 cm from the cathode on a paper sample

131 applicator. The gel was cooled at 4 ° C by a Huber HS 40 cryostat during all the experiment and the running conditions were as follows: current and power unlimited, voltage going from 500 V to 1500 V. After a 40 min run, the pH gradient was estimated along a control track which has been loaded with a sample of the p I calibration kit. This track was then stained with Coomassie brilliant blue G250 according to Vesterberg [20]. Tracks containing the labeled receptor samples were either cut into 1 mm slices for radioactivity counting or used for further chromatographic characterization. In some experiments a rather large complex was applied nearly right across the agarose gel. After focusing, the position of the labeled receptor was checked on a control track and the bulk of the agarose corresponding to this position was scraped of and layered onto the top of the various minicolumns used for a further characterization of focused receptor (see below).

Mini-column chromatographic receptor assay We followed a procedure inspired by Holbrook et al. [21] to determine rapidly the various forms of the [3 H]steroid-receptor complexes. The various gel media, hydroxyapatite, DNA-cellulose and DEAE-trisacryl, were equilibrated in Tris buffer containing 10 mM sodium molybdate. Two Seraclear T M plastic tubes were cut short at a 2 cm length and filled with 0.5 ml DNA-cellulose and 0.5 ml DEAE-trisacryl, respectively. The bottom of each tube was then connected to the top of a second Seraclear tube containing 0.5 ml hydroxyapatite to form the first two columns. A third column was made of a single Seraclear tube filled with 0.5 ml hydroxyapatite. All the following chromatographic receptor assays were performed in strictly parallel conditions. Agarose gel strips containing the focused steroidreceptor complexes were layered directly on the top of each column (DNA-cellulose connected with hydroxyapatite, DEAE-trisacryl connected with hydroxyapatite and hydroxyapatite). The three columns were washed with about 10 ml of Tris buffer containing 10 mM sodium molybdate. The columns were then dismantled, the hydroxyapatite part of each column was dried by suction and poured in a scintillation vial for tritium counting.

Covalent labelling with dexamethasone mesylate The procedure proposed by Simons [22] was followed with only minor modifications. Briefly, labelling was performed overnight with 100 nM tritiated dexamethasone mesylate in 20 mM tricine, 1 mM EDTA, 10 mM sodium molybdate and 10% glycerol (pH 7.8) at 4°C. Parallel control samples were incubated in presence of a 500-fold excess of unlabeled dexamethasone mesylate (control 1) or of unlabeled dexamethasone (control 2). All samples were submitted to isoelectric focusing; the gel slab was then fixed according to Versterberg [20] and impregnated for one hour with EN 3H ANCE T M (New England Nuclear Research Products, Dreieich, FRG). The radioactivity was revealed by fluorography after a 1-2 months exposure to a Kodak XAR-5 film at - 7 0 ° C.

Miscellaneous Radioactivity was measured in an Intertechnique SL 4000 liquid spectrometer using Aqualyte (Baker Chemicals, Deventer, The Netherlands) as scintillation cocktail (36% tritium efficiency). Results

Isoelectric focusing In each experiment, receptor samples incubated in duplicate with either triamcinolone acetonide or RU 486 were run in parallel. In all cases the bulk of the bound radioactivity was focused on a rather acidic region with a pI laying between 4.3 and 5.5. A careful analysis of this region by cutting the gel tracks into 1 mm slices revealed the presence of three distinct radioactive peaks focused at pI 5.3 _+ 0.2 (n = 17), 4.83 +_ 0.04 (n = 17) and 4.4 + 0.1 (n = 17): Representative experiments are depicted in Fig. 1. The middle peak with p I 4.83 appeared the most constant one, whereas the p I 4.4 peak was far more variable. Moreover all these three peaks consisted mainly of tritiated steroid bound to specific protein(s), since they were not observed on tracks loaded with control cytosol samples incubated with 30 nM labelled steroid (either triamcinolone acetonide or RU 486) in presence of a 1000-fold molar excess of the non-labelled steroid (in this case no radioactivity was found outside the sample laying area). When the samples were treated by dextran-coated charcoal before use for

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Fig. I. Effect of heat-treatment of glucocorticoid and antiglucocorticoid-receptor complexes on their isoelectric focusing pattern. After incubation for 4 h at 0 ° C with 30 n M of either [3H]triamcinolone acetonide or [3H}RU 486, rat liver cytosol samples were heated at 2 5 ° C for 30 rain. 50 /L1 aliquots of non-transformed and transformed samples were applied onto the agarosc gel slab and submitted to isoelectric focusing for 40 min at 4°C. Each sample track was cut into 1 m m slices which were laid into scintillation vials for direct counting. Solid lines represent the non-transformed cytosolic receptor incubated

the focusing experiment, in order to eliminate most of the free labelled steroid, there was as expected a sharp decrease of the radioactivity in the laying area with no change in the radioactive peaks observed outside this area. In our previous work on the molybdate-stabilized glucocorticoid receptor, either partly purified by protamine sulfate precipitation of highly purified by affinity chromatography [12], we reported an isoelectric point of 5.1 with no mention of the heterogeneity depicted here. However, these previous results were obtained after cutting the gel tracks in rather coarse 5 mm slices and this has precluded the individualization of the three peaks mentioned here, which therefore have appeared as a single broad peak. In order to characterize these three peaks further, we performed experiments using either transformed or non-transformed steroid receptor complexes to see if the isoelectricfocusing pattern would change after receptor transformation. When incubated with tritiated triamcinolone acetonide and submitted to a 30 rain transformation step at 25 ° C the glucocorticoid receptor yielded only two peaks, at p l 5.2 and 4.9, whereas the non-transformed sample displayed a third variable peak at p l 4.4. This peak at p l 4.4 was always strikingly reduced after transformation and in most cases persisted only as a shoulder (Fig. la). In a sharp contrast to this result, the heated complex obtained after incubation with the tritiated antiglucocorticoid RU 486 displayed an only partial decrease of the peak at pI 4.4 (Fig. lb) which also appeared higher and far more constant in the non-transformed sample than in the case of an incubation in presence of triamcinolone acetonide. Since cytosolic RU 486 glucocorticoid receptor complexes have been shown to undergo only partial and impaired transformation after heat treatment [15 18], the results of our isoelectrofocusing experiments performed on both the RU 486 and the triamcinolone acetonide receptor complexes supported the hypothesis that the species focused with either [3H]triamcinolone acetonide (panel a) or [3H]RU 486 (panel b), whereas dashed lines represent the complexes heated prior to isoclectric focusing. The pH gradient was determined along a control track using the low p l calibration kit (pl 2.5-6.5) from Pharmacia Fine Chemicals (Uppsala, Sweden).

133

at p I 4.4 could be the non-transformed steroid receptor complexes. However, the rather variable importance of the peak at p I 4.4 in the case of the triamcinolone acetonide receptor complex suggested that a partial transformation could have occurred during the focusing experiment itself and that the final receptor state observed after focusing could therefore be quite different from its initial cytosol state. Thus a control of the functional properties of the steroid receptor complexes after focusing appeared highly desirable to shed further light on this point.

Minicolumn chromatographic assay of the steroid receptor complexes after isoelectricfocusing Our preliminary attempts to characterize the steroid receptor complex after the isoelectricfocusing step by usual DNA cellulose or phosphocellulose assays failed owing to the instability and fast dissociation of focused complexes. This result was not surprising since we indeed checked that in an acidic medium the dissociation rate constant of the steroid receptor complex progressively increased as the pH fell, and that below pH 5.4 protein precipitation occurred in cytosol samples (data not shown). Thus we turned to the rapid microchromatographic assay recently reported by Holbrook [21]. The resort to the three-column variant described in this paper could appear more complicated than the original technique of Holbrook. However, our procedure avoids the counting of either the DNA-cellulose or the DEAE-Trisacryl part of the columns, and these media are far more expensive than hydroxyapatite. Moreover the complete elution of the receptor bound to either DNA-cellulose or DEAETrisacryl was impossible to obtain in the very low volume, i.e., less than 0.5 ml, which was needed to avoid the dilution of samples containing a very small amount of receptor. Since DNA-cellulose and DEAE-Trisacryl T M bind the transformed and the non-transformed steroid receptor complexes, respectively, and since hydroxyapatite retains all the steroid binding forms of the receptor (i.e., both the transformed and untransformed complexes together with the so-called meroreceptor which is unable to bind either DNA-cellulose or DEAE-Trisacryl T M in the conditions described here) the repartition of these various receptor

forms in the sample could be assessed from the tritium content of the hydroxyapatite part of each of the three columns used in parallel for the chromatographic assay. The DNA-cellulose hydroxyapatite column yielded the sum of the untransformed complex plus the meroreceptor, whereas the DEAE-Trisacryl-hydroxyapatite column gave the sum of the transformed complex plus the meroreceptor and the hydroxyapatite column gave the total steroid receptor complexes. Therefore, a simple computation led to the quantitative determination of each receptor form present in the original sample and results were expressed as percent of the total steroid receptor complexes. Representative data are summarized in Table I. The comparison of the results obtained with triamcinolone acetonide-receptor complexes either transformed or not and analyzed before and after isoelectric focusing allows several comments. First, only a limited part (about 33%) of the complexes, whatever their initial state - transformed or not survived the focusing step. However, it remained abundant enough to permit a reliable characterization of the receptor state after focusing. The most striking feature which emerged from the results was the confirmation of the hypothesis made previously: the non-transformed triamcinolone acetonide receptor underwent a near-complete transformation during the focusing step, with a shift of the level of the DNA binding receptor form from 15 to 55%, a final value quite similar to the 62% level observed with the truly heat-transformed sample. Moreover the rate of meroreceptor increased significantly after isoelectric focusing of both samples and was in the range 18-30%, whereas the initial samples contained less than 2% of this receptor form. Here again the lack of stability of the steroid-receptor complexes during the focusing probably accounts for the partial degradation observed. On the other hand when incubated with tritiated RU 486, the receptor predominantly remained in the non-transformed form. Even after heat treatment and isoelectric focusing no more than 16% of the complex appeared to have been transformed. These results are in good agreement with the low transformation level previously reported in rat thymus and liver cytosolic samples [15,18].

134 TABLE I ASSAY OF T H E V A R I O U S F O R M S ( N O N - T R A N S F O R M E D , T R A N S F O R M E D A N D M E R O R E C E P T O R ) PRESENT IN G L U C O C O R T I C O I D A N D A N T I G L U C O C O R T I C O I D R E C E P T O R COMPLEXES BEFORE A N D A F T E R ISOELECTRIC FOCUSING Three 50 #1 rat liver cytosol aliquots preincubated with 30 nM of either [3H]tfiamcinolone acetonide or [~H]RU 486 and transformed or not by heating at 2 5 ° C for 30 rain were submitted to isoelectric focusing in an agarose gel slab. For each track the entire area corresponding to the focused receptor (pl 4.0 to 6.0) was then scraped off and the three agarose samples obtained were respectively layered onto the top of a minicolumn of DNA-cellulose connected to a 1 ml hydroxyapatite minicolumn 1, a minieolumn of DEAE-Trisacryl connected to a 1 ml hydroxyapatite minicolumn 2 and 1 ml minicolumn of hydroxyapatite 3. After washing with 10 ml of 50 m M Tris-HCl, 1 m M EDTA, 10 m M sodium molybdate and 10% glycerol (pH 7.4), columns were dismantled and their hydroxyapatite part was assayed for tritium content. The same minicolumn assay was also carried out before focusing in case of cytosolic triamcinolone acetonide receptor complexes. Results are expressed as follows: column (A) refers to the total activity obtained by counting hydroxyapatite from minicolumn 3, column (B) to the non-transformed receptor complexes calculated by subtracting the counts retained on minicolumn 2 from those retained on minicolumn 3, column (C) to the transformed form similarly deduced by subtracting 1 from 3, and column (D), from the meroreceptor estimated by the difference total activity minus the sum of the transformed and non-transformed forms. In each case results were also expressed in percent of the total activity recovered.

Before focusing Triamcinolone acetonidereceptor complexes non-heated heated After focusing Triamcinolone acetonidereceptor complexes non-heated heated RU 486-receptor complexes non-heated heated

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324 406

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1260 1294

55.2 62.3

697 376

30.4 18.2

5200 4-57

3744 3093

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17 16

572 317

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Moreover, all the data reported in Table I could be reconciled with the interpretation proposed earlier about fig. 1. The peak at pH 4.4 probably represented the non-transformed complexes. With the samples analyzed on Table I (which were different from those used in the experiment reported in fig. 1), this peak was quite negligible in case of both transformed and nontransformed triamcinolone acetonide receptor complexes and displayed roughly the same extent in case of the RU 486 receptor complexes (data not shown). However, with RU 486, as reported in Fig. lb, this peak never exceeded 40% of the total radioactivity focused between pI 4.3 and 5.5, containing with the 72 76% level of non-activated receptor found by minichromatographic assay. A simple explanation of this apparent discrepancy

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can be proposed: the data reported on Table I described the results of the analysis of the remaining receptor-bound steroid present in the pI 4.3-5.5 area, whereas Fig. 1 depicted the repartition of all the tritiated steroid, either bound or free, present in the same area. The already mentioned instability of the steroid receptor complexes at acidic pH could account for the presence of a significant amount of free steroid in the pI 5.0 region. The steroid receptor complexes initially layered on the alkaline side of the gel (final pH after focusing in the range 7.5 8.5) probably started to migrate in a rather good health and thus displayed only minimal dissociation as long as they walked in a neutral or slightly acidic environment. However when the pH dropped below 5.5 they very likely began to struggle along and to be

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inactivated. Indeed, we have already pointed out that only 33% of the steroid receptor complexes survived the focusing strip. Thus it could be reasonably thought that the peak at p I 5.3 and 4.8 contained a significant part of free steroid in addition to the meroreceptor and the transformed receptor complexes. This last species probably predominated in the p I 4.8 peak, whereas the two former were preponderant in the p I 5.3 one, since the rare samples containing a very low amount of meroreceptor after focusing most often displayed a decreased p I 5,3 peak (Fig. 2).

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Since significant receptor dissociation was observed in acidic conditions and could have resuited in misinterpretation of the previous results, we performed isoelectric focusing experiments on cytosol samples incubated with [3H]dexamethasone mesylate, the now well-known steroidal covalent affinity label described 5 years ago by Simons [19] and since widely used for the study of glucocorticoid receptors in denaturing conditions. The isoelectric focusing pattern obtained after fluorography (Fig. 3 lane A) was in complete agreement with the results reported in the previous experiments. Three rather narrow radioactive bands were revealed at p I 4.4, 4.7 and 5.1, together with a wider trail between p l 5.25 and 5.65. All these bands were either absent from a control sample incubated with [3H]dexamethasone mesylate in presence of an excess of unlabelled mesylate (lane B) or strongly reduced when the unlabelled steroid was dexamethasone (lane C). In this last case, owing to the long incubation time

Fig. 2. Correlation between a low meroreceptor content and the focusing pattern of glucocorticoid-receptor complexes. Rat-liver cytosol samples were incubated with either [3H]triamcinolone acetonide or [3H]RU 486 and duplicated aliquots (50/~1) were submitted to isoelectric focusingin an agarose gel slab. For each sample, one track was cut into 1 mm slices and counted as for Fig. 1, whereas the other was scraped off in the pl 4.0-6.0 area and submitted to rninicolumn assay as described in Table I to quantify the various forms of receptor present in the focused complexes. Results are summarized below each panel for (a) triamcinolone acetonide-receptor complexes and (b) RU 486-receptor complexes.

136

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Fig. 3. Isoelectric focusing of glucocorticoid receptor samples incubated with [3H]dexamethasone mesylate. Rat liver cytosol was incubated overnight at 4 ° C with 100 n M [3H]dexamethasone mesylate in absence or in presence of an excess of unlabeled dexamethasone mesylate or of dexamethasone. 20 ~ 1 aliquots of each sample were focused according to Fig. 1. After focusing, the gel was fixed in a 25% acetic acid/8% methanol solution and then impregnated with E N 3 H A N C E . The gel was then thoroughly washed with water, dried, and revealed by fluorography at - 7 0 ° C using a Kodak XAR-5 film. The position of the p I markers of the Pharmacia low p I calibration kit is indicated on the top of the figure. Lane A, incubation with [3H]dexamethasone mesylate alone: lane B, same as lane A with an excess of unlabeled dexamethasone mesylate; lane C, the same as lane A with an excess of unlabeled dexamethasone.

used (16 h) a significant labelling of the receptor by [3H]dexamethasone mesylate was not unexpected, even in presence of an excess of the noncovalent ligand, and probably resulted from a progressive and irreversible exchange of dexamethasone by dexamethasone mesylate. Discussion

The rather large discrepancy displayed by the various p I values already published for the native rate liver glucocorticoid receptor complex [4-12] can be easily explained by technical considerations. The first attempts to determine the p I of this receptor were made using columns containing gradients of sucrose or glycerol and have probably encountered the technical difficulties well known to be inherent to this procedure [23]. In particular, the very long time required for focusing proteins to equilibrium (24-72 h) could appear unacceptable regarding the low stability of the steroid receptor complexes. Thus, the difference between the

values of 6.7 reported by Litwack [4] and 5.9-6.1 reported by Kalimi [5] and Eisen [6] was not unexpected. Therefore, most researchers working on glucocorticoid receptors have resorted over the past few years to non-denaturing isoelectric focusing on polyacrylamide gel slab [7 11,24,25] according to Wrange [7]. Following this technique, both Gustafsson's group and our team found the same pI value around 5.9, whether the triamcinolone acetonide receptor complex was activated or not, highly purified or not [7 10], whereas Cidlowski claimed significant higher values in the range 7.0-7.5 [11]. However, a recent report of Ben Or [13] pointing out that isoelectric focusing on polyacrylamide gel led to aggregation problems during the analysis of the glucocorticoid receptor of the chick embryo neutral retina, prompted us to turn to the flat-bed agarose technique used for this paper. We originally found a p l of 5.1 for both crude and highly purified non-activated rat liver glucocorticoid-receptor complexes [12]. As discussed in this previous paper, the discrepancy between the data obtained on the polyacrylamide matrix and on the agarose one might be explained by the high molecular weight and the likely hydrophobic character of the receptor, which could result in either steric or hydrophobic interactions with the acrylamide gel and then could slow down its focusing. Since the samples are applied on the alkaline side of the slab, an overestimated p l would be obtained. Such armful interactions would be avoided on agarose, a more polar gel with large pore size. Thus we think that the value obtained in agarose is the most reliable. A similar acidic pI, of 5.3-5.4, was also observed for the rabbit glucocorticoid receptor [26] and for one of the free distinct components of the chick glucocorticoid receptor that Ben Or separated by isoelectric focusing on a Sephadex column [13]. Heterogeneity in the isoelectric points of glucocorticoid [4,5,7,13,25-27], progesterone [23] and androgen [28] receptors has been reported, but the molecular basis for the appearance of such various charged forms has generally not been thoroughly documented. The first attempt to relate the various isoforms observed to the peculiar state of the glucocorticoid receptor sample submitted to focusing was made by Kalimi [5], which reported a p I of 7.1 for the unactivated complex and a p I of 6.1

137

for the activated one. These values were the opposite of those predicted for an increased exposure of positive charges upon activation [3]. More recently, Ben Or and Chrambach [13] claimed a p I of 7.6 for the activated chick embryo glucocorticoid receptor and p I of 5.4 and 6.5 for two distinct forms of the non-activated receptor. Here, isoelectric focusing was performed on Sephadex G-75 columns in very careful and special conditions (peculiar electrode device and cooling at - 4 ° C ) which allowed a quantitative recovery of the binding activity after focusing. The IEF fractionated steroid receptor complexes were then assayed for binding to isolated nuclei. Thus these results seemed quite reliable and may be compared to our own results. Due to resorting to a conventional flat-bed agarose technique with a slightly less efficient cooling, our receptor recovery was less quantitative, but the focusing could be performed more quickly and the rapid minicolumn assay we used allowed the distinction of the three main receptor forms, i.e., activated, nonactivated and meroreceptor. In both studies the most acidic isoform never exceed 45% of the total, even for the molybdate-stabilized sample, and was extensively decreased in heat-activated samples for which the logically expected shift towards more alkaline isoforms was indeed observed. Moreover, in the case of the rat liver glucocorticoid receptor, the isoelectric focusing heterogeneity we found was more narrow by far than in the case of the chick receptor. Finally, as hoped for, the combination of post-focusing receptor analysis and of use of RU 486 as a non-activated receptor stabilizing steroid [18] afforded substantial clues for the interpretation of this heterogeneity. However, a point recently stressed by Smith [25] deserves notice: all the data obtained in nondenaturing conditions, if they are of utmost interest for characterizing the native activated and non-activated receptor complexes, fail to permit differentiation between charge heterogeneity that ensues from direct covalent modification of the glucocorticoid receptor protein and apparent charge heterogeneity resulting from interaction of the receptor protein with some other receptor-associated components. The recently demonstrated receptor phosphorylation [24] could probably induce some isoelectric point heterogeneity and the

results reported here need to be completed by studies performed in denaturing conditions. Thus, work is now in progress to elucidate further the structure of our purified glucocorticoid receptor by affinity labelling and subsequent high-resolution two-dimensional electrophoresis.

Acknowledgments This work was supported by the University of Lille II and by grants from INSERM (CRL No. 854008) and from EEC (contract No. STZJ-0075l-B). The help of Ms. P. Toulouse and B. Masselot is gratefully acknowledged. We are also grateful to P.A. Bradawl for linguistic revision of the manuscript and to Ms. A .Morandi for her excellent secretariat assistance. We are also indebted to Dr. Philibert from Roussel-Uclaf for generous gift of unlabelled and labelled RU 486 and helpful discussions.

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