Elimination and Reconstitution of the Requirement for Hormone in Promoting Temperature-dependent Transformation of Cytosolic Glucocorticoid Receptors to the DNA-binding State*

Cytosols contain a heat-stable, chelatable, anionic, molybdate-like factor that stabilizes glucocorticoid receptors in a heteromeric complex with hsp90 (refers to the 90-kDa heat shock protein) and inhibits their transformation to the DNA-binding state (Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., and Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817). In this work, we demonstrate that removal of this factor by passage of L cell cytosol through the metal-chelating resin Chelex-100 makes the glucocorticoid receptor unstable, thus markedly facilitating both its dissociation from hsp90 and its transformation to the DNA-binding state. In normal cytosol, both temperature-mediated dissociation of hsp90 and temperature-mediated receptor transformation are hormone-dependent events. In the Chelex-treated, metal-depleted cytosol, however, temperature-mediated dissociation of hsp90 and receptor transformation occur very rapidly in a manner that is no longer hormone-dependent. When boiled L cell cytosol is added to the metal-depleted receptor system, the hormone dependence of both temperature-mediated dissociation of receptor from hsp90 and receptor transformation to the DNA-binding state is reconstituted. Like boiled cytosol, molybdate stabilizes the receptor complex and inhibits its transformation in metal-depleted cytosol, but it does not reconstitute the hormone dependence of the system. These results support the proposal that an endogenous metal anion interacts with the glucocorticoid receptor to stabilize it in the heteromeric, inactive, non-DNA-binding state in cytosol and that binding of the hormone promotes conversion of the receptor to the DNA-binding state through an effect on this metal anion center.

In this work, we demonstrate that removal of this factor by passage of L cell cytosol through the metal-chelating resin Chelex-100 makes the glucocorticoid receptor unstable, thus markedly facilitating both its dissociation from hsp90 and its transformation to the DNA-binding state. In normal cytosol, both temperature-mediated dissociation of hsp90 and temperature-mediated receptor transformation are hormonedependent events.
In the Chelex-treated, metal-depleted cytosol, however, temperature-mediated dissociation of hsp90 and receptor transformation occur very rapidly in a manner that is no longer hormonedependent.
When boiled L cell cytosol is added to the metal-depleted receptor system, the hormone dependence of both temperature-mediated dissociation of receptor from hsp90 and receptor transformation to the DNA-binding state is reconstituted. Like boiled cytosol, molybdate stabilizes the receptor complex and inhibits its transformation in metal-depleted cytosol, but it does not reconstitute the hormone dependence of the system. These results support the proposal that an endogenous metal anion interacts with the glucocorticoid receptor to stabilize it in the heteromeric, inactive, non-DNA-binding state in cytosol and that binding of the hormone promotes conversion of the receptor to the DNA-binding state through an effect on this metal anion center.
In 1977, our laboratory published that a heat-stable system in L cell cytosol was capable of converting glucocorticoid receptors from a nonsteroid-binding to a steroid-binding form (1). The heat-stable system was found to contain two activities (2,3); a receptor reducing factor that was subsequently identified as thioredoxin (4,5), and a small receptor stabilizing factor (3). We recently showed that the endogenous receptor stabilizing factor, which is present in boiled cytosol prepared from a wide variety of sources, produces all of the effects on cytosolic glucocorticoid receptors that are produced by the * This investigation was supported by Grant DK31573 from the National Institutes of Health. The costs of oublication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.
group VI-B transition metal oxyanions molybdate, vanadate, and tungstate (6). This factor stabilizes the receptor in its 9 S heteromeric form in association with hsp901 and inhibits transformation' of the glucocorticoid receptor to the DNAbinding state (6). This receptor stabilizing factor has the same effect on the receptor as an endogenous inhibitor of glucocorticoid receptor transformation reported by the laboratories of Litwack et al. (7,8) and Milgrom et al. (9) and subsequently shown by Sato et al. (10) to inhibit the transformation of androgen and estrogen receptors. The factor behaves as a strong anion with an apparent M, of 340 on Bio-Gel P-2, it is stable to heating at 320 "C for 1 h, and it binds tightly to Chelex-100, a metal chelating resin (6). Thus, we have proposed that the factor is a metal anion and that molybdate and vanadate may exert their effects on the glucocorticoid receptor by interacting with the binding site for this endogenous metal anion. Bodine and Litwack (11,12) have also published the purification of a cytosol factor with the same molybdate-like actions on glucocorticoid receptors but have proposed an organic composition of a phosphoglyceride nature. If this endogenous factor plays a role in stabilizing cytosolic glucocorticoid receptors in their untransformed complex with hsp90, then removal of the factor from cytosol should result in complexes that are less stable and more readily transformed to their DNA-binding state. In this paper we show that removal of endogenous metals from L cell cytosol with Chelex-100 resin facilitates both dissociation of the glucocorticoid receptor from hsp90 and conversion of the glucocorticoid receptor to the DNA-binding state. It has previously been shown that glucocorticoid receptor transformation in normal cytosol preparations is both temperature-dependent and hormone-dependent (13,14). In contrast, in metal-free cytosol, glucocorticoid receptor dissociation from hsp90 and transformation to the DNA-binding state are still temperature-dependent events but they are no longer hormone-dependent. We show that readdition of heat-stable components of L cell cytosol to this system reconstitutes the requirement for hormone for promoting both dissociation of hsp90 and generation of the DNA-binding state. To our knowledge, this is the first whole cytosol refers to cytosol that was not treated with Chelex-100 resin. * The term transformation will be used throughout this paper to describe the process whereby the receptor is converted from a non-DNA-binding to a DNA-binding form.
time that the requirement for the ligand has been eliminated and reconstituted in a cell-free system in which receptors are converted from an inactive to an active conformation. at 2% of the final volume, the mixture was incubated for 2 h at 0 "C, and each sample was added to a protein-A-Sepharose pellet (10-J pellet/O.1 ml of L cell cytosol). Samples were mixed by rotation for 3 h at 4 "C and protein-A-Sepharose pellets were washed three times by resuspension in l-ml aliquots of TEGM buffer. Both glucocorticoid receptor and receptor-associated hsp90 were assayed by SDS-PAGE and immunoblotting.
Gel Electrophoresis and Immunoblotting-SDS-polyacrylamide gel electrophoresis was performed in 7% slab gels according to Laemmli (18). Gels were cooled to 4 "C during electrophoresis. Western immunoblot analysis with a '*"I-labeled second antibody probe as described above. Preparation of Boiled Cytosol-L cell cytosol was boiled for 15 min in a boiling water bath, and centrifuged at 12,000 x g for 5 min. The supernatant was collected and lyophilized to a 20-fold concentrated stock solution.
To prepare metal-free boiled cytosol, 0.25 ml of 20fold concentrated boiled cytosol was loaded onto a column of Chelex-100 resin (1.5 x 10 cm) which was equilibrated at pH 7.2 with 10 mM Hepes buffer. The column was subsequently eluted with 10 mM Hepes buffer, pH 7.2, and the dropthrough fractions were collected, lyophilized to dryness, and reconstituted to the original volume in 10 mM Hepes buffer, pH 7.2.

Passage of Cytosol through Chelex-100 Promotes Transformation of the Gluocorticoid
Receptor-In our previous publication (6), we showed that passage of boiled cytosol through Chelex-100 resin removes endogenous metals, such as molybdenum, zinc, and aluminum and also removes the endogenous receptor stabilizing activity. Fig. 1 shows that Chelex-treatment of whole cytosol promotes glucocorticoid receptor transformation. In the first several experiments shown in this paper, transformation will be brought about by elevating the cytosolic pH as originally described by Bailly et al. (19 Cytosol containing steroid-bound receptors was adjusted to pH 7.2 or 8.2, and half of each portion was passed through a bed of Chelex-100 equilibrated at the same pH as described under "Materials and Methods." The cytosol preparations were incubated on ice, and at various times duplicate aliquots were removed for determination of steroid binding and binding to DNA-cellulose. Steroid binding at zero time was the same in pH 7.2 and 8.2 cytosols, and it did not vary more than 15% during the course of an experiment. Each value represents an average of two experiments with the range indicated by the uertical line through the symbols. 0, non-chelextreated cytosol at pH 7.2; 0, Chelex-treated cytosol at pH 7.2; q , untreated cytosol, pH 8.2; n , Chelex-treated cytosol, pH 8.2. Cytosol containing steroid-bound receptors was adjusted to pH 8.2 and treated with pH 8.2 Chelex-100 resin. Aliquots of the alkaline Chelex-treated cytosol were incubated for 9 h on ice with buffer, with 10 mM sodium molybdate, or with 3 relative units of the factor preparation, and steroid binding and binding to DNA-cellulose were assaved.  (Table I).
Alkaline pH-mediated transformation of glucocorticoid recep-tor in Chelex-treated cytosol is accompanied by a change in the size of the receptor complex as reflected by a reduction in the sedimentation coefficient from 9 S to 4 S, and this reduction in apparent receptor size is also inhibited by either the stabilizing factor or molybdate (not shown).
There is considerable evidence that both glucocorticoid receptor transformation to the DNA-binding state and its conversion from the 9 S to 4 S form is due to the dissociation of the receptor from hsp90 (see Refs. 20 and 21 for review). In the experiment in Fig. 2, we show that receptors in Chelextreated, pH 8.2, cytosol dissociate from hsp90 at 0 "C. To interpret this data it is important to know (as documented later in Fig. 5) that Chelex treatment does not alter the ability of the receptor to be recognized by the BuGR antibody. Both sodium molybdate (lane 4) and the heat stable factor (lane 5) are able to inhibit the alkaline pH-mediated loss of hsp90 from the Chelex-treated receptor complexes. Thus, it appears that removal of the endogenous stabilizing factor by Chelex treatment of cytosol facilitates both dissociation of the receptor from hsp90 and generation of the DNA-binding state caused by elevation of pH.
Temperature-dependent Transformation of GR in Chelextreated Cytosol Is No Longer Steroid-dependent-When L cell cytosol containing hormone-free glucocorticoid receptor is incubated at 25 "C, the receptors lose their ability to bind steroid, a similar incubation of steroid-bound glucocorticoid receptor converts receptors to the DNA-binding state (22). The experiment of Fig. 3 presents the rates of inactivation of steroid binding capacity of unliganded receptors (panel A) and the rates of transformation of steroid-bound receptors (panel B) in both Chelex-treated and untreated cytosol. It is clear that the rate of temperature-dependent inactivation of steroid binding capacity and the rate of transformation to the DNA-binding state are more rapid in the metal-depleted, Chelex-treated cytosol than in whole cytosol. In Chelextreated cytosol, both glucocorticoid receptor inactivation and transformation proceed very rapidly at 25 "C and the experiments of this paper are carried out at 15 or 20 "C to slow down both events.
In our usual L cell cytosol preparations containing all of the endogenous metals, binding of glucocorticoid to the receptor promotes temperature-dependent transformation of the receptor to the DNA binding form (13). To determine if steroid was still required after removal of metals, we performed the experiments of Fig. 4 Replicate aliquots of pH 8.2, Chelex-treated cytosol containing steroid-bound receptors were incubated for 10 h on ice with additions indicated below. Samples were then immunoadsorbed to protein A-Sepharose with the BuGR anti-receptor antibody and resolved by SDS-polyacrylamide gel electrophoresis as described under "Materials and Methods." The samples were analyzed by the Western blot procedure using the AC88 monoclonal antibody to probe for the receptor-associated hsp90. Lane 1, untreated cytosol immunoadsorbedwith nonimmune mouse IgG. Lane 2, untreated cytosol immunoadsorbed with BuGR. Panel A, inactivation of steroid binding capacity. Either untreated cytosol or cytosol passed through Chelex resin at pH 7.2 was incubated at 0 or 15 "C!. At various times, aliquots were removed and the steroid binding capacity was determined by binding with [3H]triamcinolone acetonide. The data are presented as the percent of zero time binding capacity. Panel B, receptor transformation. Either untreated cytosol or pH 7.2 Chelex-treated cytosol containing steroid-bound receptors was incubated at 0 or 15 "C. At various times, aliquots were removed for determination of both steroid and DNA binding. Data are presented as the percent of total receptor bound to DNA-cellulose. In both panels the values represent the mean and standard error of determinations from three separate experiments. Where no error bars are shown, the error lies within the symbols. The conditions in this and following figures are indicated as follows: R, steroid-free receptor in untreated cytosol; RS, steroidbound receptor in untreated cytosol; CR, steroid-free receptor in Chelex-treated cytosol; CRS, steroid-bound receptor in Chelex-treated cytosol. Samples are 0, untreated cytosol incubated at 0 "C; 0, untreated cytosol incubated at 15 "C; A, Chelex-treated cytosol incubated at 0 "C; A, Chelex-treated cytosol incubated at 15 "C. Whole cytosol was divided into two portions, one of which was passed through Chelex resin (pH 7.2). Half of each portion was then incubated 2 h at 0 "C with 100 nM nonradioactive dexamethasone to form steroid-bound receptors. Portions of each condition were then incubated at 0 or 15 "C, and at various times, aliquots were removed and incubated with DNA-cellulose at 0 "C. The amount of DNA-bound receptor was assayed by the quantitative Western immunoblot procedure using a 'ZSI-labeled second antibody probe as described under "Materials and Methods." Each value in the diagram represents the mean and standard error of determinations from three separate experiments. Autoradiograms of Western blots of glucocorticoid receptor from one experiment are presented above the diagrams. To obtain the values plotted in the diagram each band on the Immobilon transfer membrane was excised and counted. To permit normalization of values from different experiments, the amount of DNA-bound receptor is expressed in both panels as a percent of the highest DNA-binding value achieved in Chelex-treated cytosol. Panel A represents values from the Chelex-treated portion of cytosol and panel i3 from the untreated whole cytosol. A, unliganded (steroid-free) receptor in whole (R) or Chelex-treated (CR) cytosol at 0 "C; A, unliganded receptor in whole or Chelex-treated cytosol at 15 "C; 0, steroid-bound receptor in whole (RS) or Chelex-treated (CSR) cytosol at 0 "C; 0, steroid-bound receptor in whole or Chelex-treated cytosol at 15 "C.
in Chelex-treated (panel A) and whole (panel B) cytosol. In tor. Autoradiograms of immunoblots from one experiment are these experiments the DNA-bound receptor has been assayed shown above the diagrams, which present average values from by Western blotting in order to permit us to follow the three experiments. The maximum DNA binding achieved in behavior of the unliganded as well as the steroid-bound recep-these experiments varies between 40 and 60% of the total Whole cytosol and Chelex-treated (pH 7.2) cytosol were divided into two portions and receptors in half were bound with nonradioactive dexamethasone. Each sample was incubated at 20 "C and at various times aliquots were removed and receptors were immunoadsorbed with the BuGR anti-receptor antibody. The immunoadsorbed proteins were extracted from the pellet and resolved by SDS-PAGE. The amount of hsp90 was quantitated by lz51 Western immunoblot analysis using the AC88 monoclonal anti-hsp90 antibody as the probing agent. The amount of receptor-associated hsp90 in each sample is presented as a percent of the zero time value. The autoradiogram of the hsp90 is shown above the graph on the left. The amount of receptor in each condition was also quantitated by probing the Western blots with BuGR and the autoradiogram on the right shows that the amount of receptor in each sample remained constant. 0, steroid-free receptor in untreated cytosol, R; 0, steroid-bound receptor in untreated cytosol, RS; A, steroid-free receptor in Chelex-treated cytosol, CR; A, steroid-bound receptor in Chelex-treated cytosol, CM. number of receptors (as estimated from immunoblotting small aliquots of whole cytosol to determine the total amount of receptor/sample).
To permit normalization of values from different experiments, the values for both whole cytosol and Chelex-treated cytosol are expressed as a percent of the highest DNA binding value achieved in each experiment (in this case, the 30 min, 15 "C value presented in panel A).
As shown in panel A of Fig. 4, Chelex-treated cytosol is rapidly transformed to the DNA-binding state in a temperature-dependent manner regardless of whether it is steroidbound or not. At 0 "C, neither liganded nor unliganded Chelex-treated cytosol is transformed to the DNA-binding state. In the case of whole cytosol (i.e. non-Chelex-treated), the steroid-bound receptor is transformed to the DNA-binding state but at a much slower rate than in Chelex-treated cytosol. Under identical conditions, unliganded receptor in whole cytosol is not transformed to the DNA binding form at 15 "C (panel B). Thus, in the normal cytosol temperaturedependent transformation is hormone-dependent, whereas in the metal-depleted cytosol transformation is still temperature-dependent but it proceeds rapidly in a hormone-independent manner.
We have previously shown that both the temperature- Untreated cytosol containing steroid-bound glucocorticoid receptor and Chelex-treated (pH 7.2) cytosol containing unliganded glucocorticoid receptor were incubated at 20 "C. At the indicated times, aliquots were removed and assayed for receptorassociated hsp90 by immunoadsorption with BuGR and immunoblotting with AC88 (autoradiogram on the left) or for binding to DNAcellulose by immunoblotting DNA-cellulose-bound proteins with BuGR (autoradiogram on the right). The bands in the immunoblot were excised and counted for I?. Receptor-associated hsp90 (solid lines) is presented as percent of the zero time value. After subtraction of the zero time values, the amount of glucocorticoid receptor binding to DNA (dashed lines) was plotted as a percent of the highest DNA binding achieved in each series. 0, steroid-bound receptors in whole cytosol; A, unliganded (steroid-free) receptors in Chelex-treated cy-toso1. dependent loss of steroid binding capacity and transformation of the glucocorticoid receptor to the DNA-binding state correlate with dissociation of hsp90 from the receptor (13,17). The experiment of Fig, 5 examines the dissociation of hsp90 from glucocorticoid receptor in both whole cytosol and Chelex-treated cytosol at 20 "C. In whole cytosol, the receptorspecific hsp90 is lost only when the receptor is bound by steroid. In Chelex-treated cytosol, however, hsp90 dissociates from the receptor at the same rapid rate regardless of whether the receptor is steroid bound or not. At 0 "C, the glucocorticoid receptor-hsp90 complex in Chelex-treated cytosol remains intact over the course of several hours. (data not shown).
If dissociation of hsp90 is associated with transformation of the glucocorticoid receptor to the DNA-binding state, then the rates of the two processes should be similar. The experiment shown in Fig. 6 compares the rates of both hsp90 dissociation and transformation in the same samples of Chelex-treated and whole cytosol incubated at 20 "C. Both transformation and hsp90 dissociation occur at a similar rapid rate in metal-depleted cytosol and at the same slow rate in whole cytosol.
Reconstitution of the Hormone Requirement for Temperature-dependent Dissociation of hsp90 and Transformation-The above experiments suggest that elimination of a metal component of cytosol by chelation removes the requirement for hormone in promoting thermal dissociation of hsp90 from the receptor. In the experiment of Fig. 7, boiled L cell cytosol was added back to Chelex-treated cytosol to see if the requirement for the hormone could be reconstituted.
Boiled L cell Aliquots of Chelex-treated cytosol were incubated for 2.5 h on ice in the presence of the indicated combinations of buffer, one relative unit of boiled L cell cytosol (the concentration of heat-stable components normally present in whole cytosol), Chelex-treated-boiled cytosol, and nonradioactive dexamethasone. This preincubation time ensures occupancy of all steroid-binding sites. Samples were then incubated at 20 "C and at various times aliquots (400 pl) were removed and immunoadsorbed to protein A-Sepharose with BuGR2 anti-receptor antibody. Proteins were extracted from the pellet and resolved by SDS-PAGE. The amount of receutor-snecific hsn90 was auantitated by '251 Western immunoblot analysis using AC88 monocional anti-hsp90 antibody as the probing agent. The amount of receptor-specific hsp90 in each sample is presented as the % of the time zero value. 0, steroid-free whole cytosol, R; 0, steroid-bound whole cytosol, RS; A, Chelex-treated cytosol incubated with buffer, CR; A, Chelex-treated cvtosol incubated with steroid. CRS: 0. Chelex-treated cvtosol incubated with boiled cytosol, CR g f; n , Chelex-treated cytosol incubated with boiled cytosol and steroid, CR + f + S; 0, Chelex-treated cytosol incubated with Chelex-treated boiled cytosol, CR + Cf.
cytosol, which is indicated as f (for heat-stable factor preparation) in the figures, contains the endogenous receptor stabilizing factor (2, 3, 6), and as shown in Fig. 7, it inhibits thermal dissociation of hsp90. Chelex-treated boiled cytosol does not inhibit dissociation, suggesting that a metal component of boiled cytosol is responsible for stabilization of the glucocorticoid receptorhsp90 complex. Most importantly, after readdition of the heat-stable components of cytosol to the Chelex-treated system, binding of the hormone is again required to promote dissociation of the receptor from hsp90.
The experiments of Fig. 8 demonstrate that the hormone requirement for receptor transformation to the DNA-binding state is also reconstituted when the heat-stable components of cytosol are added back to the system. In these experiments, the highest value of transformation is achieved by unbound receptors in Chelex-treated cytosol at 60 min, and this value has been set as 100% DNA binding in Fig. 8 to permit normalization of data from separate experiments. It is clear that addition of the heat-stable components of cytosol to the Chelex-treated system inhibits transformation and that dex-amethasone again promotes transformation in the reconstituted system.

DISCUSSION
Because a number of steroid hormone receptors are associated with hsp90 when they are in their non-DNA-binding form, but not after conversion to their DNA-binding form, the concept has arisen that dissociation from hsp90 is required to derepress the DNA binding function of the receptor (13,14,(23)(24)(25)(26)(27). This could occur either via a simple unmasking of the DNA-binding site or more likely via a major conformational change in the receptor that occurs when hsp90 dissociates (17, 28). The site of association of the glucocorticoid receptor with hsp90 and the site(s) of interaction with the metal oxyanions (e.g. molybdate, vanadate) that stabilize the receptor-hsp90 association lie within the steroid-binding domain of the receptor (29,30). A number of studies have shown that the steroid-binding domain acts as a "regulatory cassette" in the sense that it can endow the property of glucocorticoid regulation (e.g. 28). These types of observations have led to the model that steroid occupancy of the receptor somehow promotes dissociation of the glucocorticoid receptor from hsp90 as the initial hormone event (see Refs. 20, 21 for reviews).
It is important to realize that this notion that steroidmediated dissociation of the receptor complex is the initiating event in the hormone action is a working model. It is not known if the steroid acts only as a trigger and subsequent events are a consequence of this dissociation (20) or if the receptor must be bound with steroid for subsequent events in transcriptional activation to occur. The model already has a major exception. The receptor for thyroid hormone is a member of the same receptor family (31) and it differs from glucocorticoid, progesterone, and most other steroid receptors in that it is not recovered in the cytosolic fraction because it is very tightly associated with nuclear-binding sites prior to hormone binding. In contrast to the glucocorticoid receptor, where newly translated receptor is already bound to hsp90 and is in a non-DNA binding form (32, 33), the receptor for triiodothyronine is translated in a DNA binding form and is not bound to hsp90.3 In the case of the thyroid hormone receptor, and perhaps also in the case of the retinoic acid receptor, it would seem that there must be a hormone-dependent event, probably at the level of the regulated genes, that does not depend upon dissociation of a receptor-hsp90 complex.
Despite the uncertainty regarding the place of the dissociation model in steroid hormone action in the intact cell, we can certainly learn much that is of importance to a fundamental understanding of the information transduction mechanism if we can study and control rapid hormone-dependent events under cell-free conditions. Several laboratories have set up conditions, such as those used here for whole L cell cytosol, in which both dissociation of the heteromeric glucocorticoid receptor complex and transformation of the receptor to the DNA-binding state occur in a manner that requires both the presence of hormone and elevated temperature (e.g. 13,14). It is known that glucocorticoid receptors in intact cells also acquire DNA binding activity if they are exposed to steroid at elevated temperatures but not if cells are kept at 0 "C (e.g. 34, 35). Hormone-dependent, temperature-dependent transformation of cytosolic receptors to the DNA-binding state has been used as a cell-free model of the initial event in glucocorticoid action. It is of course inherent to the concept CHELEX nIlnut.. Aliquots of Chelex-treated cytosol or whole cytosol were preincubated at 0 "C with additions as described in the legend to Fig. 8. Samples were then heated at 15 "C, and at various times aliquots (100 ~1) were removed and incubated with DNA-cellulose on ice. The DNA-bound receptor was quantitated by Y-Western immunoblot analysis using the BuGR2 antireceptor antibody. The data are averaged from three experiments and are presented as a percent of the highest DNA-binding achieved at 60 min by receptors in steroid-free, Chelex-treated cytosol (CR). Panel A, A, DNA binding of receptors in Chelex-treated cytosol incubated with buffer; 0, Chelex-treated cytosol incubated with boiled cytosol; n , Chelex-treated cytosol incubated with boiled cytosol and steroid; 0, Chelex-treated cytosol incubated with Chelex-treated boiled cytosol. Panel B, 0, unliganded whole cytosol; 0, steroid-bound whole cytosol.
of hormone action that the binding of a hormone somehow activates its receptor and the process of cytosolic receptor transformation is often called receptor "activation" to reflect the impression that the receptor is converted from a biologically inactive to an active state when it acquires DNA binding activity.
The experiments of this paper are intended to address the problem of how hormone binding initiates the transformation (i.e. "activation") event. The fact that receptors in metaldepleted cytosol are particularly sensitive to both dissociation from hsp90 and transformation to the DNA-binding state suggests that an endogenous metal (or metals) must play a role in determining the stability of the untransformed glucocorticoid receptor in normal cytosol preparations. Addition of boiled cytosol, the partially purified endogenous stabilizing factor, or molybdate to the metal-depleted cytosol stabilizes the glucocorticoid receptor complex. Addition of zinc does not (data not shown). Although both molybdate and boiled L cell cytosol stabilize receptors in Chelex-treated cytosol, only the latter reconstitutes the system such that steroid binding again drives both dissociation of the receptor complex and transformation to the DNA-binding state. In contrast to boiled cytosol, we obtain only low and inconsistent reconstitution of hormone-dependent transformation with our factor preparation. This leads us to think that another heat stable component of cytosol is required for reconstitution of hormone dependence in addition to the metal that is removed by Chelex.
Transformation of cytosolic glucocorticoid receptors can be brought about without the presence of hormone by diluting cytosol or by increasing the pH or the ionic strength. These are conditions that also promote dissociation of the receptor from hsp90, and they may serve to counteract the stabilizing effect of the endogenous metal anion on the receptor-hsp90 complex. Transformation can also be brought about by heating cytosol. When the temperature of L cell cytosol is raised as high as 37 "C, the glucocorticoid receptor rapidly dissociates from hsp90 and acquires DNA binding activity even when it is not bound by steroid (data not shown). It is only at lower temperatures where one can easily demonstrate in whole cytosol that steroid binding and thermal energy are both required for receptor transformation.
The fact that hormone-free glucocorticoid receptor in metal-depleted cytosol undergoes temperature-dependent dissociation and transformation at the same rate as hormonebound glucocorticoid receptor (Figs. 4 and 5) suggests that there is a temperature-dependent component of transformation that is independent of the steroid-dependent component. The observations of this paper also suggest that the steroiddependent transformation of receptors that occurs at temperatures where this event is usually measured in cytosol preparations (20-27 "C) may reflect an affect of steroid binding on the ability of the endogenous metal anion to stabilize the complex. The binding of steroid to its receptor site could affect such a metal center either directly through a conformational change that affects the positioning of the metalbinding ligands or perhaps indirectly by affecting the redox state of metal-binding ligands, such as cysteine thiol groups located in the steroid-binding domain.
A model that could explain the observations of this paper and the concept of independent temperature-dependent and ligand-dependent events assumes two principal centers of interaction between hsp90 and the glucocorticoid receptor, with only one of these sites being involved in metal binding. One site that has been suggested for hsp90 binding is a highly conserved hydrophobic region that lies within the steroidbinding domain (29,36). This region may provide the major contribution to the binding forces that are disrupted in a temperature-dependent manner when metal is no longer present to stabilize the second site of interaction with hsp90. We know from the study of mutant mouse receptors in which this region is deleted that the metal anion stabilization site is situated outside of the conserved domain, most probably toward the carboxyl terminus of the steroid-binding domain.4 'P. R. Housley, E. R. Sanchez, G. M. Ringold, and W. B. Pratt, manuscript in preparation.