Purification and characterization of two novel phosphoglycerides that modulate the glucocorticoid-receptor complex. Evidence for two modulator binding sites in the occupied/unactivated steroid hormone receptor

Modulator is a novel ether aminophosphoglyceride that is commonly known as the low-molecular weight inhibitor of glucocorticoid-receptor complex activation. An ultra-large scale purification of modulator has been performed from 1000 rat livers. This purification was similar to our previous one (Bodine, P. V., and Litwack, G. (1988) J. Biol. Chem. 263, 3501-3512), but involved the chromatography of heated rat liver cytosol on a 7-liter bed volume Sephadex G-15 gel filtration column. Two peaks of modulator activity eluted from the giant gel-filtration column, and these two modulators (peak-1 and peak-2) were chromatographed separately on Dowex-1 anion-exchange columns. Both modulators were determined to be homogeneous after this step by analytical high-performance thin-layer chromatography, analytical high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy. Furthermore, although peak-1 and peak-2 differed in molecular weight, the two modulators co-chromatographed by anion-exchange, high-performance thin-layer, and high-performance liquid chromatography. These results suggest that the two modulators have similar structures and therefore appear to be isoforms of each other. In addition, both of the modulators are organic molecules that are devoid of molybdenum and 62 other metals. Activity assays indicated that the larger peak-1 modulator was five times more potent than the smaller peak-2 modulator at inhibiting receptor activation and at stabilizing the steroid-binding ability of the occupied and unoccupied receptors. Mixing experiments indicated that the activities of the two modulators were synergistic for both receptor activation inhibition and for occupied receptor steroid-binding stabilization. However, the effects of peak-1 and peak-2 modulator on unoccupied receptor steroid-binding stabilization were additive. Thus, although the two modulators have similar chemical structures, the biological potencies of the two compounds are different. Moreover, these results suggest that although the unoccupied/unactivated receptor has only one modulator binding site, the occupied/unactivated receptor has two modulator binding sites, one site for each of the isoforms.     

Identification of a macromolecular inhibitor of glucocorticoid-receptor complex activation in rat liver cytosol

We have identified an endogenous regulator of the glucocorticoid receptor following fractionation of dialyzed rat liver cytosol on DEAE-cellulose. The macromolecular regulator, purified approximately 20-fold as judged by Lowry-reactive material, inhibits activation of glucocorticoid-receptor complexes when assayed by DNA-cellulose binding and by chromatography on DEAE-cellulose minicolumns. In addition the active DEAE-cellulose fraction stabilizes the unoccupied glucocorticoid receptor against heat inactivation. Evidence is presented that the observed inhibition of activation by the active DEAE-cellulose fraction is not due to concentration of cytosolic proteases or RNA. The inhibitory molecule in the active fraction is not stable to heating at 90 degrees C (15 min) and is partially inactivated at 45 degrees C (15-60 min).     

Modulators of the glucocorticoid receptor also regulate mineralocorticoid receptor function.

Modulators are proposed to be novel ether aminophosphoglycerides that stabilize unoccupied and occupied glucocorticoid receptor steroid binding and inhibit glucocorticoid receptor complex activation. Two isoforms, modulator 1 and modulator 2, have been purified from rat liver cytosol [Bodine, P.V., & Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554]. Since the mineralocorticoid receptor is relatively resistant to activation, modulator's effect on rat distal colon mineralocorticoid receptor function was examined. Warming of unoccupied receptor decreased residual specific [3H]aldosterone binding by 86 +/- 2%. Both modulator isoforms completely prevented this destabilization with Km's of 2 +/- 1 microM modulator 1 and 24 +/- 5 microM modulator 2. Warming of occupied mineralocorticoid receptors decreased [3H]aldosterone binding by 56 +/- 3%. Modulator only partially stabilized occupied receptor binding with Km's of 10 +/- 2 microM modulator 1 and 68 +/- 8 microM modulator 2. Modulator inhibited receptor activation with Km's of 3 +/- 1 microM modulator 1 and 33 +/- 10 microM modulator 2. Double-reciprocal analysis showed linear kinetics, and mixing modulator isoforms together had additive effects on unoccupied and occupied receptor steroid binding stabilization and activation inhibition. Colon cytosol contained a low molecular weight, heat-stable factor(s) which inhibited receptor activation and stabilized occupied receptor steroid binding. Molybdate completely stabilized unoccupied mineralocorticoid receptor steroid binding and inhibited activation with half-maximal effects at 3-4 mM but only stabilized occupied receptor binding by approximately 40%. These data indicate that (i) apparent physiologic concentrations of modulator stabilize mineralocorticoid receptor steroid binding and inhibit receptor activation, (ii) an aldosterone-responsive tissue contains a modulator-like activity, and (iii) molybdate mimics the effects of modulator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the glucocorticoid-receptor complex

A crucial step in the interaction of glucocorticoids with target cells is the activation step, which involves a conformational change in the cytoplasmic glucocorticoid-receptor protein complexes and facilitates their binding to the cell nucleus. Activation can be quantified by measuring the ability of glucocorticoid-receptor complexes to bind to polyanions, such as DNA-cellulose, and unactivated complexes can be separated from activated complexes by rapid ion exchange chromatography using diethylaminoethyl (DEAE)-Sephadex or DEAE-cellulose. Activation occurs in vivo under physiological conditions and the rate of activation of cytoplasmic glucocorticoid-receptor complexes can be enhanced in vitro by physical manipulations (elevated temperature, increased ionic strength, dilution). In vitro studies suggest that activation is a regulated process and a low molecular weight component termed modulator, which has been identified in rat hepatic cytosol, inhibits activation. Additional studies employing phosphatase inhibitors, such as molybdate, and purified calf intestinal alkaline phosphatase suggest that either the receptor protein or a regulatory component is dephosphorylated during activation. Results obtained with specific chemical probes suggest that activation results in the exposure of basic amino acid residues consisting minimally of lysine, arginine, and histidine. Pyridoxal 5'-phosphate, a specific probe for lysine residues, exerts dual effects on glucocorticoid-receptor complexes, since it stimulates the rate of activation and also inhibits the binding of previously activated complexes to nuclei or DNA-cellulose. The ability of 1,10-phenanthroline, a metal chelator, to inhibit the DNA-cellulose binding of activated complexes suggests that a metal ion(s) located at or near the DNA binding site may become exposed as a consequence of activation. Collectively, the results of these various experiments suggest that activation is a regulated biochemical phenomenon with physiological significance.     

Degradation without apparent change in size of molybdate-stabilized nonactivated glucocorticoid-receptor complexes in rat thymus cytosol

We have investigated the stability of the [3H]dexamethasone 21-mesylate-labeled nonactivated glucocorticoid-receptor complex in rat thymus cytosol containing 20 mM sodium molybdate. Cytosol complexes were analyzed under nondenaturing conditions by gel filtration chromatography in the presence of molybdate and under denaturing conditions by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. When analyzed under nondenaturing conditions, complexes from fresh cytosol and from cytosol left for 2 h at 3 degrees C eluted from gel filtration as a single peak of radioactivity with a Stokes radius of approximately 7.7 nm, suggesting that no proteolysis of the complexes had occurred in either cytosol. When analyzed under denaturing conditions, however, whereas the fresh cytosol gave a receptor band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr approximately 90,000 (corresponding to the intact complex), the cytosol that had been left for 2 h at 3 degrees C gave only a fragment (Mr approximately 50,000). This fragment, just as the intact complex, could be thermally activated to a DNA-binding form. Proteolysis of the receptor could be blocked by preparing the cytosol in the presence of EGTA, leupeptin, or a heat-stable factor present in the cytosol of rat liver and WEHI-7 mouse thymoma cells. From these results we conclude: (i) 20 mM molybdate does not protect the nonactivated glucocorticoid-receptor complex present in rat thymus cytosol against proteolysis under conditions which are commonly used for cell-free labeling of the receptor, and (ii) the demonstration of a Stokes radius of approximately 8 nm for the nonactivated glucocorticoid-receptor complex is not sufficient to indicate that the receptor complex is present in its intact form.     

Partial purification of the glucocorticoid receptor from rat liver: a rapid, two-step procedure using DNA-cellulose.

Glucocorticoid receptor from rat liver was purified 1800-fold by a rapid two-step procedure using DNA-cellulose. The procedure is based on increasing the affinity of the glucocorticoid-receptor complex for DNA by heating the complex. During a first chromatography step, unheated glucocorticoid-receptor complex is separated from cytosol proteins that bind to DNA-cellulose with high affinity. During a second chromatographic step, heat-treated glucocorticoid-receptor complex is separated from proteins with low affinity for DNA. The partially purified complex is functionally competent in that it is taken up by isolated rat liver nuclei.     

Evidence that the endogenous heat-stable glucocorticoid receptor-activating factor is thioredoxin

Extraction of rat liver cytosol with 10% charcoal at 4 degrees C inactivates specific glucocorticoid-binding capacity. The steroid-binding capacity of extracted cytosol can be restored by adding dithiothreitol or by incubating with boiled liver cytosol at 20 degrees C in the presence of 10 mM sodium molybdate. Two components of boiled cytosol are required for receptor activation: NADPH and an endogenous heat-stable protein with an apparent Mr of 12,300 by Sephadex G-50 chromatography. This endogenous receptor-activating protein coelutes on Sephadex G-50 chromatography with endogenous thioredoxin activity, and it can be replaced in the activating system by purified Escherichia coli thioredoxin. These observations suggest that glucocorticoid receptors in cytosol preparations are maintained in a reduced, steroid-binding state by a NADPH-dependent, thioredoxin-mediated reducing system.     

Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex

The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.     

Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.     

Inactivation of rat liver glucocorticoid receptor by molybdate. Effects on both non-activated and activated receptor complexes.

When freshly prepared glucocorticoid-receptor complex from rat liver cytosol was incubated at 23 degrees C in the presence of sodium molybdate, its subsequent binding to isolated nuclei, DNA-cellulose and ATP-Sepharose was blocked. In addition, binding to these acceptors by cytosol receptor complex fractionated with (NH4)2SO4 was also blocked by incubation of the complexes with 50 mM-sodium molybdate. However, molybdate had no effect on the binding of activated receptor complexes to ATP-Sepharose. Molybdate was also effective in extracting the nuclear- and DNA-cellulose-bound glucocorticoid-receptor complexes in a dose-dependent manner. Molybdate appears to exert its effects directly on the receptor by interacting with both non-activated and activated receptor forms.     

Maximizing the purification of the activated glucocorticoid receptor by DNA-cellulose chromatography.

With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.     

Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.     

The molybdate-stabilized nonactivated glucocorticoid receptor contains a dimer of Mr 90,000 non-hormone-binding protein

A glucocorticoid receptor-associated Mr approximately 90,000 non-hormone-binding protein was purified and characterized. The molybdate-stabilized nonactivated rat liver glucocorticoid-receptor complex (Mr approximately 300,000) was immunoadsorbed on cyanogen bromide-activated Sepharose 4B to which a monoclonal IgG 2a antibody directed against the activated rat glucocorticoid receptor (Mr approximately 94,000) had been coupled. Following removal of molybdate and thermal activation of the receptor immobilized on the immunoaffinity matrix, an Mr approximately 90,000 non-hormone-binding protein was specifically eluted. This protein was further purified to homogeneity using high performance ion exchange chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, sucrose gradient ultra-centrifugation, and high performance size-exclusion chromatography. Hydrodynamic characterization under nondenaturing conditions revealed that the purified glucocorticoid receptor-associated protein represents a molecular species with a sedimentation coefficient of 6.1 S, a Stokes radius of 6.9 nm, and a calculated Mr approximately 184,000. These results, combined with analysis on denaturing electrophoresis indicate that, under certain conditions, the Mr approximately 94,000 steroid-binding protein is associated with a dimer of Mr approximately 90,000 non-hormone-binding protein.     

Interconversion of androgen receptor forms by divalent cations and 8 S androgen receptor-promoting factor. Effects of Zn2+, Cd2+, Ca2+, and Mg2+

The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor.     

Differential effects of molybdate on the hydrodynamic and DNA-binding properties of the non-activated and activated forms of the androgen receptor in calf uterus

Calf uterine cytosol contains an androgen receptor with a relative molecular mass of approx. 90,000. In this study we have analysed the structure and aggregation properties of the androgen receptor, using sucrose density gradient centrifugation on a vertical rotor (VTi65). In the presence of 10 mM NaCl the androgen receptor in whole cytosol sedimented at 8 S irrespective of the presence of molybdate. In 400 mM NaCl the receptor dissociated to a 4.3 S entity. In whole cytosol molybdate promoted a partial shift of the 4.3 S receptor into the aggregated 8 S state. The time of exposure of the receptor to molybdate and NaCl determined the proportion of receptor sedimentating at 8 S and 4.3 S. The DNA-binding form of the uterine androgen receptor when analysed under the conditions of the DNA-cellulose binding assay, sedimented at 6.5 S. Increasing concentrations of molybdate shifted its sedimentation coefficient gradually from 6.5 S to 4.5 S and in parallel reduced the DNA-binding capacity. Molybdate added to a partially purified, DNA-binding form of the androgen receptor did not promote receptor aggregation to faster sedimentating forms. This suggests that such preparations are devoid of an androgen receptor-aggregation factor. Indirect evidence for such a factor was obtained from reconstitution experiments with whole cytosol. Our results indicate that the DNA-binding form of the androgen receptor interacts with a cytosol factor to form the 8 S receptor complex. Molybdate has diverse effects: in the presence of the cytosol factor it stabilizes the 8S complex; in its absence molybdate prevents in a concentration-dependent way DNA-binding as well as reaggregation of the monomeric 4.3 S form.     

Interaction of rat liver glucocorticoid receptor with sodium tungstate.

Effects of sodium tungstate on various properties of rat liver glucocorticoid receptor were examined at pH7 and pH 8. At pH 7, [3H]triamcinolone acetonide binding in rat liver cytosol preparations was completely blocked in the presence of 10--20 mM-sodium tungstate at 4 degrees C, whereas at 37 degrees C a 30 min incubation of cytosol receptor preparation with 1 mM-sodium tungstate reduced the loss of unoccupied receptor by 50%. At pH 8.0, tungstate presence during the 37 degrees C incubation maintained the steroid-binding capacity of unoccupied glucocorticoid receptor at control (4 degrees C) levels. In addition, heat-activation of cytosolic glucocorticoid-receptor complex was blocked by 1 mM- and 10 mM-sodium tungstate at pH 7 and pH 8 respectively. The DNA-cellulose binding by activated receptor was also inhibited completely and irreversibly by 5 mM-tungstate at pH 7, whereas at pH 8 no significant effect was observed with up to 20 mM-tungstate. The entire DNA-cellulose-bound glucocorticoid-receptor complex from control samples could be extracted by incubation with 1 mM- and 20 mM-tungstate at pH 7 and pH 8 respectively, and appeared to sediment as a 4.3--4.6 S molecule, both in 0.01 M- and 0.3 M-KCl-containing sucrose gradients. Tungstate effects are, therefore, pH-dependent and appear to involve an interaction with both the non-activated and the activated forms of the glucocorticoid receptor.     

Immunochemical characterization of the rat kidney glucocorticoid receptor. The role of proteolysis in the formation of corticosteroid binder IB

Glucocorticoid receptors of rat kidney and liver were compared by physicochemical and immunochemical methods to investigate the role of proteolysis in the formation of corticosteroid binder IB. Kidney cytosol prepared in the presence of sodium molybdate contained receptor forms comparable to rat liver glucocorticoid receptor; [3H]triamcinolone acetonide-labeled receptors eluted from Sephacryl S-300 as a multimeric 6.1 nm component in the presence of molybdate and as a monomeric 5.7 nm component in the absence of molybdate. Both forms were recognized by the monoclonal antibody BUGR-1 which was raised against rat liver glucocorticoid receptor. When kidney cytosol was prepared in the absence of molybdate, labeled receptor complexes eluted from Sephacryl S-300 as a 5.8 nm component in the presence of molybdate. However, in the absence of molybdate, the receptor eluted as a smaller 3.4 nm component which was identical with the size of activated kidney glucocorticoid receptor chromatographed in either the presence or absence of molybdate. The 3.4 nm activated kidney glucocorticoid receptor did not bind to DEAE-cellulose under conditions where activated liver receptor was retained. These properties of the activated kidney receptor are characteristic of corticosteroid binder IB. Incubation of the activated kidney receptor complex with BUGR-1 resulted in a shift in apparent Stokes radius from 3.4 nm to 5.4 nm, indicating immunochemical similarity with rat liver receptor. Identification of the immunoreactive receptor subunit by Western blotting demonstrated that kidney cytosol prepared in the presence of molybdate contained a major 94-kDa immunoreactive component which co-migrated with rat liver glucocorticoid receptor, while cytosol prepared in the absence of molybdate contained principally a 44-kDa immunoreactive species. These results suggest that corticosteroid binder IB can be generated by in vitro proteolysis and does not represent a polymorphic form of the glucocorticoid receptor.     

Subunit composition of the molybdate-stabilized non-activated glucocorticoid receptor from rat liver

A monoclonal IgG 2a antibody directed against the activated rat liver glucocorticoid receptor (GR) was used to prepare an immunoaffinity matrix of high capacity. The molybdate-stabilized GR from rat liver cytosol was immunoadsorbed on this gel. A non-hormone-binding protein of Mr approximately 90,000, as determined after denaturing gel electrophoresis, was eluted from this matrix following removal of molybdate and exposure to heat (25 degrees C) and salt (0.15 M NaCl). Subsequently, the Mr approximately 90,000 protein was purified to homogeneity using high-performance ion-exchange chromatography, covalently radiolabelled, and analyzed by high-performance size-exclusion chromatography and sucrose gradient ultracentrifugation. Hydrodynamic characterization indicates that, under our experimental conditions, the molybdate-stabilized rat liver GR (Rs approximately 7.4 nm, s20,w approximately 9.1 S, calculated mol. wt Mr approximately 285,000) includes one steroid-binding unit (Rs approximately 5.5 nm, S20,w approximately 4.3 S, calculated Mr approximately 100,000) and a dimer of Mr approximately 90,000 non-hormone-binding protein (Rs approximately 6.9 nm, S20,w approximately 6.1 S, calculated native Mr approximately 180,000).     

Evidence that the endogenous heat-stable glucocorticoid receptor stabilizing factor is a metal component of the untransformed receptor complex

Boiled cytosols prepared from a wide variety of sources contain a low Mr factor that inhibits glucocorticoid receptor transformation to the DNA-binding state (Leach, K.L., Grippo, J.F., Housley, P.R., Dahmer, M.K., Salive, M.E., and Pratt, W.B. (1982) J. Biol. Chem. 257, 381-388). In this work, we show that this endogenous factor, which is partially purified from rat liver, produces all of the effects of the group VI-A transition metal oxyanions molybdate and vanadate on the structure and function of glucocorticoid receptors in cytosol preparations. Like molybdate, the endogenous factor behaves as a strong anion with an apparent Mr of 340 on Bio-Gel P-2, and it binds to both hydroxylapatite and Chelex 100 resins. The receptor stabilizing activity of the factor is completely stable to heating at 320 degrees C for 1 h. The small size, profound heat stability, and absorption by a metal chelating resin strongly suggest that the factor is an endogenous metal anion. As reduction of the concentration of the factor in cytosol promotes generation of the DNA-binding form of the receptor, we suggest that this endogenous metal anion interacts with the receptor to stabilize the 9 S complex and maintain the receptor in its untransformed, non-DNA-binding state. We propose that molybdate and vanadate may exert their effects on the untransformed receptor by interacting with the binding site for the endogenous metal anion.     

Interaction of the Mr = 90,000 heat shock protein with the steroid-binding domain of the glucocorticoid receptor

We have investigated the physiochemical characteristics of trypsin-treated, molybdate-stabilized glucocorticoid-receptor complexes from rat liver in the presence of 10 mM sodium molybdate by high performance ion-exchange chromatography, high performance size-exclusion chromatography, and sedimentation analysis. Trypsin treatment was performed under conditions previously reported to degrade the monomeric Mr approximately 94,000 steroid-binding protein to an Mr approximately 27,000 ligand-binding entity (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865). Also in the presence of molybdate, an Mr approximately 27,000 steroid-binding fragment was obtained by limited trypsinization. However, no major differences in the tested physicochemical parameters were seen when trypsin-treated glucocorticoid-receptor complexes were compared with crude cytosolic complexes. Furthermore, the Mr approximately 27,000 steroid-binding fragment generated in the presence of molybdate could be immunoprecipitated by antibodies specific for the glucocorticoid receptor-associated Mr approximately 90,000 heat shock protein. These results provide direct evidence for an interaction of the Mr approximately 90,000 heat shock protein with the steroid-binding domain of the glucocorticoid receptor, known to correspond to the C-terminal third of the receptor protein.