Effects of molybdate on steroid receptors in intact GH1 cells. Evidence for dissociation of an intracellular 10 S receptor oligomer prior to nuclear accumulation

Treatment of intact GH1 cells with sodium molybdate inhibits the subsequent rate of nuclear accumulation of hormone-occupied glucocorticoid and estrogen receptors. Cells were incubated at 23 degrees C for 1 h with 30 mM molybdate and then for up to 30 min with [3H]triamcinolone acetonide or [3H]estradiol in the continued presence of molybdate. Although molybdate did not affect the rate of receptor occupancy with either steroid, cells treated with molybdate had more occupied cytosolic and fewer occupied nuclear receptors than control cells. For the glucocorticoid receptor, cells treated with molybdate had more 10 S and fewer 4 S cytosolic receptors than control cells. In low salt cytosol molybdate inhibits the temperature-mediated subunit dissociation of occupied 10 S glucocorticoid receptor. These results suggest that a hormone-mediated dissociation of an intracellular 10 S oligomeric glucocorticoid receptor form to its 4 S subunits is required prior to accumulation of occupied receptors in the nuclear fraction. In cells incubated at 37 degrees C for 1 h or longer with [3H]triamcinolone acetonide, molybdate shifts the steady state intracellular distribution of receptor toward the 10 S cytosolic receptor form, consistent with the interpretation that molybdate affects the rapidly exchanging subunit equilibrium between the 10 S and 4 S cytosolic forms by slowing the rate of 10 S receptor dissociation. Molybdate prevents loss of glucocorticoid-occupied 10 S but not 4 S receptors in heated cytosol by stabilizing the relatively protease-resistant 10 S receptor. Since molybdate stabilizes 10 S oligomeric steroid receptors in vitro, the effects of molybdate on nuclear accumulation of occupied receptors in intact cells support the intracellular existence and physiological relevance of 10 S glucocorticoid and estrogen receptors. These results support a general model for steroid receptor activation in which binding of hormone promotes dissociation of intracellular 8-10 S oligomeric receptors to their DNA-binding subunits.     

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.     

Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reduction agents, ATP, and heat-stable factors.

The specific glucocorticoid binding capacity in cytosol preparations of rat thymocytes decays with a half-life of 4 h at 0 degrees C or 20 min at 25 degrees C. Phosphatase inhibitors (molybdate, fluoride, glucose 1-phosphate) added alone do not prevent this inactivation. Dithiothreitol (2 mM) has a large stabilizing effect on the binding capacity at 0 degrees C but only a small effect at 25 degrees C. Addition of 10 mM molybdate plus 2 mM dithiothreitol totally prevents inactivation for at least 8 h at 25 degrees C as well as at 0 degrees C. Fluoride (100 mM) also retards the inactivation if added with dithiothreitol. Addition of dithiothreitol at 25 degrees C to inactivated cytosol receptors results in partial activation of the binding capacity. Addition of dithiothreitol to receptors inactivated at 25 degrees C in the presence of molybdate allows total reactivation of the binding capacity to the maximum zero time value. If binding capacity is inactivated by preincubation of the cytosol at 25 degrees C, addition of ATP with dithiothreitol enhances the activation observed with only dithiothreitol. This ATP stimulated activation is optimal at 1 to 3 mM. ATP (10 mM) is required when molybdate is added to prevent simultaneous inactivation. ADP, GTP, CTP, and UTP have some activating capacity but the effects of all nucleotides are inhibited by the ATP analog, adenyl-5'-yl (beta, gamma-methylene)diphosphonate. ATP-dependent activation can also be prevented with 50 mM EDTA, and addition of magnesium partially overcomes the EDTA inhibition. Dithiothreitol activation of thymocyte glucocorticoid binding capacity can also be enhanced by addition of a heat-stable preparation from thymocytes, L cells, or liver. Sephadex G-25 chromatography, assay of ATP, and inhibition of the activation with adenyl-5'-yl (beta, gamma-methylene)diphosphonate suggest that these preparations contain varying amounts of endogenous reducing equivalents and ATP as well as a larger heat stable factor. Maximum activation is obtained by adding dithiothreitol, ATP, molybdate, and the larger heat-stable factor. These results suggest that stabilization and activation of glucocorticoid binding capacity in thymocytes requires phosphorylation as well as reduction of the receptor itself or of some other component required for the steroid binding reaction.     

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.     

The effect of molybdate on glucocorticoid receptor transformation and translocation in intact, viable AtT-20 mouse pituitary tumor cells

The effect of 20 mM molybdate on the transformation and translocation of glucocorticoid receptors in intact AtT-20 mouse pituitary tumor cells was investigated. To test whether transformation of the receptor was inhibited during in vivo incubations with both molybdate and glucocorticoid, the DEAE cellulose elution profile of extracted receptor was determined. It was found that receptors from both high speed cytosols and nuclear extracts were transformed. To test whether translocation was affected by molybdate, the fraction of glucocorticoid-receptor complexes found in the nucleus was determined. At 37 degrees C, in the absence of molybdate, 55-60% of the glucocorticoid receptor complexes were in the nuclear compartment. Molybdate did not effect the magnitude of nuclear translocation. Control studies suggested that the agent entered the cells, however. Cold exposure (0 degrees C) reduced nuclear translocation to 20-25%. It is concluded that in vivo, either molybdate is not able to interact with the receptor or transformation in vivo is not mediated by the same molybdate-sensitive mechanisms currently being studied using broken cell-preparations.     

Molybdate inhibition of glucocorticoid receptor inactivation and transformation.

The inactivation of glucocorticoid receptors that occurs when cytosol is heated at 25 degrees C is blocked reversibly by molybdate and slowed by some other phosphatase inhibitors such as fluoride and glucose 1-phosphate. Molybdate is also capable of preventing nonenzymatic inactivation of unbound receptors caused by exposure to salt or precipitation with ammonium sulfate at 0 degrees C. Inactivation of unbound receptors caused by Sephadex G-50 gel filtration is prevented by all three inhibitors. Both molybdate and tungstate block temperature-dependent transformation of glucocorticoid.receptor complexes to the DNA-binding state, where fluoride and glucose 1-phosphate have no effect. Transformation brought about at 0 degrees C by salt, ammonium sulfate precipitation, or gel filtration is also blocked by both molybdate and tungstate. Tungstate differs from molybdate in that it has little or no effect on receptor inactivation. Fluoride and glucose 1-phosphate do not inhibit transformation. These observations support the proposal that molybdate and tungstate are interacting through a reversible association with the glucocorticoid receptor itself. We propose that they may act by forming a complex with a phosphate moiety on the receptor.     

Evidence that removal of an endogenous metal that stabilizes the untransformed glucocorticoid receptor in cytosol allows ligand-independent receptor transformation

Cytosol preparations contain an endogenous heat-stable factor which stabilizes the glucocorticoid receptor in its untransformed, non DNA-binding form. Elution of a partially purified preparation of this stabilizing factor through a metal chelating resin (Chelex-100) leads to the loss of its ability to inhibit temperature-mediated transformation of the receptor. Sodium molybdate mimicks the ability of this endogenous metal to stabilize the untransformed receptor, and it too is adsorbed by Chelex resin. When an L-cell cytosol preparation containing the glucocorticoid receptor is passed through a column of Chelex-100 resin and then incubated at 15 degrees C, the receptor is rapidly transformed to the DNA-binding state, regardless of whether it is steroid-bound or not. In contrast, whole cytosol containing endogenous metals is transformed to the DNA-binding state only when the receptor is both steroid-bound and exposed to elevated temperature. these data suggest that a metal (or metals) may be involved in conferring the property of ligand-dependency to the transformation process.     

Hepatic Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Partial stabilization by molybdate

In structure and general mode of action, the Ah receptor is very similar to the receptors for steroid hormones. Molybdate previously has been shown to be highly effective at preserving ligand-binding function in steroid receptors during their exposure to elevated temperature or high ionic strength and at stabilizing steroid receptors as high molecular weight oligomeric complexes. Since such stabilization by molybdate can be very useful during characterization and purification of receptors, we tested the effects of molybdate on the Ah receptor to determine if the Ah receptor, like the receptors for steroid hormones, might be stabilized. In hepatic cytosols from C57BL/6N mice and Sprague-Dawley rats, molybdate concentrations up to 30 mM in homogenizing and analysis buffers did not alter the concentration of specific Ah receptor sites detected by binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. However, inclusion of 20 mM molybdate in the homogenizing buffer did significantly protect unliganded Ah receptor from thermal inactivation at 20 degrees C and from KCl-induced loss of ligand-binding ability. In accord with previous reports, 20 mM molybdate in homogenizing and analysis buffers greatly increased the concentration of detectable glucocorticoid receptor in rat hepatic cytosol and estrogen receptor in rat uterine cytosol. Exposure to 0.4 M KC1 caused the glucocorticoid receptor from rat liver to shift sedimentation from approximately equal to 8 S to approximately equal to 4 S and caused a severe loss of specific glucocorticoid binding. Presence of 20 mM molybdate stabilized the glucocorticoid receptor as a single discrete peak sedimenting at approximately equal to 8 S. In contrast, the Ah receptor from rat liver exposed to 0.4 M KC1 in the presence of molybdate sedimented as biphasic peaks; one peak (approximately equal to 9.5 S) corresponded to the form of Ah receptor observed at low ionic strength, while the other peak (approximately equal to 5.5 S) corresponded to the form of Ah receptor seen in cytosol treated with 0.4 M KC1 in the absence of molybdate. Addition of heparin to hepatic cytosols from mice or rats shifted sedimentation of Ah receptor from approximately equal to 9.5 S to approximately equal to 5.5 S. Molybdate, again, provided stabilization in the approximately equal to 9.5 S form, but only for about one-half the total Ah receptor content in both rat and mouse hepatic cytosols. In sum, molybdate is far less effective at stabilizing rodent Ah receptors than it is at stabilizing steroid receptors in the same species.     

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.     

Nontransformed rabbit liver glucocorticoid receptor: purification, characterization and transformation

The molybdate-stabilized nontransformed form of the glucocorticoid receptor from rabbit liver has been purified approximately 8,000-fold by a three-step procedure. The first step involved protamine sulfate precipitation which allowed a 5-6-fold purification with 85% yield. The second step, affinity chromatography using a N-(12-dodecyl-amino) 9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxamide substituted Sepharose gel, purified the receptor 1,500-2,000-fold as calculated by specific radioactivity. The third step involved high performance liquid chromatography resulting in overall purification near 8,000-fold. The final glucocorticoid receptor appeared about 60% pure. The purified nontransformed glucocorticoid receptor had a sedimentation coefficient of 9 S in 0.16 M phosphate containing 5-20% sucrose gradients and the Stokes radius was 6.1-6.3 nm as determined by low pressure gel filtration and HPLC. Binding specificity of the purified receptor was identical to that previously reported in crude rabbit liver cytosol. Isoelectricfocusing and ion-exchange chromatography showed that the purification procedure affected the net charge of the receptor protein. This phenomenon could be related to interactions between the glucocorticoid receptor and cytosolic factors. SDS polyacrylamide gel electrophoresis showed a major Mr = 94,000 protein band which is in good agreement with previously reported values for glucocorticoid receptors. Transformation of the purified receptor was achieved after removal of molybdate by exposure at 25 degrees C to 0.4 M KCl. Characterization of the molecular forms was performed by means of incorporation into isolated nuclei, affinity towards polyanionic exchangers and high pressure size exclusion chromatography. Results show that about 40% of the receptor is in the transformed state.     

Chemical differentiation of type I and type II receptors for adrenal steroids in brain cytosol

Studies outlined here compare the properties of mineralocorticoid (Type I) and glucocorticoid (Type II) receptors in cytosol from adrenalectomized mouse brain. Pretreating cytosol with dextran-coated charcoal (DCC) produced a 4.7-fold increase in the subsequent macromolecular binding of the mineralocorticoid, [3H]aldosterone (20 nM ALDO, in the presence of a 50-fold molar excess of the highly specific synthetic glucocorticoid, RU 26988), whereas it produced a 55% decrease in the binding of the glucocorticoid, [3H]triamcinolone acetonide (20 nM TA). Scatchard analyses revealed that DCC pretreatment had no effect on the affinity or maximal binding of Type I receptors for [3H]ALDO (in the presence of a 0-, 50- or 500-fold excess of RU 26988), whereas it produced a 3- to 6-fold increase in the Kd, and an 8-43% decrease in the maximal binding, of Type II receptors for [3H]TA and [3H]dexamethasone. Optimal stability of unoccupied Type I receptors at 0 degree C was found to be achieved in buffers containing glycerol, but lacking molybdate. Although the addition of molybdate was found to reduce the loss in Type I receptor binding observed after incubating unlabelled cytosol at 12 or 22 degrees C, this stabilization was accompanied by a concentration-dependent reduction in the binding of [3H]ALDO at 0 degree C. Scatchard analyses showed that this reduction was due to a shift in the maximal binding, and not the affinity, of the Type I receptors for [3H]ALDO. The presence or absence of dithiothreitol in cytosol appeared to have little effect on the stability of Type I receptors. In contrast to our finding for Type I receptors, it was possible to stabilize the binding capacity of unoccupied Type II receptors, even after 2-4 h at 12 or 22 degrees C, if the glycerol containing buffers were supplemented with both molybdate and dithiothreitol. In summary, these results indicate distinct chemical differences between Type I and Type II receptors for adrenal steroids.     

Characterization of the purified molybdate-stabilized glucocorticoid receptor from rat liver. An in vitro transformable complex

Rat liver glucocorticoid receptor was purified in the presence of molybdate by a three-step procedure comprising protamine sulfate precipitation, affinity chromatography on a dexamethasone matrix and high-performance size-exclusion chromatography (HPSEC) on a TSK G 3000 SW column. The [3H]triamcinolone-acetonide-receptor complex was obtained in 20% yield with an overall 11 800-fold purification. The dissociation rate constant of this complex was 1.6 X 10(-4) min-1. The purified receptor sedimented at 8.3 S in high-salt and 9.4 S in low-salt sucrose gradients containing molybdate. A 7.0-nm Stokes radius was determined by HPSEC on a TSK G 4000 column in high-salt buffer. The calculated Mr was 278000. Dodecyl sulfate/polyacrylamide gel electrophoresis revealed an almost homogeneous 90 000-Mr band. Three minor bands with Mr of 78 000, 72 000 and 48 000 were also inconstantly seen. An apparent pI = 5.1 was observed for the [3H]steroid complex by isoelectric focusing in agarose gel. Furthermore high-performance ion-exchange chromatography of the purified complex on a DEAE 545 LKB column (DEAE HPLC) yielded a sharp peak eluted at a 315 mM potassium ion concentration. This peak was shown to contain almost all the 90 000-Mr protein. Moreover the purified receptor complex appeared to be transformable to a DNA-binding state after molybdate removal followed by warming 30 min at 25 degrees C in presence of 0.2% bovine serum albumin: 50-78% transformation yield could be demonstrated by DNA-cellulose chromatography. Partial transformation could also be obtained at 0 degrees C in the absence of any added protein and was followed by DEAE HPLC. The transformed complex was eluted by 180 mM potassium.     

Molybdate effect on the glucocorticoid receptor in cell-free systems and intact lymphocytes

The effect of sodium molybdate on the stability and activation of the glucocorticoid receptor from chick and rat thymus were investigated. Molybdate, at a concentration range of 1–10 mM, blocked denaturation of the cytosol receptor by elevated (25 and 37°C) temperatures. This effect could be observed only with the aggregated (low-salt) form of the receptor. Molybdate also inhibited transformation of the receptor-hormone complex to the DNA-binding state which occurs either with incubation at 25°C or with salt treatment. The inhibitory effect of molybdate could be observed only on the non-activated receptor; nuclear- and DNA-binding of the activated receptor was not significantly changed by molybdate. Both effects were concentration-dependent. Molybdate had no effect on the activation of the partially purified glucocorticoid receptor. Molybdate effect was also examined using intact lymphocytes. Sodium molybdate had no effect either on the steroid binding of whole cells or on the nuclear transfer of the hormone-receptor complex. While the mechanism of molybdate action remains unknown the results of experiments on purified receptor suggest that molybdate does not act directly on the receptor molecule; rather through some cytosol factor(s). However, these effects could only be seen in cell-free experiments, and not during the conditions of the living cell.     

Interactions of the nuclear thyroid hormone receptor with core histones

These studies concern the interactions of the rat liver thyroid hormone nuclear receptor with histones and factors influencing the receptor's assay and stability. Heating certain crude receptor preparations at 50 degrees C produces a selective loss of triiodothyronine (T3) but not thyroxine (T4) binding activity, whereas, with more purified preparations, such heating decreases both T3 and T4 binding. The selective T3-binding loss in crude preparations was found to be due to the simultaneous denaturation of the receptor's high-affinity hormone-binding activity for both T3 and T4 and generation of new low-affinity T4-binding sites. The fraction in which T4 binding can be activated could be separated from the receptors by Sephadex G-100 chromatography. Core histones stimulated both T3- and T4-binding activity of 6-fold-purified receptor preparations, and data from several different experimental approaches suggest that this stimulation is due to the capability of the core histones to prevent the receptor from binding to or being denatured by Sephadex G-25 assay columns. The core histones were also found to stabilize 500-fold-purified but not 6-fold-purified or crude receptor preparations. A number of other acidic or basic proteins had little or none of these stimulatory effects, whereas a few proteins (such as the insulin B chain and histone H1) did have activity, although it was less than that of the core histones. There were no significant differences between the purified core histone subfractions (H2A, H2B, H3, and H4). That core histones can interact with the thyroid hormone receptors was demonstrated more directly by the finding that the receptors bind to histone-Sepharose but not Sepharose or insulin- or ovalbumin-Sepharose columns and that this binding was blocked by core histones at concentrations suggestive of an affinity for the receptor-core histone interaction of around 3 microM at 0.15 M salt concentration. The results demonstrate the utility of the histones in the assay and stabilization of purified thyroid hormone receptors, but they fail to support our previous hypothesis of a receptor subunit where T3- but not T4-binding activity is regulated selectively by histones. However, the results indicate that histones may interact with the receptors with some degree of specificity, and they raise the possibility that the histones participate in the nuclear localization of the receptors.     

Physicochemical characteristics of the interaction of the glucocorticoid antagonist RU38486 with glucocorticoid receptors in vitro, and the role of molybdate

The physicochemical properties of complexes formed between the glucocorticoid antagonist, RU38486, and the glucocorticoid receptor in rat thymus cytosol were investigated and compared with those of complexes formed with the potent agonist, triamcinolone acetonide. The equilibrium dissociation constant for the interaction of [3H]RU38486 with the molybdate-stabilized glucocorticoid receptor was lower than that for [1,2,4-3H]triamcinolone acetonide at 0 degree C but higher at 25 degrees C, suggesting that hydrophobic interactions play a major role in the binding of RU38486. Differences in equilibrium constants were reflected in corresponding differences in dissociation rate constants; association rate constants for the two steroids were similar. The rate of dissociation of [3H]RU38486 from the glucocorticoid receptor was higher in the absence of molybdate than in its presence both at 0 degree C and at 25 degrees C, suggesting that molybdate modifies the physical state of the antagonist-receptor complex, but other physical properties were similar both in the presence and in the absence of molybdate. The rate of inactivation of the unoccupied glucocorticoid receptor at 25 degrees C in the absence of molybdate was lower in phosphate buffer than in Tris-HCl buffer but the rate of dissociation of [3H]RU38486 was the same in both buffers. The binding of RU38486 afforded little, if any, protection against inactivation in either buffer; [3H]RU38486 dissociated irreversibly from the inactivated receptor at the same rate as from the non-inactivated complex but molybdate had no effect on the dissociation kinetics of the inactivated complex. It is concluded that RU38486 interacts with the ground state of the glucocorticoid receptor in a manner which neither promotes receptor transformation nor prevents receptor inactivation.     

Sodium molybdate converts the RNA-associated transformed, oligomeric form of the glucocorticoid receptor into the transformed, monomeric form

The glucocorticoid receptor from rat liver cytosol prepared in 2 ml buffer/g tissue sedimented at approximately 10 S in low salt density gradient centrifugation without molybdate. When the receptor was heated at 25 degrees C, both approximately 10 S and approximately 7 S forms were seen in low salt gradient. The approximately 10 S form was not capable of binding to DNA-cellulose and was stabilized by sodium molybdate, namely it corresponded to untransformed receptor. The approximately 7 S form was capable of binding to DNA-cellulose and regarded as transformed receptor. On the other hand, partially-purified transformed receptor labeled with [3H]dexamethasone-21-mesylate sedimented at approximately 5 S, which migrated as a approximately 94 kDa species in SDS-polyacrylamide gel electrophoresis. The reconstitution analysis of this partially-purified approximately 5 S receptor and liver cytosol, showed the shift to approximately 7 S form. RNase A or T1 converted approximately 7 S transformed form into approximately 5 S but it did not affect approximately 10 S untransformed form. 5-20 mM sodium molybdate also shifted approximately 7 S to approximately 5 S. These results indicate that the approximately 7 S transformed form of the glucocorticoid receptor observed in low salt conditions might be an oligomer, probably including both approximately 5 S steroid-binding component and RNA/ribonucleoprotein, and that molybdate dissociates these interactions in a specific manner.     

Relationship between glucocorticoid receptor steroid-binding capacity and association of the Mr 90,000 heat shock protein with the unliganded receptor

Treatment of rat liver cytosol with hydrogen peroxide (H2O2) or sodium molybdate (MoO4(2-)) inhibits thermal inactivation of glucocorticoid receptor steroid-binding capacity at 25 degrees C. Dithiothreitol (DTT) prevents the stabilization of receptors by H2O2. Heating (25 degrees C) of immune pellets formed by immunoadsorption of L-cell murine glucocorticoid receptor complexes to protein-A-Sepharose with an anti-receptor monoclonal antibody (BuGR2) results in dissociation of the M 90,000 heat shock protein (hsp90) from the steroid binding protein. Such thermal-induced dissociation of hsp90 is inhibited by H2O2. Pretreatment of immunoadsorbed receptor complexes with the thiol derivatizing agent, methyl methanethiosulfonate (MMTS) prevents the ability of H2O2 to stabilize the hsp90-receptor interaction. These data suggest a role for hsp90 in maintaining an active steroid-binding conformation of the glucocorticoid receptor.     

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.     

Stabilization of glucocorticoid receptors in vitro using divalent cations plus cation chelators

The specific glucocorticoid receptor binding of rat liver cytosol was very unstable in vitro at 25 and 4 degrees C. However, 5 mM CaCl2 added with 5 mM EDTA to cytosol prior to incubation markedly stabilized unbound glucocorticoid receptors at both temperatures. Optimal effectiveness was achieved using equimolar (5 mM) amounts of CaCl2 and EDTA. On the other hand, 5 mM CaCl2 (added alone) further destabilized the unbound glucocorticoid receptor, while 5 mM EDTA (added alone) had no effect at 25 degrees C. EGTA (in lieu of EDTA) added with CaCl2 stabilized hepatic receptor binding at 25 degrees C. On the other hand, citrate added with calcium was ineffective in stabilizing the hepatic glucocorticoid receptor. MgCl2 effectively replaced CaCl2 as a stabilizing agent at 25 degrees C if added with 5 mM EDTA. When added alone, MgCl2 slightly destabilized the unbound receptor. Sucrose density gradient analysis (in low salt) revealed that CaCl2 plus EDTA enhanced the steroid-receptor complex sedimentation coefficient from 7 S to about 10 S. Unlike molybdate, CaCl2 plus EDTA had no apparent effect on steroid-receptor complex thermal transformation into a nuclear binding form, while MgCl2 plus EDTA partially reduced transformation. These results suggest a novel means to chemically stabilize unbound hepatic glucocorticoid receptors in vitro which may be of particular importance for receptor purification studies.     

Transformation of glucocorticoid and progesterone receptors to the DNA-binding state

This brief review explores some recent observations relating to the structure of untransformed glucocorticoid and progesterone receptors and the mechanism by which the receptors are transformed to the DNA-binding state. In their molybdatestabilized, untransformed state, progesterone and glucocorticoid receptors exist as a heteromeric 8-9S complex containing one unit of steroid binding phosphoprotein and one or two units of the 90 kD heat shock protein hsp90. When the receptors are transformed, the steroid-binding protein dissociates from hsp90. In cytosol preparations, temperature-mediated dissociation proceeds much more rapidly in the presence of hormone. The dissociated receptor binds to DNA with high affinity, regardless of whether it is in the hormone-bound or the hormone-free state. These observations raise the possibility that the primary, and perhaps the only, role for the hormone is to promote dissociation of the receptor-hsp90 complex. Molybdate, vanadate, and tungstate inhibit receptor transformation to the DNA-binding form, an effect that appears to reflect the ability of these transition metal oxyanions to stabilize the complex between the steroid receptor and hsp90. By promoting the formation of disulfide bonds, hydrogen peroxide also stabilizes the glucocorticoid receptor-hsp90 complex and prevents receptor transformation. A small, heat-stable factor present in all cytosol preparations inhibits receptor transformation, and, when the factor is removed, glucocorticoid receptors are rapidly transformed. This ubiquitous factor has the physical properties of a metal anion, and it is proposed that molybdate and vanadate affect steroid receptor complexes by interacting with a metal anion-binding site that is normally occupied by this endogenous receptor-stabilizing factor.