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.     

In vitro activation of rat cardiac glucocorticoid antagonist-versus agonist-receptor complexes

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [³H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25°C for 30 min or at 15°C for 30 min in the presence of 5 mM pyridoxal 5′-phosphate. Once thermally activated, the cardiac [³H]triamcinolone acetonide and [³H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215–250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.     

In vitro activation and DNA binding affinity of human lymphoid (CEM-C7) cytoplasmic receptors labeled with the antiglucocorticoid RU 38486

The data reported here demonstrate that the synthetic steroid RU 38486 functions as an optimal antagonist in the glucocorticoid-sensitive human leukemic cell line CEM-C7. This steroid blocks the ability of the potent agonist triamcinolone acetonide (TA) to induce glutamine synthetase activity and to ultimately cause cell lysis, but when given alone does not exhibit partial agonist activity. Both [3H]RU 38486 and [3H]TA bind with high affinity and specificity to cytosolic glucocorticoid receptors in this cell line. However, under a variety of in vitro conditions (elevated temperature and presence of exogenous ATP), [3H]TA promotes receptor activation more effectively than [3H]RU 38486. This difference in the extent of activation was verified by two independent techniques: DEAE-cellulose chromatography and DNA-cellulose binding. [3H]RU 38486 and [3H]TA dissociate at the same rate from the unactivated receptors but at 25 degrees C (not 0 degree C) [3H]RU 38486 dissociates slightly more rapidly from the activated receptors. The defective receptors in the glucocorticoid-resistant subclone 3R7 appear to be "activation labile" (rapid dissociation of ligand from activated form) using either tritiated steroid. Once activated in vivo, the CEM-C7 [3H]TA- and [3H]RU 38486-receptor complexes undergo similar nuclear translocation and those activated complexes generated in vitro appear to bind to nonspecific DNA-cellulose with the same relative affinities. Thus the precise mechanism(s) by which RU 38486 exerts its potent antiglucocorticoid effect in this human cell line cannot be easily explained in terms of a defect in one of the crucial steps (specific high affinity binding, activation, translocation, DNA binding) required to elicit a physiological response. However, the data presented here do suggest that when comparing an antagonist and agonist which both bind to receptors with the same relative high affinity, the agonist may be more effective in facilitating the conformational change associated with in vitro activation.     

In vitro activation of rat cardiac glucocorticoid antagonist- versus agonist-receptor complexes

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [3H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25 degrees C for 30 min or at 15 degrees C for 30 min in the presence of 5 mM pyridoxal 5'-phosphate. Once thermally activated, the cardiac [3H]triamcinolone acetonide and [3H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215-250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Analysis of the glucocorticoid receptor during differentiation of 3T3-F442A preadipocyte cell line in culture

A pure glucocorticoid agonist RU 28362 and the potent antagonist RU 38486 were compared with dexamethasone for the evolution and the molecular nature of the GR during insulin-dependent conversion of 3T3-F442A preadipocytes into mature cells. In the whole cell assay system, the affinity for preadipocyte GR was observed in the order RU 38486 greater than RU 28362 greater than dexamethasone. The GR complex was most stable in presence of dexamethasone followed by the antagonist RU 38486 = the agonist RU 28362. Similar results were obtained in mature adipocytes but the binding of RU 38486 was more equivocal. An insulin-dependent differentiation process did not alter any of these parameters but increased the number of GR nearly fivefold over a 2-week period. Ion-exchange analysis of the cytosolic receptor revealed that the differentiation process was not accompanied by the appearance of any novel or new forms of GR, contrary to the situation in the liver, since both RU 38486 and dexamethasone were bound to identical molecular species of GR. These data provide a defined system for further analysis of cellular receptor as a function of steroid, tissue, and species, contrary to the classical dogma where GR is generally thought to be identical as a passive vehicle for the steroid in all circumstances, and affinity for steroid is generally equated with receptor stability.     

Hepatic glucocorticoid receptor behaves differently when its hormone binding site is occupied by agonist (triamcinolone acetonide) or antagonist (RU486) steroid ligands

We have examined the influence of sulfhydryl (SH)-group modifying agents on the interaction of the rat liver glucocorticoid receptor (GR) with its known agonist triamcinolone acetonide (TA) and the newly synthesized antagonist mifepristone (RU486). In the freshly prepared cytosol, [3H]TA or [3H]RU486 bound to macromolecule(s) which sediment as 8-9 moieties: the binding of either ligand can be competed with radioinert TA or RU486. The presence of 2-10 mM dithiothreitol (DTT), beta-mercaptoethanol (beta-MER), and monothioglycerol (MTG) caused a 2-3 fold increase in the [3H]TA and [3H]RU486 binding to GR. Iodoacetamide (IA) and N-ethylmaleimide (NEM) decreased the agonist binding significantly. In contrast, the [3H]RU486 binding to GR increased by 50 percent in the presence of IA. IA and NEM inhibited the binding of the heat-transformed [3H]TA-receptor complex to DNA-cellulose by 70-90 percent whereas DNA binding of [3H]RU486-bound GR was inhibited only slightly. These results indicate that either a) the interaction of GR with the agonist or antagonist steroid ligands causes differential structural alterations, which are more readily detectable in the presence of SH-modifying agents or b) the agonist and the antagonist interact with distinct steroid binding sites.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Activation of rat liver glucocorticoid receptor bound to the antiglucocorticoid RU38486

The kinetics of steroid binding to rat liver glucocorticoid receptor (GR) and receptor denaturation were dependent upon the nature of the molecule occupying GR. Both the agonist [triamcinolone acetonide (TA)] and the antagonist (Ru38486) however competed for the same saturable binding site. Despite opposing physiological action, both steroid analogues permitted receptor activation as evident by binding to DNA-cellulose and 9S to 4S shift on sucrose gradient sedimentation. It therefore seems necessary to reevaluate a current notion that antagonist action of RU38486 in rat liver is a result of impaired receptor activation.     

Mechanism of action of a steroidal antiglucocorticoid in lymphoid cells

We compared the biochemical properties of receptors extracted from mouse lymphoma cells and complexed with the glucocorticoid, triamcinolone acetonide, or with the high affinity antiglucocorticoid RU 38486 [17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propynyl)-estra- 4,9-diene-3-one]. Upon salt treatment the high molecular weight receptor complexes of both types yielded dissociated forms that had the same affinity for DNA. Increased temperature caused subunit dissociation of the agonist complex but ligand dissociation of the antagonist complex. The latter was prevented if subunit dissociation was blocked by sodium molybdate but not by chemical cross-linking of the heteromeric receptor. Immunochemical studies suggest that the instability of the RU 38486 complex only affects the level of bound ligand but not the integrity of the receptor polypeptide. In intact cells at 37 degrees C the receptor polypeptide associated with nuclei only in the presence of hormone but not in its absence or if the antihormone was present. Cells incubated at 37 degrees C with RU 38486 retained in the cytosol the high molecular weight receptor in its ligand bound form. The data suggest that in intact cells under physiological conditions the antagonist binds to the heteromeric receptor and blocks its dissociation into subunits thus preventing nuclear receptor translocation.     

Evidence for separate binding sites for progesterone and RU486 in the chick oviduct

Binding characteristics of synthetic steroid, mifepristone (RU38486 - also referred to as RU486), were examined in cytosol prepared from the chick oviduct and the calf uterus, and were compared with those of progesterone and synthetic progestin R5020. Unlike [³H]progesterone binding, the [³H]RU486 binding in the oviduct cytosol did not saturate at 50 nM ligand concentration. The [³H]progesterone binding could not be eliminated in the presence of excess RU486, and [³H]RU486 binding was seen to be indisplaceable upon pretreatment of the chick oviduct cytosol with a 1000-fold excess progesterone. It is apparent that the chick oviduct cytosol is endowed with two separate sets of sites which interact with progesterone and RU486 independently. Furthermore, [³H]RU486 binding in the chick oviduct cytosol remained intact when incubated for 60 min at 37°C; it exhibited a single ionic form upon elution from DEAE-Sephacel and the [³H]RU486-associated radioactivity sedimented in the 4 S region both in salt-free and 0.3 M KCl-containing 5–20% sucrose gradients. In the calf uterus cytosol, both steroids exhibited comparable binding profiles. Our results provide evidence that chick oviduct possesses distinct binding sites that accept either progesterone or RU486, but not both, as is the case in the calf uterus.     

The Mitochondrion as a Primary Site of Action of Glucocorticoids: Mitochondrial Nucleotide Sequences,Showing Similarity to Hormone Response Elements,Confer Dexamethasone Inducibility to Chimaeric Genes Transfected in LATK−Cells

The hypothesis of a primary action of steroid hormones on mitochondrial gene expression has been supported by the detection of the glucocorticoid receptor in liver mitochondria and the demonstration of the interaction of the receptor with putative mitochondrial HREs. We now show that two putative mitochondrial glucocorticoid response elements present within the cytochrome oxidase subunit I gene (GREI and GREII), linked to a thymidine kinase promoter and to the CAT gene, transfected to LATK⁻cells, confer dexamethasone inducibility to the CAT gene. As the plasmids were stably transfected, hormone induction was analysed in the nuclear background. This effect is dose dependent and is abolished by the glucocorticoid antagonist RU38486.     

Regulation of arginase production by glucocorticoid in three human gastric cancer cell lines.

Gastric cancer tissues have high levels of glucocorticoid receptors (GR) and arginase. To investigate the interrelation of glucocorticoid, GR and arginase, three human gastric cancer cell lines (AZ-521, NUGC-3, KATO-III) were treated with hydrocortisone in the presence or absence of a glucocorticoid antagonist RU38486. GR were found to be present in all three lines, and hydrocortisone significantly increased the production of total arginase in all 3 lines. The induction of arginase production by hydrocortisone was inhibited by RU38486. These findings suggest that the regulation of arginase production by hydrocortisone in gastric cancer cells is mediated through GR.     

Expression of the 11beta-hydroxysteroid dehydrogenase type II enzyme in breast tumors and modulation of activity and cell growth in PMC42 cells

Manipulating the metabolism of glucocorticoids may serve as a useful adjunct in the treatment of breast cancer. The 11β-hydroxysteroid dehydrogenase type 2 enzyme (11βHSD2) potently inactivates glucocorticoids thereby protecting the non-selective mineralocorticoid receptor (MR) in fluid transporting tissues. In the present study, Western blot analysis showed the presence of 11βHSD2 in 66% of the breast tumor samples. The 11βHSD2 and MR are also present in the breast tumor cell line PMC42. Glycyrrhetinic acid abolished glucocorticoid metabolism and inhibited cell growth by 40%, the latter at concentrations consistent with glucocorticoid receptor (GR) and MR binding studies. Metabolism was increased by glucocorticoids, the anti-glucocorticoid RU 38486 and anti-mineralocorticoid spironolactone, while aldosterone had no effect. Neither cortisol nor aldosterone affected cell proliferation, but both RU 38486 and spironolactone caused a significant decrease in cell number. The effects of RU 38486 were only observed at micromolar concentrations and are inconsistent with an action via GR or progesterone receptor (PR). This study shows that 11βHSD2 activity and cell proliferation of PMC42 cells can be modulated via steroid receptors.     

Progestins stimulate the differentiation of 3T3-L1 preadipocytes

The effect of progesterone on the differentiation of the 3T3-L1 preadipocytes was investigated and compared with other sex steroids (estradiol and testosterone), with cortisol, with the synthetic progestin R5020 and with the progestin/glucocorticoid antagonist RU38486. At 10⁻⁸ M, progesterone stimulated the activity of glycerol-3-phosphate dehydrogenase and triglyceride deposition. Progesterone, R5020, cortisol, and RU38486 increased triglycerides about 2-fold at 10⁻⁷ M. Only minimal effects were observed with testosterone and estradiol even at 10⁻⁶ M. When the cells were cultured in presence of 10⁻⁵ M metyrapone the effect of progesterone was unchanged, suggesting that the progesterone was not metabolized to a glucocorticoid. Progesterone, R5020 and RU38486 competed efficiently with [³H]dexamethasone for the glucocorticoid receptor in 3T3-L1 cytosol. These results indicate a significant, reproducible dose-dependent effect of progestins on differentiation of the preadipocytes, which appears to be mediated via the glucocorticoid receptor.     

Characterization of an antiserum against the glucocorticoid receptor

An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor from rat liver cytosol contained specific antibodies to glucocorticoid receptor. This was shown following incubation of the [³H]triamcinolone acetonide-glucocorticoid receptor (TA-GR) complex with the IgG fraction by (I) adsorption of the [³H]TA-GR-antibody complex to protein A linked to Sepharose, (II) an increased sedimentation rate of the [³H]TA-GR-antibody complex compared to that of the [³H]TA-GR complex, and (III) an increased molecular size of the [³H]TA-GR-antibody complex when compared to that of the [³H]TA-GR complex as judged from gel filtration. The antibody fraction was characterized with regard to titer, cross-reactivity and specificity. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus), as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. In the rat liver, the antibody bound to both the nuclear and the cytosolic glucocorticoid receptor (Stokes radius 6.1 nm). It did not cross-react with the proteolytic fragments of the glucocorticoid receptor, the 3.6 nm complex or the 1.9 nm complex. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat to rat serum transcortin. With an indirect competitive ELISA (enzyme-linked immunosorbent assay) combined with various separation techniques, based on different physiocochemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. These findings also indicate an immunochemical similarity between glucocorticoid receptors in different tissues as well as in different species, but not between glucocorticoid receptors and other steroid hormone receptor proteins. The cytosolic and nuclear glucocorticoid receptors in rat liver were shown to be immunochemically similar.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[³H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90 000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [³H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [³H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37°C. SDS-polyacrylamide gel electrophoresis of [³H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37°C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei aas well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

Interactions Between the Glutamate and Glycine Recognition Sites of the N-Methyl-d-Aspartate Receptor from Rat Brain, as Revealed from Radioligand Binding Studies

Abstract: The N-methyl-d -aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P₂ membranes we have investigated the effect of glutamate recognition site ligands on [³H]glycine (agonist) and (±)4-trans-2-car-boxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]l -689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on l -[³H]glutamate (agonist), dl -3-(2-carboxypiperazin-4-yl)-[³H]propyl-1 -phosphonate ([³H]-CPP; “C-7” antagonist), and cis-4-phosphonomethyl-2-[³H]piperidine carboxylate ([³H]CGS-19755; “C-5” antagonist) binding to the glutamate site. “C-7” glutamate site antagonists partially inhibited [³H]l -689,560 binding but had no effect on [³H]glycine binding, whereas “C-5” antagonists partially inhibited the binding of both radioligands. Glycine, d -serine, and d -cycloserine partially inhibited [³H]CGS-19755 binding but had little effect on l -[³H]-glutamate or [³H]CPP binding, whereas the partial agonists (+)-3-amino-1-hydroxypyrrolid-2-one [(+)-HA-966], 3R-(+)cis-4-methyl-HA-966 (l -687,414), and 1-amino-1-carboxycyclobutane all enhanced [³H]CPP binding but had no effect on [³H]CGS-19755 binding, and (+)-HA-966 and l -687,414 inhibited l -[³H]glutamate binding. The association and dissociation rates of [³H]l -689,560 binding were decreased by CPP and d -2-amino-5-phosphonopentanoic acid (“C-5”). Saturation analysis of [³H]l -689,560 binding carried out at equilibrium showed that CPP had little effect on the affinity or number of [³H]l -689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [³H]l -689,560 is discussed.     

Extraction of DNA-cellulose-bound glucocorticoid-receptor complexes with sodium tungstate

Glucocorticoid-receptor complex from rat liver cytosol, activated by warming at 23°C or fractionation with (NH₄)₂SO₄, was adsorbed over DNA-cellulose. This DNA-cellulose-bound [³H]triamcinolone acetonide-receptor complex was extracted in a dose-dependent manner by incubation with different concentrations of sodium tungstate. A 50% recovery of receptor was achieved with 5 mM sodium tungstate. Almost the entire glucocorticoid-receptor complex bound to DNA-cellulose could be extracted with 20 mM sodium tungstate. The [³H]triamcinolone acetonide released from DNA-cellulose following tungstate and molybdate treatment was found to be associated with a macromolecule, as seen by analysis on a Sephadex G-75 column. The glucocorticoid-receptor complex extracted by both the compounds sedimented as a 4 S entity of 5–20% sucrose gradients under low- and high-salt conditions. Addition of tungstate or molybdate to the preparations containing activated receptor had no effect on the sedimentation rate of receptor. However, addition of tungstate to non-activated receptor preparation caused aggregates of larger size. The tungstate-extracted glucocorticoid-receptor complex failed to rebind to DNA-cellulose even after extensive dialysis, whereas receptor in molybdate-extract retained its DNA-cellulose binding capacity.