Effects of ATP and pyrophosphate on properties of glucocorticoid-receptor complexes from rat thymus cells

Cytosols from rat thymus cells incubated with glucocorticoid contain nonactivated and activated receptors and mero-receptor complexes, in relative amounts that depend on the incubation conditions. These forms can be separated by a rapid minicolumn chromatographic technique based on their differential affinities for DNA, DEAE, and hydroxylapatite. We have used this method to examine the effects of ATP, pyrophosphate (PPi), and related compounds on cytosolic complexes. In addition to ATP, already known to promote activation at 0 degrees C, PPi, ADP, and other triphosphates at millimolar concentrations promoted activation of nonactivated complexes. AMP and Pi had little effect. ATP and PPi at millimolar concentrations also reduced binding of activated complexes to DNA. Characterization of the ATP- and PPi-activated complexes by gel filtration and ion exchange chromatography revealed two DNA-binding forms. One was essentially identical (Stokes radius of approximately 5.4 nm, elution from DEAE at approximately 50 mM KCl) to the normal activated complex obtained directly from cells incubated at 37 degrees C. The other had a Stokes radius of approximately 3.1 nm and had no affinity for DEAE. Analysis by minicolumns and gel filtration showed that ATP and PPi prevented formation of mero-receptor complexes, a process which occurs relatively rapidly in untreated thymus cytosols. These compounds did not alter properties of preformed mero-receptor. The accumulation of 3.1-nm complexes in thymus cytosols in which formation of mero-receptor is prevented suggests that this form is an intermediate, normally short-lived, in the conversion of 5.4 nm complexes to mero-receptor.     

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.     

Nonactivated and activated glucocorticoid-receptor complexes in WEHI-7 and rat thymus cells

Our own results and those of others have indicated that nonactivated glucocorticoid-receptor complexes are oligomeric proteins with Stokes radius R_s = 8–9 nm, and that activation is accompanied by a reduction in size to R_s = 5–6 nm. The most convincing evidence for the large size of the nonactivated compared to the activated complex has been obtained with cytosols stabilized with molybdate. It has been suggested, however, that molybdate causes aggregation of complexes. Here we show that nonactivated rat thymus complexes in cytosols with molybdate and 400 M KCl have R_s = 8 nm. Furthermore, cytosols from WEHI-cells, which are exceptionally stable, show clear indications of 8 nM nonactivated complexes even without molybdate.The principal complexes in thymus cells under physiological conditions are the nonactivated, activated and nuclear-bound forms. We have studied the rapid kinetics of formation and interconversion of these complexes in intact cells at 37°C, using our newly-developed mini-column procedure to assay nonactivated and activated complexes. These kinetic results, along with many earlier results, can be accounted for quantitatively with a simple cyclic (irreversible) model in which the dissociation rate constant of the steroid plays a key role. The model predicts correctly the different degrees of activation in the cell with glucocorticoids such as triamcinolone acetonide and dexamethasone on the one hand, and cortisol and corticosterone on the other, without assuming steroid-specific allosteric influences of each of these steroids on the receptor.     

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.     

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.     

Stabilization of glucocorticoid-receptor complexes in rat thymus cytosol by a factor from WEHI-7 cells

Using a variety of physico-chemical techniques we have recently characterized three distinct forms of glucocorticoid-receptor complexes present in the cytosol from rat thymus cells incubated with glucocorticoid; the relative proportions of these complexes are dependent on the conditions to which the cells or cytosols are exposed. Two of these complexes correspond to the well established nonactivated and activated receptor forms, while the third has properties consistent with mero-receptor. Based on their differential affinities for DNA- and DEAE-cellulose we have developed a rapid mini-column chromatographic procedure for separating these three forms and have used it to examine the stability of complexes in cytosol preparations. We have found that activated glucocorticoid-receptor complexes from rat thymus cells are relatively unstable under cell-free conditions in that they undergo time-dependent losses in DNA binding and are converted to mero-receptor. In contrast, cytosolic glucocorticoid-receptor complexes prepared from WEHI-7 mouse thymoma cells are remarkably stable under similar conditions. Mixing experiments with equal portions of rat thymus and WEHI-7 cytosol revealed that the difference between the two tissues cannot be accounted for merely by differences in amounts of proteolytic enzymes, since addition of rat thymus cytosol to WEHI-7 cytosol containing activated glucocorticoid-receptor complexes does not result in their conversion to mero-receptor. However, the WEHI-7 cytosol affords considerable protection to activated glucocorticoid-receptor complexes in thymus cytosol. The stabilizing factor from WEHI-7 cytosol is heat stable (survives 100 degrees C for 30 min), insensitive to pH over a wide range (4.0-10.0), and appears to be macromolecular. It does not inhibit activation, and thus appears distinct from the previously described endogenous glucocorticoid receptor stabilizing factor responsible for stabilization of thymocyte receptor binding capacity (Leach et al., J. Biol. Chem. 257: 381-388, 1982). We propose that the factor is an endogenous inhibitor of the protease(s) responsible for mero-receptor formation.     

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.     

Comparison of pyridoxal phosphate and 0.4 M KCl-extracted nuclear glucocorticoid receptors in HeLa S3 cells

A comparison of the physicochemical properties between pyridoxal 5'-phosphate- and 0.4 M KCl-extracted nuclear glucocorticoid receptors has been made utilizing HeLa S3 cells as a source of receptor. Both pyridoxal 5'-phosphate/NaBH4-reduced and 0.4 M KCl-extracted receptors sedimented as approximately 3.5-4.5 S species in 5-20% sucrose gradients containing 0, 0.15, and 0.4 M KCl. Under low-ionic-strength buffer conditions, pyridoxal 5'-phosphate-extracted receptor elutes close to the void volume of a Sephacryl S-300 gel-exclusion column. Increasing the [KCl] of the column to 0.4 M resulted in the elution of receptor with a Stokes radius of 58 A and calculated Mr = 96,000. Nuclear receptors extracted with 0.4 M KCl also formed a large-molecular-weight complex which eluted close to the void volume of the gel-exclusion column. Increasing the [KCl] to 0.4 M had the effect of shifting this receptor form to a species which had a Stokes radius of 62 A and calculated Mr = 89,700. Ion-exchange analysis of nuclear-extracted receptors revealed that 0.4 M KCl-extracted receptors exhibited considerable charge heterogeneity, whereas pyridoxal 5'-phosphate-extracted receptors did not. Pyridoxal 5'-phosphate-extracted receptors (approximately 86%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M; approximately 14% of the receptors had little affinity for DEAE-cellulose. Pyridoxal phosphate-treated receptors had little affinity for hydroxylapatite, phosphocellulose, and DNA-cellulose. The predominant form of 0.4 M KCl-extracted nuclear receptors (approximately 78%) eluted from DEAE-cellulose between 0.05 and 0.15 M KCl, a position coincident with "activated" glucocorticoid receptors. The remaining receptor fraction (approximately 22%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M, a position coincident with "unactivated" glucocorticoid receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physicochemical characterization of the nuclear form of Ah receptor from mouse hepatoma cells exposed in culture to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Molecular properties of nuclear aromatic hydrocarbon (Ah) receptor from Hepa-1c1c9 (Hepa-1) cells were assessed by velocity sedimentation on sucrose gradients and by gel permeation chromatography on Sephacryl S-300. Nuclear Ah receptor was obtained by exposing intact cells to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 1 h at 37 degrees C in culture followed by extraction of receptor from nuclei with buffers containing 0.5 M KCl. The nuclear Ah receptor was compared to the cytosolic Ah receptor from the same cells. Under conditions of low ionic strength, the Ah receptor from Hepa-1 cytosol sedimented as a single 9.4 +/- 0.63 S binding peak that had a Stokes radius of 7.1 +/- 0.12 nm and an apparent relative molecular mass of 271,000 +/- 16,000. After prolonged (24 h) exposure to high ionic strength (0.5 M KCl), cytosol labeled with [3H]TCDD exhibited two specific binding peaks. The large form of cytosolic Ah receptor seen under high ionic strength conditions sedimented at 9.4 +/- 0.46 S, had a Stokes radius of 6.9 +/- 0.19 nm, and an apparent Mr 267,000 +/- 15,000. The smaller ligand-binding subunit generated by exposing cytosol to 0.5 M KCl sedimented at 4.9 +/- 0.62 S, had a Stokes radius of 5.0 +/- 0.14 nm, and an apparent Mr 104,000 +/- 12,000. Nuclear Ah receptor, analyzed under high ionic strength conditions, sedimented at 6.2 +/- 0.20 S, had a Stokes radius of 6.8 +/- 0.19 nm, and an apparent Mr 176,000 +/- 7000. Nuclear Ah receptor from rat H4IIE hepatoma cells was analyzed and found to have physicochemical characteristics identical to those of nuclear Ah receptor from the mouse Hepa-1 cells. The molecular mass of Hepa-1 nuclear Ah receptor was found to be statistically different from both the Mr approximately 267,000 cytosolic Ah receptor and the Mr approximately 104,000 subunit which were present in cytosol under high ionic strength conditions. Hepa-1 nuclear Ah receptor could not be converted to a smaller ligand-binding subunit by treatment with alkaline phosphatase, ribonuclease, or sulfhydryl-modifying reagents or prolonged exposure to 1.0 M KCl. Cytosolic Ah receptor from Hepa-1 cells was "transformed" by heating at 25 degrees C in vitro into a form with high affinity for DNA-cellulose. The transformed cytosolic Ah receptor, when analyzed under conditions of high ionic strength, sedimented at approximately 6 S, had a Stokes radius of approximately 6.7 nm, and an apparent Mr approximately 167,000.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the progesterone receptor solubilized by micrococcal nuclease and DNase I digestion

In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion. The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM [3H]ORG 2058. The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000. About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000. A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction. DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000. About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000. These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA. Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion. Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I.     

Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. A comparison with the glucocorticoid receptor

The interaction of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) with immobilized heparin (heparin-Sepharose) or DNA (DNA-cellulose) has been compared to the polyanionic-binding properties of the rat hepatic glucocorticoid receptor. Both the nonoccupied and in vitro occupied forms of the receptors interacted with heparin-Sepharose but with varying strength, as determined by ligand binding assays or an enzyme-linked immunosorbent assay based on a monoclonal antibody against the steroid- and DNA-binding Mr approximately 94,000 glucocorticoid receptor protein. In the absence of ligand, both the dioxin and glucocorticoid receptors eluted from heparin-Sepharose at 0.1-0.2 M KCl, in contrast to the in vitro occupied receptor forms which eluted at 0.3-0.4 M KCl. Following elution of the in vitro occupied dioxin receptor from heparin-Sepharose, it was efficiently retained on DNA-cellulose and eluted at an ionic strength of approximately 0.2 M KCl. In the presence of 20 mM sodium molybdate which is known to inhibit the activation of steroid hormone receptors to a DNA-binding form, both the dioxin and glucocorticoid receptors eluted at 0.1-0.2 M KCl from heparin-Sepharose. In analogy to what has previously been shown for the glucocorticoid receptor, sodium molybdate stabilized a large dioxin-receptor complex with a sedimentation coefficient, S20,w, of 9-10 S, a Stokes radius of approximately 7.5 nm, and a calculated Mr of 290,000-310,000. Limited proteolysis of both the dioxin and glucocorticoid receptors with trypsin which is known to eliminate the DNA-binding property of both receptor forms also resulted in a decreased strength in the interaction of both in vitro occupied receptors with heparin-Sepharose (elution at 0.1-0.2 M KCl). In line with these data, calf thymus DNA in solution competed for receptor binding to heparin-Sepharose. In conclusion, the chromatographic properties of the dioxin receptor on heparin-Sepharose are indistinguishable from those of the glucocorticoid receptor, and both receptors appear to be structurally and functionally closely related proteins.     

Purification of the glucocorticoid receptor from rat liver cytosol.

The [3H]-triamcinolone acetonide-labeled glucocorticoid receptor from rat liver cytosol was purified to 85% homogeneity according to sodium dodecyl sulfate gel electrophoresis. It consisted of one subunit with a molecular weight of 89,000 and had one ligand-binding site per molecule. The purification involved sequential chromatography on phosphocellulose, DNA-cellulose twice, and Sephadex G-200. Between the two chromatography steps on DNA-cellulose, the receptor was heat activated. The receptor was affinity eluted from the second DNA-cellulose column with pyrodixal 5'-phosphate. The purification achieved in the first three chromatographic steps varied between 60 and 95% homogeneity in different experiments. After chromatography on the second DNA-cellulose column, the steroid.receptor complex had a Stokes radius of 6.0 nm and a sedimentation coefficient of 3.4 S in 0.15 M KCl. In the absence of KCl, the sedimentation coefficient was 3.6 S. After concentration on hydroxylapatite, the steroid.receptor complex was analyzed by isoelectric focusing in polyacrylamide gel. The radioactivity was shown to focus together with the major protein band with pI 5.8. Following limited proteolysis with trypsin, the radioactivity, together with the major protein band, focused at pI 6.2 as previously described for the unpurified steroid.receptor complex.     

Activation of the chick oviduct progesterone receptor by heparin in the presence or absence of hormone.

Activation (transformation) of the chick oviduct progesterone receptor was found to be induced at 0 degrees C by heparin free in solution as well as by chromatography on a column of heparin linked to acrylamide/agarose. The transformed molecule displayed properties of the activated form of [3H]progesterone-receptor complex obtained by heat treatment or by high ionic strength: smaller size (s20,w = 3.9 S, Stokes radius = 5.2 nm), lower rate of dissociation (t 1/2 approx. 50 h at 0 degrees C compared with approx. 20 h for the 'native' form) and increased binding to phosphocellulose. In all cases, molybdate was an effective inhibitor of transformation and stabilized a large 'native' form (s20,w = 7.9 S, Stokes radius = 7.6 nm). Transformation by neither KCl nor heparin depended on the presence of ligand bound to the receptor, and the properties of the receptor molecule produced by treatment of ligand-free receptor with high ionic strength or with heparin were identical with those of the activated progesterone-receptor complex, demonstrating that receptor activation can be obtained experimentally in the absence of hormone. Our data are compatible with a model in which activation implies separation of the 4 S units, which compose the approx. 8 S 'native' form.     

Application of high-performance ion-exchange chromatography to the analysis of cytosolic glucocorticoid receptor

The use of high-performance ion-exchange chromatography (HPIEC) on a Mono Q column was investigated for the analysis of glucocorticoid receptor. In the presence of 10 mM sodium molybdate, both liganded and unliganded glucocorticoid receptor were eluted as a single and sharp peak (0.32 M NaCl). In the absence of molybdate and after exposure to heat and salt, another peak of specifically bound radioactivity was eluted with 0.08 M NaCl. When HPIEC was performed in the absence of molybdate, two molecular forms of the liganded receptor were detected which eluted with 0.08 M NaCl (Stokes' radius Rs = 5.1 nm, s20,w = 4.6 S, calculated mol. wt Mr approximately 100,000) and 0.32 M NaCl (Rs = 7.3 nm, S20,w = 9.0 S, calculated Mr approximately 280,000). Analysis of both forms with mini-columns of DNA-Ultrogel, DEAE-Trisacryl and hydroxylapatite (HA-Ultrogel) confirmed the identity of the two peaks with transformed and non-transformed glucocorticoid-receptor complexes. These results suggest that HPIEC may provide a useful tool for the rapid resolution and quantification of receptor molecular forms.     

Partial purification and characterisation of the human skin fibroblast androgen receptor: detection of abnormal receptor complexes in cells from patients with androgen insensitivity syndromes

After incubation of hGSF with [3H]5 alpha-dihydrotestosterone, 17 beta-hydroxy-7 alpha, 17 alpha-dimethyl-4-estrene-3-one, or 17 beta-hydroxy-17 alpha-methyl-4,9,11-estrien-3-one, androgen-receptor complexes were extracted with 0.5 M KCl and precipitated by 35% ammonium sulphate. Receptor complexes from control hGSF sedimented at approximately 4S on linear 5-20% sucrose gradients. The 4S peak was diminished or absent in cells from androgen insensitive patients exhibiting absent, deficient or unstable binding of androgens in intact hGSF. This procedure may be a useful means of distinguishing quantitative and qualitative defects in androgen binding to receptor, since one cell line found to have normal levels of androgen receptor complexes in whole cell assays had a profile resembling that of receptor negative cells on sucrose gradients. The complexes from one patient with complete androgen insensitivity having normal androgen binding in intact hGSF were indistinguishable from control complexes after sucrose gradient analysis and ADP-Sepharose chromatography. Receptor complexes were eluted from the ADP-Sepharose between 0.5-1.0 M KCl. HPLC-gel filtration of androgen receptor complexes at 22 degrees C revealed two peaks, the larger had a Mr of 60-65K, Stokes radius of 3.16 nm and a frictional ratio between 1.21 and 1.43. The second peak, Mr of 15K, was believed to represent a fragment of the receptor containing the steroid binding domain. On gel filtration at 22 degrees C the complexes from a patient with partial androgen insensitivity, who showed a diminished 4S receptor peak on sucrose gradients, revealed only the small "meroreceptor" fragment, suggesting that the mutation in this individual might render the androgen receptor more susceptible to proteolysis in vitro.     

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.     

Chromatographic separation of nontransformed and transformed glucocorticoid-receptor complexes from rat liver cytosol. Use of tungstate in the purification, inactivation, and resolution of receptor forms

Aliquots of rat liver cytosol glucocorticoid-receptor complexes (GRc) were transformed by an incubation with 8-10 mM ATP at 0 degrees C and were compared with those transformed by an exposure to 23 degrees C. The extent of receptor transformation was measured by chromatography of the samples over columns of DEAE-Sephacel. The ATP-transformed complexes, like those which were heat-transformed, exhibited lower affinity for the positively charged ion-exchange resin and were eluted with 0.12 M KCl (peak-I): the nontransformed complexes appeared to possess higher affinity and required 0.21 M KCl (peak II) for their elution. As expected, the receptor in the peak-I exhibited the DNA-cellulose binding capacity and sedimented as 4S in sucrose gradients. Peak II contained an 8-9S glucocorticoid receptor (GR) form that showed reduced affinity for DNA-cellulose. Presence of sodium tungstate (5 mM) prevented both heat and ATP transformation of the GRc resulting in the elution of the complexes in the region of nontransformed receptors. When parallel experiments were performed, binding of the cytosol GRc to rat liver nuclei or DNA-cellulose was seen to increase 10-15 fold upon transformation by heat or ATP: tungstate treatment blocked this process completely. The transformed and nontransformed GRc were also differentially fractionated by (NH4)2SO4: tungstate-treated (nontransformed) receptor required higher salt concentration and was precipitated at 55% saturation. In addition, the GRc could be extracted from DNA-cellulose by an incubation of the affinity resin with sodium tungstate resulting in approximately 500-fold purification of the receptor with a 30% yield. These studies show that the nontransformed, and the heat-, salt-, and ATP-transformed GRc from the rat liver cytosol can be separated chromatographically, and that the use of tungstate facilitates the resolution of these different receptor forms. In addition, extraction of the receptor from DNA-cellulose by tungstate provides another new and efficient method of partial receptor purification.     

Activation of cytosolic glucocorticoid-receptor complexes in intact WEHI-7 cells does not dephosphorylate the steroid-binding protein

In order to determine the ratio of phosphates to hormone-binding sites on nonactivated (non-DNA-binding) glucocorticoid receptors in WEHI-7 mouse thymoma cells, we have extracted these receptors from cells grown to a steady state with 32P, labeled them with a saturating concentration of [3H]dexamethasone 21-mesylate, purified them using a monoclonal antibody, and analyzed them by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The complexes contained approximately 5 mol of phosphate/mol of bound steroid. Only half of the phosphates were associated with the approximately 100-kDa protein which is labeled with [3H]dexamethasone 21-mesylate. The remaining phosphates were associated with the approximately 90-kDa non-steroid-binding component of the nonactivated complex. Dual label studies, using [35S]methionine to measure receptor protein and 32P to measure receptor phosphates, have enabled us to determine the phosphate content, relative to receptor protein, of both nonactivated and activated cytosolic complexes generated in intact WEHI-7 cells exposed to triamcinolone acetonide at 37 degrees C. The total amount of phosphate associated with the activated complex is roughly half of that associated with the nonactivated complex, the decrease being accounted for by dissociation of the approximately 90-kDa phosphoprotein which accompanies activation. However, the ratio of 32P to 35S counts associated with the approximately 100-kDa steroid-binding protein is the same for the activated and nonactivated complexes. These results indicate that there is no net change in the phosphorylation of the approximately 100-kDa steroid-binding component of the cytosolic glucocorticoid-receptor complex upon activation in the intact cell.     

Activation of the rat liver cytosol glucocorticoid receptor by sephacryl S-300 filtration in the presence and absence of molybdate. Physical properties of the receptor and evidence for an activation inhibitor

The DNA-binding and physical properties of the rat liver cytosol glucocorticoid receptor were determined before and after Sephacryl S-300 filtration in the presence or absence of molybdate. Cytosol was prepared and labeled with [3H]triamcinolone acetonide in buffer containing molybdate. Prior to gel filtration, only 5 +/- 3% (mean +/- S.E.) of labeled receptors bound to DNA-cellulose. After gel filtration in the presence and absence of molybdate, the per cent of labeled receptors binding to DNA-cellulose was 57 +/- 10% and 83 +/- 1%, respectively. Nonreceptor fractions from the Sephacryl S-300 column contained a heat-stable factor which blocked receptor activation but did not block the binding of activated receptors to DNA-cellulose. The activation inhibitor eluted from the column in the region of the albumin standard, but after heating its size was considerably reduced (Mr less than 3500). Receptors activated by Sephacryl S-300 filtration underwent the same size changes in the presence or absence of molybdate. Prior to gel filtration, the S20,w of labeled receptors in the presence of molybdate was 9.2 +/- 0.2 S. After filtration in the presence and absence of molybdate, the S20,w of labeled receptors was 4.2 +/- 0.2 and 4.4 +/- 0.1 S, respectively. The Stokes radius (Rs) of labeled receptors after gel filtration in either the presence or absence of molybdate was 65 +/- 1 A. From the Rs and S20,w values, the molecular weight (Mr) of activated receptors was calculated to be 115,000 to 121,000, which was in close agreement with the Mr of affinity-labeled receptors determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Different forms of the oxysterol-binding protein. Binding kinetics and stability

Based upon measurements of the sedimentation coefficient and the Stokes radii, three forms of the oxysterol-binding protein were identified. The unliganded binding protein was the largest (7.7 S, Stokes radius = 71.6 A, Mr = 236,000) was relatively asymmetric (f/f0 = 1.7), and was composed of at least three subunits. Binding of 25-hydroxycholesterol was associated with a reduction in the size of the protein (7.5 S, Stokes radius = 50 A, Mr approximately 169,000) and an increase in symmetry (f/f0 = 1.4), due to the loss of a subunit of Mr approximately 67,000. At pH 6 or lower, the Mr = 169,000 sterol-protein complex was altered so that reversible dissociation to give a smaller (4.2 S, Stokes radius = 53 A, Mr = 97,000) more asymmetric (f/f0 = 1.8) sterol-protein complex occurred when it was sedimented in a sucrose gradient buffered at pH 7.4 containing 0.3 M KCl and 2.5 M urea. Irreversible dissociation of the 7.5 S, Mr = 169,000 form to a 4.2 S form occurred spontaneously when the complex in whole cytosol buffered at pH 7.8 was allowed to stand overnight at 0 degree C, or when the partially purified complex was incubated at pH 5.5 at 0 degree C for several days. The partially purified, unliganded binding protein was unstable at 0 degree C (approximately 75% loss of binding activity in 24 h) whereas the liganded protein was stable for 7 days at 0 degree C although irreversible conversion to a 4.2 S form occurred under some conditions. Rates of sterol binding and dissociation were increased in the presence of 2.5 M urea at pH 7.4 or when the pH was lowered to 5.5 Kd values were not greatly altered under the various incubation conditions.