Covalent stabilization of the nontransformed chick oviduct cytosol progesterone receptor by chemical cross-linking

The nontransformed forms of the chick oviduct cytosol progesterone receptor of sedimentation coefficient approximately 8 S (8S-PR) are heterooligomers including one hormone binding molecule, either B, approximately 110,000, or A, approximately 79,000, and two non-hormone binding subunits recently identified as heat-shock protein Mr approximately 90,000 (hsp 90) [Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., & Baulieu, E. E. (1984) Biochemistry 23, 6016-6023]. In the crude cytosol, bisimidates reacted under mild conditions and gave rise to complexes, binding progesterone and reacting with BF4, an anti-hsp 90 monoclonal antibody. These complexes have a sedimentation coefficient of 8.4 S and Rs of 8.1 nm in the presence of 0.4 M KCl and in the absence of molybdate ions, i.e., in conditions that would transform non-cross-linked 8S-PR to Rs approximately 5 nm forms of approximately 4-S sedimentation coefficient. All bisimidates tested, of an effective reagent length between 0.73 and 1.09 nm, gave comparable results in the cytosol prepared with or without molybdate ions, confirming that the latter were not responsible for the formation of the cross-linked 8S complexes. It was found that the dimethyl pimelimidate cross-linked 8S-PR was more resistant to inactivating conditions, urea, or heat treatment than the non-cross-linked 8S-PR. The 8S-PR cross-linked in the cytosol was purified by affinity chromatography in the absence of molybdate ions. After purification, it also reacted with the monoclonal antibody BF4 and had the same Rs (8.0 nm), sedimentation coefficient (approximately 8.5 S), and thus Mr (approximately 290,000) as the original cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit composition of the molybdate-stabilized "8-9 S" nontransformed estradiol receptor purified from calf uterus

The structure of the calf uterus nontransformed molybdate-stabilized estradiol receptor (ER) has been investigated using affinity labeling with tamoxifen aziridine and several monoclonal antibodies directed either against the steroid binding protein (Mr approximately 65,000) or against the heat shock protein of Mr approximately 90,000 (hsp 90). The purification was performed using affinity chromatography and a DEAE-Sephacel column. The [3H] estradiol-ER complex was obtained as a well-defined radioactive peak, the specific activity varying between 1,600 and 3,400 pmol/mg of protein. The purified ER sediments in glycerol gradients at 9.4 S +/- 0.2 (n = 5) and at 8.1 S +/- 0.2 (n = 15) in a 0.15 M KCl containing gradient ("8-9 S" ER). From a measured Stokes radius of 7.4 +/- 0.2 nm (n = 12), an Mr of approximately 300,000 has been calculated. Studies of the purified 8-9 S ER by glycerol gradient centrifugation and by "twin antibody" assay with the JS34/32 anti-ER monoclonal antibody suggest the presence of two binding subunits in the nontransformed molecular complex. Results of immunological analysis with polyclonal and several monoclonal antibodies against hsp 90 suggest the association of two molecules of this protein to the two steroid binding subunits. In high salt medium (0.4 M KCl), the purified ER sediments at 5.2 +/- 0.3 (n = 8), has a Stokes radius of 5.7 nm +/- 0.1 (n = 2) and the Mr is approximately 129,000, values expected for a homodimer consisting of two hormone-binding subunits (Mr approximately 65,000), a result confirmed by glycerol gradient centrifugation experiments, using the monoclonal antibody JS34/32. The relationship between the nontransformed 8-9 S ER and the transformed 5 S-ER forms are discussed, the simplest possibility being the release of the already formed homodimeric ER from 8-9 S ER during transformation.     

Characterization of the nontransformed and transformed androgen receptor and heat shock protein 90 with high-performance hydrophobic-interaction chromatography

The hydrophobicity of the nontransformed and transformed androgen receptor from rat submandibular gland and heat shock protein 90 (hsp90) from rat submandibular gland and liver was characterized by using high-performance hydrophobic-interaction chromatography on TSK gel Ether-5PW. In the absence of molybdate, cytosol [3H]R1881-androgen receptor complexes were mainly eluted in the 1.3 M region (Peak 1) with a small peak in the 0.8 M region (Peak 2) of a descending salt gradient (2 to 0 M) of ammonium sulfate. In the presence of molybdate, Peak 2 was predominant. When labeled-cytosol was applied after being heated at 25 degrees C for 30 min, a third peak (Peak 3) at around 0.64 M ammonium sulfate was newly observed. Peaks 2 and 3 were observed, while Peak 1 completely disappeared with the labeled-cytosol precipitated at 40% saturated ammonium sulfate. The Stokes radius of Peak 1 was 7 nm, and of Peak 2 was 8 nm. Both peaks were retained poorly by DNA-cellulose but bound rather well to DEAE-cellulose. These results suggest that these two peaks represent the nontransformed receptor, indicating that there are isoforms of the nontransformed androgen receptor which are distinguished by their hydrophobic properties and Stokes radii. Peak 3 had a Stokes radius of 5 nm and preferentially bound to DNA-cellulose, suggesting that this peak corresponds to the transformed receptor. These results indicated that the transformation of the androgen receptor accompanies the enrichment of the hydrophobicity of the receptor molecule. Hsp90 purified from rat livers and hsp90 in the cytosol both from livers and submandibular glands were eluted from Ether-5PW at 0.8 M ammonium sulfate, at almost the same position as Peak 2. This finding suggests that the enrichment of hydrophobicity on transformation is due to dissociation of hsp90 from the nontransformed androgen receptor.     

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.     

A protein kinase copurified with chick oviduct progesterone receptor

A magnesium-dependent protein kinase activity was copurified with both the molybdate-stabilized 8S form of the chick oviduct progesterone receptor (PR) and its B subunit. In each case, purification was performed by hormonal affinity chromatography followed by ion-exchange chromatography. The Km(app) values of the phosphorylation reaction for [gamma-32P]ATP and calf thymus histones were approximately 1.3 X 10(-5) M and approximately 1.6 X 10(-5) M, respectively, and only phosphorylated serine residues were found in protein substrates, including PR B subunit. Physicochemical parameters of the enzyme [pI approximately 5.3, Stokes radius approximately 7.2 nm, sedimentation coefficient (S20,w) approximately 5.6 S, and Mr approximately 200,000] were compared to those of purified forms of PR (B subunit, pI approximately 5.3, Stokes radius approximately 6.1 nm, and Mr approximately 110,000; 8S form, Stokes radius approximately 7.7 nm and Mr approximately 240,000). The results suggest that most of the protein kinase activity copurified with both oligomeric and monomeric forms of PR belongs to an enzyme distinct from currently known receptor components. Its physiological significance remains unknown.     

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

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

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)     

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.     

Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor.

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the 'B' subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the 'A' subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.     

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.     

Characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: use of chemical crosslinking and a monoclonal antibody directed against a 59-kDa protein associated with steroid receptors

The Ah receptor regulates induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) by "3-methylcholanthrene-type" compounds and mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons. Hepatic Ah receptor from untreated rodents is localized in the cytosol and has an apparent molecular mass of 250 to 300 kDa. This large form can be dissociated into a smaller ligand-binding subunit upon exposure to high ionic strength. The Ah receptor displays many structural similarities to the receptors for steroid hormones. Two non-ligand-binding proteins have been identified to be associated with the cytosolic forms of the steroid hormone receptors. The first is a 90-kDa heat shock protein (hsp 90); the second is a 59-kDa protein (p59) of unknown function. The cytosolic Ah receptor ligand-binding subunit previously has been shown to be associated with hsp 90. In the present study, we used a monoclonal antibody, KN 382/EC1, generated against the 59-kDa protein which is associated with rabbit steroid receptors to determine if p59 also is a component of the large cytosolic Ah receptor complex. Cytosolic forms of rabbit progesterone receptor, glucocorticoid receptor, and Ah receptor were analyzed by velocity sedimentation on sucrose gradients under low-ionic-strength conditions and in the presence of molybdate. Progesterone receptor from rabbit uterine cytosol and glucocorticoid receptor from rabbit liver each had a sedimentation coefficient of approximately 9 S. In the presence of KN 382/EC1 antibody the progesterone receptor and the glucocorticoid receptor both underwent a shift in sedimentation to a value of approximately 11 S. The increase in sedimentation velocity is an indication that the receptor-protein complexes are interacting with the antibody. Under low-ionic-strength conditions the Ah receptors from rabbit uterine cytosol and liver cytosol had a sedimentation coefficient of approximately 9 S. However, in contrast to the steroid receptors, the Ah receptor showed no change in its sedimentation properties in either tissue in the presence of KN 382/EC1, indicating that the antibody is not interacting with the Ah receptor. Multimeric Ah receptor complexes that were chemically crosslinked still did not show any interaction with KN 382/EC1. These data indicate that the 59-kDa protein either is not associated with the Ah receptor or is present in an altered form which the antibody cannot recognize.     

Mineralocorticosteroid receptor of the chick intestine. Oligomeric structure and transformation

The binding of [3H]aldosterone in the chick intestine cytosol was analyzed in terms of affinity and specificity. In this tissue, aldosterone binds to the mineralocorticosteroid receptor, with a high affinity (Kd approximately 0.3 nM) and low capacity (approximately 50 fmol/mg protein), and to the glucocorticosteroid receptor. The selective labeling of the mineralocorticosteroid receptor was achieved by incubating the cytosol with [3H]aldosterone in the presence of RU 486. This synthetic steroid completely inhibited the binding of [3H]aldosterone to the glucocorticosteroid receptor and did not bind to the mineralocorticosteroid receptor. The oligomeric structure of the mineralocorticosteroid receptor was studied by using BF4, a monoclonal antibody which reacts with the 90-kDa heat shock protein (hsp 90), a nonhormone-binding component of nontransformed steroid receptors. The mineralocorticosteroid receptor sedimented at 8.5 +/- 0.4 S (n = 8) in a 15-40% glycerol gradient. This peak was shifted to 11.2 +/- 0.6 S (n = 5) after incubation with BF4, indicating that, in the cytosol, hsp 90 was associated with the mineralocorticosteroid receptor. Dissociation of the complex was observed on gradients containing 0.4 M KCl, as judged by the absence of displacement by BF4 of the 4.3 +/- 0.4 S (n = 10) peak. The effect of molybdate and tungstate ions, and of dimethyl pimelimidate, an irreversible cross-linking agent, on the stability of the hsp 90-receptor complex was investigated. Complexes recovered in the presence of 20 mM molybdate ions dissociated on gradients containing 0.4 M KCl (5.2 +/- 0.6 S (n = 4), whereas complexes prepared in the presence of 20 mM tungstate ions sedimented at 8.5 +/- 0.4 S (n = 7). Similarly, complexes prepared in the presence of molybdate ions dissociated during high pressure liquid chromatography (HPLC) gel filtration analysis performed in 0.4 M KCl (RS (Stokes radius) = 3.9 +/- 0.5 nm (n = 3) versus 7.3 +/- 0.2 nm (n = 3) in the presence of 20 mM molybdate ions), whereas complexes prepared in the presence of tungstate ions did not dissociate (RS = 6.9 +/- 0.2 nm (n = 3]. As observed for the tungstate-stabilized receptor, the cross-linked receptor dissociated neither on gradient containing 0.4 M KCl (9.5 +/- 0.1 S (n = 3] nor during HPLC performed in 0.4 M KCl (RS = 6.5 +/- 0.3 (n = 4]. Furthermore, the cross-linked receptor was more resistant to the inactivating effect of urea on aldosterone binding than the noncross-linked receptor prepared in the presence of either molybdate or tungstate ions.     

The Mr 90,000 heat shock protein-free androgen receptor has a high affinity for steroid, in contrast to the glucocorticoid receptor

Transformed and bacterially expressed glucocorticoid receptors free from Mr 90,000 heat shock protein (hsp90) have a 100-fold lower steroid-binding affinity than the hsp90-bound nontransformed receptor, suggesting that hsp90 is needed for high-affinity steroid binding [Nemoto, T., Ohara-Nemoto, Y., Denis, M., & Gustafsson, J.-A. (1990) Biochemistry 29, 1880-1886]. To investigate whether or not this phenomenon is common to all steroid receptors, we investigated the steroid-binding affinities of bacterially expressed and transformed androgen receptors. The C-terminal portion of the rat androgen receptor containing the putative steroid-binding domain was expressed as a fusion protein of protein A in Escherichia coli. The recombinant protein bound a synthetic androgen, [3H]R1881, with high affinity (Kd = 0.8 +/- 0.3 nM). Glycerol gradient analysis revealed that the recombinant protein sedimented at around the 3S region irrespective of the presence of molybdate, indicating that the receptor is present in monomeric form. The steroid-free transformed androgen receptor was obtained by exposure of rat submandibular gland cytosol to 0.4 M NaCl in the absence of steroid. High-performance ion-exchange liquid chromatography analysis showed that the transformed androgen receptor bound to [3H]R1881 with high affinity. Thus these observations indicate that, in contrast to the glucocorticoid receptor, hsp90 is not required for the high-affinity steroid binding of the androgen receptor. In addition, the hsp90-free androgen receptor prebound with radioinert R1881 was efficiently relabeled with [3H]R1881, while the triamcinolone acetonide-bound, transformed glucocorticoid receptor failed in ligand exchange. The inability to achieve ligand exchange probably reflects the low steroid-binding affinity of this entity.     

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.     

Chick oviduct progesterone receptor phosphorylation: characterization of a copurified kinase and phosphorylation in primary cultures

A protein kinase activity was copurified with the chick oviduct progesterone receptor. The enzyme is magnesium dependent and can use the B subunit of progesterone receptor or histones as substrates. The physiochemical parameters of the kinase were determined [pI approximately 5.3; Stokes radius approximately 7.2 nm; sedimentation coefficient (S 20,w) approximately 5.6] and compared to those of the purified B subunit. The results were consistent with the presence of an unique enzyme distinct from the receptor itself. The physiological significance of receptor phosphorylation was investigated in oviduct cells grown in primary culture. Cells were labeled with [32P]orthophosphate in presence or absence of progesterone and the receptor components were immunoprecipitated with a specific polyclonal antibody. Although progesterone treatment lead to the attachment of most of the receptor (approximately 80%) to nuclear structures, the 32P-labeled B subunit was only recovered in the cytosol fraction. Different procedures to extract the nuclear receptor did not allow detection of any 32P-labeled form in the nuclear-soluble fractions, suggesting that the B subunit was not further phosphorylated upon the exposure of cells to progesterone.     

Nuclear localization of two steroid receptor-associated proteins, hsp90 and p59

It has been proposed that the unliganded nontransformed form of steroid hormone receptor is a heterooligomer comprising, in addition to the hormone-binding subunit, two associated proteins: a heat shock protein of MW 90,000 (hsp90) and another protein of MW 59,000 (p59). Using monoclonal antibodies, we demonstrate immunocytochemically the presence of both hsp90 and p59 in cell nuclei of progesterone target cells of the rabbit uterus. While steroid receptors (e.g., progesterone receptors) appear to be exclusively nuclear, we find p59 predominantly in the cell nuclei and hsp90 in both the nucleus and the cytoplasm. In addition, Western blotting of high-salt extracts of nuclear proteins detects the presence of hsp90 and p59 in the nuclei of rabbit uterus. These observations are consistent with the presence of the untransformed heterooligomeric form of steroid hormone receptors in the nuclei of target cells.     

The receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the mouse hepatoma cell line Hepa 1c1c7. A comparison with the glucocorticoid receptor and the mouse and rat hepatic dioxin receptors

The molecular properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the mouse hepatoma cell line Hepa 1c1c7 were investigated. The receptor was found to represent a highly asymmetrical molecule with a sedimentation coefficient, s20,w, of approximately 8 S, a Stokes radius of 7-8 nm, and a calculated Mr approximately equal to 260,000-300,000. In comparison, the Hepa 1c1c7 glucocorticoid receptor in analogy to the glucocorticoid receptor in general as well as the C57BL/6 mouse and rat hepatic dioxin receptors are molecules with an s20,w value of 4-5 S, a Stokes radius of approximately 6 nm, and a calculated Mr approximately equal to 100,000. In the presence of 20 mM sodium molybdate, a large Mr approximately equal to 270,000-310,000 form of the Hepa 1c1c7 glucocorticoid receptor is stabilized which is hydrodynamically indistinguishable from the Mr approximately equal to 260,000-300,000 Hepa 1c1c7 dioxin receptor. Sodium molybdate does not have any effect on the molecular properties of the Hepa 1c1c7 dioxin receptor. In conclusion, the large form of dioxin receptor present in Hepa 1c1c7 mouse hepatoma cells in the absence of sodium molybdate is strikingly similar to molybdate-stabilized steroid hormone receptors as well as the molybdate-stabilized form of the dioxin receptor previously demonstrated in rat hepatic cytosol. Therefore, the Hepa 1c1c7 dioxin receptor might offer an interesting model for studies on the structure and function of Mr approximately equal to 300,000 forms of soluble receptors.     

A new affinity matrix for mineralocorticoid receptors

The behavior of mineralocorticoid and glucocorticoid receptors of rabbit kidney cytosol was investigated on two affinity gels: a new affinity matrix prepared with a 3-O-derivative of carboxymethyloxime deoxycorticosterone (deoxycorticosterone gel) and a gel linked to a 17 beta-dexamethasone derivative (dexamethasone gel). Deoxycorticosterone gel was highly specific, since it retained mineralocorticoid but not glucocorticoid receptors, and dexamethasone gel exhibited high selectivity for glucocorticoid receptors since it did not bind mineralocorticoid receptors. The use of these two matrices allowed separation of mineralocorticoid and glucocorticoid receptors and further characterization of each type of cytosolic receptors after its isolation. Cytosolic mineralocorticoid and glucocorticoid receptors stabilized by tungstate were found to have a Stokes radius of approximately 6 nm, as determined by high performance size exclusion chromatography and a sedimentation coefficient of approximately 9 S, determined on a glycerol density gradient containing tungstate, under either high or low salt conditions. The hydrodynamic parameters, binding characteristics, and specificity of mineralocorticoid receptors were the same in the untreated and dexamethasone gel-treated cytosol. Similarly glucocorticoid receptor characteristics remained unchanged after deoxycorticosterone gel treatment, indicating biochemical independence of cytosolic mineralocorticoid and glucocorticoid receptors. The [3H]aldosterone receptor complex eluted from deoxycorticosterone gel was recovered with a 30-40% yield and a purification factor of about 1000. Purified mineralocorticoid receptors had the same sedimentation coefficient as cytosolic mineralocorticoid receptors (9 S) but a different Stokes radius (4 versus 6 nm). The decrease in the Stokes radius of the purified mineralocorticoid receptors was probably due to the gel filtration method. These results indicate that the newly synthesized matrix specific for mineralocorticoid receptors constitutes a powerful tool for their extensive purification.     

Characterization of non-liganded glucocorticoid receptor in rat liver cytosol using indirect competitive enzyme-linked immunosorbent assay

We have previously shown that the purified or unfractionated cytosolic, activated glucocorticoid receptor of rat liver consists of a polypeptide with a Stokes radius of approximately 6 nm, a sedimentation coefficient of 4S and a molecular mass of approximately 90,000 Daltons. We have confirmed previous observations by other authors that if sodium molybdate is introduced into the cytosol preparation buffer the non-activated glucocorticoid receptor appears as an 8 nm, 9S species with an apparent molecular mass of 330,000 Daltons. In order to study the physicochemical parameters of the glucocorticoid receptor prior to ligand binding, we have used an enzyme-linked immunosorbent assay (ELISA) based on antibodies raised in rabbits against the purified activated glucocorticoid receptor. In isotonic buffer, the non-liganded glucocorticoid receptor was shown to have a Stokes radius of 6 nm in the absence and 8 nm in the presence of molybdate. Furthermore, experimental conditions known to result in activation of the glucocorticoid receptor complex (increased ionic strength, increased temperature) did not lead to activation of the 6 nm non-liganded glucocorticoid receptor as judged from the lack of binding of the treated, non-liganded receptor to DNA-cellulose. The existence of both 6 and 8 nm forms of nonactivated, non-liganded glucocorticoid receptor in vitro suggests that dissociation of an 8 nm form to a 6 nm form, if it occurs in vivo, is probably not the only molecular event constituting the activation of the glucocorticoid receptor.