Progesterone-binding components of chick oviduct. VIII. Receptor activation and hormone-dependent binding to purified nuclei.

A cell-free system prepared from the estrogen-primed chick oviduct was developed and used to study the uptake of cytoplasmic progesterone-receptor complex by isolated nuclei. The receptor and purified nuclei were shown to be stable at 25 degrees, but not at 37 degrees. Thus, nuclear incubations were routinely performed at 25 degrees. Such incubations revealed greater nuclear uptake of the cytoplasmic hormone-receptor complex as compared to control incubations performed at 0 degrees. The uptake process showed a quantitative preference for oviduct nuclei. No net uptake occurred during 0 degrees incubations when the nuclei were preincubated in the absence of cytoplasmic components at 25 degrees. In contrast, the temperature requirement was partially removed by preincubation of the hormone-receptor complex at 25 degrees prior to incubation with nuclei at 0 degrees. Nuclear uptake was not accompanied by measurable alterations in the sedimentation properties of the progesterone receptor. The activation and nuclear uptake of receptor was clearly dependent upon prior binding of steroid hormone to the receptor indicating that the active nuclear form of the receptor could not be generated in the absence of the hormone. Receptor precipitation with ammonium sulfate also partially removed the temperature requirement for nuclear binding. In contrast to temperature activation, ammonium sulfate precipitation activated the receptor in the absence of hormone. It thus seemed likely that temperature and salt activation of receptor occurred via different mechanisms. Although we were able to destroy up to 60% of the nuclear DNA content by treatment with DNase prior to nuclear incubation, some 80 to 85% of the receptor-binding capacity was still present in the treated nuclei. Thus, chick progesterone receptors apparently bind to a relatively DNase-resistant portion of the oviduct genome. The properties of this system indicate its value for further investigation into the initial events of progesterone action in the chick oviduct.     

Glucocorticoid receptors in lung. Comparison between nonactivated and activated forms of the cytoplasmic glucocorticoid binding protein and their relationship to the nuclear binding protein of fetal rabbit lung.

In the absence of salt the cytoplasmic glucocorticoid receptor of fetal rabbit lung sediments at 7 S while the nuclear receptor sediments at 4 S. However, if nuclear extracts are mixed with receptor-depleted cytosol preparations in dilute buffer solutions without added salt, the nuclear 4 S receptor sediments as a 7 S species similar to that observed for the cytoplasmic form under the same conditions suggesting an interaction of the nuclear receptor with other cytosol proteins rather than with itself. In addition, both cytoplasmic and nuclear receptors sediment at 4 S in 0.4 M KCl and a major fraction of the nuclear receptor has an agarose elution profile identical to that of the cytoplasmic receptor. Thus a major fraction of the nuclear receptors is indistinguishable from the cytoplasmic receptors by the methods used. Since the cytoplasmic receptor sediments at 4 S in 0.15 M KCl, it is suggested that in vivo the glucocorticoid receptor may exist as a 4 S species and that the 7 S form described previously may result from an interaction of the 4 S component with other cytosol proteins in hypotonic media. About 25% of the receptor present in nuclear extracts has an agarose elution profile different from that of the cytoplasmic receptor in 0.4 M KCl. This suggests that either the nuclear receptor associates with itself or other nuclear proteins or that more than one form of nuclear receptor exists. Earlier observations suggested that in the absence of hormone the glucocorticoid receptor is localized exclusively in the cytoplasm of lung cells and that the nuclear receptor is formed by a transfer of the cytoplasmic steroid-receptor complex into the nucleus. A prerequisite for this transfer seems to be a modification of the receptor to an active form which can bind to nuclei. This receptor transfomration, referred to in this paper as activation of the receptor, can occur in the absence of nuclei and is highly dependent on temperature and ionic strength. Cytoplasmic receptors activated either by heating or by exposure to high ionic strength are indistinguishable from nonactivated receptors by sucrose density gradient analysis or by agarose gel filtration in solutions containing 0.4 M KCl. Simiarly, no significant difference in the absence of salt is observed after activation by heating. These results suggest that activation of the cytoplasmic glucocorticoid receptor involves conformational changes which favor its transfer and/or binding to nuclear sites rather than conversion of a 4 S species to a faster-sedimenting form by dimerization or by addition of another protein unit as has been proposed for the activation of the estrogen receptor of the rat uterus.     

Binding of progesterone receptor by nuclear preparations of rabbit and guinea pig uterus.

Guinea pig and rabbit uterine nuclei bound [3H] progesterone in vitro only in the presence of cytosol from estrogen-stimulated uteri. Nuclei from unstimulated and estrogen-stimulated uteri bound progesterone equally well. Nuclei of nontarget tissues also bound progesterone, but to a lesser extent. The rate of nuclear bindins increased with temperature from 0-30 degrees. At 25 degrees nuclear binding remained stable for at least 3 h, but at temperatures of 30 degrees and greater, nuclear binding decreased rapidly after 15 min. Activation of the progesterone-cytoplasmic receptor complex (the change in the complex that enables it to bind quickly to nuclei at 0 degrees) took place slowly at temperatures from 0-5 degrees and rapidly at 10-25 degrees. Activation was facilitated by dilution of the cytosol. Some activation occurred in diluted cytosol in the absence of added progesterone. The cytoplasmic progesterone receptor had a sedimentation coefficient of 7 S when concentrated cytosol (20 mg of protein/ml) was incubated with progesterone at 0 degrees in 5 mM phosphate buffer. Diluting the cytosol and increasing the temperature to 20 degrees caused the sedimentation coefficient to decrease to 5.5 S. Gel filtration of guinea pig uterine cytosol on Sephadex G-100, in the absence of progesterone, yielded a progesterone-binding fraction in the void volume, with a sedimentation coefficient of 5.5 S. The complex of progesterone with the material in the void volume was taken up by nuclei at 0 degrees more rapidly than the complex of progesterone and crude cytosol. The nuclear uptake of progesterone was decreased in phosphate buffer of concentrations greater than 80 mM. Under conditions that favor the nuclear binding of progesterone, the sedimentation coefficient of the cytoplasmic progesterone receptor was 5.5 S. This may be the form of the preceptor which is taken up by nuclei. In decreasing order of effectiveness, unlabeled progesterone, 5 alpha-pregnane-3,20-dione, corticosterone 20 alpha-hydroxy-4-pregnen-3-one, testosterone, estradiol-17 beta, and cortisol competed with [3H] progesterone for binding to nuclei.     

Specific binding of 1alpha,25-dihydroxycholecalciferol to nuclear components of chick intestine.

Specific binding of 1alpha,25-dihydroxycholecalciferol to macromolecular components of small intestinal mucosa nuclei is demonstrated in vitamin D-deficient chicks. The nuclear 1alpha,25-dihydroxycholecalciferol-macromolecule complex was isolated on sucrose density gradients and sediments at 3.7 S in the presence of 0.3 M KCl. Agarose gel filtration of the nuclear component indicated an apparent molecular weight of 47,000. The nuclear receptor complexes could not be distinguished from previously described cytoplasmic 1alpha,25-dihydroxycholecalciferol-binding components by the ultracentrifugation and chromatographic procedures employed. The association of the 3-H-sterol with the nuclear component is thermolabile and is destroyed by treatment with pronase, but not by nucleases; the receptor component is therefore presumed to be a protein. The macromolecular-1alpha,25-dihydroxycholecalciferol complex formed in vivo or in vitro at 25 degrees can be extracted from intestinal nuclei by 0.3 M KCl, but not by low salt buffers. Smaller amounts of the 3.7 S binding component can be detected in isolated purified chromatin or after incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin at 0 degrees. Following incubation of the labeled hormone with reconstituted cytosol-chromatin at 0 degrees, 1alpha,25-dihydroxy[3-H]cholecalciferol is primarily associated with the cytoplasmic receptor, After shifting the incubation temperature to 25 degrees, a progressive increase in the concentration of the nuclear receptor complex and a concomitant decrease in the concentration of the cytoplasmic binding component occur. Thus the 1alpha,25-dihydroxycholecalciferol binding molecules appear to exist primarily in the cytoplasm, where they presumably function to transport the hormone into the nucleus. Experiments employing incubation of 1alpha,25-dihydroxy[3-H]cholecalciferol with reconstituted cytosol-chromatin from nontarget tissues indicate a requirement for both intestinal cytosol and chromatin for maximal formation of the nuclear hormone-receptor complex. These results suggest that the nuclear-binding component arises from hormone-dependent transfer of the cytoplasmic 1alpha,25-dihydroxycholecalciferol receptor to intestinal chromatin acceptor sites.     

Activation of glucocorticoid cytoreceptors in rat heart]

Accumulation of 3H-dexamethasone by the nuclei in a cell-free system was studied. The increase in temperature from 0 degrees to 20 degrees C and treatment of cytosol by KCl (0.4 M) or theophylline (10mM) significantly increased the absorption the bound hormone by the nuclear fraction. Activation of the steroid-protein complex induced by temperature and addition of theophylline did not change its size. The increase in the ionic strength decreased the Stokes radius from 53 A down to 39.5 A and the sedimentation coefficient value from 7S down to 4S. It is concluded that the heart tissue cytoplasm contains a glucocorticoid receptor.     

Recycling and desensitization of glucocorticoid receptors in v-mos transformed cells depend on the ability of nuclear receptors to modulate gene expression

In v-mos transformed cells, glucocorticoid receptor (GR) proteins that bind hormone agonist are not efficiently retained within nuclei and redistribute to the cytoplasmic compartment. These cytoplasmic desensitized receptors cannot be reutilized and may represent trapped intermediates derived from GR recycling. We have used the glucocorticoid antagonist RU486 to examine whether v-mos effects can be exerted on any ligand-bound GR. In the rat 6m2 cell line that expresses a temperature-sensitive p85gag-mos oncoprotein, RU486 is a complete antagonist and suppresses dexamethasone induction of metallothionein-1 mRNA at equimolar concentrations. Using indirect immunofluorescence, we observe efficient nuclear translocation of GR in response to RU486 treatment in either the presence or absence of v-mos oncoproteins. However, in contrast to the redistribution of agonist-bound nuclear receptors to the cytoplasm of v-mos-transformed cells, RU486-bound GRs are efficiently retained within nuclei. Interestingly, withdrawal of RU486 does not lead to efficient depletion of nuclear GR in either nontransformed or v-mos transformed cells. It is only after the addition of hormone agonist to RU486 withdrawn v-mos-transformed cells that GRs are depleted from nuclei and subsequently redistributed to the cytoplasm. Thus, only nuclear GRs that are agonist-bound and capable of modulating gene activity can be subsequently processed and recycled into the cytoplasm.     

Rat liver chromatin non-histone proteins and glucocorticoid binding

We have previously characterized a specific corticosterone binding protein in chromosomal non histone proteins (NHP) from rat liver. In this paper, we present evidence that a relationship exists between this protein and the cytoplasmic glucocorticoid receptor. The binding capacity of NHP is reduced by 40 p. cent when this fraction is isolated from adrenalectomized animals. Incubation of isolated nuclei with the glucocorticoid hormone receptor complex results in a decrease in the specific radioactivity of the cytoplasmic proteins and simultaneously in a rapid uptake of the isotope by the nucleus; radioactive hormone was extracted along with the NHP. Evidence is presented that the NHP component binding the hormone is closely related or identical to the cytoplasmic receptor-proteins. Progesterone and corticosterone compete similarly for the binding of dexamethasone to nuclear and cytoplasmic forms of the receptor. However the nuclear form of the receptor has a higher affinity for corticosterone (K_a : 6 × 10⁹ M⁻¹) than for dexamethasone (K_A : 10⁸ M⁻¹) in vitro.A mixture of rat liver NHP and cytosol was shown to bind specifically more corticosterone than when the two proteins were incubated separately with the hormone. The Scatchard analysis shows that the enhancement of binding is due to an interaction of nuclear and cytoplasmic proteins leading to the appearance of a stable protein-protein complex which has a high affinity for the hormone (K_a : 2 × 10⁸ M⁻¹). KCl prevented this interaction. Complex formation does not require the presence of the hormone. The experiments presented here favor the hypothesis of the existence of a regulatory protein in the nucleus. This protein associated with the binding protein to reveal or enhance the active form of the receptor.     

Subcellular distribution of glucocorticoid receptor in the neoplastic epithelial duct cell line from human salivary gland

Neoplastic epithelial duct cell line from human salivary gland (HSG cell line) contains the specific glucocorticoid receptor. The time course study on the uptake of [3H]triamcinolone acetonide (TA), a synthetic glucocorticoid, by intact HSG cells in a growing monolayer culture showed that translocation of glucocorticoid receptors into nuclei occurred at 37 degrees C, but not at 0 degrees C. To elucidate the subcellular distribution of glucocorticoid receptor from HSG cells, a scaled-up-culture was employed. When the cells were incubated with [3H]TA at 0 degrees C, 94% of the receptors were found in the cytosol fraction, while only 6% of the receptors existed in the nuclei. When the cells were incubated at 37 degrees C, 49% of the receptor complexes were distributed in the nuclei and 74% of these nuclear receptor complexes were extractable with 5 mM pyridoxal phosphate.     

Glucocorticoid receptor phosphorylation, transformation, and DNA binding

Glucocorticoid receptors were isolated by immunoadsorption from cytosol of L cells that were cultured for 18 h in the presence of [32P]orthophosphate, and the phosphorylation state of the receptor was examined before and after transformation to the DNA-binding state. Temperature-mediated transformation of the glucocorticoid receptor under cell-free conditions results in no change in receptor size or degree of phosphorylation. When cytosol containing transformed receptors is incubated with DNA-cellulose, 30-50% of the receptors are able to bind to DNA and the remainder do not bind to DNA. Both the heated receptors that bind to DNA and the receptors that do not bind to DNA are phosphorylated to the same degree. When intact cells containing 32P-labeled receptors are incubated for 2 h at 0 degree C with triamcinolone acetonide and then for 20 min at 37 degrees C in the presence of the hormone, 80% of the receptor becomes tightly associated with the nucleus in a manner that is both temperature-dependent and ligand-dependent. Approximately 80% of the nuclear-bound receptor is extracted with 0.4 M NaCl. Both the cytosolic receptor from cells incubated at 0 degree C and the salt-extracted nuclear receptor from cells incubated at 37 degrees C have been resolved by immunoadsorption to protein A-Sepharose with the BuGR1 monoclonal antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting and autoradiography of the immunoblots. In addition, direct measurements of the amounts of 32P contained per unit of receptor protein were performed for receptors transformed both in the intact cell and in cell-free lysates. The results demonstrate that the untransformed receptor and the nuclear-bound transformed receptor are labeled with 32P to the same extent.     

ATP-dependent activation of glucocorticoid receptor from rat liver cytosol.

The glucocorticoid--receptor complex from freshly prepared rat liver cytosol is in a non-activated form, with very little affinity to bind to isolated nuclei. When such preparations were incubated with 5--10 mM-ATP at 4 degrees C, the receptor complex acquired the properties of an 'activated' transformed form, which readily bound to nuclei, ATP--Sepharose, phosphocellulose and DNA--cellulose. This transformation was comparable with the activation achieved by warming the steroid--receptor complex at 23 degrees C. The effect of ATP was specific, as it was more effective than ADP, whereas AMP had no such effect on activation. The process of receptor activation was sensitive to the presence of 10 mM-sodium molybdate; the latter blocked activation by both ATP and heat. Bivalent cations had no observable effect on the receptor activation at low temperature, but they decreased the extent of activation by ATP. The steroid-binding properties of glucocorticoid receptor remained intact under the above conditions. However, a significant increase in steroid binding occurred when ATP was preincubated with cytosol receptor before the addition of [3H]triamcinolone acetonide. ATP also stabilized the glucocorticoid--receptor complexes at 23 degrees C. These results suggest a role for ATP in receptor function and offer a convenient method of studying the activation process of glucocorticoid receptor under mild assay conditions.     

Interaction of the activated cytoplasmic glucocorticoid hormone receptor complex with the nuclear envelope

Highly purified activated cytoplasmic glucocorticoid hormone receptor binds with high affinity to sites in the nuclear envelope. Nuclear envelope fragments can be isolated from purified chromatin. They bind activated cytoplasmic glucocorticoid receptor with the same equilibrium constant as nuclear envelopes. The presence of envelope components in chromatin is confirmed by the virtual identity of the gel electrophoretic glycoprotein pattern of nuclear envelope, chromatin nonhistones, and nuclear envelope fragments from chromatin.     

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

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

Glucocorticoid-receptor interaction and induction of murine mammary tumor virus.

The relationship between the cellular uptake of glucocorticoid hormones, the binding of these hormones to specific in vitro receptors, and the induction of mouse mammary tumor viruses in an established mouse mammary tumor cell line was highly correlated. These results suggest that the induction of mouse mammary tumor virus by glucocorticoid hormones is a physiological process acting through a mechanism of high affinity, saturable steroid-receptors. A temperature-sensitive or salt-dependent step following glucocorticoid-receptor interaction was required for nuclear uptake of the steroid. Induction studies with different adrenocorticoids indicate that the synthetic glucocorticoid, dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), is the most potent inducer of mouse mammary tumor viruses and all steroids which caused significant induction were glucocorticoids. Other glucocorticoids appear to stimulate murine mammary tumor virus production by a mechanism similar to that of dexamethasone; for example, corticosterone competes with dexamethasone for binding to the glucocorticoid receptor and blocks the uptake of dexamethasone into cells. Progesterone also blocks the cellular uptake of dexamethasone and can bind to the glucocorticoid receptor at low concentrations (10-7 to 10-8 M) but progesterone does not consistently induce virus at hormone concentrations even as high as 10-4 M. Thus, in this system, binding to a cytoplasmic receptor is necessary but not sufficient for induction by glucocorticoids. Estrogens and androgens interfere with receptor binding and cellular uptake of dexamethasone but only at much higher concentration (10-4 M) than progesterone, and do not induce mammary tumor virus production. Although there was a positive correlation between steroid structure, binding, and biologic induction, other factors clearly affect the physiological manifestations of steroid actions. Mouse cells with comparable cytoplasmic receptor levels and comparable nuclear uptake differed absolutely in their degree of murine mammary tumor virus induction following hormone treatment. Although all mouse cells examined contain comparable levels of murine mammary tumor virus DNA, only cells producing constitutive levels of murine mammary tumor virus RNA could be induced to higher levels by a variety of glucocorticoids.     

Transformation of the chick oviduct progesterone receptor in the absence of hormone

Chicken oviduct progesterone receptor in cytosol was found to be transformed from the 8S to 4S form by incubation at 25 degrees C as well as by 0.3 M KCl in the absence of hormone. Heat transformation of ligand-free receptor took place at a much slower rate than that of ligand-bound receptor. The eventual percentage of transformation, however, was almost the same. The 4S form of the receptor transformed by KCl in the absence of hormone could bind to DNA-cellulose, but not to nuclei. However, upon exposure it acquired the ability to bind to nuclei. It was shown that the transformed ligand-free receptor could bind to progesterone to form the normal activated steroid-receptor complex. Conversely, when activated 4S progesterone-receptor complex was treated with DCC to peel off the hormone, a resulting ligand-free receptor was formed which behaved just like the KCl-transformed receptor in the absence of hormone.     

Glucocorticoid receptors in cytosol and nuclear extract of human leukocytes

Cortisol binding by cytosol and 0.4 M KCl extract of the nuclear fraction of human leukocytes were studied by gel chromatography and ion exchange filtration on DEAE cellulose. The cytoplasmic cortisol binding protein has a molecular weight 95 000 and the soluble nuclear binding protein 50 000. The absence of the uptake of radioactive cortisol by isolated nuclei and the apparent requirement of the cytosol for glucocorticoid specific binding in nuclear receptor sites was observed. The association constant characterising the binding of cortisol to cytosol was KA = 3.5 . 10(9) l/mol.     

Cyclic AMP-binding protein and estrogen receptor: antagonism during nuclear translocation in a hormone-dependent mammary tumor

Incubation of nuclei from hormone-dependent rat mammary tumors with its cytosol activated with 5 nM 17β-estradiol resulted in a 4-fold increase of nuclear estrogen binding activity over the control nuclei. The presence of 100 nM cAMP in the activated cytosol inhibited this nuclear uptake of estrogen receptor by 50%. Conversely, incubation of the nuclei with cytosol activated with 100 nM cAMP increased nuclear cAMP binding and cAMP-dependent protein kinase activity 4-fold, while the presence of 5 nM 17β-estradiol in the activated cytosol inhibited the nuclear cAMP binding and the protein kinase activity by 50%. No competition was found between estrogen and cAMP for each other's cytoplasmic binding proteins or the nuclear acceptor sites. These data suggest that a mutual antagonism exists between the cAMP-binding protein and estrogen receptor during their nuclear translocation.     

Evidence that invivo estradiol receptor translocated into nuclei is dephosphorylated and released into cytoplasm

Most of the estradiol binding activity is lost after the receptor-hormone complex migrates into the nuclei. We report that at the same time an equivalent amount of receptor unable to bind hormone appears in cytosol. ATP incubated with uterine cytosol from 17β-estradiol injected mice causes an increase in hormone binding activity. This activation is catalyzed by the ATP-dependent enzyme which reactivates hormone binding of estradiol receptor previously inactivated by a nuclear phosphatase (1). The inactive receptor retains the property to be activated by the ATP-dependent enzyme after a partial purification by heparin-Sepharose. A large dose of cycloheximide does not inhibit the appearance of ATP-activated cytosol receptor. Apparently the 17β-estradiol receptor which migrates into the nucleus is inactivated by the nuclear phosphatase and then released in an inactive, dephosphorylated form into the cyoplasm.     

Glucocorticoid receptors in mouse mammary tumors: Specific binding to nuclear components.

The specific interaction of glucocorticoids with nuclei of mouse mammary tumor was studied in vitro by incubation of the tissue with [3H]dexamethasone at 25 degrees. It was demonstrated that the mammary tumors contain a limited number of specific nuclear binding sites which were saturated with low hormone concentrations (10-8 M)9 The concentrations of specific binding sites in the nuclei were related to the concentration of cytoplasmic binding sites of unincubated tissues and varied between individual tumors. The binding component in the nuclei appeared to be a protein and was easily solubilized with 0.4 M KCl containing buffers. The ability of various corticoids to block the nuclear localization of the steroid correlated well with their glucocorticoid potency. Estradiol and progesterone at concentrations of 10-6 M were also effective in competing for the glucocorticoid receptor binding sites. However, while the glucocorticoids such as hydrocortisone and corticosterone translocated to nuclear sites also specific for dexamethasone, estradiol and progesterone competed for the cytoplasmic binding sites and did not translocate to the nucleus. The possible significance of the interaction of various steroids with the glucocorticoid receptors in mammary tumors is discussed.     

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.     

v-mos oncoproteins affect the nuclear retention and reutilization of glucocorticoid receptors

Expression of the p85gag-mos oncoprotein in temperature sensitive transformed 6m2 cells results in desensitization of glucocorticoid induction of metallothionein-1 mRNA. Indirect immunofluorescence analyses demonstrate that hormone insensitivity in v-mos transformed cells is associated with inefficient nuclear retention of glucocorticoid receptor (GR) protein. Desensitized receptors that accumulate in the cytoplasm of transformed 6m2 cells do not regain the capacity for hormone-dependent nuclear translocation after turnover of the thermo-labile p85gag-mos oncoprotein. Although ligand induced down-regulation of immunoreactive GR protein occurs in transformed 6m2 cells, desensitized receptors appear to retain some capacity to bind hormone in vivo. Thus alterations in the intracellular partitioning of GR protein in v-mos-transformed cells result in the generation of a novel desensitized receptor that is apparently trapped in the cytoplasm and incapable of being reutilized.