Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species

The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross-reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity.     

Mercury inhibits rat liver and kidney glucocorticoid receptor hormone binding activity

The present study was focused on the influence of mercury on the rat liver and kidney glucocorticoid receptor (GR) binding properties. The time-course and dose-dependence of mercury effects, as well as possible involvement of thiol groups were examined after in vivo and in vitro administration of the metal in the form of HgCl2. Mercury led to reduction of the liver and kidney GR hormone binding capacity. In both examined tissues maximal reduction was noticed 4 h after administration of the metal at 2 and 3 mg Hg/kg bw, but the effect was more prominent in kidney as compared to liver. On the other hand, binding affinity in the two tissues was similar. The complete reversal of mercury effects on GR binding capacity by 10 mmol/L DTT was achieved in liver and partially in kidney. The reversal by DTT suggested that mercury caused the decrease of GR binding activity by interacting with thiol groups. The difference in the response of the two tissues reflected the fact that kidney contained a higher mercury concentration and a lower thiol content in comparison to liver. The implicated thiols probably belong to GR, since when applied in vitro at 0 degrees C, mercury produced reduction of the receptor binding activity similar to that observed in vivo. GR protein level examined by quantitative Western blot was either unchanged, when determined by polyclonal antibody, or reduced, when determined by BuGR2 antibody, suggesting that Hg might affect BuGR epitope availability.     

Localization of phosphorylation sites with respect to the functional domains of the mouse L cell glucocorticoid receptor

Digestion of the rat liver glucocorticoid receptor with chymotrypsin results in the generation of a 42-kDa fragment which contains the steroid-binding and DNA-binding domains and the antigenic site for the BuGR anti-glucocorticoid receptor monoclonal antibody, while digestion with trypsin generates a 15-kDa receptor fragment containing only the DNA-binding function and the BuGR epitope (Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R. W., and Eisen, H.J. (1985) J. Biol. Chem. 260, 11805-11810). In this paper, glucocorticoid receptor of mouse L cells that were grown in the presence of [32P]orthophosphate was digested with trypsin or chymotrypsin (either before or after immune purification with BuGR antibody) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and Western blotting. The receptor is endogenously phosphorylated only on serine residues. Chymotrypsin digestion results in a 32P-labeled 42-kDa receptor fragment which contains steroid-binding, DNA-binding, and BuGR-reactive sites. Trypsin digestion generates a 27-kDa steroid-bound fragment (meroreceptor) which is not labeled with 32P and a 32P-labeled 15-kDa fragment which contains both the DNA-binding domain and the BuGR epitope. We have calculated that there are 4 times as many phosphate residues in the intact receptor than in the 42-kDa chymotrypsin fragment. From examination of 32P-labeled receptor fragments, we have deduced that one phosphate is located between amino acids 398 and 447, a region containing the BuGR epitope and about one-third of the DNA-binding domain, and the remaining three phosphates appear to be clustered just to the amino-terminal side of the BuGR epitope in a region defined by amino acids 313 to 369. Treatment of intact 32P-labeled receptor in cytosol with alkaline phosphatase removes these three phosphates, but it does not remove the phosphate from the DNA-binding-BuGR-reactive fragment and it does not affect the ability of the transformed receptor to bind to DNA-cellulose.     

Interactions of glucocorticoids with the AtT-20 cell: effect on protein accumulation

In order to investigate the mechanism through which glucocorticoids downregulate the number of their own receptors in the AtT-20 cell, the effect of glucocorticoids on cell protein metabolism was studied. Glucocorticoids were found to inhibit cellular protein accumulation when included in long-term cultures. The concentrations of agonists that cause a mid-maximal effect are similar to those needed to half-saturate the glucocorticoid receptor, suggesting that the growth-inhibiting effect is receptor-mediated. Two-dimensional electrophoresis of cytosolic extracts of treated and control cells suggested that the effect reflected a general suppression of overall protein accumulation rather than a selective effect on certain classes. Comparison of the protein to DNA ratio of control and dexamethasone-treated cells showed that the latter have higher ratios suggesting that cell composition may be altered by agonists. However, time-course studies of this effect indicated that this is basically an expression of a glucocorticoid effect on cell growth rather than a selective effect on protein metabolism. It is concluded that glucocorticoids inhibit overall AtT-20 cell growth and that this, in turn, manifests itself as a decrease in the rate of protein accumulation. It is suggested that this change in protein metabolism may be a minor component in the mechanism through which glucocorticoids decrease AtT-20 cell ACTH secretion and glucocorticoid receptor number.     

Kinetic pulse-chase labeling study of the glucocorticoid receptor in mouse lymphoma cells. Effect of glucocorticoid and antiglucocorticoid hormones on intracellular receptor half-life

A kinetic pulse-chase labeling technique was used to measure the intracellular half-life of the glucocorticoid receptor in S49 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine for 30 min and then cultured in the presence of unlabeled methionine (chase). Labeled receptors were quantitated at periodic time points during the chase by immunoadsorption to protein A-Sepharose using the BuGR2 monoclonal antireceptor antibody. The decay of labeled receptors during the chase was linear on a semilog plot, consistent with first order kinetics. Receptor half-life was 9 h when cells were cultured in either phenol red-containing medium supplemented with fetal calf serum or in phenol red free-medium supplemented with charcoal extracted serum, indicating that endogenous steroids do not affect receptor half-life. Receptor half-life was also unchanged when cells were cultured in the presence of 0.1 microM dexamethasone, a glucocorticoid hormone, or 0.1 microM RU486 (11 beta-(4-dimethylamino-phenyl)-17 beta-hydroxy-17 alpha-(propynylestra-4,9- diene-3-one), an antiglucocorticoid hormone. We conclude that the intracellular half-life of the glucocorticoid receptor in S49 mouse lymphoma cells is not regulated by either glucocorticoid or antiglucocorticoid hormones.     

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

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

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.     

Hyperthermic stress stimulates the association of both constitutive and inducible isoforms of 70 kDa heat shock protein with rat liver glucocorticoid receptor

Glucocorticoid hormone receptor exists in the cytoplasm of target cells in the form of dynamic multiprotein heterocomplexes with heat shock proteins Hsp90 and Hsp70, and additional components of the molecular chaperone machinery. Whole body hyperthermic stress was previously shown to induce alterations in protein composition of these complexes increasing the share of Hsp70, but participation of individual Hsp70 family members was not investigated. In the present study the association of glucocorticoid receptor with constitutive and inducible forms of Hsp70 in the liver cytosol of rats exposed to 41 degrees C whole body hyperthermic stress was examined. Immunoprecipitation of glucocorticoid receptor heterocomplexes by monoclonal anti-receptor antibody (BuGR2) followed by quantitative immunoblotting revealed the presence of both nucleocytoplasmic Hsp70 family members, constitutive--Hsc70 and inducible--Hsp72, within the complexes. Immediately after the stress only Hsc70 was found in association with glucocorticoid receptor. However, after the induction of Hsp72 by stress, its appearance within the glucocorticoid receptor heterocomplexes was also recorded and the presence of both Hsp70 forms within the heterocomplexes was evident by the end of examined 24h period after the stress. This study confirms that heat stress affects protein composition of rat liver glucocorticoid receptor heterocomplexes increasing the share of Hsp70 and shows that this increase could be equally ascribed to constitutive and inducible forms of Hsp70.     

Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor

The glucocorticoid receptor (GC-R) isolated from the mouse AtT-20 pituitary tumor cell line exists in three forms. The untransformed (non-DNA-binding), 9.1S species (319K) can be converted into two transformed (DNA-binding) species. One of these (5.2 S, Mr 132K) appears to be composed of one molecule of the hormone-binding, monomeric protein (96K) plus a small RNA, while the second transformed species is the monomeric, hormone-binding subunit (3.8 S, 96K) itself. We wished to determine whether the untransformed GC-R contains RNA or if the monomer binds to RNA subsequent to subunit dissociation (which occurs during receptor transformation). Kinetic studies using both the crude and purified untransformed GC-R show that the untransformed, 9.1S GC-R dissociates into 3.8S monomeric subunits, without forming a transient 5.2S complex. The untransformed receptor was then purified with affinity chromatography, gel filtration, and DEAE-cellulose chromatography. One major protein band, corresponding in size to the GC-R monomer (94K-96K), was observed on sodium dodecyl sulfate-polyacrylamide gels upon silver staining or fluorography of [3H]dexamethasone mesylate covalently labeled receptor. In vivo 32P-labeling of AtT-20 cells, followed by purification of the untransformed GC-R, yielded two major 32P-labeled components (94K-96K and 24K). Both of these bands were protease-sensitive, contained phosphoserine, and were unaffected by ribonuclease treatment. We conclude that the untransformed mouse GC-R is wholly proteinaceous and contains no RNA. Thus, RNA binding occurs subsequent to dissociation of the oligomeric, untransformed GC-R complex into monomers.     

The physico-chemical properties of the AtT-20 cell's glucocorticoid receptor during depletion

Glucocorticoid agonists decrease the number of glucocorticoid receptors in the cloned AtT-20 mouse pituitary tumor cell. To investigate whether the structure of the receptor is altered during this process, we monitored the physico-chemical properties of the nuclear and cytosolic receptors undergoing depletion. Agarose chromatography, DEAE-cellulose chromatography and sucrose gradient ultracentrifugation were employed. Cells were sampled after 2, 24, 48 and 96 h incubation with 10 nM tritiated triamcinolone acetonide. Agarose chromatography yielded, in each case, a single receptor-containing peak that had a Stokes radius of 5.8 nm. Nuclear and cytosolic glucocorticoid receptors from each preparation eluted from DEAE-cellulose as a single, symmetric peak at a KCl concentration of 0.075 M. Sucrose gradient ultracentrifugation of all samples also yielded only a single peak. For each technique the amount of receptor recovered was inversely related to the length of intact cell incubation. Thus, depletion of the glucocorticoid receptor is not accompanied by observable changes in its size, surface charge or hydrodynamic properties. These results suggest that the first step of agonist-induced glucocorticoid receptor depletion in the AtT-20 cell involves the loss or alteration of the receptor's steroid-binding site.     

Regulation of the glucocorticoid receptor in fetal rat lung during development

The effect of the synthetic glucocorticoid betamethasone on the regulation of the glucocorticoid receptor mRNA and on receptor protein was studied in fetal rat lung during development. Using a glucocorticoid receptor cRNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and by solution hybridization. A monoclonal antibody against the glucocorticoid receptor was used to study regulation of the receptor protein by the Western immunoblotting technique. In fetal rat lungs, of 16-21 days of gestation, as well as in adult lungs, betamethasone treatment resulted in a significant decrease of glucocorticoid receptor mRNA to 50-65% of the control level. In contrast, betamethasone treatment did not down-regulate the receptor protein in rat lungs of 16-19 days of gestation, whereas a decrease of glucocorticoid receptor protein to 40-60% of control was seen in lungs of 21 days of gestation, in postnatal and adult lung. These results provide data for a change in regulation in vivo of the glucocorticoid receptor by its homologous ligand in fetal rat lung during development.     

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

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

Evidence from pulse-chase labeling studies that the antiglucocorticoid hormone RU486 stabilizes the nonactivated form of the glucocorticoid receptor in mouse lymphoma cells

A pulse-chase labeling technique was used to determine the properties of glucocorticoid receptors occupied by the antiglucocorticoid hormone RU486 in S49.1 mouse lymphoma cells. Cells were pulse-labeled with [35S]methionine and then at the beginning of the chase, either no hormone (control), dexamethasone, or RU486 was added to cells. At 4 h into the chase, cytosol was prepared and receptors were immunoadsorbed to protein A-Sepharose using the BuGR2 antireceptor antibody. Immunoadsorbed proteins were resolved by gel electrophoresis and analyzed by autoradiography. The 90 kDa heat shock protein (hsp90) coimmunoadsorbed with receptors from control cells when protein A-Sepharose pellets were washed with 250 mM NaCl but not when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that hsp90-receptor complexes are disrupted by a high concentration of salt in the absence of molybdate. hsp90 coimmunoadsorbed with receptors from RU486-treated cells even when protein A-Sepharose pellets were washed with 500 mM NaCl, indicating that RU486 stabilizes the association of hsp90 with the glucocorticoid receptor. In contrast, hsp90 did not coimmunoadsorb with receptors from dexamethasone-treated cells, consistent with earlier evidence that hsp90 dissociates from the receptor when the receptor binds glucocorticoid hormone. Dexamethasone induced a rapid quantum decrease in the amount of normal receptor recovered from cytosol but did not induce a decrease in the amount of nuclear transfer deficient receptor recovered from cytosol, consistent with tight nuclear binding of normal receptors occupied by dexamethasone. In contrast, RU486 did not induce a quantum decrease in the recovery of normal receptors from cytosol, indicating that receptors occupied by RU486 are not tightly bound in the nuclear fraction. We conclude that the antiglucocorticoid hormone RU486, in contrast to the glucocorticoid hormone dexamethasone, stabilizes the association between the glucocorticoid receptor and hsp90. The decreased affinity of receptors occupied by RU486 for the nuclear fraction may be due to their association with hsp90 and may account for the failure of RU486 to exert agonist activity.     

Characterization and immunocytochemical demonstration of glucocorticoid receptor using antisera specific to transformed receptor

Cytosolic and nuclear forms of the glucocorticoid receptor were characterized using immunochemical techniques. Antibodies were raised in rabbits to an M_r 58,000 fragment of the transformed (DNA-binding) glucocorticoid receptor purified from rat liver cytosol by DNA-cellulose chromatography and polyacrylamide gel electrophoresis. Antibodies reacted with the transformed receptor form in a radioimmunoassay for glucocorticoid receptor. Western blot analysis of antibody reactivity revealed a single M_r 185,000 receptor form in rat liver cytosol but a smaller M_r 85,000 form in nucleosol, indicating the M_r 85,000 form is the transformed receptor. Furthermore, western blot analysis indicates that the M_r 185,000 receptor undergoes proteolysis during receptor purification and in vitro transformation processes by generating immunochemically similar proteins of smaller molecular weights. An identical M_r 185,000 glucocorticoid receptor was detected in cytosols of four rat tissues; liver, brain, adrenal medulla, and thymus. The glucocorticoid receptor was localized to the cytoplasm and nucleus of rat adrenal medulla cells by immunohistochemistry, demonstrating the existence in vivo of the transformed receptor and translocation of the receptor from cytoplasm to nucleus.     

Secretion of Carboxypeptidase E from Cultured Astrocytes and from AtT-20 Cells, a Neuroendocrine Cell Line: Implications for Neuropeptide Biosynthesis

Cultured astrocytes have recently been shown to produce certain neuropeptides, as well as neuropeptide processing enzymes. To characterize the secretory pathway in cultured astrocytes, we used the neuropeptide processing enzyme carboxypeptidase E (CPE) as a marker for neuropeptide secretion. Cultured astrocytes and AtT-20 cells, a mouse pituitary-derived neuroendocrine cell line, were labeled with [35S]Met for 15 min and then chased with unlabeled Met. CPE was isolated from either medium or cell extracts using a substrate affinity column. The time course of secretion of radiolabeled CPE was significantly different for cultured astrocytes as compared with AtT-20 cells. CPE was rapidly secreted from the astrocytes after a 30-min lag time, presumably reflecting transport through the endoplasmic reticulum and Golgi apparatus, followed by constitutive secretion. The secretion of radiolabeled CPE was essentially complete by 2 h. In contrast, only a portion of the radiolabeled CPE was secreted from AtT-20 cells over a 2-3-h period, indicating that the majority of newly synthesized CPE is stored, presumably in secretory granules within the AtT-20 cells. The regulation of CPE secretion from astrocytes was also examined. CPE secretion is stimulated two- to threefold by prolonged treatment (3-48 h) with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) but not by treatment with other secretagogues that stimulate CPE secretion from AtT-20 cells (forskolin, isoproterenol, A23187, and vasoactive intestinal peptide) or short (less than 3 h) exposure to TPA. Taken together, these results indicate that the secretory pathway for CPE, and presumably neuropeptides, is substantially different in astrocytes than the secretory pathway for CPE in neuroendocrine cells.     

Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors

We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc