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

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

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

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

Analysis of the glucocorticoid antagonist action of dexamethasone 21-mesylate in HeLa S3 cells

Several properties of human glucocorticoid receptors complexed to the synthetic glucocorticoid agonists dexamethasone (DEX) and triamcinolone acetonide (TA) and the antagonist dexamethasone 21-mesylate (DM) are compared in an attempt to define the mode of action of DM. Both DEX and TA induce an increase in alkaline phosphatase activity in HeLa S3 cells. Not only is DM without effect on alkaline phosphatase activity at concentrations as great as 10(-7) M, it blocks the action of DEX and TA on enzyme induction, thus acting as a pure antagonist in this system. DM-receptor complexes, like agonist-receptor complexes, are recovered in the cytosol when cells are incubated with ligand at 0 degrees C but are recovered from the nucleus when incubation is shifted to 37 degrees C, suggesting that activation of the antagonist-receptor complex occurs in vivo. The molecular species that undergoes this temperature-dependent shift from the cytosolic compartment to the nuclear compartment exhibits saturable binding to the antagonist. Both the cytosolic and nuclear species exhibit a relative molecular mass of approximately equal to 94,000 Daltons when analysed by SDS-polyacrylamide gel electrophoresis. Receptors labeled in intact cells with [3H]DM at 0 degrees C sediment at approximately 8S in sucrose gradients, shifting to 4S when the gradients contain 0.4 M KCl. DEX- and TA-labeled receptors show the same sedimentation behavior, which has been accepted as one criterion of receptor subunit dissociation, or activation.     

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.     

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

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

Effects of molybdate on steroid receptors in intact GH1 cells. Evidence for dissociation of an intracellular 10 S receptor oligomer prior to nuclear accumulation

Treatment of intact GH1 cells with sodium molybdate inhibits the subsequent rate of nuclear accumulation of hormone-occupied glucocorticoid and estrogen receptors. Cells were incubated at 23 degrees C for 1 h with 30 mM molybdate and then for up to 30 min with [3H]triamcinolone acetonide or [3H]estradiol in the continued presence of molybdate. Although molybdate did not affect the rate of receptor occupancy with either steroid, cells treated with molybdate had more occupied cytosolic and fewer occupied nuclear receptors than control cells. For the glucocorticoid receptor, cells treated with molybdate had more 10 S and fewer 4 S cytosolic receptors than control cells. In low salt cytosol molybdate inhibits the temperature-mediated subunit dissociation of occupied 10 S glucocorticoid receptor. These results suggest that a hormone-mediated dissociation of an intracellular 10 S oligomeric glucocorticoid receptor form to its 4 S subunits is required prior to accumulation of occupied receptors in the nuclear fraction. In cells incubated at 37 degrees C for 1 h or longer with [3H]triamcinolone acetonide, molybdate shifts the steady state intracellular distribution of receptor toward the 10 S cytosolic receptor form, consistent with the interpretation that molybdate affects the rapidly exchanging subunit equilibrium between the 10 S and 4 S cytosolic forms by slowing the rate of 10 S receptor dissociation. Molybdate prevents loss of glucocorticoid-occupied 10 S but not 4 S receptors in heated cytosol by stabilizing the relatively protease-resistant 10 S receptor. Since molybdate stabilizes 10 S oligomeric steroid receptors in vitro, the effects of molybdate on nuclear accumulation of occupied receptors in intact cells support the intracellular existence and physiological relevance of 10 S glucocorticoid and estrogen receptors. These results support a general model for steroid receptor activation in which binding of hormone promotes dissociation of intracellular 8-10 S oligomeric receptors to their DNA-binding subunits.     

Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

The physicochemical nature of 37 degrees C cytoplasmic glucocorticoid receptors in HeLa S3 cells

The physicochemical properties of size, shape and surface charge have been determined for the soluble fraction of cytoplasmic glucocorticoid receptors which are located in the HeLa S3 cell cytoplasm after incubation of whole cells with glucocorticoid at 37 degrees C. Under hypotonic buffer conditions approximately 80% of the total recovered [3H]triamcinolone acetonide receptor complexes sedimented through a 5-20% density gradients to the tube bottom, and approximately 90% eluted from a Sephacryl S-300 gel exclusion column in the void volume. Increasing the [KCl] of the buffer in the sucrose density gradients, and gel exclusion columns to 0.15 M caused a reduction in the percentage of this large aggregate to approximately 64% and approximately 75%, respectively. Further increases in the [KCl] during analysis to 0.4 M reduced the percentage of rapidly sedimenting receptors to approximately 62%, and shifted the sedimentation coefficient of the slower sedimenting receptors from approximately 5.2 S to 3.9 S. These conditions also decreased the fraction of receptor in the void volume of gel exclusion columns to 67%. Ion exchange analysis of receptor binding to DEAE cellulose, hydroxylapatite, phosphocellulose, and DNA cellulose revealed heterogenous populations of receptor species; comprising both "unactivated" and "activated" receptor forms. The ratios of unactivated/activated receptors was highly dependent on the matrix employed and differed substantially among those evaluated. For example, by the criteria of DEAE cellulose and phosphocellulose chromatography approximately 60% of the total 37 degrees C cytoplasmic receptors were in the "activated" state. A large fraction of these receptors, however, failed to bind to DNA cellulose. These results demonstrate that the glucocorticoid receptors which remain in the HeLa S3 cytoplasm at 37 degrees C do not bind to ion exchange materials, which are used as indexes of receptor "activation," in a uniform manner. We hypothesize that the diminished DNA binding capability of these receptors accounts for their cellular localization in the HeLa S3 cell cytoplasm at 37 degrees C.     

Differential solubilization of nuclear glucocorticoid receptors by DNAase I and DNAase II

This study analyzes the sensitivity of nuclear bound glucocorticoid receptors to solubilization from nuclei by DNAase I and DNAase II. Thymocytes were incubated with 10(-8) M [3H]dexamethasone, [3H]cortisol or [3H]triamcinolone acetonide, without or with 10(-6) M unlabelled dexamethasone, for 30 min at 37 degrees C and nuclei from these cells were digested with either DNAase I and DNAase II. DNAase I for 2 h at 3 degrees C leads to solubilization of 60% of the nuclear DNA and release of 10--20% triamcinolone acetonide-receptor, 30--40% dexamethasone-receptor and 85--90% cortisol-receptor. DNAase II at the same enzymatic concentration solubilizes only 10--20% of the nuclear DNA, but releases 40--50% triamcinolone-receptor, 60--70% dexamethasone-receptor and 100% cortisol-receptor. Release of nuclear bound dexamethasone-receptor by DNAase I parallels the solubilization of DNA, reaching maximum values by 2 h at 3 degrees C, whereas maximal release by DNAase II is obtained within 45 min when DNA solubilization is not complete. When nuclei initially extracted with DNAase I are re-extracted with DNAase II, greater than 65% of the DNAase I residual dexamethasone-receptors are solubilized, whereas DNAase I is ineffective in solubilizing DNAase II residual dexamethasone-receptors. DNAase I solubilizes only 30% of the 0.4 M KCl residual dexamethasone-receptor whereas DNAase II digests over 90% of this fraction. DNAase I extracts of nuclear dexamethasone-receptor chromatograph on G-100 Sephadex as a single radioactive peak just after the void volume, whereas DNAase II extracts of nuclear dexamethasone-receptor chromatograph as two peaks of radioactivity, one which is similar to the DNAase I solubilized receptor and a second broad peak of macromolecular bound radioactivity which is smaller in size.     

Comparative study of nuclear binding sites for oestradiol in rat testicular and uterine tissue. Determination of low amounts of specific binding site by an [3H] oestradiol-exchange method.

1. An [3H]oestradiol-exchange method was developed for the determination of oestradiol-receptor complexes in the nuclear fraction of immature rat testicular tissue. This method permits the determination of nuclear oestradiol-receptor sites in the presence of a relatively large amount of non-specific oestradiol binding present in testicular nuclei. After incubation of nuclei for 60min at 20 degrees C in the presence of [3H]oestradiol with or without a 1000-fold excess of non-radioactive diethylstilboestrol, specific binding can be determined quantitatively in the KCl-extractabe fraction, which contains 40% of the total receptor population. 2. The amount of receptor-bound steroid present in the 0.4m-KCl extract of testicular neclei remained constant during incubation at 20 degrees C. For uterine nuclei incubated with [3H]oestradiol at 37 degrees C a shift of specifically bound [3H]oestradiol occurred from the KCl-soluble fraction to the KCl-insoluble fraction. 3. In intact rat testis, about 20% of the total receptor concentration was present in its nuclear form. Hypophysectomy 5 days before measurement resulted in a twofold decrease in the amount of receptor, which was present mainly in the cytosol. After injection of choriogonadotropin to intact animals, the total receptor concentration increased threefold. 4. This nuclear exchange method might be useful for determination of occupied specific receptor sites in tissues with relatively low contents of specific receptors.     

Characterization of glucocorticoid receptor in HeLa-S3 cells

Glucocorticoid receptor of the human cell line HeLa-S3 has been characterized and has been compared to rat and to mouse glucocorticoid receptors. If HeLa cells were lysed in the absence of glucocorticoid, glucocorticoid receptor was isolated in a nonactivated form, which did not bind to DNA-cellulose. If HeLa cells were preincubated with glucocorticoid, glucocorticoid receptor was isolated in an activated, DNA-binding form. HeLa cell glucocorticoid receptor bound [3H]triamcinolone acetonide with a dissociation constant (KD = 1.3 nM at 0 degrees C) that was similar to those of mouse and rat glucocorticoid receptors. Similarly, the relative binding affinities for steroid hormones decreased in the order of triamcinolone acetonide greater than dexamethasone greater than promegestone greater than methyltrienolone greater than aldosterone greater than or equal to moxestrol. Nonactivated and activated receptors were characterized by high-resolution anion-exchange chromatography (FPLC), DNA-cellulose chromatography, and sucrose gradient centrifugation. Human, mouse, and rat nonactivated glucocorticoid receptors had very similar ionic and sedimentation properties. Activated glucocorticoid receptors were eluted at similar salt concentrations from DNA-cellulose columns but at different salt concentrations from the FPLC column. A monoclonal mouse anti-rat liver glucocorticoid receptor antibody [Westphal, H.M., Mugele, K., Beato, M., & Gehring, U. (1984) EMBO J. 3, 1493-1498] did not cross-react with HeLa cell glucocorticoid receptor. Glucocorticoid receptors of HeLa, HTC, and S49.1 cells were affinity labeled with [3H]dexamethasone and with [3H]dexamethasone 21-mesylate. The molecular weights of [3H]dexamethasone 21-mesylate labeled glucocorticoid receptors (MT 96 000 +/- 1000) were undistinguishable by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing [3H]triamcinolone acetonide [( 3H]TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, [3H]TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. When similar experiments were conducted under conditions of large receptor excess, using 3' [32P]-MMTV LTR DNA, the trace quantity of DNA formed a stable 10-14S complex with DNA-cellulose pretreated cytosols or with untreated cytosols in the presence of excess Escherichia coli competitor DNA. If trace quantities of the 3' [32P]-MMTV LTR DNA were incubated with untreated crude cytosols, much larger complexes were formed, indicating the association of other cytosolic proteins with the MMTV LTR DNA fragment. Activated [3H]TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA, but with lower apparent affinities in the order MMTV LTR DNA fragment much greater than pBR322 fragment containing a single GRE DNA consensus sequence greater than non-GRE-containing pBR322 fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects

The translocation of occupied surface insulin receptors to the nuclei of isolated hepatocytes was studied using the biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). When hepatocytes were photolabeled at 4 degrees C, extensively washed, and then further incubated at 37 degrees C for 1 h, photolabeled insulin receptors, which were initially localized to the cell surface, accumulated in the subsequently isolated nuclei. When the isolated nuclei were solubilized and subjected to polyacrylamide gel electrophoresis and radioautography, labeled proteins with Mr identical to the cell surface insulin receptor were detected. Light microscopic radioautography of nuclei isolated from cells incubated for 1 ha at 37 degrees C demonstrated that 28% of these nuclei were specifically labeled with one or more grains. Electron microscopic radioautography of intact cultured hepatocytes, incubated 60 min at 37 degrees C, revealed that 26% of the thin-sectioned nuclei contained at least a single grain and 8.3% of the total cell-associated associated grains were located over the nuclei. Only 1.6% of grains were localized to lysosomes. In contrast, if photolabeled hepatocytes were incubated at 4 degrees C for up to 2 h, negligible accumulation of nuclear radioactivity was observed by polyacrylamide gel electrophoresis on light or electron microscopic radioautography. Conclusions are as follows. Occupied cell surface insulin receptors can internalize and translocate to the nucleus of intact hepatocytes by a time- and temperature-dependent mechanism. Accumulation and possible degradation of insulin receptors in lysosomes involves only a small percentage of the receptors internalized. Nuclear translocation of occupied cell surface insulin receptors may be a mechanism which mediates insulin's long term effects.     

The glucocorticoid antagonist 17 alpha-methyltestosterone binds to the 10 S glucocorticoid receptor and blocks agonist-mediated dissociation of the 10 S oligomer to the 4 S deoxyribonucleic acid-binding subunit

The glucocorticoid antagonist 17 alpha-methyltestosterone inhibits binding of the agonist [3H]triamcinolone acetonide ot the glocucorticoid receptor in cytosol prepared from rat pituitary tumor GH1 cells. Competitive binding studies indicate that the dissociation constant for 17 alpha-methyltestosterone is about 1 microM. After incubation of intact GH1 cells with 10 nM [3H]triamcinolone acetonide at 37 C and subsequent cell fractionation at 4 C, three glucocorticoid receptor forms are observed: cytosolic 10 S receptor, cytosolic 4 S receptor, and nuclear receptor. Concurrent incubation with 17 alpha-methyltestosterone reduces the amount of [3H]triamcinolone acetonide bound to each of these receptor forms. Ligand-exchange assays performed at 0 C in intact cells using [3H]triamcinolone acetonide show that the exchangeable antagonist is associated predominantly with cytosolic 10 S receptor. Immunochemical analysis using monoclonal antibody BuGR2 indicates that 17 alpha-methyltestosterone does not cause substantial accumulation of glucocorticoid receptors in GH1 cell nuclei and, when present together with agonist, reduces nuclear accumulation of receptor seen with agonist alone. Results from dense amino acid labeling studies show that unlike [3H]triamcinolone acetonide, 17 alpha-methyltestosterone does not reduce the total amount of cellular glucocorticoid receptor and does not reduce receptor half-life. These results are consistent with a model for glucocorticoid receptor transformation in which binding of agonist promotes the dissociation of an oligomeric 10 S cytosolic receptor protein to its DNA-binding 4 S subunit. The antagonist 17 alpha-methyltestosterone competes with agonist for binding to the 10 S cytosolic receptor but does not appear to promote dissociation of the oligomer, thus inhibiting agonist-mediated nuclear actions of the glucocorticoid receptor.     

Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.     

Chemical differentiation of type I and type II receptors for adrenal steroids in brain cytosol

Studies outlined here compare the properties of mineralocorticoid (Type I) and glucocorticoid (Type II) receptors in cytosol from adrenalectomized mouse brain. Pretreating cytosol with dextran-coated charcoal (DCC) produced a 4.7-fold increase in the subsequent macromolecular binding of the mineralocorticoid, [3H]aldosterone (20 nM ALDO, in the presence of a 50-fold molar excess of the highly specific synthetic glucocorticoid, RU 26988), whereas it produced a 55% decrease in the binding of the glucocorticoid, [3H]triamcinolone acetonide (20 nM TA). Scatchard analyses revealed that DCC pretreatment had no effect on the affinity or maximal binding of Type I receptors for [3H]ALDO (in the presence of a 0-, 50- or 500-fold excess of RU 26988), whereas it produced a 3- to 6-fold increase in the Kd, and an 8-43% decrease in the maximal binding, of Type II receptors for [3H]TA and [3H]dexamethasone. Optimal stability of unoccupied Type I receptors at 0 degree C was found to be achieved in buffers containing glycerol, but lacking molybdate. Although the addition of molybdate was found to reduce the loss in Type I receptor binding observed after incubating unlabelled cytosol at 12 or 22 degrees C, this stabilization was accompanied by a concentration-dependent reduction in the binding of [3H]ALDO at 0 degree C. Scatchard analyses showed that this reduction was due to a shift in the maximal binding, and not the affinity, of the Type I receptors for [3H]ALDO. The presence or absence of dithiothreitol in cytosol appeared to have little effect on the stability of Type I receptors. In contrast to our finding for Type I receptors, it was possible to stabilize the binding capacity of unoccupied Type II receptors, even after 2-4 h at 12 or 22 degrees C, if the glycerol containing buffers were supplemented with both molybdate and dithiothreitol. In summary, these results indicate distinct chemical differences between Type I and Type II receptors for adrenal steroids.     

Effect of the 90 kDa heat shock protein, HSP90, on glucocorticoid receptor binding to DNA-cellulose

Glucocorticoid receptors in the IM-9 human lymphoblastoid cell line were affinity labeled with [3H]dexamethasone 21-mesylate and activated to a DNA-binding form by filtration through a Bio-Gel A-1.5m column. The 90 kDa heat shock protein, HSP90, was identified by labeling IM-9 cells with 35S-methionine at both 37 degrees C and 42 degrees C and purified to near homogeneity by sequential chromatography through DE52 and hydroxyapatite. Addition of purified HSP90 to activated, affinity labeled glucocorticoid receptors in a molecular ratio of 16 to 1 inhibited the binding of the receptors to DNA-cellulose. HSP90 did not affect the binding of other proteins to DNA-cellulose, indicating that the inhibitory effect of HSP90 was specific for the glucocorticoid receptor. These results suggest that HSP90 may associate with the glucocorticoid receptor, masking its DNA-binding site and thereby inhibiting receptor interaction with DNA.     

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line, BCL1

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL1 cells to sequentially deproteinized BCL1 chromatin-cellulose was investigated. [3H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAE-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [3H]TA-receptor complexes to chromatin-cellulose extracted with 0-8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocorticoid receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [3H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL1 cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL1 cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6-8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.     

Interaction of rat liver glucocorticoid receptor with a newly synthesized antisteroid ZK98299

Steroid receptor antagonists are important biochemical probes for understanding the mode of steroid hormone action. We have studied the interaction between rat liver glucocorticoid receptor and a newly synthesized antisteroid ZK98299 (13-antigestagen; [11-beta-(4-dimethylaminophenyl)-17a-hydroxy-17 beta-(3- hydroxypropyl)-13 alpha-methyl-4,9-gonadien-3-one]). Glucocorticoid receptor from freshly prepared hepatic cytosol bound [3H]ZK98299 with affinity approximately equal to that of [3H]triamcinolone acetonide. The binding of both steroids reached a maximum at 4 h at 0 degrees C. Both ligands were able to compete for the steroid binding site but progesterone, estradiol and dihydrotestosterone (DHT) failed to compete for the [3H]ZK98299 and [3H]triamcinolone acetonide binding. While [3H]ZK98299 binding to glucocorticoid receptor could occur in the presence of iodoacetamide and N-ethylmaleimide (NEM), [3H]triamcinolone acetonide binding capacity was completely abolished following such treatments. The [3H]ZK98299-receptor complexes sedimented as 9 S and 4 S molecules under control (4 degrees C) and receptor transforming (23 degrees C) conditions, and exhibited a faster rate of dissociation at 23 degrees C when compared with [3H]triamcinolone acetonide-receptor complexes. These results indicate that ZK98299 interacts with hepatic glucocorticoid receptor. The differential effects of iodoacetamide and NEM on the interaction of glucocorticoid receptor with ZK98299 and triamcinolone acetonide, and the faster rate of dissociation of [3H]ZK98299-receptor complexes suggest that treatment with these agents (NEM and iodoacetamide) results in distinct conformational changes in glucocorticoid receptor structure with respect to triamcinolone acetonide and ZK98299 binding. Alternatively, ZK98299 may be interacting with a site which is distinct from one which accepts triamcinolone acetonide.