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

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

Examination of exchange assay for glucocorticoid receptor

We examined a method for the measurement of total, activated and non-activated glucocorticoid receptors using sodium-p-hydroxymercuribenzoate (PHMB) and dithiothereitol (DTT) developed by Banerji and Kalimi (1981). Since the concentration of PHMB required for dissociation of the ligand from the receptors varied with the concentration of protein in the reaction mixture and the rate of reassociation of the ligand to the ligand-liberated receptors was sensitive to the concentration of PHMB used, it was necessary to find the minimum concentration of PHMB which was required for complete dissociation of the ligand. When the optimum concentration of PHMB was selected based on the concentration of protein in the cytosol, almost 100% exchange was attained in the non-heated dexamethasone (Dex)-receptor complexes by this method. However when Dex-receptor complexes were heated at 25 degrees C for 30 min, the amount of 3H-Dex reassociated with the glucocorticoid receptors dropped to 60% of that of the non-heated ones. DEAE-cellulose chromatography of the heated sample revealed that approx. 40% of the bound receptors were activated (eluted with 0.05 M KCl) during the heating period. After DEAE cellulose column chromatography of the exchanged 3H-Dex receptor, complexes reassociated with 3H-Dex were observed only in the fraction of unactivated receptor complexes (eluted with 0.2 M KCl). Furthermore, the fraction eluted with 0.05 M KCl in the DEAE cellulose chromatography of liver cytosol bound to unlabelled Dex did not exchange significantly with 3H-Dex with the method used in the present study.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Physicochemical characterization of hepatic glucocorticoid receptors from pre- and post-weaned mice

The physicochemical parameters viz., molecular weight, stokes radius and ionic state of hepatic glucocorticoid receptors from pre-(10-day) and post-(60-day) weaned mice were studied. Gel permeation studies of the crude receptors showed a molecular mass of approximately 290 kDa for the unactivated receptors from both the age groups while the thermally activated receptors showed a molecular mass of approximately 90 kDa. The stokes radii were approximately 5.8 and 3.6 for the unactivated and activated receptors, respectively from both the age groups studied. Elution of the bound glucocorticoid receptors from anion-exchanger did not reveal any charge difference in the two age groups; the unactivated receptors eluted at approximately 250 mM KCl whereas the activated receptors eluted at approximately 100 mM KCl. Salt extraction of thermally activated nuclear bound receptors and immunological studies on the unactivated receptors revealed no age-related variation in the two groups of mice. Our findings confirm that the physicochemical properties of hepatic glucocorticoid receptors remain unchanged at these developmental stages of mice.     

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)     

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.     

Arsenite and cadmium(II) as probes of glucocorticoid receptor structure and function

Low concentrations of arsenite, but not arsenate, and Cd2+ blocked steroid binding to the glucocorticoid receptors of HTC cells. Inhibition by arsenite was faster and occurred at lower concentrations than for Cd2+. Half-maximal inhibition of [3H]dexamethasone binding was seen after a 30-min preincubation with approximately 7 microM arsenite. The effect of arsenite and of Cd2+ appears to be mediated by a reaction with vicinal dithiols of the receptor as shown by (a) the reversal of arsenite inhibition by much lower concentrations of dithiothreitol (approximately 0.1 mM) than of beta-mercaptoethanol (approximately 10 mM); (b) the ability of both arsenite and Cd2+ to block [3H]dexamethasone 21-mesylate labeling of receptors but not of other thiol-containing proteins; and (c) the known selectivity of arsenite and of Cd2+ for reactions with vicinal dithiols. Arsenite forms a tight complex with these vicinal dithiols since the removal of loosely associated arsenite by gel exclusion chromatography did not reverse the inhibition of steroid binding. The effect of other ions on steroid binding was also examined. Half-maximal inhibition of binding occurred with approximately 5 microM selenite, whereas up to 300 microM Zn2+ was without effect. Much higher concentrations of arsenite were required for effects on unactivated and activated complexes. Arsenite slowly induced a loss of unactivated complexes but rapidly inhibited a portion of the DNA binding of activated complexes. Any effect on activation occurred at arsenite concentrations equal to or higher than those that inhibited DNA binding. In contrast, Cd2+ concentrations similar to those that block steroid binding caused a biphasic loss of unactivated complexes and a marginal loss of activated complexes. This is the first report of effects of arsenite on glucocorticoid receptors. These results confirm directly our earlier hypothesis that steroid binding to rat glucocorticoid receptors involves a vicinal dithiol (Miller, N. R., and Simons, S. S., Jr. (1988) J. Biol. Chem. 263, 15217-15225) and show that arsenite is a potent new reagent for probing receptor structure and function.     

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

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

Salt-induced activation of 1,25-dihydroxyvitamin D3 receptors to a DNA binding form

In this report we examine the DNA-cellulose binding and sedimentation properties of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptors from rat intestine and cultured human mammary cancer cells (MCF-7) extracted in nonactivating (low salt) buffers. Receptors prepared in hypotonic buffer had low DNA binding (13%) compared to receptors extracted with 0.3 M KCl (50%). Treatment of low salt receptor preparations with KCl significantly increased (approximately 3-fold) DNA-binding (activation), demonstrating that receptors can be "activated" in vitro. Activated receptors eluted from DNA-cellulose at 0.18 M KCl. Sedimentation analysis followed by DNA-cellulose binding indicated that activated receptors are approximately 3.2 S and unactivated receptors 5.5 S in size. These results suggest that dissociation of an aggregated moiety may lead to receptor activation. Treatment of unactivated receptor with RNase did not alter DNA binding or sedimentation properties of the aggregated receptor. Treatment of unactivated receptor complexes with heat did not increase DNA binding, and molybdate did not block subsequent salt activation. In summary these results suggest that 1,25(OH)2D3 receptors undergo a salt-induced activation step similar to that described for other steroid receptor systems. However, 1,25(OH)2D3 receptors differ from other steroid receptors in not exhibiting heat activation nor having salt activation blocked by molybdate.     

The Effect of Including Molybdate in the Eluting Buffer Used for Deae-Cellulose Chromatography on the Quantitation of Glucocorticoid Receptor Transformation

Abstract

This study analyzes the effect of including molybdate in the elution buffers used in DEAE cellulose chromatography on the fraction of glucocorticoid receptor which elutes as the transformed species. Inclusion of molybdate leads to a significant decrease in the fraction of receptor eluting as transformed; samples which appear to be nearly 50% transformed if eluted in the absence of molybdate were found to be less than 10% transformed when analyzed using a buffer which contained 5 mM molybdate. This decrease was not caused by loss of receptor or a reversion of transformation. DEAE cellulose accelerates receptor transformation. It is concluded that DEAE cellulose should not be used to quantitate transformation unless molybdate is included in all buffers.     

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.     

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.     

Purification and characterization of the untransformed androgen receptor in rat prostate

The isolation and characterization of the untransformed form of androgen receptors has not yet been successful, owing to their inherent lability as well as to their ready proteolysis. In this study, we have stabilized rat prostate androgen receptors by sodium molybdate and by rapid filtration on phosphocellulose. Proteases were inhibited by bacitracin, aprotinin, leupeptin and PMSF. Under these conditions the untransformed complex was purified approx 3000-fold, corresponding to 18% yield, by differential chromatography on DEAE cellulose and phosphocellulose gels. The partially purified receptor has the same ionic characteristics as the original untransformed receptor of crude cytosol; in addition, it possesses a Stokes' radius of 75 A, as determined by Sephacryl S-300 gel filtration, a sedimentation coefficient of 8.8S, a calculated molecular weight of 275 kDa and a friction coefficient of 1.6. The [3H]R1881 receptor complex was specific to androgens since unlabelled R1881 and dihydrotestosterone were able to completely displace bound [3H]R1881, whereas estradiol, cortisol, and triamcinolone acetonide did not compete. The purified complex was a multimer dissociable by 0.6 M KCl, resulting in a form migrating in the 4S area on sucrose density gradient. After treatment with 0.5% formaldehyde, three forms were obtained, migrating in the areas of 8-9, 5-6 and 3-4S respectively, of a sucrose density gradient containing 0.6 M KCl. This is the first step towards the purification to homogeneity of the untransformed androgen receptor.     

Isolation of ribonucleic acid from the unactivated rat liver glucocorticoid receptor

We have reported that the 7-8S form of the rat liver glucocorticoid receptor is associated with RNA. Whether the unactivated 9-10S form of the glucorticoid receptor is also associated with RNA is less clear. Here we provide evidence that the unactivated 9-10S receptor is indeed associated with RNA. Unactivated 9-10S receptor was partially purified by diethylaminoethyl (DEAE)-cellulose chromatography in the presence of molybdate, an activation inhibitor. This preparation was then bound to BuGR-2, a mouse monoclonal antibody of the immunoglobulin G (IgG)-2 class to the rat liver glucocorticoid receptor, or to nonspecific mouse IgG-2. The antibody-antigen complex was then bound to protein A sepharose and washed to remove extraneous RNA. When the receptor was dissociated from the antibody and the RNA extracted and end-labeled, a distinct band of approximately 170 nucleotide (nt) was found that was specific for the BuGR-2 purified receptor. This band could also be found in DEAE-cellulose receptor that had been isolated from sucrose gradients. The DEAE-cellulose receptor was then cross-linked with formaldehyde before mixing with BuGR-2 in order to permit more vigorous washing of the antigen-antibody complex. In addition to the 170 nt RNA band, another distinct band at approximately 400 nt was seen that was specific to the BuGR-2 derived isolate. These results provide evidence that the 9-10S form of the glucocorticoid receptor from rat liver is associated with RNA.     

Localization of pyrodoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 μg/ml) or chymotrypsin (100 μg/ml) digestion of intact receptors sediment as 2.6 S species on 5–20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocoritcoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.     

Effects of Temperature,Nucleotides and Sodium Molybdate on Activation and DNA Binding of Estrogen,Glucocorticoid, Progesterone and Androgen Receptors In MCF-7 Human Cancer Cells

Abstract

We studied the effects of temperature, ribonucleotides and sodium molybdate on the activation and DNA cellulose binding of estrogen, glucocorticoid, progesterone and androgen receptor complexes in MCF-7 cells. Using DNA cellulose binding as a measure of receptor activation, we found that ribonucleotides activated all four of these receptor complexes. Temperature also activated glucocorticoid receptor complexes efficiently but activated progesterone and androgen receptor complexes less well. Temperature did not activate estrogen receptor complexes. Sodium molybdate blocked either ATP or temperature induced activation of glucocorticoid, progesterone and androgen receptor complexes but only partially blocked estrogen activation. Sodium molybdate also prevented the formation of multiple forms of estrogen and glucocorticoid receptor complexes seen on DEAE cellulose and hydroxylapatite chromatography of crude cytosol. The mechanism by which ribonucleotide enhances and molybdate inhibits activation are discussed.     

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

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

Hepatic Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Partial stabilization by molybdate

In structure and general mode of action, the Ah receptor is very similar to the receptors for steroid hormones. Molybdate previously has been shown to be highly effective at preserving ligand-binding function in steroid receptors during their exposure to elevated temperature or high ionic strength and at stabilizing steroid receptors as high molecular weight oligomeric complexes. Since such stabilization by molybdate can be very useful during characterization and purification of receptors, we tested the effects of molybdate on the Ah receptor to determine if the Ah receptor, like the receptors for steroid hormones, might be stabilized. In hepatic cytosols from C57BL/6N mice and Sprague-Dawley rats, molybdate concentrations up to 30 mM in homogenizing and analysis buffers did not alter the concentration of specific Ah receptor sites detected by binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. However, inclusion of 20 mM molybdate in the homogenizing buffer did significantly protect unliganded Ah receptor from thermal inactivation at 20 degrees C and from KCl-induced loss of ligand-binding ability. In accord with previous reports, 20 mM molybdate in homogenizing and analysis buffers greatly increased the concentration of detectable glucocorticoid receptor in rat hepatic cytosol and estrogen receptor in rat uterine cytosol. Exposure to 0.4 M KC1 caused the glucocorticoid receptor from rat liver to shift sedimentation from approximately equal to 8 S to approximately equal to 4 S and caused a severe loss of specific glucocorticoid binding. Presence of 20 mM molybdate stabilized the glucocorticoid receptor as a single discrete peak sedimenting at approximately equal to 8 S. In contrast, the Ah receptor from rat liver exposed to 0.4 M KC1 in the presence of molybdate sedimented as biphasic peaks; one peak (approximately equal to 9.5 S) corresponded to the form of Ah receptor observed at low ionic strength, while the other peak (approximately equal to 5.5 S) corresponded to the form of Ah receptor seen in cytosol treated with 0.4 M KC1 in the absence of molybdate. Addition of heparin to hepatic cytosols from mice or rats shifted sedimentation of Ah receptor from approximately equal to 9.5 S to approximately equal to 5.5 S. Molybdate, again, provided stabilization in the approximately equal to 9.5 S form, but only for about one-half the total Ah receptor content in both rat and mouse hepatic cytosols. In sum, molybdate is far less effective at stabilizing rodent Ah receptors than it is at stabilizing steroid receptors in the same species.     

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.