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 DEAF-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 glucocortical 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.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

Binding of glucocorticoid receptors to mammary chromatin acceptor sites

We have recently characterized the interaction of mouse mammary estrogen receptors (ER) with mammary chromatin acceptor sites and demonstrated that ER from estrogen resistant lactating mammary glands do not bind to chromatin. In this study we have characterized the chromatin binding of the glucocorticoid receptor from mouse mammary glands isolated from nulliparous and lactating mice in order to better understand the relationship between receptor binding to chromatin and steroidogenic sensitivity of the tissue. Mammary chromatin was linked covalently to cellulose and deproteinized sequentially by 0-8 M Gdn-HCl. Binding to intact chromatin as well as to chromatin deproteinized by Gdn-HCl was determined using partially purified [3H]dexamethasone labelled glucocorticoid-receptor complexes (GR) obtained by fractionation on DEAE-cellulose columns. The binding of [3H]GR from mammary glands of nulliparous mice to chromatin fractions from the same tissue revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 5-6 M Gdn-HCl. Binding of [3H]GR was of high affinity (Kd = 0.2 nM) and saturable. A simultaneous comparison of the chromatin binding patterns for [3H]ER and [3H]GR isolated from mammary glands of nulliparous mice revealed that the chromatin subfractions obtained with 4-6 M Gdn-HCl extraction contained acceptor sites for both [3H]ER and [3H]GR; however, while the [3H]ER bound to a 4.5 M and a 5.5 M site, the [3]GR bound a 5 M and a 6 M site. Competition experiments supported the steroid receptor specificity of the chromatin acceptor sites. Thus, the 4-6 M chromatin fractions contain distinct acceptor sites for the glucocorticoid receptor and for the estrogen receptor. In addition our studies reveal that the binding patterns of [3H]GR isolated from mammary glands of nulliparous and lactating mice to their homologous chromatin is essentially similar. Thus, in contrast to estrogen receptors, glucocorticoid receptors from lactating mammary glands are able to effectively bind to chromatin acceptor sites which supports our previous suggestion that the estrogenic insensitivity of lactating mouse mammary glands may at least be in part due to the impeded interaction of ER with chromatin acceptor sites.     

The binding of [3H]oestradiol-receptor complexes to calf uterine chromatin.

Various aspects of the interaction of oestrogen-receptor complexes with calf uterine chromatin covalently coupled to cellulose were analysed. Partially purified [3H]oestradiol-receptor complexes were bound to intact, or partially deproteinized, chromatin resins. Proteins were removed from the chromatin-cellulose resins by extraction with high molarities of salt, including NaCl/urea, guanidine hydrochloride and guanidine thiocyanate. After extensive washing to remove the salt, [3H]oestradiol-receptor-complex solutions were added to the resins and the degree of binding was determined. The extent of [3H]oestradiol-receptor-complex binding to chromatin was enhanced by extraction of chromosomal proteins. By varying the molarity of the salt, and consequently the extent of protein removal, it was possible to resolve [3H]oestradiol-receptor-complex binding to guanidine thiocyanate-extracted chromatin into two components. Similarly, [3H]oestradiol-receptor-complex binding to guanidine hydrochloride-treated chromatin included three regions of enhanced binding capacity. The [3H]oestradiol-receptor-chromatin interaction was saturable with respect to both intact and salt-extracted resins. Thus uterine chromatin may contain three or more specific classes of acceptors for the oestrogen-receptor complex.     

Acceptor sites for the oestrogen receptor in hen oviduct chromatin.

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.     

Activation of the human glucocorticoid receptor: evidence for a two-step model

The relationship between glucocorticoid receptor subunit dissociation and activation was investigated by DEAE-cellulose and DNA-cellulose chromatography of monomeric and multimeric [3H]triamcinolone acetonide ([3H]TA)-labeled IM-9 cell glucocorticoid receptors. Multimeric (7-8 nm) and monomeric (5-6 nm) complexes were isolated by Sephacryl S-300 chromatography. Multimeric complexes did not bind to DNA-cellulose and eluted from DEAE-cellulose at a salt concentration (0.2 M KCl) characteristic of unactivated steroid-receptor complexes. Monomeric [3H]TA-receptor complexes eluted from DEAE-cellulose at a salt concentration (20 mM KCl) characteristic of activated steroid-receptor complexes. However, only half of these complexes bound to DNA-cellulose. This proportion could not be increased by heat treatment, addition of bovine serum albumin, or incubation with RNase A. Incubation of monomeric complexes with heat inactivated cytosol resulted in a 2-fold increase in DNA-cellulose binding. Unlike receptor dissociation, this increase was not inhibited by the presence of sodium molybdate. Fractionation of heat inactivated cytosol by Sephadex G-25 chromatography demonstrated that the activity responsible for the increased DNA binding of monomeric [3H]TA-receptor complexes was macromolecular. These results are consistent with a two-step model for glucocorticoid receptor activation, in which subunit dissociation is a necessary but insufficient condition for complete activation. They also indicate that conversion of the steroid-receptor complex to the low-salt eluting form is a reflection of receptor dissociation but not necessarily acquisition of DNA-binding activity.     

The binding of 3H-labelled androgen-receptor complexes to hypothalamic chromatin of neonatal mice: effect of sex and androgenization

The binding of 3H-labelled androgen-receptor complexes, prepared by (NH4)2SO4 precipitation from the 105,000 g supernatant of hypothalamic cytosol, to hypothalamic chromatin of neonatal mice covalently coupled to cellulose was measured in vitro. Saturation binding was also determined after extraction of histones and the masking of acidic proteins with high molarities of guanidine hydrochloride. This investigation showed the presence of high-affinity, low-capacity acceptor sites for [3H]-testosterone-receptor complexes in male hypothalamic chromatin (Kd value = 0.39 x 10(-10) M and binding sites of 41 fmol per mg of DNA). Acceptor activity seems to be associated with the acidic protein fraction of chromatin. No specific acceptor sites of similar nature were found in chromatin taken from the hypothalami of female mice. On the basis of these results, it is suggested that the androgen-unresponsiveness of female mice is related to the absence of acceptors for the androgen-receptor in female mice hypothalami.     

Physicochemical characteristics of the interaction of the glucocorticoid antagonist RU38486 with glucocorticoid receptors in vitro, and the role of molybdate

The physicochemical properties of complexes formed between the glucocorticoid antagonist, RU38486, and the glucocorticoid receptor in rat thymus cytosol were investigated and compared with those of complexes formed with the potent agonist, triamcinolone acetonide. The equilibrium dissociation constant for the interaction of [3H]RU38486 with the molybdate-stabilized glucocorticoid receptor was lower than that for [1,2,4-3H]triamcinolone acetonide at 0 degree C but higher at 25 degrees C, suggesting that hydrophobic interactions play a major role in the binding of RU38486. Differences in equilibrium constants were reflected in corresponding differences in dissociation rate constants; association rate constants for the two steroids were similar. The rate of dissociation of [3H]RU38486 from the glucocorticoid receptor was higher in the absence of molybdate than in its presence both at 0 degree C and at 25 degrees C, suggesting that molybdate modifies the physical state of the antagonist-receptor complex, but other physical properties were similar both in the presence and in the absence of molybdate. The rate of inactivation of the unoccupied glucocorticoid receptor at 25 degrees C in the absence of molybdate was lower in phosphate buffer than in Tris-HCl buffer but the rate of dissociation of [3H]RU38486 was the same in both buffers. The binding of RU38486 afforded little, if any, protection against inactivation in either buffer; [3H]RU38486 dissociated irreversibly from the inactivated receptor at the same rate as from the non-inactivated complex but molybdate had no effect on the dissociation kinetics of the inactivated complex. It is concluded that RU38486 interacts with the ground state of the glucocorticoid receptor in a manner which neither promotes receptor transformation nor prevents receptor inactivation.     

Interaction of sodium thiocyanate with rat hepatic glucocorticoid-receptor complexes

0.1–0.3 M sodium thiocyanate greatly enhanced the rate of inactivation of unbound rat hepatic glucocorticoid receptors in vitro at 4°C. Prior treatment of the unbound glucocorticoid receptor with 10 nM molybdate (at 25°C for 30 min) protected the receptor from 0.3 M KCl, but not from 0.3 M NaSCN inactivation. When the [³H]dexamethasone-receptor complex was examined on sucrose density gradients containing 0.1 M NaSCN, the receptor sedimented as a 4 S complex rather than the 7 S form observed in 0.1 M KCl gradients. NaSCN was found to be more effective in the extraction of both in vivo and in vitro nuclear-bound [³H]dexamethasone-receptor complexes than KCl. At a concentration of 0.3 M, NaSCN extracted most of the specific nuclear-bound receptor. 50 mM NaSCN significantly blocked the thermal activation of preformed [³H]dexamethasone-receptor complexes. The chaotropic salt, NaSCN, appears therefore to have significant effects on glucocorticoid receptors in vitro. In addition, NaSCN appears to be a useful agent in quantitative extraction of steroid from nuclear-bound steroid-receptor complexes.     

Cytosol glucocorticoid receptor in the neoplastic epithelial duct cell line from human salivary gland (HSG)

Neoplastic epithelial duct cell line from human salivary gland (HSG cell) contained cytosol glucocorticoid receptor. Scatchard analysis of cytosol indicated that the dissociation constant (Kd) was 5.6-6.5 nmol/l and the number of binding sites was 83-92 fmol/mg protein. A competitive assay showed that the binding sites for [3H]triamcinolone acetonide were specific to glucocorticoid. Glycerol density gradient centrifugation displayed that the [3H]triamcinolone acetonide receptor complexes sedimented in the 8.5 S region under low salt conditions and in the 4.2 S region under high salt condition (0.4 M KCl). The same high salt conditions induced an increased binding of [3H]triamcinolone acetonide complexes for DNA-cellulose.     

Binding of the estradiol-receptor complex to reconstituted nucleoacidic protein from calf uterus

Non-histone protein-DNA complexes with acceptor activity for estradiol-receptor complexes were reconstituted from fractionated calf uterine chromatin. Acceptor activity had tissue specificity with target tissue binding exceeding non-target tissue binding. The binding of estradiol-receptor complexes to acceptor sites was dependent on intact non-histone protein-DNA complexes, reconstituted select non-histone proteins, and protein equivalent: DNA reconstitution ratios. [³H]Estradiol-receptor complexes were bound to reconstituted non-histone protein-DNA complexes (i.e., nucleoacidic protein) with a high affinity and with a limited number of binding sites. Fractionation of uterine chromatin non-histone proteins identified two major sets of non-histone proteins which had acceptor activity when reconstituted with DNA. Thus, it seems possible to reconstitute nucleoacidic protein fractions with specific acceptor activity for the calf uterine estrogen receptor.     

The distribution and properties of the glucocorticoid receptor from rat brain and pituitary

The distribution and properties of cytoplasmic binding sites for the synthetic glucocorticoid dexamethasone and the natural glucocorticoid corticosterone in the brain and the pituitary were studied in detail. Cortisol-17 beta acid, a derivative which does not bind to the glucocorticoid receptor but is a competitor of corticosterone binding to plasma, was used to overcome plasma interference. In vitro competition assays in the presence of excess cortisol acid reveal that dexamethasone is as effective a competitor for [3H]corticosterone binding as corticosterone itself. Scatchard analysis of equilibrium experiments with both steroids, using cytosol from various brain areas and from the pituitary yielded linear plots, suggesting one class of binding sites. The quantitative distribution of the sites follows the pattern: cortex greater than hippocampus greater than or equal to pituitary greater than hypothalamus greater than brain stem white matter. Furthermore, kinetic analysis of corticosterone dissociation showed a first order reaction, thus indicating the presence of one type of receptor in all brain areas examined. Rat brain cytosolic receptors for corticosterone and dexamethasone elute from DEAE-Sephadex A-50 anion exchange columns at 0.3 M NaCl in the presence of stabilizing sodium molybdate and at 0.15 M NaCl and/or in the buffer wash when heat-activated, thus exhibiting the characteristic activation pattern of rat liver cytosolic glucocorticoid receptor. The ratio of the buffer wash to the 0.15 M NaCl form is low for dexamethasone and very high for corticosterone. Receptor complexes from various brain parts showed the same activation pattern. In our experiments, brain corticosterone and dexamethasone receptors stabilized by sodium molybdate are indistinguishable by a number of techniques, thus indicating that it is unnecessary to evoke specific binding sites for each glucocorticoid.     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

Influence of molybdate, ionic strength and pH on ligand binding to the glucocorticoid receptor

The addition of molybdate to rabbit liver cytosol increased significantly the affinity of the glucocorticoid receptor for [3H] dexamethasone without influencing the concentration of binding sites. This effect was concentration dependent. Analysis of the binding data by curve-fitting and Scatchard plot revealed the occurrence of a complex binding process in the presence of molybdate. The pH-dependence curve of the binding was shifted towards alkaline values by the oxyanion. Taken together, these data suggest that molybdate exerts its effects via an interaction with the receptor molecule.     

Characterization of cytosolic glucocorticoid receptor of fetal rat epiphyseal chondrocytes

The cytosolic glucocorticoid receptor of 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 x 10(-8) M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 +/- 1.43 x 10(-9) M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinities of steroid for receptor were found to be triamcinolone acetonide greater than corticosterone greater than hydrocortisone greater than progesterone greater than medroxyprogesterone acetate much greater than 17 alpha-hydroxyprogesterone much greater than testosterone greater than 17 beta-estradiol. Cytosolic preparations activated in vitro by warming (25 degrees C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4 degrees C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.     

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

Thermal activation of the purified rat hepatic glucocorticoid receptor. Evidence for a two-step mechanism

Thermal "activation" or "transformation" of rat hepatic [6,7-3H]triamcinolone acetonide (TA)-receptor complexes purified in the unactivated state to near homogeneity (Grandics, P., Miller, A., Schmidt, T. J., Mittman, D., and Litwack, G. (1984) J. Biol. Chem. 259, 3173-3180) has been further investigated. The data generated in reconstitution experiments demonstrate that warming (25 degrees C for 30 min) of the purified unactivated complexes promotes their activation as judged by an increase in DNA-cellulose binding, but to a lower extent than that observed after warming of glucocorticoid-receptor complexes in crude cytosols. However, maximal DNA-cellulose binding capacity can be detected in reconstituted systems (also heated at 25 degrees C for 30 min) consisting of purified unactivated [3H]TA-receptor complexes and a cytoplasmic "stimulator(s)." This cytoplasmic factor(s), which does not copurify with the receptor, is heat-stable (90 degrees C for 30 min), excluded from Sephadex G-25, and trypsin-sensitive and stimulates DNA-cellulose binding in a dose-dependent manner. The ability of Na2MoO4 to block thermal activation of the highly purified receptor complexes suggests that this transition metal anion interacts directly with the receptor protein itself. The fact that the cytoplasmic stimulator(s) enhances DNA-cellulose binding of the [3H]TA-receptor complexes without increasing the proportion of those complexes eluted in the activated (low salt) position from DEAE-cellulose is consistent with a proposed two-step model of in vitro activation. During the Na2MoO4-sensitive Step 1, elevated temperature (25 degrees C for 30 min) may directly alter the conformation of the purified receptor complexes (i.e. subunit dissociation or disaggregation), resulting in the appropriate shift in the elution profile of the [3H]TA-receptor complexes on DEAE-cellulose but only in a minimal (approximately 2-3-fold) increase in the binding of these complexes to DNA-cellulose. During the Na2MoO4-insensitive and temperature-independent Step 2, a heat-stable cytoplasmic protein(s) may interact with these thermally activated [3H]TA-receptor complexes and enhance their ability to bind to DNA-cellulose without further increasing the percentage of those complexes which elute from DEAE-cellulose in the activated position. In crude cytosols these two steps would presumably occur simultaneously, and addition of Na2MoO4 prior to warming would block Step 1 and hence Step 2 would not occur.     

Aflatoxin inhibition of glucocorticoid finding capacity of rat liver nuclei.

The effect of aflatoxin B1 on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [3H]desamethasone-cytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B1. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 26 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B1 dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant. Administration of [3H]dexamethasone to alfatoxin B1 -treated rats produced a similar pattern of glucocorticoid binding distribution in vivo to that observed in vitro. No differences in [3H]dexamethasone-cytoplasmic receptor binding between control and alfatoxin B1 -treated rats were found, whereas nuclear [3H]dexanthasone binding was reduced 34% by alfatoxin B1 -treatment.     

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

A specific fraction of avian oviduct chromosomal proteins can be reannealed to pure avian DNA to reconstitute nativelike specific nuclear binding sites (acceptor sites) for the oviduct progesterone receptor (PR). These specific nuclear binding sites represent the difference between the binding to the reconstituted NAP and that to pure DNA. The specific fraction of chromatin protein which contains the acceptor activity, fraction CP-3, is very tightly bound to hen DNA in a complex termed nucleoacidic protein (NAP). Removal of the CP-3 fraction from NAP results in a loss of specific PR binding sites. Resins containing chromatin adsorbed to hydroxylapatite are used as a rapid method to isolate the CP-3 fraction. Reconstitution of the CP-3 fraction to DNA by the described method involving a regressing gradient of 6-0 M guanidine hydrochloride (Gdn-HCl) results in a reconstituted NAP which displays specific PR binding sites identical with those in native (undissociated) NAP and whole chromatin. Optimal conditions and potential problems for reconstituting these nucleoproteins are described. Only partially purified receptor preparations were used in these cell-free binding analyses since they have been shown to bind with similar properties and patterns as the nuclear binding in vivo. Therefore, the binding of PR to the reconstituted NAPs was demonstrated to be receptor dependent, saturable, and of high affinity. Further, the pattern of binding to the reconstituted sites mimics those which are observed in vivo. Thus, nonfunctional receptors that cannot translocate and bind to the nuclear acceptor sites in vivo also failed to bind to the acceptor sites on the reconstituted NAPs generated by the acceptor proteins. In contrast, the binding to pure DNA does not reflect these receptor differences in receptor bindings. Specific binding of PR to reconstituted NAP can be reversed by again removing the protein fraction. Moreover, the specific binding can be destroyed by proteases and protected by protease inhibitors, indicating that acceptor activity is proteinaceous in nature. The reconstitution of the activity is both a concentration-dependent and time-dependent process. During the reconstitution, acceptor activity appears to reconstitute on the DNA when the Gdn-HCl concentration reaches 2.0 M. By use of the reconstitution method as an assay for acceptor activity, the activity in the CP-3 fraction was shown by molecular sieve chromatography to elute in a relatively broad molecular weight range between 13 000 and 25 000. The activity also focuses in isoelectric focusing resins with apparent pI's of 5.2 and 6.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of the radiolabelled high-affinity anti-oestrogen [3H]H1285 with the cytoplasmic oestrogen receptor.

The high-affinity triarylethylene anti-oestrogen H1285 [4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between [3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with [3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. [3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. [3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with [3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of [3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for [3H]oestradiol. Because of the extremely slow dissociation of [3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of [3H]H1285-receptor complexes with those of [3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. [3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with [3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of [3H]H1285-receptor complexes and [3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of [3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for [3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor.