Nanomolar concentrations of untransformed glucocorticoid receptor in nuclei of intact cells

The subcellular distribution of untransformed glucocorticoid-receptor complex in vivo has been studied by chemical crosslinking of intact cells, and using a procedure adequate for correction of experimental errors due to redistribution of components between cytosolic and nuclear fractions. We found that in HeLa S₃ cells 85.4% of total glucocorticoid-receptor complexes are located in nuclei, and 14.6% are cytosolic. If measurements were performed with MCF-7 cells, we determined that the nuclear pool of glucocorticoid-receptor complexes accounts for 75.2% of the total cellular content, whereas the remaining 24.8% are cytosolic. When the subcellular distribution of estrogen-receptor complexes was determined, instead, we found that they are almost exclusively located in nuclei of MCF-7 cells, which contain 88.9% of the total. In order to estimate the molar concentration of receptors in cytosol and nuclei of intact cells, we determined the free water content of the two compartments. The volume of solvent was found to vary in the three cell lines we have studied, and our data showed that these variations are due to the cytosolic fractions, as the free water content of nuclei is essentially the same in those cells. When the free water content and the levels of glucocorticoid-receptor complexes we have measured were used to estimate the molar ncentrations of receptors, we found that these range between 0.4 and 18.9 nM in cytosols, and between 3.9 and 6.3 nM in nuclei of the three cell lines we have studied. We then concluded that the relative distribution of untransformed glucocorticoid-receptor complexes between cytosol and nuclei is cell-specific but their molar concentration in the nuclear compartment does not greatly vary among different cells.     

Detection of glucocorticoid-receptor complex oligomers in nuclear extracts from cells exposed to hormone at physiological temperature.

When HeLa cells were incubated with tritiated dexamethasone mesylate at 2 degrees C, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytosolic and nuclear extracts revealed the presence of two monomeric receptor complex forms with estimated molecular masses of about 98 and 87 kDa. If cells were subjected to crosslinking with glutaraldehyde, a third form consisting of a 250 kDa oligomer was also detected. When HeLa cells were treated with dexamethasone mesylate at 37 degrees C, and were subjected to crosslinking, electrophoresis of cytosolic glucocorticoid-receptor complexes was drastically reduced, whereas their levels in nuclear extracts were not appreciably altered.     

Glucocorticoid receptors are associated with particles containing DNA and RNA in vivo

Chemical crosslinking of glucocorticoid-receptor complexes to associated components in living cells was performed by the use of formaldehyde. Glucocorticoid binding sites were predominantly located in nuclei, and could not be efficiently extracted by 0.3 M NaCl. Sonication was found to cause the release of about 40% of nuclear receptor complexes. By sucrose density gradient centrifugation of soluble extracts from nuclear sonicates, crosslinked receptor complexes were found in oligomeric forms under high salt conditions. Treatment of these extracts with hydrolytic enzymes showed that DNA and RNA were associated with crosslinked receptor complexes.     

Characterization of nonactivated and activated glucocorticoid-receptor complexes from intact rat thymus cells

In cells exposed to glucocorticoids at 37 degrees C activated glucocorticoid-receptor complexes (complexes with affinity for nuclei and DNA) are formed after nonactivated complexes. Activation thus appears to be an obligatory physiological process. To investigate this process we have characterized cytoplasmic complexes formed in rat thymocytes at 0 and 37 degrees C. Complexes in cytosols stabilized with molybdate were analyzed by sucrose gradient centrifugation and by chromatography on DNA-cellulose, DEAE-cellulose, and agarose gels. Two major complexes were observed: the nonactivated complex, eluted from DEAE at approximately 200 mM KCl, was formed at 0 and 37 degrees C, gave S20,w = 9.2 S, Stokes radius = 8.3 nm, and calculated Mr = 330,000; the activated complex, eluted from DEAE at approximately 50 mM KCl, appeared only at 37 degrees C, gave S20,w = 4.8 S, Stokes radius = 5.0 nm, and Mr = 100,000. A third, minor complex, probably mero-receptor, which appeared mainly at 37 degrees C, bound to neither DNA nor DEAE, and gave S20,w = 2.9 S, Stokes radius = 2.3 nm, and Mr = 27,000. With three small columns in series (DNA-cellulose, DEAE-cellulose and hydroxylapatite), the three complexes can be separated in 5-10 min. By this method we have examined the stability of complexes under our conditions. We conclude that in intact thymus cells glucocorticoid-receptor complexes occur principally in two forms, nonactivated and activated, and that activation is accompanied by a large reduction in size. The origin of the mero-receptor complex remains uncertain.     

Chemical crosslinking: a useful tool for evaluations of steroid receptor structures and their functional states in intact cells

A procedure of chemical crosslinking of intact cells with glutaraldehyde was employed to contribute to the understanding of glucocorticoid receptor structures and their functional states in vivo. Under optimal experimental conditions, glucocorticoid binding sites were found almost equally distributed between cytosolic and nuclear fractions of crosslinked cells. Sedimentation properties of crosslinked receptor complexes in cytosolic and nuclear extracts revealed that these entities were oligomers, which heterogeneously sedimented between 11 and 4S in the presence of 0.3 M NaCl. By anion exchange chromatography, we could establish that these receptor complex oligomers behaved as untransformed forms.     

The effect of alkaline phosphatase on the activation of glucocorticoid-receptor complexes in rat liver cytosol

Calf intestinal alkaline phosphatase was found to stimulate the rate of in vitro activation of rat liver glucocorticoid-receptor complexes. This effect was registered both at 0 and 25 degrees C and could be prevented by sodium molybdate. The resulting change in sedimentation behaviour (shift of sedimentation coefficient from 9.6 S to 4.8 S for molybdate-stabilized and alkaline phosphatase-treated complexes, respectively) was similar to that observed after heat activation.     

Oligomeric structures of cytosoluble estrogen-receptor complexes as studied by anti-estrogen receptor antibodies and chemical crosslinking of intact cells

The structure of estrogen-receptor complexes recovered in cytosolic extracts of MCF-7 cells treated with hormone at 2°C was probed by chemical crosslinking of intact cells and sample analysis with four monoclonal anti-estrogen receptor antibodies. When MCF-7 cells were treated with either glutaraldehyde or dithiobis(succinimidyl propionate), cytosoluble estrogen-receptor complexes consisted of two major forms sedimenting as 4 S monomers and 8–9 S salt-resistant oligomers. By high salt sucrose density gradient centrifugation, we could observe that the four monoclonal anti-estrogen receptor antibodies bound different forms of receptor complexes from crosslinked cells. While H222 and H226 antibodies could interact with any form we detected, the D75 and D547 monoclonals could only recognize those showing sedimentation coefficients lower than 7 S. When cytosolic extracts from [³⁵S]-methionine-labeled cells were subjected to immunoprecipitation with H222 and D75 anti-estrogen receptor antibodies, electrophoretic analysis of material extracted from immunoprecipitates revealed the presence of 65 kDa estrogen receptors. If extracts were prepared from crosslinked cells, instead, two more components with estimated molecular masses of 220 and 100 kDa were specifically immunoprecipitated by the H222 antibody, whereas only the 100 kDa component and the estrogen receptor were found in immunoprecipitates obtained with the D75 monoclonal. When estrogen-receptor complexes were immunopurified from extracts prepared after cells had been crosslinked with dithiobis(succinimidyl propionate), and the oligomers were dissociated by treatment with β-mercaptoethanol, electrophoretic analysis of our samples showed that only the 65 kDa estrogen receptor and a 50 kDa protein were selectively immunoprecipitated by anti-estrogen receptor antibodies. We concluded that the structures of cytosoluble estrogen-receptor complexes in MCF-7 cells treated with hormone at 2°C, include oligomeric forms which contain a 50 kDa non-steroid binding protein.     

Extraction of DNA-cellulose-bound glucocorticoid-receptor complexes with sodium tungstate

Glucocorticoid-receptor complex from rat liver cytosol, activated by warming at 23°C or fractionation with (NH₄)₂SO₄, was adsorbed over DNA-cellulose. This DNA-cellulose-bound [³H]triamcinolone acetonide-receptor complex was extracted in a dose-dependent manner by incubation with different concentrations of sodium tungstate. A 50% recovery of receptor was achieved with 5 mM sodium tungstate. Almost the entire glucocorticoid-receptor complex bound to DNA-cellulose could be extracted with 20 mM sodium tungstate. The [³H]triamcinolone acetonide released from DNA-cellulose following tungstate and molybdate treatment was found to be associated with a macromolecule, as seen by analysis on a Sephadex G-75 column. The glucocorticoid-receptor complex extracted by both the compounds sedimented as a 4 S entity of 5–20% sucrose gradients under low- and high-salt conditions. Addition of tungstate or molybdate to the preparations containing activated receptor had no effect on the sedimentation rate of receptor. However, addition of tungstate to non-activated receptor preparation caused aggregates of larger size. The tungstate-extracted glucocorticoid-receptor complex failed to rebind to DNA-cellulose even after extensive dialysis, whereas receptor in molybdate-extract retained its DNA-cellulose binding capacity.     

Tight binding of glucocorticoid-receptor complexes to histone-agarose

"Activated" glucocorticoid-receptor complexes purified about 3,000-fold from rat liver were found to bind to histone-agarose. Because of their tight binding, they could not be eluted from the column by high salt solution (3 M KCl) or low salt plus polyol buffer (50% ethylene glycol), but their binding could be disrupted by pyridoxal 5'-phosphate; more than 70% recovery of the "activated" receptor complexes was achieved with buffer containing 20 mM pyridoxal 5'-phosphate. This interaction of "activated" glucocorticoid-receptor complexes of rat liver with histone-agarose suggests a role of histones in the mechanism of action of steroid hormone.     

Stabilization of glucocorticoid-receptor complexes in rat thymus cytosol by a factor from WEHI-7 cells

Using a variety of physico-chemical techniques we have recently characterized three distinct forms of glucocorticoid-receptor complexes present in the cytosol from rat thymus cells incubated with glucocorticoid; the relative proportions of these complexes are dependent on the conditions to which the cells or cytosols are exposed. Two of these complexes correspond to the well established nonactivated and activated receptor forms, while the third has properties consistent with mero-receptor. Based on their differential affinities for DNA- and DEAE-cellulose we have developed a rapid mini-column chromatographic procedure for separating these three forms and have used it to examine the stability of complexes in cytosol preparations. We have found that activated glucocorticoid-receptor complexes from rat thymus cells are relatively unstable under cell-free conditions in that they undergo time-dependent losses in DNA binding and are converted to mero-receptor. In contrast, cytosolic glucocorticoid-receptor complexes prepared from WEHI-7 mouse thymoma cells are remarkably stable under similar conditions. Mixing experiments with equal portions of rat thymus and WEHI-7 cytosol revealed that the difference between the two tissues cannot be accounted for merely by differences in amounts of proteolytic enzymes, since addition of rat thymus cytosol to WEHI-7 cytosol containing activated glucocorticoid-receptor complexes does not result in their conversion to mero-receptor. However, the WEHI-7 cytosol affords considerable protection to activated glucocorticoid-receptor complexes in thymus cytosol. The stabilizing factor from WEHI-7 cytosol is heat stable (survives 100 degrees C for 30 min), insensitive to pH over a wide range (4.0-10.0), and appears to be macromolecular. It does not inhibit activation, and thus appears distinct from the previously described endogenous glucocorticoid receptor stabilizing factor responsible for stabilization of thymocyte receptor binding capacity (Leach et al., J. Biol. Chem. 257: 381-388, 1982). We propose that the factor is an endogenous inhibitor of the protease(s) responsible for mero-receptor formation.     

Hormone dependency of transformation of rat liver glucocorticoid receptor in vitro: effects of heat, salt, and nucleotides

A majority of the untransformed glucocorticoid-receptor complexes (GRc) from rat liver cytosol sedimented in the 9S region in 5-20% sucrose gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of the cytosol at 23 degrees C, or at 0 degree C with 10 mM ATP or 0.3 M KCl caused appearance of a slower migrating (4S) form which exhibited an increased affinity toward DNA-cellulose and ATP-Sepharose. Presence of 20 mM Na2MoO4 blocked this 9S to 4S transformation of GRc. A complete conversion of the 9S to the 4S form occurred upon a 2 h incubation of GRc with 10 mM ATP at 0 degree C. Other nucleoside triphosphates (GTP, CTP, and UTP), ADP and PPi (but not AMP or cAMP) were also effective in transforming the 9S form. The heat transformation occurred in a time-dependent manner and was complete within 1 h at 23 degrees C; presence of 10 mM ATP during this 23 degrees C incubation period allowed a complete 9S to 4S alteration in 10-20 min. Addition of ATP also accelerated the rate of salt activation of the GRc; a 50% conversion to the 4S form occurred in 20 min or 3 min in the absence or the presence of 10 mM ATP during the 0 degree C incubation of GRc with 0.15 M KCl. An absolute requirement of the hormone for 9S to 4S transformation of glucocorticoid receptor (GR) was evident, as no conversion of the 9S form to the 4S form could be achieved with the ligand-free GR under any of the above conditions. Incubation of cytosol preparations at 23 degrees C or at 0 degree C with KCl or ATP caused dissociation of the GRc and reduced the steroid binding capacity of GR. Although aurintricarboxylic acid, pyridoxal 5'-phosphate, Na2MoO4, Na2WO4, o-phenanthroline, Rifamycin AF/013 and heparin inhibited the ATP-Sepharose and DNA binding of the GRc, only Na2MoO4 and Na2WO4 selectively blocked the 9S to 4S conversion. We suggest that the 9S to 4S transformation in vitro of rat liver GRc represents an acquisition of DNA and ATP-Sepharose binding ability and may involve a separation of subunits from an oligomeric receptor structure.     

Nucleoprotein Complexes Containing Replicating Simian Virus 40 DNA: Comparison with Polyoma Nucleoprotein Complexes

Procedures for isolating nucleoprotein complexes containing replicating polyoma DNA from infected mouse cells were used to prepare short-lived nucleoprotein complexes (r-SV40 complexes) containing replicating simian virus 40 (SV40) DNA from infected monkey cells. Like the polyoma complexes, r-SV40 complexes were only partially released from nuclei by cell lysis but could be extracted from nuclei by prolonged treatment with solutions containing Triton X-100. r-SV40 complexes sedimented faster than complexes containing SV40 supercoiled DNA (SV40 complex) in sucrose gradients, and both types of SV40 nucleoprotein complexes sedimented ahead of polyoma complexes containing supercoiled polyoma DNA (py complex). The sedimentation rates of py complex and SV40 complex were 56 and 61S, respectively, based on the sedimentation rate of the mouse large ribosomal subunit as a marker. r-SV40 complexes sedimented as multiple peaks between 56 and 75S. Sedimentation and buoyant density measurements indicated that protein is bound to all forms of SV40 DNA at about the same ratio of protein to DNA (1-2/1) as was reported for polyoma nucleoproteins.     

Transformation of glucocorticoid-receptor complex from rat thymocytes and its accumulation in chromatin in a cell-free system.

The accumulation of glucocorticoid-receptor complex from rat thymocyte cytosol in a thymocyte chromatin preparation has been studied. A thymocyte 100 000 X g supernatant was prepared and the receptor and the receptor stabilized by the addition of glycerol until 40%. Tritiated glucocorticoid-receptor complex was formed by incubation of this solution with tritiated glucocorticoids at -5 degree C. The chromatin accumulated part of the complex at incubations at 4 degrees C. Receptor without hormone was not accumulated in the chromatin. The accumulation from cytosol diluted and preincubated at 4 degrees C prior to the addition of the chromatin occurred with a high rate, whereas a low rate was seen without preincubation. This indicated a transformation of the complex during the preincubation. This transformation was found to be obligatory for the accumulation and to be promoted by dilution of the supernatant and by high ionic strength. The transformed and the untransformed complexes differed with respect to partition coefficients in an aqueous dextran-polyethylene glycol two-phase system and in their behaviour during adsorptions with dextran-coated charcoal, where great loss of transformed complex was observed. The accumulation of complex in the chromatin was found to be unsaturable in the concentration interval studied (0.07--0.25 nM).     

Interaction of the hepatic glucocorticoid-receptor complex with Affigel blue

Summary Unlike the unactivated glucocorticoid-receptor complex, the thermally activated glucocorticoid-receptor complex was able to bind to Affigel blue (a matrix previously shown to bind proteins containing a dinucleotide fold region) under low ionic conditions (0.05 M KCl). Glucocorticoid-receptor complex binding capacity to Affigel blue was enhanced by increasing salt concentration. Optimal binding was obtained at 0.15 M KCl and remained at a plateau level up to 0.4 M KCl. In contrast to Affigel blue binding, glucocorticoid-receptor complex binding to nuclei was optimum at low ionic strength buffer, declined at 0.15 M KCl and became negligible at 0.4 M KCl. Interestingly, at physiological ionic strength (0.15 M KCl) both nuclei and Affigel blue bound to the glucocorticoid-receptor complex with almost identical capacity. Glucocorticoid-receptor complexes incubated 45 min at 25 °C (activation conditions) in the presence of 10 mM molybdate were unable to bind to Affigel blue (or isolated nuclei) as expected. The results obtained suggest that Affigel blue mimics isolated nuclei in the binding of activated glucocorticoid-receptor complexes under physiological (0.15 M KCl) conditions. In addition, Affigel blue may provide a rapid and easy method to study glucocorticoid-receptor complex activation and interaction with nuclear acceptor sites.     

Chromatographic separation of nontransformed and transformed glucocorticoid-receptor complexes from rat liver cytosol. Use of tungstate in the purification, inactivation, and resolution of receptor forms

Aliquots of rat liver cytosol glucocorticoid-receptor complexes (GRc) were transformed by an incubation with 8-10 mM ATP at 0 degrees C and were compared with those transformed by an exposure to 23 degrees C. The extent of receptor transformation was measured by chromatography of the samples over columns of DEAE-Sephacel. The ATP-transformed complexes, like those which were heat-transformed, exhibited lower affinity for the positively charged ion-exchange resin and were eluted with 0.12 M KCl (peak-I): the nontransformed complexes appeared to possess higher affinity and required 0.21 M KCl (peak II) for their elution. As expected, the receptor in the peak-I exhibited the DNA-cellulose binding capacity and sedimented as 4S in sucrose gradients. Peak II contained an 8-9S glucocorticoid receptor (GR) form that showed reduced affinity for DNA-cellulose. Presence of sodium tungstate (5 mM) prevented both heat and ATP transformation of the GRc resulting in the elution of the complexes in the region of nontransformed receptors. When parallel experiments were performed, binding of the cytosol GRc to rat liver nuclei or DNA-cellulose was seen to increase 10-15 fold upon transformation by heat or ATP: tungstate treatment blocked this process completely. The transformed and nontransformed GRc were also differentially fractionated by (NH4)2SO4: tungstate-treated (nontransformed) receptor required higher salt concentration and was precipitated at 55% saturation. In addition, the GRc could be extracted from DNA-cellulose by an incubation of the affinity resin with sodium tungstate resulting in approximately 500-fold purification of the receptor with a 30% yield. These studies show that the nontransformed, and the heat-, salt-, and ATP-transformed GRc from the rat liver cytosol can be separated chromatographically, and that the use of tungstate facilitates the resolution of these different receptor forms. In addition, extraction of the receptor from DNA-cellulose by tungstate provides another new and efficient method of partial receptor purification.     

Effect of dextran-charcoal treatment on the dissociation kinetics of glucocorticoid-receptor complexes: possible involvement of lipid

Dissociation kinetics were determined at 0 degrees C for molybdate-stabilized glucocorticoid-receptor complexes in rat thymus cytosol. Exposure of complexes to dextran-coated charcoal had no effect on their chromatographic properties or transformation status, but dissociation rates measured after charcoal treatment were significantly lower than those determined by displacement with excess competing steroid. The dissociation rate of the [2,4,6,7-3H]prednisolone-receptor complex was similarly modified by chromatography on Lipidex 1000, but not by chromatography on Sephadex G-25 or G-75. It is concluded that treatment of glucocorticoid-receptor complexes with dextran-charcoal or Lipidex 1000 brings about a change in dissociation rate as a consequence of the removal of a lipid component from the complex.     

Transformation of glucocorticoid-receptor complex from rat thymocytes and its accumulation in chromatin in a cell-free system

The accumulation of glucocorticoid-receptor complex from rat thymocyte cytosol in a thymocyte chromatin preparation has been studied. A thymocyte 100 000 × g supernatant was prepared and the receptor stabilized by the addition of glycerol until 40%. Tritiated glucocorticoid-receptor complex was formed by incubation of this solution with tritiated glucocorticoids at −5°C. The chromatin accumulated part of the complex at incubations at 4°C. Receptor without hormone was not accumulated in the chromatin. The accumulation from cytosol diluted and preincubated at 4°C prior to the addition of the chromatin occurred with a high rate, whereas a low rate was seen without preincubation. This indicated a transformation of the complex during the preincubation. This transformation was found to be obligatory for the accumulation and to be promoted by dilution of the supernatant and by high ionic strength. The transformed and the untransformed complexes differed with respect to partition coefficients in an aqueous dextran-polyethylene glycol two-phase system and in their behaviour during adsorptions with dextran-coated charcoal, where great loss of transformed complex was observed. The accumulation of complex in the chromatin was found to be unsaturable in the concentration interval studied (0.07–0.25 nM).     

Characterization of nontransformed and transformed androgen receptor from rat submandibular gland

Rat submandibular gland cytosol contained androgen receptor which had a single class of specific binding and an apparent dissociation constant of (1.1-1.2) X 10(-9) M. The process of transformation was investigated by a slightly modified minicolumn method in which the transformed receptor complexes were separated from the nontransformed receptor and meroreceptor. 10 mM ATP or pyrophosphate at 0 degrees C induced transformation of androgen receptor as did heat or salt treatment. 20 mM of sodium molybdate completely inhibited transformation that resulted from ATP, heat or salt treatment. The nontransformed androgen receptor complexes sedimented at 8 S and eluted at 250-260 mM KCl from DEAE-Sephacel, and its molecular weight was found to be 220 000 on Sephacryl S300 gel chromatography. On the other hand, the transformed androgen receptor complexes sedimented at 4.1-4.3 S (ATP or KCl treatment) or 3.5-3.8 S (heat treatment) and eluted at 60-80 mM KCl from DEAE-Sephacel. The molecular weight of the transformed androgen receptor complexes was 80 000-85 000 (ATP or KCl treatment) or 70 000-80 000 (heat treatment). These results suggest that the transformation of androgen-receptor complexes from rat submandibular gland was induced by the subunit dissociation and that salt bridges may be involved in the subunit interaction.