Activated steroid-receptor complex. Comparison of assays using DNA-cellulose or homologous nuclei.

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [3H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the later only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.     

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

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

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.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Interaction of glucocorticoid · receptor complexes with rat liver nuclei

Some previous reports on acellular binding of glucocorticoid · receptor complexes to rat liver nuclei have pointed to the conclusion that there exists a small number of high affinity nuclear “receptor” sites. Various investigations lead us to the opposite conclusion and suggest that these results were actually due to the presence, in the cytosol, of one or several macromolecules which inhibited the binding to nuclei of steroid · receptor complexes. The mechanism of this inhibition was examined. It appeared to be due not to a competition between both molecules for the same nuclear acceptor site but to an interaction in the cytosol between teh inhibitor and the steroid · receptor complex which prevented the binding of the latter to the nuclei. The search for high affinity specific acceptor sites was also negative for physiological saline conditions and for the non-salt-extractable fraction of the nuclear receptor. When 940-fold purified receptor · steroid complexes were used, very high concentrations of complexes could be achieved and saturation of nuclei was then observed, but only under physiological ionic strength conditions. However, the interaction was of relatively low affinity (K_A = 3.8 · 10⁷ M^?1) and to a great number of acceptor sites (N = 26.2 pmol/mg DNA), largely exceeding the cellular concentration of receptor (5.8 pmol/mg DNA).These results suggested that saturation of nuclei by steroid · receptor complexes should not occur in the intact liver cell. They were confirmed by studies on the distribution of steroid · receptor complexes in liver slices incubated with various concentrations of [³H]dexamethasone. For all hormone concentrations a constant proportion (90%) of the complexes was found in the nuclei, thus showing no saturation of the nuclear acceptor sites.     

Sensitivity of progesterone receptors to aurintricarboxylic acid: Inhibition of nuclear uptake,ATP and DNA binding

When hen oviduct cytosol samples containing progesterone receptor complexed to [³H]progesterone were included with isolated nuclei in presence of 0.2 mM aurintricarboxylic acid, more than 50% inhibition occurred in the uptake of progesterone receptor by the nuclei. The activated form of progesterone receptor appeared to be more sensitive to the presence of aurintricarboxylic acid since pretreatment of non-activated progesterone receptor with the inhibitor and the subsequent removal of the latter prior to activation did not result in the inhibition of receptor uptake by the nuclei. Also, the binding of progesterone receptor to columns of DNA-cellulose or ATP-Sepharose was abolished under simmilar conditions. When nuclei, ATP-Sepharose or DNA-cellulose were preincubated with the inhibitor prior to the addition of receptor preparations, no such inhibition resulted indicating that the inhibitor may be interacting with the receptor protein and not complexing to ATP, DNA or sites in the nuclei. The steroid binding properties of progesterone receptor, however, remained intact under these conditions. Both A and B forms of progesterone receptor are equally sensitive to aurintricarboxylic acid presence when tested for their nuclear uptake. Aurintricarboxylic acid was also found to be very effective at low concentrations (0.25 mM) in eluting the receptor complexes off ATP-Sepharose columns without disrupting the steroid binding properties of progesterone receptor. Our results suggest that auintricarboxylic acid is an effective inhibitor of progesterone receptor and that it may be acting by interfering with a site(s) on progesterone receptor which may be exposed upon activation and are involved in such processes as ATP binding, nuclear uptake and DNA binding. These observations suggest the use of aurintricarboxylic acid as a chemical probe for the analysis of progesterone receptor.     

Assessment of “activation” of estrogen receptors in lactating mammary gland using DNA-cellulose binding

To circumvent the need for isolated nuclei in studies on activation of estrogen-receptor complexes in mammary gland of the rat, a DNA-cellulose binding assay was employed using a cell-free system. Incubation at 28°C for 30 min of receptors previously charged with [³H]estradiol markedly enhanced their association with DNA-cellulose. Once activated, estrogen-receptor complexes bound maximally to DNA-cellulose within 20–30 min. The temperature optimum for activation was 28 ± 2°C using cytosol preparations. The temperature-induced activation required the presence of both steroid and cytosolic receptors simultaneously. Density gradient centrifugation revealed that, unlike those of uterus, both activated and charged estrogen-receptor complexes of lactating mammary tissue sedimented as a 4 S species in sucrose gradients containing 0.4 M KCl.     

Author index

Binding of dexamethasone · receptors with isolated nuclei, DNA-cellulose and cellulose has been compared with respect to dependence on salt concentration and resistance to KCl extraction and DNAase I digestion. A solution of cytoplasmic dexamethasone-receptor complexes was prepared by the incubation of rat thymus cells with steroid at 3°C and breaking the cells by hypotonic lysis. Activation of the complexes was accomplished by warming the solution at 25°C for 15 min. Activation significantly increased the ability of dexamethasone · receptors to bind to nuclei and DNA-cellulose but not to cellulose. Dexamethasone-receptor complexes bound to nuclei at 3°C are completely resistant to extraction with 0.1 M KCl, 76% resistant to 0.2 M KCl and 20% resistant to 0.4 M KCl. Dexamethasone · receptors bound to DNA-cellulose are 45% resistant to extraction with 0.1 M and 0.2 M KCl and 29% resistant to 0.4 M KCl extraction. Cellulose-bound dexamethasone · receptors are not resistant to any of these extractions. DNAase I treatment releases 60% of the dexamethasone · receptors bound to DNA-cellulose but only 13% of those bound to nuclei, though at least 60% of the nuclear DNA is solubilized. The presence of 0.15 M KCl decreases binding of activated dexamethasone · receptors to nuclei by 73% but to DNA-cellulose by only 17%. Pretreatment of nuclei with 0.1–0.4 M KCl reduces their capacity to bind activated dexamethasone · receptors by 90% whereas similar treatment reduces the capacity of DNA-cellulose to bind dexamethasone · receptors by only 29%. Nuclei extracted with 0.1 M KCl appear to have a limited capacity to accept dexamethasone · receptors. These studies demonstrate that binding of dexamethasone · receptors to nuclei and DNA-cellulose differs by (a) the higher resistance of nuclear complexes to KCl and DNAase I treatment; (b) the much greater sensitivity of nuclei to KCl treatment.     

Factors modifying equilibrium between activated and non-activated forms of steroid-receptor complexes.

Steroid-receptor complexes formed in concentrated cytosol at low temperature, low ionic strength and neutral pH are unable to bind to nuclei. Various procedures are known to promote their 'activation'. In the present work it is shown that an increase in temperature only enhances the rate of the reaction whereas no change in the equilibrium between activated and non-activated complexes is observed. On the contrary an increase in ionic strength or pH, as well as a removal of a low-molecular-weight inhibitor, not only accelerate the reaction but also increase the concentration of activated complexes at equilibrium. Using two steroids differing 3-fold in their affinity for the receptor, no difference was seen in the effect of the bound steroid on receptor activation. When combining various activation procedures it was observed that they acted independently of each other and additively. In all cases they retained their property of either modifying only the rate of the reaction or both its rate and equilibrium. Using changes in pH, it was also possible to induce shifts in the equilibrium between activated and non-activated complexes. After activation at pH 6.5, a first equilibrium was attained. When the pH was increased to 8 the equilibrium was displaced towards higher concentrations of activated complexes. A lowering of the pH resulted in a reversal of steroid-receptor complexes from the activated to the non-activated state. To clearly establish that this was not due to irreversible damage of the receptor, which would render it unable to bind to nuclei, it was shown that the complexes which had reverted to the non-activated state were still susceptible to activation. Regulatory events may thus exist which, for a given level of hormone and receptor, modulate the concentration of activated steroid-receptor complexes.     

The "activated" hepatic glucocorticoid-receptor complex. Its generation and properties.

The glucocorticoid receptor-glucocorticoid complex of the hepatic cytosol need undergo an "activation" to enable its binding to nuclei, chromatin, or stripped DNA. The conditions of this activation have been studied using native calf thymus DNA absorbed to cellulose. At low ionic strength, activation is very slow at 0 degrees, but, takes place rapidly at 25 degrees, reaching completion at 1 hour. Addition of 10 mm CaCl2 or 150 mm NaCl increases the rate of activation of the receptor at 0 degrees. Neither magnesium nor manganese ions can replace calcium with respect to enabling activation of the steroid-receptor complex to occur at low temperatures. Isofocusing studies reveal that the major component of the unactivated steroid-receptor complex has an isoelectric point of 7.1. Incubation of the steroid-receptor complex at 25 degrees for 30 min leads to its conversion to a form with an isoelectric point of 6.1 concurrent with the development of its ability to bind to DNA-cellulose. Sucrose density gradient analysis reveals that no detectable alteration in the sedimentation coefficient of the steroid-receptor complex occurs during its activation. MnCl2 (20mm) effeciently precipitates the unactivated hormone-receptor complex and to a lesser degree, precipitates the activated hormone-receptor complex.     

Activation of the glucocorticoid-receptor complex

A crucial step in the interaction of glucocorticoids with target cells is the activation step, which involves a conformational change in the cytoplasmic glucocorticoid-receptor protein complexes and facilitates their binding to the cell nucleus. Activation can be quantified by measuring the ability of glucocorticoid-receptor complexes to bind to polyanions, such as DNA-cellulose, and unactivated complexes can be separated from activated complexes by rapid ion exchange chromatography using diethylaminoethyl (DEAE)-Sephadex or DEAE-cellulose. Activation occurs in vivo under physiological conditions and the rate of activation of cytoplasmic glucocorticoid-receptor complexes can be enhanced in vitro by physical manipulations (elevated temperature, increased ionic strength, dilution). In vitro studies suggest that activation is a regulated process and a low molecular weight component termed modulator, which has been identified in rat hepatic cytosol, inhibits activation. Additional studies employing phosphatase inhibitors, such as molybdate, and purified calf intestinal alkaline phosphatase suggest that either the receptor protein or a regulatory component is dephosphorylated during activation. Results obtained with specific chemical probes suggest that activation results in the exposure of basic amino acid residues consisting minimally of lysine, arginine, and histidine. Pyridoxal 5'-phosphate, a specific probe for lysine residues, exerts dual effects on glucocorticoid-receptor complexes, since it stimulates the rate of activation and also inhibits the binding of previously activated complexes to nuclei or DNA-cellulose. The ability of 1,10-phenanthroline, a metal chelator, to inhibit the DNA-cellulose binding of activated complexes suggests that a metal ion(s) located at or near the DNA binding site may become exposed as a consequence of activation. Collectively, the results of these various experiments suggest that activation is a regulated biochemical phenomenon with physiological significance.     

Age-dependent activation of glucocorticoid receptors in the liver of male rats

The binding of hepatic [3H] dexamethasone-receptor complexes to DNA-cellulose and purified nuclei was studied in the immature (3-week) and mature (26-week) Long-Evans male rats to determine the age-associated changes, if any, in the physicochemical properties of glucocorticoid-receptors. Our data show that heat activation (for 45 min at 25 degrees C) significantly enhances the binding of [3H] dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both the ages, with a greater magnitude in mature rats. Cross-mixing experiments (i.e. binding of activated cytosol from mature rats to nuclei of immature and vice-versa) show receptor specificity. Ca2+ activation (20mM Ca2+ for 45 min at 0 degrees C) also enhances the nuclear and DNA-cellulose binding at both the ages but to a similar extent. These findings indicate that some of the physicochemical properties (e.g. heat activation) of glucocorticoid receptor change, while others (e.g. Ca2+ activation) remain unchanged at these phases of the life span. The observed changes may lead to functional alterations in the tissue response as a function of age.     

In vitro activation of rat cardiac glucocorticoid antagonist-versus agonist-receptor complexes

The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [³H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25°C for 30 min or at 15°C for 30 min in the presence of 5 mM pyridoxal 5′-phosphate. Once thermally activated, the cardiac [³H]triamcinolone acetonide and [³H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215–250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.     

Inactivation of rat liver glucocorticoid receptor by molybdate. Effects on both non-activated and activated receptor complexes.

When freshly prepared glucocorticoid-receptor complex from rat liver cytosol was incubated at 23 degrees C in the presence of sodium molybdate, its subsequent binding to isolated nuclei, DNA-cellulose and ATP-Sepharose was blocked. In addition, binding to these acceptors by cytosol receptor complex fractionated with (NH4)2SO4 was also blocked by incubation of the complexes with 50 mM-sodium molybdate. However, molybdate had no effect on the binding of activated receptor complexes to ATP-Sepharose. Molybdate was also effective in extracting the nuclear- and DNA-cellulose-bound glucocorticoid-receptor complexes in a dose-dependent manner. Molybdate appears to exert its effects directly on the receptor by interacting with both non-activated and activated receptor forms.     

Interaction of pyridoxal 5′-phosphate with the liver glucocorticoid receptor-DNA complex

The rat liver glucocorticoid receptor has been eluted from DNA-cellulose with pyridoxal 5′-phosphate at low ionic strength. This elution is concentration dependent with 80–90% of the receptor eluted in 30 rain at 0 °C when the concentration of pyridoxal 5′-phosphate is 10 mm. This elution is specific for the 4′-aldehyde group of pyridoxal 5′-phosphate since vitamin B₆ analogs lacking this group are inactive in eluting the steroid-receptor complex from DNA-cellulose. Receptor has also been eluted from rat liver nuclei with similar results. The receptor eluted with pyridoxal 5′-phosphate has been compared with the receptor eluted with 0.45 m NaCl. Both methods of elution yield a steroid-receptor complex which sediments at about 3.7 S. The pyridoxal 5′-phosphate-eluted receptor however, is less prone to aggregation at low ionic strength and more stable with respect to steroid binding than the 0.45 m NaCl-eluted steroid-receptor complex. The complement of proteins eluted from DNA-cellulose with pyridoxal 5′-phosphate is very similar to that eluted with NaCl as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

A low molecular weight inhibitor of steroid receptor activation.

Dilution at 0 degrees of rat liver cytosol incubated with [3H]triamcinolone acetonide provoked an enhanced binding of steroid-receptor complexes to nuclei. The explanation of this phenomenon was found to be an "activation" of the complexes. Dilution acted by decreasing the concentration of a cytosol inhibitor. This reaction was irreversible at 0 degrees: once activated the complexes could not be reversed to the nonactivated state by the addition of inhibitor. The presence of hormone was necessary, since hormone-free receptor molecules could not be activated by dilution. Removal of the inhibitor did not lead to activation of all complexes: after 24 h a "plateau" was attained where 55 to 70% of the complexes were activated. The inhibitor was shown to be a low molecular weight molecule by dialysis, Sephadex G-25 chromatography, ammonium sulfate precipitation, and ultrafiltration. Thus [3H]triamcinolone acetonide-receptor complexes present in a cytosol from which the inhibitor had been removed by Sephadex G-25 chromatography became spontaneously activated at low ionic strength and at 0 degrees. The inhibitor is not a steroid (at least of usual polarity) since it cannot be extracted by methylene chloride or adsorbed by activated charcoal. It is thermostable (resists to 30 min at 100 degrees). Its removal by incubation with a cation exchange resin suggests that it may be positively charged, however it is not complexed by EDTA. This inhibitor must be distinguished from a previously described inhibitor of steroid-receptor complexes binding to nuclei. The latter compound has been shown in various systems to be responsible for an artifactual saturation of nuclear acceptor by steroid-receptor complexes. It inhibits the binding to nuclear acceptors of already activated complexes and is probably a macromolecule. It is thus different from the low molecular weight activation inhibitor described in the present paper.     

Purification and preliminary characterization of a macromolecular inhibitor of glucocorticoid receptor binding to DNA

Rat liver cytosol contains a heat-labile macromolecule that inhibits the binding of the transformed glucocorticoid-receptor complex to nuclei or DNA-cellulose (Milgrom, E., and Atger, M. (1975) J. Steroid Biochem. 6, 487-492; Simons, S. S., Jr., Martinez, H. M., Garcea, R. L., Baxter, J. D., and Tomkins, G. M. (1976) J. Biol. Chem. 251, 334-343. We have developed a quantitative assay for the inhibitor and have purified it 600-700-fold by ammonium sulfate precipitation, ethanol precipitation, and phosphocellulose and Sephacryl S-300 chromatography. The inhibitory activity copurifies with a Mr = 37,000 protein doublet. Under low salt conditions, both the inhibitory activity and the 37-kDa protein doublet behave as high Mr aggregates that subsequently dissociate in the presence of salt. The inhibitor is positively charged at physiological pH, and it is not affected by digestion with several serine proteases or RNase. The inhibitor does not affect the transformation process, and it does not cause the release of steroid-receptor complexes that have been prebound to DNA-cellulose. The inhibitor preparation does not cleave receptors in L-cell cytosol that are covalently labeled with the site-specific affinity steroid [3H]dexamethasone 21-mesylate. If the steroid-receptor complex is first separated from the great majority of cytosol protein by transforming it and binding it to DNA-cellulose, addition of the inhibitor preparation results in receptor cleavage. Under these conditions, cleavage can be blocked with 1-chloro-3-tosylamido-7-amino-L-2-heptanone and antipain, but protease inhibitors do not affect the inhibition of DNA binding that occurs in whole cytosol. The inhibitor acts through an interaction with the receptor, not with DNA. We suggest that the inhibitor may prove to be a useful tool for studying the interaction of the steroid-receptor complex with DNA or nuclei and speculate that it may be important in determining normal events of the receptor cycle as they occur in the intact cell.     

Activation of [3H]estradiol--and [3H]H1285-receptor complexes: effect of salt versus ATP on molybdate-stabilized estrogen receptors

The activation by salt or ATP of [3H]estradiol- and [3H]H1285-receptor complexes from rabbit uterus and their binding capacity to DNA-cellulose, phosphocellulose and ATP-Sepharose has been studied. The estrogen-receptor was prepared in 1 mM molybdate which stabilized the receptor; but both salt- and ATP-transformation of estrogen receptors occurred. The binding of molybdate-stabilized cytosol [3H]estradiol-receptor complexes to the various resins revealed that salt-activation by 0.3 M KCl caused the greatest binding (5-6-fold) to DNA-cellulose as compared to other resins. However, 5 mM ATP-dependent activation of receptor-complexes resulted in preferential binding to ATP-Sepharose. Activated cytosol [3H]H1285-receptor complexes bound all the resins to a lesser degree when compared to [3H]estradiol-receptor complexes. Partially purified receptor complexes also showed different resin-binding patterns for salt- and ATP-mediated activation. These findings suggest that salt-activation is different than ATP-activation. Further, the differential magnitude of [3H]estradiol- and [3H]H1285-receptor activation suggests that estrogen-receptor complexes are "fully" activated as compared to "partially" activated antiestrogen-receptor complexes.     

Functions of calcium in the interaction of progesterone-receptor complex with nuclear components

Chick oviduct cytosol [³H]progesterone-receptor complex treated with 30 mm Ca²⁺ at 0 °C demonstrated a twofold greater binding to isolated chick oviduct nuclei or DNA-cellulose than such complexes activated thermally (25 °C). Divalent ions such as Mg²⁺ and Mn²⁺ were unable to mimic the effect of Ca²⁺ under identical conditions. The capacity of the Ca²⁺-treated progesterone-receptor complex to bind to nuclei or DNA-cellulose reached a peak within 45 min of Ca²⁺ treatment of the complex at 0 °C. This binding gradually declined as a function of incubation time and after 24 h at 0 °C no significant binding was observed. The Ca²⁺- and heat-treated chick oviduct [³H]progesterone-receptor complex was also characterized by DEAE-cellulose and agarose gel nitration chromatography. While heat-activated receptor could be resolved into A and B subunits on DEAE-cellulose, the receptor exposed to Ca²⁺ for 45 min at low temperature yielded the “A” subunit and a broad peak with poor affinity for the anion exchanger. The peak corresponding to “B” subunit was not discernible. The broad peak which eluted before the A peak was subsequently resolved by agarose gel filtration into receptor forms IV and V as described previously by Sherman et al. (M. Sherman, S. Atienza, J. Shansky, and L. Hoffman, 1974, J. Biol. Chem., 249, 5351–5363; M. Sherman, L. Pickering, F. Rollwagen and L. Miller, 1978, Fed. Proc., 37, 167–173). Again DEAE-cellulose chromatography of the progesterone-receptor complex treated as long as 24 h at 0 °C with Ca²⁺ revealed a poorly bound peak which on agarose gel filtration corresponded exclusively to form V. A correlation was apparent between an increase in form V and a gradual decrease in the binding capacity of the Ca²⁺-treated steroid-receptor complex to nuclei, DNA-cellulose, or DEAE-cellulose filters.Based on these findings, I postulate that Ca²⁺ has a functional role in the mechanism of progesterone action in chick oviduct. Firstly, it enhances a low temperature, time dependent binding of the progesterone-receptor complex to chick oviduct nuclear components, and subsequently promotes, by possible activation of endogenous protease(s) the cleavage of the receptor subunits.     

Mechanism and kinetics of the thermal activation of glucocorticoid hormone receptor complex.

Steroid-receptor complexes formed at low temperature and ionic strength do not bind to nuclei or chromatin. After a temporary exposure to high temperature, or ionic strength, or both, a fraction of them becomes activated (able to bind to nuclei). An assay of the activated form of the complex based upon titration with nuclei in excess was established. This assay was used to perform kinetic and equilibrium studies of the thermal activation of glucocorticoid-receptor complex in order to elucidate its mechanism. It was found that the reaction is of apparent first order and yields a monomolecular product. It thus probably consists of a conformational change in the steroid-receptor complex. The rate of activation is 1.37 +/- 0.06 X 10(-3) S-1 at 25 degrees. The free energy of thermodynamic activation (The word activation is used here in its usual thermodynamic meaning and not in the sense of receptor modification) of this reaction is greater than G = 21.3 Kcal. The corresponding enthalpy and entropy are respectively greater than H = 31.4 kcal and greater than S = 4 cal/degree. These positive and high values of greater than H and greater than S are very similar to those described for denaturation reactions of proteins suggesting that breakage of some noncovalent bonds could take place during activation. The reaction proceeds until approximately 60% of the complexes are activated. It was shown that this corresponds to an equilibrium between activated and nonactivated forms and not to the presence of a population of complexes unable to undergo activation. This equilibrium is not modified by temperature variations between 10 degrees and 30 degrees. It is possible to activate over 80% of the complexes when the activation is performed in the presence of excess acceptor, thus shifting the equilibrium. A similar situation is probably observed in situ in cells since 90% of the complexes are found in the nuclei when liver slices are incubated with hormone.