Activation Of Glucocorticoid-receptor Complexes Research Articles

A STUDY OF THE EFFECT OF THEOPHYLLINE ON THE ANTI-INFLAMMATORY POTENCY OF DEXAMETHASONE USING α-AMINOISOBUTYRIC ACID UPTAKE IN RAT SKIN FIBROBLASTS: A POSSIBLE MECHANISM THROUGH THE GLUCOCORTICOID RECEPTOR

The action of theophylline on the uptake of α-aminoisobutyric acid (AIB; an indicator of anti-inflammatory potency) stimulated by the glucocorticoid, dexamethasone, in cultured rat fibroblast monolayers was evaluated. Theophylline alone (0.1mm) did not show significant activity (3314±27cpm) compared with the baseline level (3186±130cpm), but in the presence of 10nmdexamethasone the stimulation of AIB uptake was increased to 5263±100cpm, approximately to the same extent as with 100nmdexamethasone alone (5397±28cpm). Activation of glucocorticoid-receptor complexes in rat fibroblast cytosol was studied by assessing the extent of their binding to DNA-cellulose. Activated and non-activated forms of glucocorticoid-receptor complexes were analysed by DEAE-Sephadex chromatography. Theophylline (1mm) was found to have a direct effect (0°C), similar to that of heat (25°C) on DNA-cellulose binding, that is approximately 64.5% and 68.7%, respectively, thus indicating that theophylline promotes activation of glucocorticoid receptors at low temperature. The effect of theophylline on the stimulation of AIB uptake by dexamethasone when considered in the light of its activation of GR receptors in the fibroblast cytosol indicates that this effect may be mediated by GR activation.

A New Synthetic Ether Aminophosphoglyceride Exhibits Partial Modulator Activity toward the Glucocorticoid Receptor

Modulator is an endogenous low-molecular weight regulator of both glucocorticoid and mineralocorticoid receptors, as well as protein kinase C. Analogs of the putative modulator structure have been synthesized. These compounds include 1-O-(3′-carboxypropyl) or (5′-carboxypentyl)-L-glycero-3-phospho-L-serine or L-threonine, and the D-glycerol stereoisomers. These compounds were tested for in vitro modulator activity using the glucocorticoid-receptor complex activation inhibition and steroid-binding stabilization assays. One of the ether phosphoglycerides, 1-O-(5′carboxypentyl)-L-glycero-3-phospho-L-threonine (H-GPT-1), partially inhibited steroid-receptor complex activation in a dose-dependent manner. However, none of the other compounds exhibited any modulator activity towards the glucocorticoid-receptor complex. Like modulator, H-GPT-1 did not inhibit activated glucocorticoid-receptor complex binding to DNA-cellulose. Surprisingly, in contrast to modulator, H-GPT-1 partially inhibited unoccupied receptor steroid-binding in a dose-dependent manner. These results suggest that although modulator is not exactly mimicked by this compound, H-GPT-I is the first synthetic organic molecule to exhibit some modulator activity towards the glucocorticoid receptor.

Modulators of the glucocorticoid receptor also regulate mineralocorticoid receptor function.

Modulators are proposed to be novel ether aminophosphoglycerides that stabilize unoccupied and occupied glucocorticoid receptor steroid binding and inhibit glucocorticoid receptor complex activation. Two isoforms, modulator 1 and modulator 2, have been purified from rat liver cytosol [Bodine, P.V., & Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554]. Since the mineralocorticoid receptor is relatively resistant to activation, modulator's effect on rat distal colon mineralocorticoid receptor function was examined. Warming of unoccupied receptor decreased residual specific [3H]aldosterone binding by 86 +/- 2%. Both modulator isoforms completely prevented this destabilization with Km's of 2 +/- 1 microM modulator 1 and 24 +/- 5 microM modulator 2. Warming of occupied mineralocorticoid receptors decreased [3H]aldosterone binding by 56 +/- 3%. Modulator only partially stabilized occupied receptor binding with Km's of 10 +/- 2 microM modulator 1 and 68 +/- 8 microM modulator 2. Modulator inhibited receptor activation with Km's of 3 +/- 1 microM modulator 1 and 33 +/- 10 microM modulator 2. Double-reciprocal analysis showed linear kinetics, and mixing modulator isoforms together had additive effects on unoccupied and occupied receptor steroid binding stabilization and activation inhibition. Colon cytosol contained a low molecular weight, heat-stable factor(s) which inhibited receptor activation and stabilized occupied receptor steroid binding. Molybdate completely stabilized unoccupied mineralocorticoid receptor steroid binding and inhibited activation with half-maximal effects at 3-4 mM but only stabilized occupied receptor binding by approximately 40%. These data indicate that (i) apparent physiologic concentrations of modulator stabilize mineralocorticoid receptor steroid binding and inhibit receptor activation, (ii) an aldosterone-responsive tissue contains a modulator-like activity, and (iii) molybdate mimics the effects of modulator.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of two novel phosphoglycerides that modulate the glucocorticoid-receptor complex. Evidence for two modulator binding sites in the occupied/unactivated steroid hormone receptor.

Modulator is a novel ether aminophosphoglyceride that is commonly known as the low-molecular weight inhibitor of glucocorticoid-receptor complex activation. An ultra-large scale purification of modulator has been performed from 1000 rat livers. This purification was similar to our previous one (Bodine, P. V., and Litwack, G. (1988) J. Biol. Chem. 263, 3501-3512), but involved the chromatography of heated rat liver cytosol on a 7-liter bed volume Sephadex G-15 gel filtration column. Two peaks of modulator activity eluted from the giant gel-filtration column, and these two modulators (peak-1 and peak-2) were chromatographed separately on Dowex-1 anion-exchange columns. Both modulators were determined to be homogeneous after this step by analytical high-performance thin-layer chromatography, analytical high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy. Furthermore, although peak-1 and peak-2 differed in molecular weight, the two modulators co-chromatographed by anion-exchange, high-performance thin-layer, and high-performance liquid chromatography. These results suggest that the two modulators have similar structures and therefore appear to be isoforms of each other. In addition, both of the modulators are organic molecules that are devoid of molybdenum and 62 other metals. Activity assays indicated that the larger peak-1 modulator was five times more potent than the smaller peak-2 modulator at inhibiting receptor activation and at stabilizing the steroid-binding ability of the occupied and unoccupied receptors. Mixing experiments indicated that the activities of the two modulators were synergistic for both receptor activation inhibition and for occupied receptor steroid-binding stabilization. However, the effects of peak-1 and peak-2 modulator on unoccupied receptor steroid-binding stabilization were additive. Thus, although the two modulators have similar chemical structures, the biological potencies of the two compounds are different. Moreover, these results suggest that although the unoccupied/unactivated receptor has only one modulator binding site, the occupied/unactivated receptor has two modulator binding sites, one site for each of the isoforms.

Glucocorticoid receptor activation during exercise in muscle

This investigation was undertaken to evaluate whether endurance running of the type known to retard the muscle atrophy associated with glucocorticoid excess inhibits activation of glucocorticoid-receptor complexes to a DNA binding state. Female adrenalectomized rats received an injection (50 microCi/100 g body wt ip) of [3H]triamcinolone acetonide and remained sedentary or were immediately exercised by endurance running at 23 m/min for up to 90 min. Receptor activation, as quantified by binding to DNA-cellulose, steadily increased from 10-20% of the receptors capable of binding DNA in uninjected controls to 25-45% by 5 min and to 53-80% by 90 min after receiving the hormone in all muscles studied (fast-twitch red vastus lateralis, fast-twitch white vastus lateralis, slow-twitch soleus, mixed gastrocnemius, and heart). Exercise did not influence the time-course changes in percent activation. When activation was determined from changes in the conformational state of the receptor as measured by diethylaminoethyl-cellulose anion exchange chromatography, there was a similar time-dependent formation of activated receptor forms in all muscle types. However, exercise did not inhibit or delay the appearance of the activated receptor from the unactivated state. These results indicate that glucocorticoid receptor activation occurs at a rate that is independent of both fiber type and delivery of steroid to working muscles during exercise. If exercise alters receptor activation, a longer time period, beyond 90 min of running, or even additional training may be needed for inhibition to be expressed.

In vitro evidence for modification of rat liver glucocorticoid receptor binding properties and transformation by hyperthermia

The physico-chemical parameters of the interaction of [ 3H]triamcinolone acetonide (TA) with the glucocorticoid receptor (GR) and the in vitro activation of glucocorticoid-receptor complexes were studied in liver cytosols of rats exposed to 41°C hyperthermia. A significant reduction in glucocorticoid binding and a slight increase in binding affinity were detected in hyperthermic rats as compared to the controls. The number of binding sites was 0.48 ± 0.02 and 0.73 ± 0.03 pmol/mg protein for heat-treated and control rats, respectively. Differences in equilibrium dissociation constants (0.52 ± 0.08 nM for hyperthermic and 0.94 ±0.13 nM for control animals) were reflected in corresponding differences in dissociation rate constants at 25°C (5.1 × 10[su−4] and 7.5 × 10 −4min −1, respectively), whereas association rate constants were similar. The inactivation kinetics of unoccupied GR at 25°C was the same in both groups. Glucocorticoid-receptor complexes in liver cytosol from hyperthermic and control rats were thermally activated to a similar extent, but the activation rate was significantly lower in the former. Mixing experiments with the control and hyperthermic rat liver cytosols suggest that this impairment of glucocorticoid-receptor complexes activation could be at least in part ascribed to heat stress-related changes in the action of activation modulator(s). Sedimentation behaviour of unactivated and activated [ 3H]TA-receptor complexes of hyperthermic and control rats was identical.

Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.

We have recently purified the modulator of the glucocorticoid-receptor complex from rat liver. Purified modulator inhibits glucocorticoid-receptor complex activation and stabilizes the steroid-binding ability of the unoccupied glucocorticoid receptor. Since these activities are shared by exogenous sodium molybdate, modulator appears to be the endogenous factor that sodium molybdate mimics. In this report, we present additional evidence for the mechanism of action of purified modulator. (i) Molybdate and modulator inhibit receptor activation as measured by DNA-cellulose binding, DEAE-cellulose chromatography, and Sepharose 4B gel filtration. (ii) The ability of molybdate and modulator to inhibit receptor activation and stabilize the unoccupied receptor appears to be additive. (iii) Scatchard analysis of heat-destabilized unoccupied receptors indicates that the number of steroid-binding sites is reduced during destabilization, whereas the steroid dissociation constant remains unchanged. Molybdate and modulator stabilize the receptor by maintaining the number of steroid-binding sites. (iv) Molybdate and modulator do not inhibit alkaline phosphatase-induced destabilization of the unoccupied receptor. However, alkaline phosphatase-induced destabilization is reversed by the addition of dithiothreitol in the presence, but not in the absence, of molybdate or modulator. These results suggest that the mechanism of action for modulator is identical to that of sodium molybdate, and we propose that modulator is the endogenous molybdate factor for the glucocorticoid receptor.

Purification and structural analysis of the modulator of the glucocorticoid-receptor complex. Evidence that modulator is a novel phosphoglyceride.

Modulator is the low molecular weight heat-stable inhibitor of glucocorticoid-receptor complex activation. We have purified modulator to apparent homogeneity from heated rat liver cytosol. This was accomplished using Sephadex G-15 gel filtration, Dowex 1 anion-exchange chromatography, and preparative silica high-performance liquid chromatography. The modulator preparation was judged to be homogeneous by analytical silica high-performance liquid chromatography, two-dimensional silica thin-layer chromatography, and proton nuclear magnetic resonance spectroscopy. The apparent concentration of modulator in rat liver cytosol is 6.5 microM. The purified modulator inhibits heat activation of the rat liver glucocorticoid-receptor complex and stabilizes the steroid binding ability of the unoccupied rat liver glucocorticoid receptor in a dose-dependent manner. At a concentration of 5-6.5 microM, modulator inhibits receptor activation and stabilizes the unoccupied receptor by 50%. At a concentration of 500-630 microM, sodium molybdate also inhibits receptor activation and stabilizes the unoccupied receptor by 50%. Thus, modulator appears to be the endogenous factor that exogenous sodium molybdate mimics in vitro. Chemical analysis of the purified modulator following two-dimensional silica thin-layer chromatography indicates that modulator is an aminophospholipid. Physical analysis of the purified modulator by infrared and nuclear magnetic resonance spectroscopy, as well as mass spectrometry, demonstrates that modulator is an ether aminophosphoglyceride.

Decreased glucocorticoid binding and receptor activation in brain of genetically diabetic (MDB/MDB) mice

Binding of [ 3H]triamcinolone acetonide (TA) to cytosolic receptors and subsequent in vitro activation of glucocorticoid-receptor complexes were studied in whole brain and liver from misty diabetic mice ( mdb/mdb) and their control littermates (??/+ +). Binding was specific for glucocorticoid receptor (GcR) since the specific glucocorticoid, RU26988, was used to compete with [ 3H]TA for binding. Reduced [ 3H]TA binding was observed in whole brain and liver in diabetic animals when compared to control animals. Within the brain, binding was significantly ( P < 0.05) decreased in cortex, hippocampus, and hypothalamus. No significant differences in binding were found in the striatum or “midbrain”. GcR binding was similar in diabetic and control animals until 2 months of age when overt diabetic symptoms appeared and the GcR binding was lower in diabetic animals. Though GcR from mdb/mdb brain cytosol could be thermally activated, the extent of activation was significantly ( P < 0.05) less than that for controls. These data indicate that GcR in liver and brain cytosol are decreased in mdb/mdb mice and that the GcR available for binding in mdb/mdb brain cytosol appears less capable of undergoing activation and binding to DNA-cellulose than GcR from control brain cytosol. Decreased GcR activation in brain cytosol from mdb/mdb mice was associated with increased dissociation of [ 3H]TA from the GcR. These results suggest that the decreased negative feedback previously observed in diabetic animals may be due to decreased binding of hormones and a decreased level of activation of hormone bound receptor complexes.

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

1. 1. Calf intestinal alkaline phosphatase was found to stimulate the rate of in vitro activation of rat liver glucocorticoid-receptor complexes. 2. 2. This effect was registered both at 0 and 25°C and could be prevented by sodium molybdate. 3. 3. 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.

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°C, 30 min), and then returning it to pH 6.5 for a second incubation (15°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.

Mechanisms of glucocorticoid hormone action

This work summarizes some of our studies of the mechanisms of glucocorticoid action, including aspects of steroid binding to receptors, the activation of glucocorticoid-receptor complexes and the regulation of expression of endogenous and transferred glucocorticoid-responsive genes. Studies of the receptor-steroid interaction support the notion that steroid entry is passive. A comparative analysis of binding in isolated cytosol and intact cells suggests that the initial receptor-steroid binding reaction and not subsequent steps such as activation and nuclear binding, is predominantly responsible for the high-affinity state that is generated. The binding is driven by entropy and enthalpy changes at low temperature; at higher temperatures it is driven by entropy changes, with enthalpy working against it. Studies of the activation of the receptor-glucocorticoid complex with the use of highly purified receptors suggest that this step is associated with a change in charge of the receptor-glucocorticoid complex (such as would occur with a dephosphorylation reaction), whereas the data do not support the notion that dissociation of a bound RNA or of receptor oligomers is responsible for generating the nuclear- and DNA-binding activity of the complex. Studies of the regulation by glucocorticoids of expression of the endogenous rat growth hormone (rGH) gene in cultured rat pituitary tumor (GC, GH 3D 6) cells suggest that glucocorticoids increase the expression of this gene by multiple mechanisms. First, there is a modest direct stimulation of transcription by a mechanism(s) that does not depend on protein synthesis; however, if the cells have been exposed to thyroid hormone for several hours, the steroid exerts a much greater increase in rGH pre-mRNA levels. Secondly, the steroid appears to stimulate some relatively stable function or functions that increase the ability of thyroid hormone to increase rGH levels. Thirdly, the steroid probably increases rGH mRNA stability, since the fold-increases in rGH mRNA exceed those of transcription. Finally, the steroid may, by unknown mechanisms, affect rGH mRNA polyadenylation. The gene transfer experiments utilized the rat and human (h) GH genes and hybrid genes containing either rGH and Herpes Simplex virus thymidine kinase (TK) gene sequences or the human metallothionein-II A (hMT-II A) and TK gene sequences. The steroid was found to regulate hMT-II A gene expression in all glucocorticoid-responsive cell types tested by actions on its 5'-flanking DNA. By contrast, the glucocorticoid regulated GH gene expression in some but not all glucocorticoid-responsive cell types. These data suggest that factors in addition to glucocorticoid receptors are necessary for the steroid to regulate GH gene expression. The use of hybrid hMT-II A-TK genes indicated that glucocorticoids act on the hMT-II A gene upstream from and independently of its “TATA” box to increase promoter activity. The steroid can affect multiple promoters simultaneously, even those of heterologous genes, and can affect these at a distance of at least 600 nucleotides away by a mechanism that resembles somewhat the actions of enhancer sequences described for other genes. Because of these findings and our earlier observations that glucocorticoids can modify chromatin structure in a way that is preserved in isolated nuclei, a model is presented whereby the glucocorticoid-receptor complex binding to chromatin modifies chromatin structure to make it more available over a considerable segment of DNA for RNA polymerase entry.

Identification of a macromolecular inhibitor of glucocorticoid-receptor complex activation in rat liver cytosol.

We have identified an endogenous regulator of the glucocorticoid receptor following fractionation of dialyzed rat liver cytosol on DEAE-cellulose. The macromolecular regulator, purified approximately 20-fold as judged by Lowry-reactive material, inhibits activation of glucocorticoid-receptor complexes when assayed by DNA-cellulose binding and by chromatography on DEAE-cellulose minicolumns. In addition the active DEAE-cellulose fraction stabilizes the unoccupied glucocorticoid receptor against heat inactivation. Evidence is presented that the observed inhibition of activation by the active DEAE-cellulose fraction is not due to concentration of cytosolic proteases or RNA. The inhibitory molecule in the active fraction is not stable to heating at 90 degrees C (15 min) and is partially inactivated at 45 degrees C (15-60 min).

In vitro stabilization of the unoccupied glucocorticoid receptor by adenosine 5'-diphosphate.

The addition of ATP to rat liver cytosol slows the rate of heat inactivation of the unoccupied glucocorticoid receptor (25 C) and stimulates the rate of activation of the preformed glucocorticoid-receptor complex (15 C). Dose-response curves and kinetic studies show that ADP is as effective as ATP in stabilizing the unoccupied glucocorticoid receptor against heat inactivation. ATP can also be replaced by analogs with a hydrolysis-resistant alpha, beta-pyrophosphate linkage (5'-adenylyl methylenephosphonophosphate or 5'-adenylyl methylenephosphonate); however, the hydrolysis-resistant beta, gamma analog (5'-adenylyl methylenediphosophonate) is ineffective. The addition of creatine phosphate plus creatine kinase, a condition favoring ATP formation, stimulates the rate of inactivation of the unoccupied glucocorticoid receptor, and the effect is only partially overcome by ADP. A condition that favors ADP formation, the addition of creatine plus creatine kinase, has no effect on the rate of inactivation of the unoccupied receptor and does not decrease the protective effect afforded by ATP alone. Collectively, these results suggest that ATP stabilization of the steroid-binding site in vitro is due to ADP generated from the triphosphate by endogenous enzymes and is not due to phosphorylation or adenylation of the receptor by ATP. Unlike ATP stabilization of the steroid-binding site, the ATP-stimulated increase in the rate of activation of the preformed glucocorticoid-receptor complex (15 C) does not require hydrolysis of the beta, gamma-pyrophosphate bond. Dose-response curves show that both ATP and 5'-adenylyl methylenediphosophonate stimulate the rate of activation of the glucocorticoid-receptor complex. Quantitation of nucleotide levels in unfractionated rat liver cytosol by high performance liquid chromatography shows that the effective concentration of added ATP that produces an optimal response is within the physiological range reported for intact cells.

Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate.

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

Activation of the glucocorticoid receptor

Past and ongoing approaches to the mechanism of activation of the glucocorticoid receptor have been summarized. In vivo labelling of receptors with [ 3H] triamcinolone acetonide at various times indicates a progression in appearance and disappearance of chromatographically separable activated and unactivated forms of steroid-receptor complexes, suggesting physiological relevance of the activation process. The DNA-binding site, exclusively expressed on activated receptor complexes appears to consist minimally of lys, arg and his residues as determined by chemical probes. The DNA-binding site of the activated receptor complex may contain a divalent metal. Since addition of calf intestinal alkaline phosphatase stimulates the rate of activation of glucocorticoid-receptor complexes at 0°, the activation step, at least in part, may consist of a dephosphorylation event. Two small molecule regulators have been found. One is called “modulator” and inhibits activation. The other is pyridoxal 5′-phosphate which functions in vitro and in vivo and combines with the activated form of the steroid-receptor complex, preventing nuclear binding. The products of activation are receptors called II and IB. They have a different distribution between liver and kidney. How they arise in the cell is not yet clear but both appear to be bona fide cellular products. IB and II both have receptor functions and differ in their binding avidity for single-stranded homodeoxypolymers. The specificity of homodeoxypolymer binding by II resembles DNA polymerase-α. DNA-binding of activated binder II is inhibited by rifamycin AF 013 , a known inhibitor of DNA-binding by various nucleic acid polymerases. Based on observations on the activation mechanism of glucocorticoid receptors, a speculative model is presented.

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.

Chromatographic studies on interconversions of “non-activated” and “activated” forms of glucocorticoid-receptor complexes from rat heart cytosol

These studies focus on the activation of glucocorticoid-receptor complexes in heart, a tissue that, despite its importance as a glucocorticoid target, has received relatively little study in terms of its receptors. DEAE-Sephadex A50 mini-column chromatography of 3H-dexamethasone-receptor complexes from rat heart cytosol reveals two peaks of bound hormone, which, by elution characteristics, correspond to “non-activated” and “activated” forms of liver cytosol glucocorticoid-receptor complexes. An increase in temperature to 20°C or treatment with 10 mM theophylline or 0.4 M KCl causes transformation of receptor-bound [ 3H]-dexamethasone from the peak of non-activated to the peak of activated form; 10 mM ATP does not influence this process. Molybdate ions (1−100 mM) completely block activation of glucocorticoid-receptor complexes, and fluoride ions (100 mM) do so partially. Moreover, molybdate can also transform temperature-activated complexes to the non-activated form. This inhibiting effect persists after removal of free molybdate ions from the medium. These data imply that the mechanism of glucocorticoid-receptor complex activation in heart resembles that in other glucocorticoid-responsive tissues. However, the finding that molybdate can reverse the conversion of receptors from the non-activated to the activated form, as judged by DEAE-Sephadex chromatography, differs from previous reports. This result implies that, if molybdate prevents receptor inactivation by blocking dephosphorylation (as has been proposed), the ion must also act in some other way as well in promoting the reversal.

Effects of calf intestinal alkaline phosphatase, phosphatase inhibitors, and phosphorylated compounds on the rate of activation of glucocorticoid-receptor complexes.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffects of calf intestinal alkaline phosphatase, phosphatase inhibitors, and phosphorylated compounds on the rate of activation of glucocorticoid-receptor complexesCarol A. Barnett, Thomas J. Schmidt, and Gerald LitwackCite this: Biochemistry 1980, 19, 23, 5446–5455Publication Date (Print):November 11, 1980Publication History Published online1 May 2002Published inissue 11 November 1980https://doi.org/10.1021/bi00564a046RIGHTS & PERMISSIONSArticle Views794Altmetric-Citations94LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

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.