Activation of the glucocorticoid–receptor complex

Schmidt, Thomas J.; Barnett, Carol A.; Litwack, Gerald

doi:10.1002/jcb.240200103

Cited by 34 publications

(15 citation statements)

References 49 publications

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“…Some partial activation occurred on gel filtration in the absence of molybdate (expt 9) while the presence of molybdate during gel filtration prevented transformation (expt 10). This has also been observed for unpurified receptor [6].…”

Section: Binding To Dna-cellulose and Nucleisupporting

confidence: 73%

Transformation in vitro and covalent modification with biotin of steroid-affinity-purified rat-liver glucocorticoid-hormone-receptor complex

Hapgood¹,

Holt²

1987

Eur J Biochem

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The molybdate-stabilized rat liver glucocorticoid receptor complex was purified 9000-fold with a 46% yield by steroid-affinity chromatography and DEAE-Sephacel ion-exchange chromatography. The purified glucocorticoid receptor was identified as a 90 -92-kDa protein by SDS/polyacrylamide gel electrophoresis. Raising the temperature to 25 "C in the absence of molybdate resulted in increased binding of the receptor complex to DNA-cellulose or nuclei, similar to the effect on the cytosolic complex. The purified complex has a sedimentation coefficient of 9-10 S before and after heat treatment in the absence of molybdate. The appearance of smaller 3-4-S species was unrelated to the extent of DNA-cellulose binding of the complex. The process termed 'transformation', i.e. increasing the affinity for DNA, is not concomitant with subunit dissociation or loss of RNA. Highly purified glucocorticoid receptor could be covalently modified with biotin to retain its steroid-binding activity but with a 50% decrease in nuclear binding capacity. The without losing its steroid.The hydrodynamic properties of the partially purified molybdate-stabilized glucocorticoid-hormone-receptor complex (GHRC) have been well characterized: s = 9.1 S; Stokes' radius of 8.3 nm; M , = 319000 [l]. Although the subunit composition of this complex has not been established, it appears to be heteromeric with only one steroid-binding subunit of about 90 kDa in the oligomeric 9-10-S complex [2-41. Transformation (also referred to as activation) of GHRC is usually defined as an increase in binding to DNA or nuclei (reviewed in [5, 61). This has often been demonstrated to be accompanied by a decrease in the size of GHRC assayed by gradient ultracentrifugation [5, 61. A wide variety of treatments on crude GHRC in the absence of molybdate such as heating, gel filtration, dialysis and exposure to high ionic strength have been reported to result in an decrease in size and an increase in DNA-binding of GHRC [5, dephosphorylation [15], conformational changes [16] and proteolysis [17,18] have been implicated in the mechanism of transformation of partially purified GHRC. It has also been suggested that transformation involves concomitant loss of the 90-kDa nonsteroid-binding protein [19]. However the exact molecular events associated with a change in size and charge of the 9 -10-S GHRC and their biological significance still remain to be resolved. Very few authors have investigated the transformation of highly purified GHRC [20-251. A cytosolic factor which is lost in the preparation of highly purified GHRC was found to be necessary for transformation [21,22]. This cytosolic factor was found to cause the formation of biotin-modified complex reacts with streptavidin-in solution 4-5-S complexes from purified untransformed 9 -10-S complexes, linking subunit dissociation with transformation. However it has also been reported that highly purified untransformed GHRC sediments at 3.9 -4.0 S before and after heat transformation [20] and that the purified untransformed G...

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Section: Binding To Dna-cellulose and Nucleisupporting

confidence: 73%

Transformation in vitro and covalent modification with biotin of steroid-affinity-purified rat-liver glucocorticoid-hormone-receptor complex

Hapgood¹,

Holt²

1987

Eur J Biochem

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“…The change occurs rapidly within 30 min at 25"C, results in the transition of the ligandreceptor complex from a non-DNA-binding form to a form with affinity for DNA or chromatin [34] and is thought to be essential for hormone-dependent gene expression [12]. In contrast to this, the TCDD receptor appears to exhibit some, albeit low, affinity for DNA (< 10% of specifically bound [3H]TCDD is retained on DNA-cellulose) after ligand-binding at 0-4°C alone.…”

Section: The Effect Of Proteolytic Enzymes and An Intercalating Drug mentioning

confidence: 99%

Characterization of the DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin

et al. 1986

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The DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated using chromatography on DNA-cellulose columns. A maximal binding of about 40% of the total receptor complex to DNA-cellulose was observed. In order to interact with DNA, the receptor must first bind TCDD. A heat-activation step followed by gel permeation chromatography using Sephadex G-25 increased the binding of the cytosolic receptor to DNA. The DNA-binding ability of the receptor was almost lost following mild proteolysis using trypsin or a-chymotrypsin, although these treatments did not reduce its ligand binding capacity and had no apparent effect on its size. Furthermore, pre-treatment of the DNA-cellulose column with an intercalating drug, ethidium bromide, resulted in inhibition of the binding of the TCDD-receptor complex to DNA, indicating that not only electrostatic interactions but also the configuration of DNA are of importance in receptor-DNA interactions.In analogy to the endocrine model of gene regulation by steroid hormones, several lines of evidence indicate that intracellular, soluble receptor proteins are essential mediators of the biological responses produced by certain xenobiotics, i. e. halogenated or non-halogenated aromatic hydrocarbons One particularly striking and often-studied biochemical effect produced by TCDD and related compounds is their potency to induce specific isozymes of cytochrome P-450 and the associated monooxygenase activity aryl hydrocarbon hydroxylase [l , 41 together with several non-monooxygenase activities, e. g. UDP-glucuronyltransferase, NAD(P)H dehydrogenase (quinone) (formerly called DT-diaphorase) and ornithine decarboxylase [l]. Several studies have demonstrated that administration of TCDD or TCDD-like inducers leads to a large increase in translatable mRNAs encoding the TCDDinduced isozyme(s) of cytochrome P-450 [5 -71. By in vitro nuclear transcription studies, this process has been shown to reflect an increased rate of transcription of the specific cytochrome P-450 gene(s) [8-lo]. In analogy to the 'twostep' model proposed for the mechanism of action of steroid Correspondence to J.-A. Gustafsson, Institutionen for Medicinsk Naringslara, Huddinge Sjukhus, F69, S-141 86 Huddinge, SwedenAbbreviations. TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDBF, 2,3,7,8-tetrachlorodibenzofuran; Azso -310 unit, the amount of material giving an absorbance at 280nm of 1 more than the absorbance at 310 nm in a 1 -cm pathlength cell; DCC, dextran-coated charcoal.Enzymes (IUB Recommendations 1984). Aryl hydrocarbon hydroxylase (EC 1.14.14.1); a-chymotrypsin (EC hormones [ l l , 121, an accumulation of TCDD-receptor complexes in the cell nucleus following occupation of cytosolic receptor sites has been reported by several authors [13 -171. This classical 'two-step' model has been questioned, however, and some recent reports [18, 191 suggest that unoccupied steroid hormone receptors as well as unoccupied TCDD receptors in the intact cell are contained in the nucleus, and ...

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“…Before these genomic responses can be initiated, however, the glucocorticoid-receptor complex must first undergo a transformation known as activation (e.g., Bloom et al, 1980;Markovic and Litwack, 1980;Munck and Foley, 1980). In cytosol preparations this transformation can be induced by incubation at elevated temperatures and salt concentrations, removal of small molecule inhibitors, and other manipulations (reviewed in Schmidt and Litwack, 1982). Previous work from this laboratory with mouse whole brain cytosol labeled with [3H]triamcinolone a~etonide([~H]TA) (Luttge et al, tosol to DNA-cellulose (DNA-C).…”

mentioning

confidence: 99%

“…The half-times for each of these apparent firstorder transformations ranged from 1.3 to 1.4 min for the adsorption to DEAE-cellulose and GF/C filters to 2.8 min for the binding to DNA-C. The changes in adsorption to the DEAE-cellulose and GF/C filters are thought to reflect an increase in the relative number of positively charged and hydrophobic groups on the surface of the activated steroid-receptor complex (e.g., Dahmer et al, 1981 ;Cousens and Eskin, 1982;Schmidt and Litwack, 1982). These changes in surface characteristics may also contribute to the increased affinity of the receptor for nuclear-associated acceptors on activation as reflected b y the increased binding to DNA-C.…”

mentioning

confidence: 99%

Temperature‐Dependent Kinetic Correlates of the Activation of the Glucocorticoid‐Receptor Complex

Luttge

Densmore

1984

Journal of Neurochemistry

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The effects of temperature on the kinetics of activation were studied in [3H]triamcinolone acetonide[( 3H]TA)-labeled cytosol preparations from mouse whole brain. After removal of unbound [3H]TA and molybdate (which prevents activation) from the unactivated steroid-receptor complex by gel exclusion chromatography, activation was initiated by incubation at 6-30 degrees C for 0.75-24 min and then rapidly quenched at -5 degrees C with Na2MoO4 (20 mM final concentration). The loss of the 9.2S (unactivated) form of the [3H]TA-receptor complex and the concomitant formation of the 3.8S (activated) form increased dramatically with increases in the activation temperature. These hydrodynamic changes were correlated directly with rapid time- and temperature-dependent increases in the binding of [3H]TA-labeled cytosol to DNA-cellulose (DNA-C). Further analyses of these data revealed a greater than 50-fold increase in the apparent first-order rate constant for the increased binding to DNA-C as the activation temperature was increased from 6 degrees C to 30 degrees C. An Arrhenius plot of these temperature-dependent kinetic constants revealed an energy of activation of 116 kJ. These data support a proposed model for activation of the glucocorticoid-receptor complex that includes the splitting of a 297 kDa, unactivated species into a 92 kDa, activated species.

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Activation of the glucocorticoid–receptor complex

Cited by 34 publications

References 49 publications

Transformation in vitro and covalent modification with biotin of steroid-affinity-purified rat-liver glucocorticoid-hormone-receptor complex

Transformation in vitro and covalent modification with biotin of steroid-affinity-purified rat-liver glucocorticoid-hormone-receptor complex

Characterization of the DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin

Temperature‐Dependent Kinetic Correlates of the Activation of the Glucocorticoid‐Receptor Complex

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