Activation and chromatographic properties of the AtT-20 mouse pituitary tumor cell line glucocorticoid receptor

Vedeckis, Wayne V.

doi:10.1021/bi00528a029

Cited by 43 publications

(30 citation statements)

References 61 publications

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“…The mouse AtT-20 pituitary tumor cell line was maintained in T flasks and spinner suspension culture as described previously (Vedeckis, 1981). When not used for purification, GC-R prepared in TETg (20 mM Tris-HCl, 1 mM EDTA, 12 mM 1-thioglycerol, pH 7.4 at 22 °C) buffer was labeled with 3 X 10-* M [3H]TA overnight at 0-4 °C.…”

Section: Methodsmentioning

confidence: 99%

“…binding moiety f reviewed in Vedeckis (1985)]. Our laboratory has performed a detailed series of studies to elucidate the structure of the glucocorticoid receptor (GC-R)1 *in the mouse AtT-20 pituitary tumor cell line (Vedeckis, 1981(Vedeckis, , 1983aEastman-Reks et al, 1984;Reker et al, 1985;Kovacic-Milivojevic et al, 1985;Vedeckis et al, 1985). We 1 Abbreviations: 94K, 24K, etc., M, of 94000, 24000, etc.…”

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confidence: 99%

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Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor

Kovac̆ic̆-Milivojević¹,

Vedeckis²

1986

Biochemistry

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The glucocorticoid receptor (GC-R) isolated from the mouse AtT-20 pituitary tumor cell line exists in three forms. The untransformed (non-DNA-binding), 9.1S species (319K) can be converted into two transformed (DNA-binding) species. One of these (5.2 S, Mr 132K) appears to be composed of one molecule of the hormone-binding, monomeric protein (96K) plus a small RNA, while the second transformed species is the monomeric, hormone-binding subunit (3.8 S, 96K) itself. We wished to determine whether the untransformed GC-R contains RNA or if the monomer binds to RNA subsequent to subunit dissociation (which occurs during receptor transformation). Kinetic studies using both the crude and purified untransformed GC-R show that the untransformed, 9.1S GC-R dissociates into 3.8S monomeric subunits, without forming a transient 5.2S complex. The untransformed receptor was then purified with affinity chromatography, gel filtration, and DEAE-cellulose chromatography. One major protein band, corresponding in size to the GC-R monomer (94K-96K), was observed on sodium dodecyl sulfate-polyacrylamide gels upon silver staining or fluorography of [3H]dexamethasone mesylate covalently labeled receptor. In vivo 32P-labeling of AtT-20 cells, followed by purification of the untransformed GC-R, yielded two major 32P-labeled components (94K-96K and 24K). Both of these bands were protease-sensitive, contained phosphoserine, and were unaffected by ribonuclease treatment. We conclude that the untransformed mouse GC-R is wholly proteinaceous and contains no RNA. Thus, RNA binding occurs subsequent to dissociation of the oligomeric, untransformed GC-R complex into monomers.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor

Kovac̆ic̆-Milivojević¹,

Vedeckis²

1986

Biochemistry

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“…From (32) kind of reduction must be occurring. Transformation of the glucocorticoid-receptor complex caused by salt or by precipitation with ammonium sulfate also is inhibited by molybdate (42,95), as is salt-mediated transformation of progesterone receptor and estrogen receptor (66,74).…”

Section: Effects Of Molybdate On the Stability Of Unbound Receptorsmentioning

confidence: 99%

“…In addition to inhibiting transformation of glucocorticoid-and estrogen bound receptors, molybdate prevents the shift of these complexes to a less negatively charged state that occurs during DEAE-cellulose chromatography (17,74,95). (42) found that molybdate inhibited inactiva tion and transformation of glucocorticoid receptors caused by salt or ammo nium sulfate precipitation at 0°-4 °C.…”

Section: Effects Of Molybdate On the Stability Of Unbound Receptorsmentioning

confidence: 99%

Effects of Molybdate and Endogenous Inhibitors on Steroid-Receptor Inactivation, Transformation, and Translocation

Dahmer¹,

Housley²,

Pratt³

1984

Annu. Rev. Physiol.

155

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Michigan 48 i d9 INTRODUCTIONTo exert their specific effects on the regulation of gene expression, steroid honnones must first bind to cytoplasmic receptors that are subsequently trans located to specific acceptor sites in the cell nucleus. Prior to this translocation, the steroid-receptor complex is transfonned by a temperature-dependent pro cess to a state that can bind to nuclear components. This general model of binding, followed by receptor transfonnation and translocation to the nucleus, was originally proposed in 1968 on the basis of studies in intact cells (25,37).The initial events in this pathway, honnone binding and receptor transfonna tion, can be readily studied under cell-free conditions. Although clearly valid cell-free models of nuclear translocation have not been developed, binding of transfonned receptors to nuclei or to DNA-cellulose can be assayed in a quantitative manner and is employed as an operational method of assaying translocation of receptors that have been transfonned under cell-free condi tions. Both group VI-A transition-metal oxyanions (42,66,92) and a small, heat-stable endogenous factor (7,9,24,43,77,79,80) have been found to 67 0066-4278/84/0315-0067$02. 00 Annu. Rev. Physiol. 1984.46:67-81. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/09/15. For personal use only. Quick links to online content Further ANNUAL REVIEWS 68 DAHMER, HOUSLEY & PRA 11'stabilize unoccupied steroid receptors and to inhibit the transfonnation of receptors to the DNA-binding state in cell-free assay systems. Macromolecu lar, heat-labile factors that inhibit translocation oftransfonned receptors have been identified in cytosols prepared from a variety of tissues (12,19,55,87). We review here the actions, under cell-free conditions, of transition metal oxyanions and the two endogenous inhibitors on steroid receptor stability, transfonnation, and translocation. REGULATION OF RECEPTORS BY PHOSPHATE AND SULFHYDRYL MOIETIESIn order to understand the rationale behind some of the experiments described here, it is important to understand the potential role of receptor phosphate and the clear role of receptor sulfhydryl moieties in detennining the ability of steroid receptors to bind hormone. It has recently been demonstrated that the murine glucocorticoid receptor in L929 cells (34) and the avian progesterone receptor in hen oviduct (20) are phosphorylated in vivo. In both cases the major phosphorylated species isolated by affmity chromatography is a protein ofMr == 90,000:-92,000 (90-92K) that is phosphorylated on a serine moiety (or perhaps moieties). In the case of the glucocorticoid receptor, a minor species at lOOK is also phosphorylated. In the case of the progesterone receptor, a second phosphorylated receptor species tha� migrates at about 109K in sodium dodecyl sulfate-polyacrylamide gels has been identified..Several observations in cell-free systems suggest that phosphorylation of steroid receptors may be important for determining their ability to bind the hormon...

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“…Cell Culture. AtT-20 cells were grown in Dulbecco's modified Eagle's medium F12 (DMEM F12, GIBCO, Grand Island, NY) supplemented with 10% newborn calf serum (Bio Whittaker, Walkersville, MD) as previously described (Vedeckis, 1981). L929 mouse fibroblast cells were grown in DMEM/high glucose (GIBCO) supplemented with 10% fetal bovine serum (FBS, Irvine Scientific, Santa Ana, CA).…”

Section: Methodsmentioning

confidence: 99%

Coordinate Regulation of Glucocorticoid Receptor and c-jun Gene Expression Is Cell Type-Specific and Exhibits Differential Hormonal Sensitivity for Down- and Up-Regulation

1996

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We have previously proposed a novel mechanism for the coupled regulation of glucocorticoid receptor (GR) and c-jun transcription in triamcinolone acetonide (TA)-treated AtT-20 cells. This involved transcriptional interference of AP-1 (Fos/Jun)-driven gene transcription by the formation of inactive GR/Jun heterodimers. To further elucidate the molecular mechanism for GR autoregulation, the expression of GR and c-jun mRNA and protein levels were examined in both mouse L929 fibroblast cells and human CEM-C7 acute lymphoblastic leukemia cells. A rapid down-regulation of both GR and c-jun mRNA and protein levels occurs in TA-treated L929 cells. All-trans-retinoic acid (RA) treatment of Jun-deficient, mouse F9, teratocarcinoma cells causes the induction of c-jun expression. The increased expression of both c-jun mRNA and protein is accompanied by the induction of GR expression. These data further suggest that functional cJun is needed for the expression of the GR and c-jun genes in F9 cells. CEM-C7 cells undergo apoptosis after exposure to glucocorticoids. There is a parallel up-regulation of GR and c-jun mRNA levels in TA-treated CEM-C7 cells. This is accompanied by a concomitant increase in GR and cJun protein levels. Dose-response analyses reveal the expected coordinate regulation of both GR and c-jun mRNA and protein in L929 cells (decreasing) and in CEM-C7 cells (increasing). However, approximately 20-fold less TA is required for the inhibition of GR and c-jun expression as compared to that required for the stimulation of these two genes. These data demonstrate that the coordinate regulation of GR and c-jun gene expression is dose-dependent and cell type-specific. These results, along with previously reported data, suggest that GR complex formation with itself or with another transcription factor is important for the coordinate up- and down-regulation, respectively, of the GR and c-jun genes.

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Activation and chromatographic properties of the AtT-20 mouse pituitary tumor cell line glucocorticoid receptor

Cited by 43 publications

References 61 publications

Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor

Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor

Effects of Molybdate and Endogenous Inhibitors on Steroid-Receptor Inactivation, Transformation, and Translocation

Coordinate Regulation of Glucocorticoid Receptor and c-jun Gene Expression Is Cell Type-Specific and Exhibits Differential Hormonal Sensitivity for Down- and Up-Regulation

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