Different states of glucocorticoid receptors in intact cells and cytosol preparations

Gehring, Ulrich; Spindler-Barth, Margarethe; Ulrich, Jürgen

doi:10.1016/0006-291x(82)90875-0

Cited by 10 publications

(2 citation statements)

References 22 publications

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“…These results show that CM-SM cells constitutively express receptor sites that bind glucocorticoids with high affinity and specificity. Receptor properties, such as affinity (-4.0 x M), kinetic characteristics, steroid specificity, and cytoplasm to nucleus translocability are in all respects similar to those described for glucocorticoid receptors in other continous cell lines (Cidlowski and Cidlowski, 1981;Munck and Leung, 1977;Ghering et al, 1982). Growth in media containing saturating concentrations of DEX resulted in a time-related sequence of marked effects on CM-SM cells.…”

Section: Discussionsupporting

confidence: 60%

Glucocorticoid receptors and cortico‐sensitivity in a human clonal monocytic cell line, CM‐SM

Ranelletti

Starace

Piantelli

et al. 1983

Journal Cellular Physiology

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CM-SM is a clonal line of human precursor mononuclear phagocytes inducible to macrophage differentiation in response to the tumor promoter phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Untreated CM-SM cells contain single class, high-affinity (KD = 4.0 X 10(-9) M) glucocorticoid-specific receptor sites (approximately 60,000 per cell), as measured by a whole cell assay, at 37 degrees C, using [3H]triamcinolone acetonide (TA). Exposure of CM-SM to dexamethasone (DEX) produced a progressive, dose- and time-related series of changes in CM-SM cell growth, saturation density, morphology, and functional properties, with half-maximal effects at about 10(-9) M for DEX. TA-receptor sites rapidly decreased (about 70%) after DEX treatment, without any apparent change in steroid specificity and affinity. After 5 days in culture with a saturating concentration (3.6 X 10(-8) M) of hormone, the cells reached a saturation density of about 9.0 X 10(6) viable cells/ml (about 4.0 X 10(6) viable cells/ml in the controls), while the modal volume of the resulting cell population was approximately 60%, as compared to the volume of untreated cells. DEX-treated cells appeared less differentiated than controls, as assessed by combined morphologic, antigenic, and cytoenzymatic analyses. DEX almost completely inhibited TPA activation of the following macrophage functions: adherency to the culture plate, expression of lysosomal enzymes, Fc and C3 receptors, and stimulation of phagocytosis. After removal of DEX, the cells, within a few passages, returned to a state apparently identical to the untreated controls and could be induced to macrophage differentiation in response to TPA.

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Section: Discussionsupporting

confidence: 60%

Glucocorticoid receptors and cortico‐sensitivity in a human clonal monocytic cell line, CM‐SM

Ranelletti

Starace

Piantelli

et al. 1983

Journal Cellular Physiology

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“…Considering these numbers, a dimerization upon binding could occur if the GR concentration is in the nanomolar range or less, although it should be noted that the GR dimerization constant might vary somewhat depending on temperature and buffer conditions. The GR probably exists as both monomeric and dimeric species in the cytoplasm, because the concentration of GR in the cytoplasm has been estimated to be in the range of 1-100 nM (Hansson et al, 1981;Gehring et al, 1982). However, if the free GR is monomeric at the conditions at which the footprinting experiments of Perlman et al (1990) were performed, then the observed free ligand concentration at 50% occupancy of available binding sites would be [GR]1/2 = l¡( 111).…”

Section: Discussionmentioning

confidence: 99%

Thermodynamics of the glucocorticoid receptor-DNA interaction: Binding of wild-type GR DBD to different response elements

Lundbäck¹,

Cairns²,

Gustafsson³

et al. 1993

Biochemistry

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We used fluorescence spectroscopy to study the chemical equilibria between an 82-residue protein fragment containing the core conserved region of the glucocorticoid receptor DNA-binding domain (GR DBD) and a palindromic glucocorticoid response element (GRE), a consensus GRE half-site, a consensus estrogen response element (ERE) half-site, and two intermediate half-sites (GRE2 and ERE2). Equilibrium parameters were determined at 20 degrees C and buffer conditions that approximate intracellular conditions. The association constants for GR DBD binding to the GRE (5'TGTTCT3') and GRE2 (5'TGTCCT3') half-sites at 85 mM NaCl, 100 mM KCl, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 and low concentrations of an antioxidant and a nonionic detergent are (1.0 +/- 0.1) x 10(6) M-1 and (5.1 +/- 0.2) x 10(5) M-1, respectively. The association constants for binding to the ERE (5'TGACCT3') and ERE2 (5'TGATCT3') half-sites are < 10(5) M-1. The implications of these numbers for the specificity and affinity for the binding of the intact GR to DNA are discussed. Comparison of GR DBD binding to a GRE half-site and a palindromic GRE sequence allowed us to estimate the cooperativity parameter, omega obs = 25-50, for GR DBD binding to GRE. The thermodynamics of the GR DBD interaction with a GRE half-site were also investigated by determining the temperature dependence of the observed association constant. The nonlinear dependence in ln Kobs as a function of 1/T is consistent with a change in standard heat capacity, delta Cp degree obs = 1.0 +/- 0.2 kcal mol-1 K-1.(ABSTRACT TRUNCATED AT 250 WORDS)

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Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies.

Westphal¹,

Mugele²,

Beato³

et al. 1984

The EMBO Journal

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Monoclonal antibodies raised against the rat liver glucocorticoid receptor were used to investigate receptors of wild‐type and glucocorticoid‐resistant variants of mouse lymphoma cells. Two of the variant types contained receptors of ‘nuclear transfer deficient’ (nt‐) and ‘increased nuclear transfer’ (nti) phenotypes, respectively, while the third was of the ‘receptorless’ (r‐) phenotype with negligible hormone binding activity. Three monoclonal antibodies of the IgM class and one of the IgG class reacted with both wild‐type and nt‐ receptors but not with the steroid binding form of nti receptors. Some of the antibodies bound the wild‐type and nt‐ receptors more efficiently after activation at 20 degrees C. By use of an immuno‐competition assay we were able to detect cross‐reacting material in considerable amounts in extracts of nti and r‐ cell variants. This material was further characterized by gel filtration and immunoblotting. The immunoreactive material of wild‐type, nti and r‐ cells gave a major band of mol. wt. 94 000 upon SDS‐gel electrophoresis while the steroid‐binding polypeptides of wild‐type and nti receptors have mol. wts. of 94 000 and 40 000, respectively. The data show that in S49.1 mouse lymphoma cells the products of two receptor alleles can be distinguished.

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Different states of glucocorticoid receptors in intact cells and cytosol preparations

Cited by 10 publications

References 22 publications

Glucocorticoid receptors and cortico‐sensitivity in a human clonal monocytic cell line, CM‐SM

Glucocorticoid receptors and cortico‐sensitivity in a human clonal monocytic cell line, CM‐SM

Thermodynamics of the glucocorticoid receptor-DNA interaction: Binding of wild-type GR DBD to different response elements

Immunochemical characterization of wild-type and variant glucocorticoid receptors by monoclonal antibodies.

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