Steady-state level of the specific glucocorticoid binding component in mouse fibroblasts

Ishii, Douglas N.; Pratt, William B.; Aronow, Lewis

doi:10.1021/bi00771a010

Cited by 102 publications

(19 citation statements)

References 15 publications

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“…Removal of the steroid from the incubation medium results in a decrease in nuclear-associated radioactivity and an increase in the steroid binding activity in the cytoplasm. The increase in cytoplasmic binding activity following steroid removal is an energy dependent process [41] and occurs in the absence of new protein synthesis [41,42]. These studies show that unlike proteolysis, the change in the receptor protein that occurs as a consequence of activation is reversible in vivo.…”

Section: Factors That Affect Activation In Vltromentioning

confidence: 79%

“…First, Sakaue and Thompson (personal communication) have found that a number of diverse proteolytic inhibitors including leupeptin, elastinal, antipain, phenylmethylsulfonyl fluoride, phosphoramidon, pepstatin, chymostatin, and trypsin inhibitor fail to block heat-induced activation of the rat liver glucocorticoid-receptor complex as assayed by DEAE-cellulose chromatography. Second, studies on intact cells show that following activation and translocation the receptor can be recycled from the nucleus back to the cytoplasm [41,42]. If intact cells are exposed to steroid, there is a decrease in the steroid binding activity of the cytoplasm and a concomitant increase in specifically bound steroid associated with the nucleus.…”

Section: Factors That Affect Activation In Vltromentioning

confidence: 99%

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Activation of the glucocorticoid–receptor complex

Schmidt

Barnett

Litwack

1982

J of Cellular Biochemistry

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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.

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Section: Factors That Affect Activation In Vltromentioning

confidence: 79%

Section: Factors That Affect Activation In Vltromentioning

confidence: 99%

Activation of the glucocorticoid–receptor complex

Schmidt

Barnett

Litwack

1982

J of Cellular Biochemistry

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“…However, this difference may not hold for receptor binding at specific loci where the affinity may be much higher (Yamamoto & Alberts, 1974). Ihere is also evidence for constant cycling of receptors, even those in the activated, nuclear sites (Ishi, Pratt & Aronow, 1975), and a dependence on active protein synthesis (Joss, Bassand & Dierks-Ventling, 1976). Evidence has also been presented for self-regulation of receptor levels following nuclear binding (Svec & Kudis, 1981).…”

Section: Nuclear Bindinfrmentioning

confidence: 99%

Glucocorticoid action: a mechanism involving nuclear and non-nuclear pathways

Johnson¹,

Longenecker²,

Baxter³

et al. 1982

Br J Dermatol

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“…According to the model, the glucocorticoid binds to a receptor that is normally located in the cytoplasm (16,17). As has been shown for other glucocorticoid-responsive systems and for other classes of steroid hormones, the steroid-receptor complex undergoes a temperature-dependent change to a form (RSn) that can bind to nuclear components (3,18 (2,7). Second, the ability of cell-free preparations from fibroblasts and liver to bind glucocorticoids specifically has been shown in this paper to be inactivated by alkaline phosphatase.…”

Section: Discussionmentioning

confidence: 99%

Evidence that dephosphorylation inactivates glucocorticoid receptors.

Nielsen¹,

Sando²,

Pratt³

1977

Proc. Natl. Acad. Sci. U.S.A.

135

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Highly purified alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] from calf intestine inactivates the glucocorticoidbinding capacity of soluble preparations from mouse fibroblasts (L cells) and rat liver. The unbound receptor is sensitive to inactivation whereas the steroid-bound receptor is unaffected. The ability of the enze preparation to inactivate the receptor, like its ability to dephosporylate p-nitrophenyl phosphate, is dependent on zinc and inhibited by arsenate. Both the dephosphorylating and receptor inactivating activities coelute during DEAE-cellulose purification of the enzyme. There is no detectable proteolytic activity in the purified alkaline phosphatase preparation. In a mixed system containing both glucocorticoid and estrogen receptors, the glucocorticoid receptor is selectively inactivated. Althoug these observations do not prove that the receptor molecule itself is the substrate, they are consistent with the proposal that the glucocorticoid receptor can be inactivated by dephosphorylation and that only the phosphorylated form of the molecule is capable of binding steroid. A phosphorylation-dephosphorylation mechanism may be responsible for determining the level of active receptor in the cell.The ability of intact cells to bind glucocorticoids in a specific manner is energy-dependent (1-3). Exposure of mouse fibroblasts (3), thymic lymphocytes (4), or chick embryo retina (5) to dinitrophenol results in a loss of binding capacity. When the metabolic inhibitor is removed from the medium, the ability to bind glucocorticoids returns and the return is unaffected by the presence of inhibitors of protein synthesis (3, 5,-6). Munck et al. (2) Abbreviations: PMSF, phenylmethyl sulfonyl fluoride; Tos-LysCH2Cl, N-a-p-tosyl-L-lysine chloromethyl ketone; Tos-PheCH2Cl, L-1-tosylamide-2-phenylethyl chloromethyl ketone. * The trivial names for steroids are used as follows: triamcinolone acetonide, 9a-fluoro-1 1f3,16a,17a,21-tetrahydroxypregna-1,4-diene-3,20-dione 16,17-acetonide; dexamethasone, 9a-fluoro16a-methyl-11f,17a,21-trihydroxypregna-1,4-diene-3,20-dione; estradiol-17f,, 1,3,5(10)-estratriene-3,17f-diol; diethylstilbestrol, 3,4-bis(p-hydroxyphenyl)-3-hexene.Ci/mmol prior to use. 14C-Labeled protein from algal cells came from Amersham/Searle Corp., Arlington Heights, IL. Alkaline phosphatase from calf intestinal mucosa (950-1100 units/mg at 37°, pH 10), Streptomyces griseus protease (4 units/mg of solid), dexamethasone, 1,10-phenanthroline, pnitrophenyl phosphate, p-nitrophenol, phenylmethyl sulfonyl fluoride (PMSF), N-a-p-tosyl-L-lysine chloromethyl ketone (Tos-LysCH2Cl), and L-1-tosylamido-2-phenylethyl chloromethyl ketone (Tos-PheCH2Cl) were obtained from Sigma Chemical Co., St. Louis, MO. Soybean trypsin inhibitor was purchased from Worthington Biochemical Corp., Freehold, NJ.Cell Culture and Fractionation. L 929 mouse fibroblasts were grown in monolayer culture in Joklik medium as previously described (8). Cells in the logarithmic phase of ...

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Steady-state level of the specific glucocorticoid binding component in mouse fibroblasts

Cited by 102 publications

References 15 publications

Activation of the glucocorticoid–receptor complex

Activation of the glucocorticoid–receptor complex

Glucocorticoid action: a mechanism involving nuclear and non-nuclear pathways

Evidence that dephosphorylation inactivates glucocorticoid receptors.

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