Glucocorticoid receptor phosphorylation, transformation, and DNA binding.
Glucocorticoid receptors were isolated by immunoadsorption from cytosol of L cells that were cultured for 18 h in the presence of [32P]orthophosphate, and the phosphorylation state of the receptor was examined before and after transformation to the DNA-binding state. Temperature-mediated transformation of the glucocorticoid receptor under cell-free conditions results in no change in receptor size or degree of phosphorylation. When cytosol containing transformed receptors is incubated with DNA-cellulose, 30-50% of the receptors are able to bind to DNA and the remainder do not bind to DNA. Both the heated receptors that bind to DNA and the receptors that do not bind to DNA are phosphorylated to the same degree. When intact cells containing 32P-labeled receptors are incubated for 2 h at 0 degree C with triamcinolone acetonide and then for 20 min at 37 degrees C in the presence of the hormone, 80% of the receptor becomes tightly associated with the nucleus in a manner that is both temperature-dependent and ligand-dependent. Approximately 80% of the nuclear-bound receptor is extracted with 0.4 M NaCl. Both the cytosolic receptor from cells incubated at 0 degree C and the salt-extracted nuclear receptor from cells incubated at 37 degrees C have been resolved by immunoadsorption to protein A-Sepharose with the BuGR1 monoclonal antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting and autoradiography of the immunoblots. In addition, direct measurements of the amounts of 32P contained per unit of receptor protein were performed for receptors transformed both in the intact cell and in cell-free lysates. The results demonstrate that the untransformed receptor and the nuclear-bound transformed receptor are labeled with 32P to the same extent.
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
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Tienrungroj
E R
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Sanchez
P R
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Housley
R W
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Harrison
W B
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Pratt
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D003907:Dexamethasone / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D013696:Temperature; D014222:Triamcinolone Acetonide / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006360:Heat-Shock Proteins; D010750:Phosphoproteins; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D004247:DNA; D014222:Triamcinolone Acetonide