title: A dynamic model of glucocorticoid receptor phosphorylation and cycling in intact cells.
abstract: Glucocorticoid receptors have been proposed to undergo an ATP-dependent recycling process in intact cells, and a functional role for receptor phosphorylation has been suggested. To further investigate this possibility we have examined the phosphate content of the steroid-binding protein of all glucocorticoid receptor forms which have been isolated from WEHI-7 mouse thymoma cells. By labeling of intact cells with 32Pi for 18-20 h in the absence of hormone, covalent binding of [3H]dexamethasone 21-mesylate, immunopurification and SDS-PAGE analysis, the steroid binding protein was found to contain, on average, 2-3 phosphates as phosphoserine. One third of the phosphates were associated with proteolytic fragments encompassing the C-terminal steroid-binding domain. The central DNA-binding domain was not phosphorylated, leaving the other two thirds of the phosphates localized in the N-terminal domain. The phosphate content of various receptor forms from cells incubated with 32Pi and [35S]methionine was compared using 35S to normalize for quantity of protein. In ATP-depleted cells a non-steroid-binding form of the receptor (the "null" receptor) is found tightly bound to the nucleus, even without steroid. The phosphate content of null receptors was two thirds that of cytosolic receptors from normal cells, suggesting phosphorylation-dependent cycling in the absence of hormone. Addition of glucocorticoid agonists, but not antagonist, to 32P- and 35S-labeled cells increased the phosphate content of the cytosolic steroid-binding protein up to 170%, indicating an average increase in the phosphates from about 3 to 5. After 30 min of hormone treatment the phosphate content of the steroid-binding protein of cytosolic activated (DNA-binding) and nonactivated receptors, and that of nuclear receptors extractable with high salt concentrations and/or DNase I digestion, was the same. No change in the phosphate content of the 90-kDa heat shock protein associated with unliganded and nonactivated receptors was detected following association of the free protein with the receptor and following hormone binding of the receptor. Analysis of the unextractable nuclear receptors indicated that they contained less phosphate (60% of that of cytosolic receptors), similarly to null receptors, indicating that dephosphorylation is associated with the unextractable nuclear fraction. The rate of hormone-dependent phosphorylation appeared to be much faster than the rate of dephosphorylation in the presence of hormone, the latter determined by a chase of the 32P label with unlabeled phosphate. Our results show that phosphorylation and dephosphorylation are involved in the mechanism of action of glucocorticoid receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Physiology, Dartmouth Medical School, Hanover, NH 03756.
authors.forename: E
authors.initials: E
authors.lastname: Ortí
authors.forename: D B
authors.initials: DB
authors.lastname: Mendel
authors.forename: L I
authors.initials: LI
authors.lastname: Smith
authors.forename: J E
authors.initials: JE
authors.lastname: Bodwell
authors.forename: A
authors.initials: A
authors.lastname: Munck
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D006360:Heat-Shock Proteins / Q000378:metabolism; D006854:Hydrocortisone / Q000494:pharmacology; D007700:Kinetics; D008223:Lymphoma; D008715:Methionine / Q000378:metabolism; D051379:Mice; D015735:Mifepristone / Q000494:pharmacology; D008954:Models, Biological; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D014222:Triamcinolone Acetonide / Q000494:pharmacology
chemical_list: D006360:Heat-Shock Proteins; D010710:Phosphates; D011965:Receptors, Glucocorticoid; D015735:Mifepristone; D000255:Adenosine Triphosphate; D008715:Methionine; D014222:Triamcinolone Acetonide; D006854:Hydrocortisone