Analysis of glucocorticoid receptor activation by high resolution two-dimensional electrophoresis of affinity-labeled receptor.
To determine if activation of the glucocorticoid receptor involves covalent charge modification of the steroid-binding protein, unactivated and activated IM-9 cell glucocorticoid receptors were examined by high resolution two-dimensional gel electrophoresis. As previously reported (Smith, A. C., and Harmon, J. M. (1985) Biochemistry 24, 4946-4951), two-dimensional electrophoresis of immunopurified, [3H]dexamethasone mesylate-labeled, steroid-binding protein from unactivated receptors resolves two 92-kDa isoforms (pI congruent to 5.7 and 6.0-6.5). After activation, the apparent pI of neither isoform was altered, indicating that there had been no covalent charge modification of the steroid-binding protein. Thus, the physicochemical changes observed after activation of the steroid receptor cannot be explained by dephosphorylation or other models which involve covalent charge modification of the steroid-binding protein. This conclusion was consistent with the observation that treatment of immunopurified, affinity-labeled receptors with calf intestine alkaline phosphatase did not alter the apparent pI values or distribution of the steroid-binding protein isoforms. However, chromatography of activated steroid-receptor complexes on DNA-cellulose revealed that only the more basic of the two steroid-binding protein isoforms bound to DNA. Therefore, the charge heterogeneity of the steroid-binding protein may be important in regulating the ability of the steroid-binding protein to interact with DNA.
D000345:Affinity Labels / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007526:Isoelectric Point; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D014222:Triamcinolone Acetonide / Q000378:metabolism
D000345:Affinity Labels; C033190:DNA-cellulose; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D002482:Cellulose; D004247:DNA; D000469:Alkaline Phosphatase; D014222:Triamcinolone Acetonide; C031710:dexamethasone 21-methanesulfonate