Abstract
Rat liver glucocorticoid-receptor complex (GRc) was purified 2000-fold by a combination of methods including (NH4)2SO4-fractionation and phosphocellulose and DNA-cellulose chromatography. The purified glucocorticoid receptor preparation contained a major peptide of Mr = 90,000 and the GRc sedimented as 4 S in 5-20% sucrose gradients. An additional peptide of Mr = 45,000 (45K) was also observed. Some preparations yielded only the Mr = 90,000 (90K) peptide suggesting that the 45K peptide may be a proteolyzed portion of the 90K protein. The purified GRc was incubated with [gamma-32P]ATP in the presence of cAMP-dependent kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the above preparation revealed the presence of two 32P-containing bands with apparent Mr = 90,000 and 45,000. The 32P incorporation was dependent on the availability of divalent cation (Mg2+). GRc in cytosol labeled with [3H]dexamethasone mesylate and purified as above co-migrated with 32P-containing bands. GRc was also purified from cytosol obtained from livers of rats injected with [32P]orthophosphate. Both 32P and 3H bands were associated with 90K and 45K peptides. Our results indicate that rat liver glucocorticoid receptor is a phosphoprotein and that both the phosphorylated peptides 90K and 45K also contain the steroid and the DNA binding regions of the glucocorticoid receptor.
Highlights
Rat liver glucocorticoid-receptorcomplex (GRc)was purified 2000-fold by a combination of methods including (NH4)2S04-fractionationand phosphocellulose and DNA-cellulose chromatography
Our results indicate that rat liver glucocorticoid receptor is a phosphoprotein and that both the phosphorylated peptides 90K and 45K contain the steroid and the DNA binding regions of the glucocorticoid receptor
Cellex 410, Bio-Gel A-0.5m, and other chemicals for electrophoresis were from Bio-RadLaboratories.Nonradioactivedexamethasone, DNA, monothioglycerol, glycerol, dithiothreitol, thyroglobulin, CAMP-dependent protein kinase, o-dianisidine, and peroxidase were purchased from Sigma
Summary
From the Departmentof Biological Sciences, Oakland University, Rochester, Michigan 48063. Rat liver glucocorticoid-receptorcomplex (GRc)was purified 2000-fold by a combination of methods including (NH4)2S04-fractionationand phosphocellulose and DNA-cellulose chromatography. The purified glucocorticoid receptor preparation contained a major peptide of M , = 90,000 and the GRc sedimented as 4 S in 5-20% sucrose gradients. GRc was purified from cytosol obtained from livers of rats injected with [32P]. Our results indicate that rat liver glucocorticoid receptor is a phosphoprotein and that both the phosphorylated peptides 90K and 45K contain the steroid and the DNA binding regions of the glucocorticoid receptor. We have purified transformed glucocorticoid receptor from rat liver cytosol and examined its phosphorylation in vitro using aCAMP-dependent protein kinase. Phosphorylation site(s) of glucocorticoid receptor appear to be associated with peptideosf Mr = 90,000 and 45,000 whichalso retain the steroid and DNA binding sites
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