Molybdate-stabilized nonactivated glucocorticoid-receptor complexes contain a 90-kDa non-steroid-binding phosphoprotein that is lost on activation.
We have used a monoclonal antibody to purify glucocorticoid-receptor complexes from WEHI-7 mouse thymoma cells. Molybdate-stabilized, nonactivated complexes were found to contain two distinct proteins which could be separated by polyacrylamide gel electrophoresis under denaturing and reducing conditions. One of the proteins, 100 kDa, was labeled when cytosol was incubated with the affinity ligand [3H]dexamethasone 21-mesylate. The second protein, 90 kDa, was not labeled. Several lines of evidence, including Western blot analysis of purified nonactivated complexes, indicate that only the 100-kDa protein is directly recognized by the antibody. The 90-kDa protein appears to be purified as a component of the nonactivated complex due to noncovalent association with the 100-kDa protein. Both the 100-kDa and 90-kDa components of the nonactivated complex become labeled with 35S when cells are grown in medium containing [35S]methionine. Using cells labeled in this manner, we have shown that activated (i.e. DNA-binding) cytosolic complexes, formed by warming either in intact cells or under cell-free conditions, contain only the 100-kDa protein. Complexes extracted from nuclei of warmed cells similarly contain only the 100-kDa protein. These results indicate that the 100-kDa and 90-kDa components of nonactivated complexes separate upon activation. Purification of nonactivated complexes from cells grown in medium containing [32P]orthophosphoric acid indicates that both the 100-kDa and 90-kDa components are phosphoproteins which can be labeled with 32P. Therefore, resolution of the two proteins will be essential in order to determine whether the receptor is dephosphorylated on activation.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003600:Cytosol / Q000032:analysis; D051379:Mice; D008970:Molecular Weight; D008982:Molybdenum / Q000494:pharmacology*; D010750:Phosphoproteins / Q000032:analysis*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D013945:Thymoma / Q000032:analysis
D000911:Antibodies, Monoclonal; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011965:Receptors, Glucocorticoid; C044659:molybdate; D008982:Molybdenum