Absence of detectable ribonucleic acid in the purified, untransformed mouse glucocorticoid receptor.
The glucocorticoid receptor (GC-R) isolated from the mouse AtT-20 pituitary tumor cell line exists in three forms. The untransformed (non-DNA-binding), 9.1S species (319K) can be converted into two transformed (DNA-binding) species. One of these (5.2 S, Mr 132K) appears to be composed of one molecule of the hormone-binding, monomeric protein (96K) plus a small RNA, while the second transformed species is the monomeric, hormone-binding subunit (3.8 S, 96K) itself. We wished to determine whether the untransformed GC-R contains RNA or if the monomer binds to RNA subsequent to subunit dissociation (which occurs during receptor transformation). Kinetic studies using both the crude and purified untransformed GC-R show that the untransformed, 9.1S GC-R dissociates into 3.8S monomeric subunits, without forming a transient 5.2S complex. The untransformed receptor was then purified with affinity chromatography, gel filtration, and DEAE-cellulose chromatography. One major protein band, corresponding in size to the GC-R monomer (94K-96K), was observed on sodium dodecyl sulfate-polyacrylamide gels upon silver staining or fluorography of [3H]dexamethasone mesylate covalently labeled receptor. In vivo 32P-labeling of AtT-20 cells, followed by purification of the untransformed GC-R, yielded two major 32P-labeled components (94K-96K and 24K). Both of these bands were protease-sensitive, contained phosphoserine, and were unaffected by ribonuclease treatment. We conclude that the untransformed mouse GC-R is wholly proteinaceous and contains no RNA. Thus, RNA binding occurs subsequent to dissociation of the oligomeric, untransformed GC-R complex into monomers.
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010911:Pituitary Neoplasms / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis*; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification* / Q000378:metabolism
D004268:DNA-Binding Proteins; D012334:RNA, Neoplasm; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone