In Vivo Activation and Nuclear Binding of the AtT-20 Mouse Pituitary Tumor Cell Glucocorticoid Receptor*

Abstract

AtT-20 mouse pituitary tumor cells were incubated at 25 C with the tritiated glucocorticoids triamcinolone acetonide (9 alpha-fluoro-11 beta, 16 alpha, 17,21-tetrahydroxy-pregna-1, 4-diene-3,20-dione 16,17-acetal with acetone), dexamethasone (9 alpha-fluoro-11 beta, 17,21-trihydroxy-16 alpha-methyl-pregna-1,4-diene-3, 20-dione), prednisolone (11 beta, 17,21-trihydroxypregna-1,4-diene-3,20-dione), and corticosterone (11 beta, 21-hydroxypregn-4-ene-3,20-dione) in order to examine the nuclear binding and glucocorticoid receptor activation produced in vivo. Although the total amounts of intracellular receptor labeled by each steroid were similar, each steroid caused different and characteristic percentages of occupied receptor to be translocated into the nucleus. DEAE chromatography of the nuclear receptor extracted in the presence of sodium molybdate to prevent spontaneous activation showed that, as expected, the nuclear receptor was in the activated form. Activated receptor in the cytosol was determined by DEAE chromatography of cytosol prepared from cells that had been incubated with the various labeled glucocorticoids mentioned above. Total intracellular activated receptor was determined as cytosolic activated receptor plus total nuclear receptor. The results showed that for the four agonists used, the extent of nuclear binding is proportional to the degree of activation and that both of these parameters correlate with steroid-receptor affinity. It was also found that the removal of steroid from the cell incubation medium caused the rapid return of nuclear receptor to the cytosolic compartment in an unactivated form. This reversal was not dependent on protein synthesis. These results are consistent with a model of nuclear binding in which the proportion of steroid-bound receptor that becomes activated is determined by steroid binding affinity. However, the activated receptor partitions between the cytoplasmic and nuclear compartments to the same extent regardless of the steroid used, suggesting that although the percentage of activated receptors is steroid dependent, the nuclear-binding ability of the activated receptor is an intrinsic and constant property of the protein itself.