Inactivation of mammary cytoplasmic glucocorticoid receptors under cell-free conditions.

lE2The conditions for the inactivation of glucocorticoid receptors in the cytosol from lactating mouse mammary glands have been studied in detail.The dexamethasone-binding capacity of cytosol was variable and decreased to near zero levels by 4 h when incubated without steroid at 4°C.This loss of binding activity was partially prevented by binding of the steroid and was readily reversed by sulfhydryl-reducing compounds such as dithiothreitol.Molybdate and other phosphatase inhibitors were without effect on this dithiothreitol-reversible binding loss.A second type of loss of specific binding of dexamethasone occurred in mammary cytosol exposed to high ionic strength or elevated temperature, but this loss could not be prevented or reversed by dithiothreitol; a 1-h treatment of cytosol with 0.3 M KC1 or a Yz-h exposure to 25°C eliminated most of the dexamethasone binding.Treatment with the steroid or with 10 m M molybdate partially prevented but did not reverse this inactivation.A molybdate-sensitive change of the steroidereceptor complex was inferred from analysis of the [3H]dexamethasone-labeled cytosol on high ionic strength sucrose gradients; the steroidoreceptor complex in the untreated cytosol migrated as a 4.4 S peak while that in the molybdate-treated cytosol migrated as a 5.4 S peak.Therefore, lactating mouse mammary glucocorticoid receptors are inactivated by two types of in vitro processes, one involving sulfhydryl groups and the other enhanced by high salt or elevated temperature.The possibility that similar in vivo processes may regulate glucocorticoid receptor activity remains to be explored.During pregnancy, mammary tissues undergo cellular differentiation leading to the subsequent secretion of milk components by epithelial cells during lactation.Both in uiuo and in vitro studies have demonstrated a major regulatory role for glucocorticoids in mediating these lactogenic functions (1-6) and receptors for glucocorticoids have been identified and characterized in normal rodent mammary tissues (7, 8).Additionally, previous studies have clearly established a role for the receptors in the regulation by glucocorticoids of at least one mammary gene product, mammary tumor virus (9-11).