Abstract
A majority of the untransformed glucocorticoid-receptor complexes (GRc) from rat liver cytosol sedimented in the 9S region in 5-20% sucrose gradients containing 0.15M KCl and 20mM Na2MoO4. Incubation of the cytosol at 23°C, or at O°C with 10mM ATP or 0.3M KCl caused appearance of a slower migrating (4S) form which exhibited an increased affinity toward DNA-cellulose and ATP-Sepharose. Presence of 20mM Na2MoO4 blocked this 9S to 4S transformation of GRc. A complete conversion of the 9S to the 4S form occurred upon a 2h incubation of GRc with 10mM ATP at 0°C. Other nucleoside triphosphates (GTP, CTP, and UTP), ADP and PP1 (but not AMP or cAMP) were also effective in transforming the 9S form. The heat transformation occurred in a time-dependent manner and was complete within 1h at 23°C; presence of 10mM ATP during this 23°C incubation period allowed a complete 9S to 4S alteration in 10-20min. Addition of ATP also accelerated the rate of salt activation of the GRc; a 50% conversion to the 4S form occurred in 20min or 3min in the absence or the presence of 10 mM ATP during the 0°C incubation of GRc with 0.15 M KCl. An absolute requirement of the hormone for 9S to 4S transformation of glucocorticoid receptor (GR) was evident, as no conversion of the 9S form to the 4S form could be achieved with the ligand-free GR under any of the above conditions. Incubation of cytosol preparations at 23°C or at 0°C with KCl or ATP caused dissociation of the GRc and reduced the steroid binding capacity of GR. Although aurintricarboxylic acid, pyridoxal 5'-phosphate, Na2MoO4, Na2WO4, o-phenanthroline, Rifamycin AF/013 and heparin inhibited the ATP-Sepharose and DNA binding of the GRc, only Na2MoO4 and Na2WO4 selectively blocked the 9S to 4S conversion. We suggest that the 9S to 4S transformation in vitro of rat liver GRc represents an acquisition of DNA and ATP-Sepharose binding ability and may involve a separation of subunits from an oligomeric receptor structure.
