In Vitro Stabilization of the Unoccupied Glucocorticoid Receptor by Adenosine 5'-Diphosphate*

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Abstract
The addition of ATP to rat liver cytosol slows the rate of heat inactivation of the unoccupied glucocorticoid receptor (25 C) and stimulates the rate of activation1 of the preformed glucocorticoid-receptor complex (15 C). Dose-response curves and kinetic studies show that ADP is as effective as ATP in stabilizing the unoccupied glucocorticoid receptor against heat inactivation. ATP can also be replaced by analogs with a hydrolysis-resistant α,β-pyrophosphate linkage (5'-adenylyl methylenephosphonophosphate or 5'-adenylyl methyl-enephosphonate); however, the hydrolysis-resistant β,γ analog (5'-adenylyl methylenediphosophonate) is ineffective. The addition of creatine phosphate plus creatine kinase, a condition favoring ATP formation, stimulates the rate of inactivation of the unoccupied glucocorticoid receptor, and the effect is only partially overcome by ADP. A condition that favors ADP formation, the addition of creatine plus creatine kinase, has no effect on the rate of inactivation of the unoccupied receptor and does not decrease the protective effect afforded by ATP alone. Collectively, these results suggest that ATP stabilization of the steroid-binding site in vitro is due to ADP generated from the triphosphate by endogenous enzymes and is not due to phosphorylation or adenylation of the receptor by ATP. Unlike ATP stabilization of the steroid-binding site, the ATP-stimulated increase in the rate of activation of the preformed glucocorticoid-receptor complex (15 C) does not require hydrolysis of the 0,7-pyrophosphate bond. Dose-response curves show that both ATP and 5'-adenylyl methylenediphosophonate stimulate the rate of activation of the glucocorticoid-receptor complex. Quantitation of nucleotide levels in unfractionated rat liver cytosol by high performance liquid chromatography shows that the effective concentration of added ATP that produces an optimal response is within the physiological range reported for intact cells.