Studies of the Activation Process of [3H] Dexamethasone Receptor Complex in Glucocorticoid Resistant Cell Lines
Fowler, Daniel H.
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Cortisol, the main glucocorticoid (GC) binds to GC receptors in target tissue to form a complex that cannot bind to DNA. The ultimate cellular effect of cortisol is the regulation of production of specific proteins at the transcriptional level. The "activation process" is essential to this since it changes the GC receptor from a form unable to bind to DNA to a DNA binding conformation. Upon this conformational change, the complex can translocate to the nucleus, bind to DNA, and regulate transcription. "Resistance" to glucocorticoids has been described in diseased states in man and in the entire population of marmoset monkeys, a New World Primate. This condition is characterized by high free plasma cortisol levels without the abnormalities generally associated with hypercortisolism. Previous studies suggest that a defect in the apparent dissociation constant (Kd) of the GC for GC receptors contributes to the GC resistance in both species. The process of activation was studied to further investigate the mechanism of their resistance. Permanent B lymphocyte cell lines established by transformation with EB virus provided a constant source of GC receptor from the two GC resistant cases and a human control. Binding data (Kd and receptor concentration) suggest that the EBV transformation may have resulted in alterations in the binding characteristics of the GC receptor for glucocorticoids. For this reason, studies in the Ge resistant EBV cells may not necessarily reflect the actual biochemical events in normal cells. Nevertheless, binding differences between resistant and control· cell lines suggest that the EBV cell lines are GC resistant and thus provide a model for study of the activation process. Phosphocellulose chromatography (PC) was used to study the activation process. Results from PC studies agree with other authors in that all cell lines were activated to the DNA binding form by increases in temperature and ionic strength. All cell lines were also activated with increases in ATP concentration; this was remarkable in that activation of the GC complex with ATP in:human or primate cells has not yet been reported. Although this may suggest that the conformational change resulting from activation involves a simple phosphorylation-dephosphorylation mechanism, an indirect, energy-dependent mechanism may be responsible for the activation process. PC studies indicate that the GC resistance in the patient and marmoset are probably of diverse biochemical origin. The marmoset displayed a reduced ability to activate GC receptor complex to the DNA binding form. This activation deficiency combines with the marmoset's increased Kd and decreased receptor capacity to account for its resistance to GC. The patient, however, appears to have a normal ability to activate GC complex. The mechanism of the human patient's resistance is probably due to an increased Kd and an unstable receptor complex.