Regulation of glucocorticoid receptor activity.

Four levels of regulation of glucocorticoid receptor (GR) activity have been investigated. (1) Phosphorylation of the GR was studied in NIH 3T3 cells metabolically labeled with [32P]orthophosphate. A highly specific antiserum against the GR was used to immunoprecipitate 32P-labeled GR, and protein blotting was used to determine the GR concentration. Comparison of the relative specific activities of non-activated and activated receptor revealed a 3-4-fold increase in GR phosphorylation within 60 min upon hormone activation. (2) The affinity of the GR for its hormone response element (GRE) was quantitated in in vitro binding and gel shift experiments. The comparison of monomers, dimers and trimers of the GRE showed that GR binding affinity to multimers is much higher than the affinity for a GRE monomer. (3) The concentration of the GR was determined in quantitative protein blot assays as a function of time after hormone treatment of NIH 3T3 cells. A down-regulation of GR was observed. Only 30% of the maximal GR concentration observed in the absence of hormone remained after 24 h of hormone treatment. (4) The effect of the presence of hormone on the subcellular location of the GR was studied. Hormone treatment and withdrawal experiments indicated that the presence of hormone is not only required to initiate the cascade of events resulting in transcriptional trans-activation. GR translocated to the nucleus upon hormone addition returns rapidly to the cytoplasm upon hormone withdrawal. This indicated an active role for the hormone in the tight nuclear binding of GR.