Heterogeneity of the glucocorticoid receptors: molecular transformations during activation, detected by electrofocusing.
Isoelectric focusing (IEF) of glucocorticoid receptor (GR) of the neural retina of the 14-day chick embryo was conducted under conditions that yielded quantitative recovery of binding activity. IEF of the cytosol, equilibrated with [3H]triamcinolone acetonide (TA) at 0-2 degrees C yielded three major TA-GR components with apparent isoelectric points (pI') of 5.4 +/- 0.3, 6.5 +/- 0.2, and 7.6 +/- 0.3, designated as I, II, and III, respectively. During temperature-induced activation (incubation at 30 degrees C for 60 min, in the presence of free [3H]TA and 0.15 M KCl), approximately 25% of the specifically bound TA was irreversibly lost. IEF reveals that this loss is accounted for by the complete loss of binding from I. During activation, II also decreases but correspondingly III increases, i.e., the sum of II and III remains unchanged. Only the bound TA of I is sensitive to the addition of KCl (a promoter of activation). This sensitivity of I is temperature dependent. Molybdate (an inhibitor of activation) protects the bound TA of I and suppresses the formation of III. These two effects of molybdate diminish simultaneously when the temperature is increased to 30 degrees C. III preferentially exhibits binding activity to nuclei. The data suggest that (i) the glucocorticoid-free cytosol contains two GRs, I and II, with possibly two different functions; (ii) activation involves the loss of bound TA from I and the transformation of II to III with increased pI; (iii) these two molecular events in GR activation are interdependent.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002642:Chick Embryo; D003600:Cytosol / Q000378:metabolism; D007525:Isoelectric Focusing; D011487:Protein Conformation; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification*; D011987:Receptors, Steroid / Q000302:isolation & purification*; D012160:Retina / Q000378:metabolism; D013696:Temperature
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid