Heterogeneity of nuclear glucocorticoid receptor interactions.

When thymocytes are incubated with glucocorticoids at 37 degrees, 60--70% of the receptor bound steroid is associated with the nucleus. Under conditions where the rate of steroid-receptor formation is not limiting the transfer of steroid-receptors from the cytoplasm to the nucleus occurs rapidly with a T 1/2 of 30 seconds. These observations have led us to investigate whether or not all glucocorticoid receptor complexes are associated with the nucleus in the same manner. To this end, nuclear glucocorticoid-receptor complexes have been extracted by differential salt extraction and DNase I and DNase II digeston. Of the nuclear dexamethasone receptor complex initially bound, 70--75% is resistant to 0.2 M KCl extraction (designated N2) and 25--30% is resistant to 0.4 extraction (designated N4). N2 can be further extracted with 0.4 M KCl whereas N4 is resistant to reextraction with either 0.2 M KCl, suggesting that N2-N4 (N2-4) and N4 represent distinct physical forms of nuclear dexamethasone receptor. In intact cells, N2 and N4 differ under the following physiological condition. (1) N4 binding occurs prior to N2-4; (2) a cold chase of unlabeled dexamethasone decreases N2-4 by 70% but N4 binding by only 10%; (3) N4 binding decreases more rapidly than N2-4 following a decrease in hormone concentration by dilution; (4) a cold chase of either cortexolone or progesterone preferentially decreases N2-4 and has little effect on N4. In addition, the nuclear N2-4 and N4 distribution differ for cortisol, dexamethasone and triamcinolone acetonide, three steroids having different in vitro biological potencies. DNase I treatment of nuclei solubilizes approximately 60% of nuclear DNA yet releases only 20--30% of nuclear receptor, whereas DNase II solubilizes only 10% of nuclear DNA and releases 76--80% of nuclear receptor. As seen with salt extraction, the resistance of nuclear glucocorticoid-receptor complexes to a DNase I and II is dependent on the steroid molecule which is associated with the receptor. Of the steroids we have tested, nuclear triamcinolone acetonide and dexamethasone receptor complexes are most resistant to nuclease attack. Nuclear cortisol receptor complexes are readily solubilized by either DNase I or II under conditions where little dissociation of steroid from receptor occurs. These data represent evidence for physiologically distinct forms of nuclear glucocorticoid receptor interaction. In addition, they demonstrate the importance of the steroid portion of the steroid receptor in directing the nature and/or location of steroid receptors within or on the nucleus.