Activated and non-activated glucocorticoid-receptor complexes in rat thymus cells: Kinetics of formation and relation to steroid structure

Abstract

Previously we showed that when glucocorticoids initially enter rat thymus cells incubated at 37°C. non-activated hormone-receptor complexes are formed within 15 s and are then rapidly replaced by activated complexes. Activated and non-activated forms were identified in cytosols from the cells by DEAE-cellulose chromatography, where they are eluted with progressively higher salt concentrations in what are referred to as Peaks I and II. On DNA-cellulose columns dexamethasone-receptor complexes in cytosols that on DEAE-cellulose give mainly Peak II (the non-activated complex, referred to as Complex II) are not bound significantly: they are eluted at the lowest salt concentration along with free steroid, from which they can be separated by binding to hydroxyapatite. This DNA-cellulose peak is referred to as Peak a. Complexes in cytosols that give only Peak I on DEAE, however, on DNA separate into two components. One of these (Complex Ia) is not bound significantly and appears in Peak a. The other (Complex Ib, presumably the true activated complex) appears in a later Peak b. These three complexes have been measured by first adsorbing Ib with DNA-cellulose, then separating Complexes Ia and II. which are not adsorbed on DNA. by means of a DEAE-cellulose column. When [ 3H]-dexamethasone is added to cells at 37°C. II is formed within 15 s and is rapidly replaced by Ib, in agreement with our earlier results. Complex Ia accounts for 15–20% of total complexes at all times, and is also present after 120 min at 0°C. Various steroids have been compared on DNA-cellulose after 30 min at 37°C. when the amount of Complex II is negligible. The ratio of Ib to Ia is highest for dexamethasone and triamcinolone acetonide, intermediate for cortisol and corticosterone and lowest for cortexolone (which seems to form only Ia), roughly in proportion to intrinsic glucocorticoid activity. Complex Ia may thus be a third normal form of extranuclear glucocorticoid-rcceptor complex, but the possibility remains that it is an artifact formed after cells are broken. The relation of Complexes Ia, Ib and II to glucocorticoid activity is discussed.