Dexamethasone‐Binding Proteins in Cytosol and Nucleus of Rat Thymocytes

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Abstract
Dexamethasone-binding proteins from the cytosol and the nucleus of rat thymocytes were analyzed by ion-exchange chromatography on DEAE-cellulose. Three dexamethasone-binding proteins were revealed in cytosol, in the flow-through (DE-1) and 2 (DE-2 and DE-3) eluting from the column with 0.13 M and 0.23 M NH4Cl, respectively. In nuclear extracts, only 1 receptor fraction, present in the flow-through, could be detected. By a combination of affinity chromatography on Cl-Sepharose to which dexamethasone 21-methanesulfonate was linked through a disulfide bond and DEAE-cellulose chromatography. 3 receptor proteins were highly purified from cytosol, with MW of 45,000, 72,000 and 90,000 and 1 from nuclear extracts with MW of 72,000. Antibodies to the 45,000-MW and 90,000-MW proteins were elicited in rabbits. The antibodies to the 45,000-MW protein cross-react with the 90,000-MW protein. The antibodies to the 90,000-MW protein cross-react with the 45,000-MW protein. Antibodies to either of the 2 proteins immunoprecipitate 60-70% of the dexamethasone-binding activity of rat thymus cytosol. Immunoaffinity chromatography of cytosol and nucleosol on columns of Sepharose linked to the IgG against either the 45,000-MW or the 90,000-MW protein leads to binding of these proteins on the columns but not of the 72,000-MW species. Two nuclear polypeptides with MW of 36,000 and 38,000 remain attached to the immunoaffinity column; these polypeptides may represent degradation products of the cytoplasmic receptor upon entrance into the nucleus. Antibodies against 2 dexamethasone-binding proteins from rat liver cytosol immunoprecipitate the 45,000-MW and 90,000-MW cytosol receptors from rat thymus.