Characterization and immunocytochemical demonstration of glucocorticoid receptor using antisera specific to transformed receptor

Cytosolic and nuclear forms of the glucocorticoid receptor were characterized using immunochemical techniques. Antibodies were raised in rabbits to an M r 58,000 fragment of the transformed (DNA-binding) glucocorticoid receptor purified from rat liver cytosol by DNA-cellulose chromatography and polyacrylamide gel electrophoresis. Antibodies reacted with the transformed receptor form in a radioimmunoassay for glucocorticoid receptor. Western blot analysis of antibody reactivity revealed a single M r 185,000 receptor form in rat liver cytosol but a smaller M r 85,000 form in nucleosol, indicating the M r 85,000 form is the transformed receptor. Furthermore, western blot analysis indicates that the M r 185,000 receptor undergoes proteolysis during receptor purification and in vitro transformation processes by generating immunochemically similar proteins of smaller molecular weights. An identical M r 185,000 glucocorticoid receptor was detected in cytosols of four rat tissues; liver, brain, adrenal medulla, and thymus. The glucocorticoid receptor was localized to the cytoplasm and nucleus of rat adrenal medulla cells by immunohistochemistry, demonstrating the existence in vivo of the transformed receptor and translocation of the receptor from cytoplasm to nucleus.