Analysis of Subunit Interactions in Steroid Receptors Using Chemical Cross-Linking

The question of subunit composition as well as the roles of different subunits within steroid receptors can be properly addressed by use of bifunctional reagents, covalent cross-linkers. Bisimidates were found most useful in experiments of this kind, owing to their specificity and the mild reaction conditions required for work with them. Oligomeric glucocorticoid receptor protein (GR) from different sources, when treated with dimethyl suberimidate, displayed hydrodynamic and ion exchange properties resembling those of the native non-transformed GR and was recognized by a monoclonal anti-GR antibody. At the same time, cross-linking prevented receptor activation upon heat or salt treatment as measured by DNA-cellulose binding or reduction in size. Heat inactivation of the cross-linked GR was much slower than that of the native receptor both in the unliganded and liganded states, either in the presence or absence of molybdate ions or KCl. However, if the oligomeric receptor was transformed by salt first, and treated with bisimidates thereafter, no protection against heat inactivation was seen. These results are in harmony with our recent data on chick oviduct progesterone receptor (Aranyi et al. Biochemistry, 27. 1330–1336, 1988). Our findings suggest that subunit interactions within oligomeric steroid receptor proteins stabilize the ligand binding site.