Conformational change in the human glucocorticoid receptor induced by ligand binding is altered by mutation of isoleucine 747 by a threonine

Limited proteolysis experiments were performed to study conformation change s induced by ligand binding on in vitro produced wild-type and 1747T mutant glucocorticoid receptors, Dexamethasone-induced conformational changes wer e characterized by two resistant proteolysis fragments of 30 and 27 kDa. Al though dexamethasone binding affinity was only slightly altered by the 1747 T substitution (Roux, S., Terouanne, B., Balaguer, P., Loffreda-Jausons, N. , Pons, M., Chambon, P., Gronemeyer, H., and Nicolas, J,-C, (1996) Mot. End ocrinol, 10, 1214-1226), higher dexamethasone concentrations were required to obtain the same proteolysis pattern. This difference was less marked whe n proteolysis experiments were conducted at 0 degrees C, indicating that a step of the conformational change after ligand binding was affected by the mutation. In contrast, RU486 binding to the wild-type receptor induced a di fferent conformational change that was not affected by the mutation. Analys is of proteolysis fragments obtained in the presence of dexamethasone or RU 486 indicated that the RU486-induced conformational change affected the C-t erminal part of the ligand binding domain differently. These data suggest t hat the ligand-induced conformational change occurs via a multistep process . In the first step, characterized by compaction of the ligand binding doma in, the mutation has no effect, The second step, which stabilizes the activ ated conformation and does not occur at 4 degrees C, seems to be a key elem ent in the activation process that can be altered by the mutation. This ste p could involve modification of the helix H12 position, explaining why the conformation induced by RU486 is not affected by the mutation.