Abstract
Limited proteolysis experiments were performed to study conformation changes induced by ligand binding on in vitro produced wild-type and I747T mutant glucocorticoid receptors. Dexamethasone-induced conformational changes were characterized by two resistant proteolysis fragments of 30 and 27 kDa. Although dexamethasone binding affinity was only slightly altered by the I747T substitution (Roux, S., Térouanne, B., Balaguer, P., Loffreda-Jausons, N., Pons, M., Chambon, P., Gronemeyer, H., and Nicolas, J.-C. (1996) Mol. Endocrinol. 10, 1214-1226), higher dexamethasone concentrations were required to obtain the same proteolysis pattern. This difference was less marked when 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 different conformational change that was not affected by the mutation. Analysis of proteolysis fragments obtained in the presence of dexamethasone or RU486 indicated that the RU486-induced conformational change affected the C-terminal part of the ligand binding domain differently. These data suggest that the ligand-induced conformational change occurs via a multistep process. In the first step, characterized by compaction of the ligand binding domain, the mutation has no effect. The second step, which stabilizes the activated conformation and does not occur at 4 degrees C, seems to be a key element in the activation process that can be altered by the mutation. This step could involve modification of the helix H12 position, explaining why the conformation induced by RU486 is not affected by the mutation.
Highlights
In its inactivated form, the glucocorticoid receptor (GR),1 a member of the steroid receptor family [1,2,3,4], is part of a large heterooligomeric complex that includes hsp90 and other heat shock proteins [5]
The 30- and 27-kDa fragments contained all or part of the ligand binding domain (LBD), as previously reported for various nuclear receptors because they were obtained with a ⌬A/B-GRwt and were recognized by a specific antibody directed against the C-terminal region of this domain
Conformational changes observed after ligand binding are assumed to induce a decrease in the accessibility of cleavage sites within the receptor molecule, which can be detected by modifications of proteolysis patterns
Summary
In its inactivated form, the glucocorticoid receptor (GR),1 a member of the steroid receptor family [1,2,3,4], is part of a large heterooligomeric complex that includes hsp90 and other heat shock proteins [5]. We recently described a mutant (substitution of human GR isoleucine 747 by a threonine, hereafter referred to as I747T) with no significant alteration of ligand binding affinity but which required higher dexamethasone concentrations than the wild-type GR (wt GR) to induce reporter gene transactivation [42].
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