To learn more about the mechanism(s) by which the EC50 and percent partial agonist activity of GR complexes are modulated for glucocorticoid-regulated gene induction, we have been conducting our studies both with saturating and subsaturating concentrations of agonists and with saturating concentrations of antisteroids.
One aim of this project is to understand what causes an antagonist to be unable to induce gene expression. The conventional model is that bulky steroids are antisteroids because they prevent the folding of the ligand binding domain (LBD) into the conformation that permits strong binding of members of the p160 family of coactivators (SRC-1, TIF2/GRIP1, and AIB1/pCIP/ACTR/RAC3/TRAM1). By this criterion, deacylcortivazol (DAC) would be expected to be an antagonist due to the presence of the bulky phenylpyrazole group that is attached to the A-ring of the glucocorticoid-like steroid. In fact, DAC is about the most potent glucocorticoid agonist known (Simons Jr. et al., 1979, Biochem. Biophys. Res. Comm., 86, 793-800). In collaboration with Eric Xu (Van Andel Research Institute, Grand Rapids, MI), we have determined the x-ray structure of DAC bound to the GR ligand binding domain (LBD). The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 (SRC-1) reveals that the GR ligand binding pocket is continuously extended into the top half of the LBD, thereby expanding to a size of 1,070 3. This reorganization of the LBD effectively doubles the size of the GR dexamethasone-binding pocket of 540 3 and yet leaves the structure of the coactivator binding site intact. DAC occupies only 50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. This dramatic expansion of the DAC-binding pocket highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.? ? Guided by this X-ray structure of DAC bound to the GR LBD, we prepared several point mutants in the LBD that are found to have little effect on the binding of either DAC or the classical agonist steroid dexamethasone (Dex). However, these same mutations dramatically alter the Amax and/or EC50 of exogenous and endogenous genes in a manner that depends on steroid structure. In some cases, Dex is no longer a full agonist. These properties appear to result from a preferential inactivation of the AF2 activation domain in the GR LBD of Dex-bound, but not DAC-bound, receptors. The Dex-bound receptors display normal binding to, but a greatly reduced response to, the coactivator TIF2, thus indicating a defect in the transmission efficiency of GR-steroid complex information to the coactivator TIF2. This reduced efficacy of associated TIF2 is further demonstrated by a major reduction in the percent partial agonist activity of antisteroids. In addition, all GR mutants that are active in gene induction with either Dex or DAC have greatly reduced activity in gene repression. This contrasts with the reports of GR mutations preferentially suppressing GR-mediated induction. The properties of these GR mutants in gene induction support the hypothesis that the Amax and EC50 of GR-controlled gene expression can be independently modified, indicate that the receptor can be modified to favor activity with a specific agonist steroid, and suggest that new ligands with suitable substituents may be able to affect the same LBD conformational changes and thereby broaden the therapeutic applications of glucocorticoid steroids.? ? As a result of the above studies, we have gained new molecular information both about the determinants of glucocorticoid steroid activity and about the modulation of the dose-response curve of agonists and the percent partial agonist activity of antisteroids by cofactors. These modulatory factors permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.
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