To learn more about the mechanism(s) by which the EC50 and 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. The two immediate aims of this project are 1) to understand what causes an antagonist to be unable to induce gene expression and 2) to examine the mechanism by which Ubc9 modulates GR induction properties. Ubc9 is the human homolog of the E2 ubiquitin-conjugating enzymes of yeast that can transfer an ubiquitin-like molecule, called small ubiquitin-like modifier-1 (SUMO-1), to proteins in vertebrate cells. However, Ubc9 also displays non-enzymic activities, such as the modulation of the EC50 and partial agonist activity of GR complexes (Kaul et al., 2002, J. Biol. Chem., 277, 12541-12549; Cho et al., 2005, Mol. Endo., 19, 290-311). ? ? With regard to the first aim, 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, TIF2GRIP1, and AIB1pCIPACTRRAC3TRAM1). 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). Guided by this structure, we have prepared several point mutants in the LBD that would be expected to preferentially alter Dex vs. DAC binding, or visa versa. Some of the mutants eliminate both Dex binding, as determined by Scatchard analysis, and DAC binding, as determined by the inability of the mutant receptors to activate a transiently transfected reporter gene, GREtkLUC. Surprisingly, other mutations have little effect on the binding of either Dex or DAC but dramatically alter the Vmax andor EC50 of exogenous and endogenous genes in a manner that depends on steroid structure. This response, in which Dex is no longer a full agonist for glucocorticoid activity, is currently being actively investigated as it offers a window into the determinants of agonist vs. antagonist activity. Furthermore, the properties of these GR mutants support our hypothesis that the Vmax and EC50 of GR-mediate gene expression can be independently modified (Kim et al., 2006, J. Steroid Biochem. Molec. Biol., 100, 3-17).? ? With regard to the second aim, the mechanism of Ubc9 modulatory action, we are conducting a two-pronged attack: identifying Ubc9 deletionsmutations that eliminate Ubc9 modulatory activity and using ChIP assays to examine the recruitment of potential transcriptional cofactors. Given the small size of Ubc9 (158 amino acids), it is perhaps not surprising that most deletion mutants were not only inactive but also poorly expressed if at all. We are currently examining whether selected point mutations that appear to influence the modulatory activity show any change in their ability to bind to GR in pull-down assays. At the same time, we are conducting ChIP assays under the same conditions of transiently transfected proteins and reporter gene that afford changes in the EC50 and partial agonist activity of GR complexes. In this manner, we can directly compare the results of the two assays. We have found that Ubc9 also modulates the EC50 and partial agonist activity of GR-mediated induction of an endogenous gene. After obtaining the ChIP results with the transiently transfected reporter gene, we will determine whether the same behavior is maintained with the endogenous induced gene.? ? As a result of the above studies, we have gained new molecular information both about the determinants of antiglucocorticoid activity and about the modulation of the dose-response curve of agonists and the 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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