The corepressors NCoR and SMRT have been documented to have opposite effects on the EC50 of agonists, and the PAA, for GR and PR induction of the same gene in the same cells. These inverted responses depend upon the joint actions of the N- and C-terminal domains of each receptor (Song et al., 2001, J. Biol. Chem., 276, 24806-24816). These results are consistent with the demonstration that corepressors interact with N-terminal regions of both GRs and PRs (Wang et al., 2007, Biochemistry, 48, 8036-8049;Wang and Simons Jr., 2005, Mol. Endo., 19, 1483-1500) in addition to the initially defined sites in the C-terminal sequences of receptors. The importance of cofactor binding to the N-terminal region of GR has recently been confirmed in our collaboration with Dr. Raj Kumar (The Commonwealth Medical College, PA), where we obtained the first biophysical evidence of the association of the amino terminal fragments of GR and the coactivator TIF2. Similarly, four other factors known to modulate GR activity (GME, GMEB2, Ubc9, and STAMP) were recently found either to differentially alter several induction parameters of GRs vs. PRs under otherwise identical conditions or to require different regions of each receptor for their activities (Szapary et al., 2008, Mol Cell Endocrinol, 283, 114-126). The objective of this study is to determine whether the mechanisms of action for those factors known to differentially modulate the EC50, PAA, and Amax of GR- regulated gene transcription change or remain the same during PR-regulated gene transcription. This task has just become tractable with our development of a theoretical model of steroid receptor action (see DK DK057800-21). Three novel features of this model, and its associated graphical analysis, permit an unprecedented level of mechanistic information regarding steroid receptor-regulated gene transactivation. First, it is now possible to determine the kinetically-defined type of action being displayed by the factor (competitive inhibitor, uncompetitive inhibitor, coactivator, etc.). Second, it is usually possible to define where the factor acts relative to a reference point called the concentration limiting step (CLS), which is the steady state analog of the rate limiting step of enzyme kinetics. Third, the model and its graphical analysis have recently been extended to the analysis of two competing factors in the same assay. As opposed to making the situation more obscure, this competition assay actually yields greater mechanistic information. Not only can such competition assays determine how and where each factor acts, relative to the CLS, but the site of action of the two factors relative to each other is usually revealed. Thus, one can now assemble an ordered sequence of reactions based on the biological function of cofactors, much as in epistasis analysis, even when the biochemical properties of the cofactors are not known. Initial experiments with a several cofactors have not exposed any major differences between GR and PR action. However, recent studies in our collaboration with Dr. David Levens (NCI, NIH) have uncovered one cofactor that differentially affects GR in a cell-specific manner. Studies are ongoing as to whether the effects on PR gene induction are the same or different in the various cells. Much of this information is not now available by any other means. Therefore, these studies will greatly contribute to our long-term goal of defining the action of GRs vs. PRs at a molecular level and of understanding their role in human physiology.
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