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Review
. 2012 Jan 30;348(2):373-82.
doi: 10.1016/j.mce.2011.05.030. Epub 2011 Jun 1.

The road less traveled: new views of steroid receptor action from the path of dose-response curves

S Stoney Simons Jr  1 , Carson C Chow
Affiliations
Review

The road less traveled: new views of steroid receptor action from the path of dose-response curves

S Stoney Simons Jr et al. Mol Cell Endocrinol. .

Abstract

Conventional studies of steroid hormone action proceed via quantitation of the maximal activity for gene induction at saturating concentrations of agonist steroid (i.e., A(max)). Less frequently analyzed parameters of receptor-mediated gene expression are EC(50) and PAA. The EC(50) is the concentration of steroid required for half-maximal agonist activity and is readily determined from the dose-response curve. The PAA is the partial agonist activity of an antagonist steroid, expressed as percent of A(max) under the same conditions. Recent results demonstrate that new and otherwise inaccessible mechanistic information is obtained when the EC(50) and/or PAA are examined in addition to the A(max). Specifically, A(max), EC(50), and PAA can be independently regulated, which suggests that novel pathways and factors may preferentially modify the EC(50) and/or PAA with little effect on A(max). Other approaches indicate that the activity of receptor-bound factors can be altered without changing the binding of factors to receptor. Finally, a new theoretical model of steroid hormone action not only permits a mechanistically based definition of factor activity but also allows the positioning of when a factor acts, as opposed to binds, relative to a kinetically defined step. These advances illustrate some of the benefits of expanding the mechanistic studies of steroid hormone action to routinely include EC(50) and PAA.

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Figures

Fig. 1
Fig. 1
Graphical evaluation of Amax, EC50, and PAA. (A) Raw data for agonist steroid induction of a luciferase reporter gene under two conditions (A and B). The position of the EC50 under each condition is indicated by the dashed vertical line. The maximum plateau value of luciferase activity for each condition is labeled Amax. (B) Normalized data for agonist steroid induction of a luciferase reporter gene under two conditions (A and B). The data of panel A are expressed as percent of maximal activity (Amax) under the same condition. (C) Raw data for induction of a luciferase reporter gene without or with saturating concentrations of agonist or antagonist steroid under two conditions (A and B). (D) Normalized data for agonist and antagonist steroid induction of a luciferase reporter gene under two conditions (A and B). The data of panel C are expressed as percent of maximal activity (Amax) under the same condition.
Fig. 2
Fig. 2
Level of coactivator TIF2 differentially affects the induction parameters of endogenous genes in PBMCs. (A) Transient transfection of TIF2 siRNA for 48 hr reduces endogenous TIF2 protein relative to control (Lamin siRNA). Western blotting with anti-TIF2 antibody reveals the level of endogenous TIF2 after the indicated treatments. (B) Reduced cellular levels of TIF2 alter the fold induction, EC50, and PAA of the antiglucocorticoid Dex-Mes (DM) for endogenous genes. The average values (± S.E.M.) of fold induction (approximately equal to Amax because uninduced levels are very similar), EC50, and PAA (% DM) activity of gene induction in PBMCs treated with TIF2 (+) or Lamin (−) siRNA for three endogenous genes from 6–7 independent experiments are plotted. Two-tailed p-values are * P=0.038, ** P=0.0008 (from Luo and Simons; Jr., 2009).
Fig. 3
Fig. 3
GR LBD mutations modify factor binding to GR much less than the transcriptional activity of GR-bound factor. (A) Structures of Dex and DAC. (B–D) Changes in induction parameters with receptor mutation for induction of exogenous reporter genes by different forms of GR plus added cofactors in transiently transfected CV-1 cells. EC50 of GREtkLUC (S.E.M., n = 5–8) and MMTVLUC (S.E.M., n = 2) reporters induced by full length GR in (B) is altered by mutations in LBD in a manner that is sensitive to agonist structure (Dex vs. DAC) with no added cofactor. In (C), the EC50 of a GAL-regulated reporter (FRluc) induced by wild type and mutant GAL/GR525C receptors plus Dex or DAC is selectively modified by the presence of cotransfected TIF2 (S.E.M., n = 5). Similarly, in (D), the Amax, PAA of the antiglucocorticoid DM vs. Dex, and EC50 of the FRluc reporter induced by wild type and mutant GAL/GR525C receptors plus Dex or DAC is differentially sensitive to the presence of cotransfected Ubc9 (S.E.M., n = 7). Two-tailed p-values vs. wt GR are * P<0.05, ** P<0.005, *** P<0.0005. (E) Variation in induction parameters of endogenous genes by wild type and mutant full length GRs in U2OS cells with and without exogenous Ubc9 depends upon steroid structure (Dex, DM, or DAC; S.E.M., n = 5–7 with Dex and DM and n = 4 with DAC). (from Tao et al., 2008, Lee and Simons; Jr., 2011)
Fig. 3
Fig. 3
GR LBD mutations modify factor binding to GR much less than the transcriptional activity of GR-bound factor. (A) Structures of Dex and DAC. (B–D) Changes in induction parameters with receptor mutation for induction of exogenous reporter genes by different forms of GR plus added cofactors in transiently transfected CV-1 cells. EC50 of GREtkLUC (S.E.M., n = 5–8) and MMTVLUC (S.E.M., n = 2) reporters induced by full length GR in (B) is altered by mutations in LBD in a manner that is sensitive to agonist structure (Dex vs. DAC) with no added cofactor. In (C), the EC50 of a GAL-regulated reporter (FRluc) induced by wild type and mutant GAL/GR525C receptors plus Dex or DAC is selectively modified by the presence of cotransfected TIF2 (S.E.M., n = 5). Similarly, in (D), the Amax, PAA of the antiglucocorticoid DM vs. Dex, and EC50 of the FRluc reporter induced by wild type and mutant GAL/GR525C receptors plus Dex or DAC is differentially sensitive to the presence of cotransfected Ubc9 (S.E.M., n = 7). Two-tailed p-values vs. wt GR are * P<0.05, ** P<0.005, *** P<0.0005. (E) Variation in induction parameters of endogenous genes by wild type and mutant full length GRs in U2OS cells with and without exogenous Ubc9 depends upon steroid structure (Dex, DM, or DAC; S.E.M., n = 5–7 with Dex and DM and n = 4 with DAC). (from Tao et al., 2008, Lee and Simons; Jr., 2011)
Fig. 3
Fig. 3
GR LBD mutations modify factor binding to GR much less than the transcriptional activity of GR-bound factor. (A) Structures of Dex and DAC. (B–D) Changes in induction parameters with receptor mutation for induction of exogenous reporter genes by different forms of GR plus added cofactors in transiently transfected CV-1 cells. EC50 of GREtkLUC (S.E.M., n = 5–8) and MMTVLUC (S.E.M., n = 2) reporters induced by full length GR in (B) is altered by mutations in LBD in a manner that is sensitive to agonist structure (Dex vs. DAC) with no added cofactor. In (C), the EC50 of a GAL-regulated reporter (FRluc) induced by wild type and mutant GAL/GR525C receptors plus Dex or DAC is selectively modified by the presence of cotransfected TIF2 (S.E.M., n = 5). Similarly, in (D), the Amax, PAA of the antiglucocorticoid DM vs. Dex, and EC50 of the FRluc reporter induced by wild type and mutant GAL/GR525C receptors plus Dex or DAC is differentially sensitive to the presence of cotransfected Ubc9 (S.E.M., n = 7). Two-tailed p-values vs. wt GR are * P<0.05, ** P<0.005, *** P<0.0005. (E) Variation in induction parameters of endogenous genes by wild type and mutant full length GRs in U2OS cells with and without exogenous Ubc9 depends upon steroid structure (Dex, DM, or DAC; S.E.M., n = 5–7 with Dex and DM and n = 4 with DAC). (from Tao et al., 2008, Lee and Simons; Jr., 2011)
Fig. 4
Fig. 4
Graphical analysis of how and where cofactors modulate GR transactivation. (A) Decision tree for determining factor mechanism from plots of Amax and EC50. The decision point “Approaches G” means that if the non-linear plot of EC50/Amax approaches a plateau value G at infinite factor concentration (or Amax/EC50 approaches 1/G), then one needs to determine the nature of the plot of the reciprocal of [(Amax/EC50) – (1/G)]. (B) Determination of TIF2 coactivator properties. The Amax and EC50 values for wild type GR transactivation of GREtkLUC with different concentrations of co-transfected TIF2 in U2OS cells were generated by a curve-fitting program (Kalediagraph, Synergy Software). These values were then used to plot Amax/(100×EC50). The straight line represents the computer-generated best fit (R2 = 0.94). The insert in B shows the Western blots with anti-TIF2 antibody of lysates from cells that had been transfected under the parallel conditions with 0 or 20 ng of TIF2 plasmid. From these data, it can be determined that the true y-axis is as indicated by the dashed vertical line (see text for details). (from Chow et al., 2011)
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References

    1. Biggadike K, Bledsoe RK, Coe DM, Cooper TW, House D, Iannone MA, Macdonald SJ, Madauss KP, McLay IM, Shipley TJ, Taylor SJ, Tran TB, Uings IJ, Weller V, Williams SP. Design and x-ray crystal structures of high-potency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor. Proc Natl Acad Sci U S A. 2009;106:18114–18119. - PMC - PubMed
    1. Bledsoe RK, Montana VG, Stanley TB, Delves CJ, Apolito CJ, McKee DD, Consler TG, Parks DJ, Stewart EL, Willson TM, Lambert MH, Moore JT, Pearce KH, Xu HE. Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell. 2002;110:93–105. - PubMed
    1. Cho S, Kagan BL, Blackford JA, Jr, Szapary D, Simons SS., Jr Glucocorticoid receptor ligand binding domain is sufficient for the modulation of glucocorticoid induction properties by homologous receptors, coactivator transcription intermediary factor 2, and Ubc9. Mol. Endo. 2005;19:290–311. - PubMed
    1. Chow CC, Ong KM, Dougherty EJ, Simons SS., Jr Inferring mechanisms from dose-response curves. Methods in Enzymology. 2011 in press. - PMC - PubMed
    1. Clarkson MW, Gilmore SA, Edgell MH, Lee AL. Dynamic coupling and allosteric behavior in a nonallosteric protein. Biochemistry. 2006;45:7693–7699. - PMC - PubMed

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