Modulation of induction properties of glucocorticoid receptor-agonist and -antagonist complexes by coactivators involves binding to receptors but is independent of ability of coactivators to augment transactivation

Coactivators such as TIF2 and SRC-1 modulate the positioning of the dose-response curve for agonist-bound glucocorticoid receptors (GRs) and the partial agonist activity of antiglucocorticoid complexes. These properties of coactivators differ from their initially defined activities of binding to, and increasing the total levels of transactivation by, agonist-bound steroid receptors. We now report that constructs of TIF2 and SRC-1 lacking the two activation domains (AD1 and AD2) have significantly less ability to increase transactivation but retain most of the activity for modulating the dose-response curve and partial agonist activity. Mammalian two-hybrid experiments show that the minimum TIF2 segment with modulatory activity (TIF2.4) does not interact with p300, CREB-binding protein, or PCAF, which also modulates GR activities. DRIP150 and DRIP205 have been implicated in coactivator actions but are unable to modulate GR activities. The absence of synergism by PCAF or DRIP150 with SRC-1 or TIF2, respectively, further suggests that these other factors are not involved. The ability of a TIF2.4 fragment (i.e. TIF2.37), which is not known to interact with proteins, to block the actions of TIF2.4 suggests that an unidentified binder mediates the modulatory activity of TIF2. Pull-down experiments with GST/TIF2.4 demonstrate a direct interaction of TIF2 with GR in a hormone-dependent fashion that requires the receptor interaction domains of TIF2 and is equally robust with agonists and most antiglucocorticoids. These observations, which are confirmed in mammalian two-hybrid assays, suggest that the capacity of coactivators such as TIF2 to modulate the partial agonist activity of antisteroids is mediated by the binding of coactivators to GR-antagonist complexes. In conclusion, the modulatory activity of coactivators with GR-agonist and -antagonist complexes is mechanistically distinct from the ability of coactivators to augment the total levels of transactivation and appears to involve the binding to both GR-steroid complexes and an unidentified TIF2-associated factor(s).     

Role of activation function domain-1, DNA binding, and coactivator GRIP1 in the expression of partial agonist activity of glucocorticoid receptor-antagonist complexes

The determinants of the partial agonist activity of most antisteroids complexed with steroid receptors are not well understood. We now examine the role of the N-terminal half of the glucocorticoid receptor (GR) including the activation domain (AF-1), the DNA binding site sequence, receptor contact with DNA, and coactivator binding on the expression of partial agonist activity in two cell lines for GRs bound by five antiglucocorticoids: dexamethasone mesylate (Dex-Mes), dexamethasone oxetanone (Dex-Ox), progesterone (Prog), deoxycorticosterone (DOC), and RU486. Using truncated GRs, we find that the N-terminal half of GR and the AF-1 domain are dispensable for the partial agonist activity of antiglucocorticoids. This contrasts with the AF-1 domain being required for the partial agonist activity of antisteroids with most steroid receptors. DNA sequence (MMTV vs a simple GRE enhancer) and cell-specific factors (CV-1 vs Cos-7) exert minor effects on the level of partial agonist activity. Small activity differences for some complexes of GAL4/GR chimeras with GR- vs GAL-responsive reporters suggest a contribution of DNA-induced conformational changes. A role for steroid-regulated coactivator binding to GRs is compatible with the progressively smaller increase in partial agonist activity of Dex-Mes > Prog > RU486 with added GRIP1 in CV-1 cells. This hypothesis is consistent with titration experiments, where low concentrations of GRIP1 more effectively increase the partial agonist activity of Dex-Mes than Prog complexes. Furthermore, ligand-dependent GRIP1 binding to DNA-bound GR complexes decreases in the order of Dex > Dex-Mes > Prog > RU486. Thus, the partial agonist activity of a given GR-steroid complex in CV-1 cells correlates with its cell-free binding of GRIP1. The ability to modify the levels of partial agonist activity through changes in steroid structure, DNA sequence, specific DNA-induced conformational changes, and coactivator binding suggests that useful variations in endocrine therapies may be possible by the judicious selection of these parameters to afford gene and tissue selective results.     

Modulation of transcriptional sensitivity of mineralocorticoid and estrogen receptors

Recent reports describe the ability of factors to modulate the position of the dose–response curve of receptor–agonist complexes, and the amount of partial agonist activity of receptor–antagonist complexes, of androgen, glucocorticoid (GRs), and progesterone receptors (PRs). We now ask whether this modulation extends to the two remaining steroid receptors: mineralocorticoid (MRs) and estrogen receptors (ERs). These studies of MR were facilitated by our discovery that the antiglucocorticoid dexamethasone 21-mesylate (Dex-Mes) is a new antimineralocorticoid with significant amounts of partial agonist activity. Elevated levels of MR, the co-activators TIF2 and SRC-1, and the co-repressor SMRT do modulate the dose–response curve and partial agonist activity of MR complexes. Interestingly, the precise responses are indistinguishable from those seen with GRs in the same cells. Thus, the unequal transactivation of common genes by MRs versus GRs probably cannot be explained by differential responses to changing cellular concentrations of homologous receptor, co-activators, or co-repressors. We also find that the dose–response curve of ER–estradiol complexes is left-shifted to lower steroid concentrations by higher amounts of exogenous ER. Therefore, the modulation of either the dose–response curve of agonists or the partial agonist activity of antisteroid, and in many cases the modulation of both properties, is a common phenomenon for all of the classical steroid receptors.     

Identification of constitutive and steroid-dependent transactivation domains in the mouse oestrogen receptor

We have identified two transactivation domains in the mouse oestrogen receptor whose activities depend on the target promoter. The major domain is contained within the C-terminal portion of the protein and depends upon oestrogen binding for its activity. The location and oestrogen dependence of this domain has been confirmed using chimaeric receptors containing the Lex A DNA binding domain. Although transactivation by the C-terminal domain is dependent upon ligand binding the analysis of receptor deletion mutants has demonstrated that these two functions are not entirely coincident. The second transactivation domain lies within the N-terminal region and is active in the absence of oestradiol. The differences in oestrogen requirement for the activity of the two transactivation domains may account for the partial agonist activity of certain antihormones.     

Effects of acetylation, polymerase phosphorylation, and DNA unwinding in glucocorticoid receptor transactivation

Varying the concentration of selected factors alters the induction properties of steroid receptors by changing the position of the dose-response curve (or the value for half-maximal induction=EC(50)) and the amount of partial agonist activity of antisteroids. We now describe a rudimentary mathematical model that predicts a simple Michaelis-Menten curve for the multi-step process of steroid-regulated gene induction. This model suggests that steps far downstream from receptor binding to steroid can influence the EC(50) of agonist-complexes and partial agonist activity of antagonist-complexes. We therefore asked whether inhibitors of three possible downstream steps can reverse the effects of increased concentrations of two factors: glucocorticoid receptors (GRs) and Ubc9. The downstream steps (with inhibitors in parentheses) are protein deacetylation (TSA and VPA), DNA unwinding (CPT), and CTD phosphorylation of RNA polymerase II (DRB and H8). None of the inhibitors mimic or prevent the effects of added GRs. However, inhibitors of DNA unwinding and CTD phosphorylation do reverse the effects of Ubc9 with high GR concentrations. These results support our earlier conclusion that different rate-limiting steps operate at low and high GR concentrations versus high GR with Ubc9. The present data also suggest that downstream steps can modulate the EC(50) of GR-mediated induction, thus both supporting the utility of our mathematical model and widening the field of biochemical processes that can modify the EC(50).