Equilibrium Interactions of Corepressors and Coactivators with Agonist and Antagonist Complexes of Glucocorticoid Receptors

Abstract Corepressors and coactivators can modulate the dose-response curve and partial agonist activity of glucocorticoid receptors (GRs) complexed with agonist and antagonist steroids, respectively, in intact cells. We recently reported that GR-antagonist complexes bind to the coactivator TIF2, (transcriptional intermediary factor 2), which is consistent with the whole-cell effects of coactivators being mediated by direct interactions with GR complexes. We now ask whether the whole-cell modulatory activity of corepressors also entails binding to both GR-agonist and -antagonist complexes and whether the association of corepressors and coactivators with GR complexes involves competitive equilibrium reactions. In mammalian two-hybrid assays with two different cell lines and in cell-free pull-down and whole-cell immunoprecipitation assays, the corepressors NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoid and thyroid hormone receptor) associate with agonist and antagonist complexes of GRs. Both N- and C-terminal regions of GR are needed for corepressor binding, which requires the CoRNR box motifs that mediate corepressor binding to other nuclear/steroid receptors. Importantly, whole-cell GR interactions with corepressors are competitively inhibited by excess coactivator and vice versa. However, the regions of the coactivator TIF2 that compete for GR binding to corepressor and coactivator are not the same, implying a molecular difference in GR association with coactivators and corepressors. Finally, when the whole-cell ratio of coactivators to corepressors is altered by selective cofactor binding to exogenous thyroid receptor β ± thyroid hormone, the GR dose-response-curve and partial agonist activity are appropriately modified. Such modifications are independent of histone acetylation. We conclude that mutually antagonistic equilibrium interactions of corepressors and coactivators modulate the dose-response curve and partial agonist activity of GR complexes in a manner that is responsive to the intracellular ratio of these two classes of cofactors. This modulation provides an attractive mechanism for differential control of gene expression during development, differentiation, homeostasis, and endocrine therapies. THE MECHANISM OF transcriptional activation by glucocorticoid hormones involves many associated proteins. A variety of heat shock/chaperone proteins form a foldosome that associates with glucocorticoid receptors (GRs) to allow steroid binding (1). After activation of the receptor-steroid complex, its migration into the nucleus, and its binding to glucocorticoid response elements (GREs), the DNA-bound receptor-steroid complexes are thought to recruit a variety of cofactors, including coactivators and corepressors, which exist as members of higher order complexes (2–5). These complexes appear to be replaced in a cyclical manner (6, 7) with a multiprotein complex called thyroid hormone receptor (TR)-associated protein/vitamin D receptor interacting protein/mediator that interacts with the RNA pol II complex to increase the rates of gene transcription (8, 9). Coactivators and corepressors are cofactors that have attracted considerable attention because they increase or decrease the total activity of most steroid-receptor complexes. Coactivators, such as the p160 family of coactivators steroid receptor coactivator 1 (10), transcriptional intermediary factor 2 (TIF2)/GR-interacting protein 1 (GRIP1) (11, 12), and amplified in breast cancer-1 (AIB1) (13) were originally defined as factors that increase the total amount of induced gene product with saturating, or pharmacological, concentrations of hormone (14–16). Conversely, corepressors such as nuclear receptor corepressor (NCoR) (17) and silencing mediator of retinoid and thyroid hormone receptor (SMRT) (18) are factors that are characterized by their ability to decrease the total amount of gene product. The prevailing model is that the nature of the ligand binding to receptors acts as a molecular switch, with agonist steroids causing both the dissociation of corepressors from ligand-free or antagonist-bound receptors and the association of coactivators (19–21). Corepressors were initially discovered on the basis of their ability to bind to ligand-free nuclear receptors, such as the thyroid receptor (17, 18). The conclusion that corepressors bind only to the nuclear receptors was modified when it was found that corepressors interact with antagonist-bound androgen (AR) (22, 23), estrogen (ER) (24, 25), GR (26), and progesterone (27, 28) receptors and often to the ligand-free steroid receptors (reviewed in Ref.29). The sites of interaction in steroid and nuclear receptors for both corepressors and coactivators have been identified to be in the ligand-binding domain (LBD); in fact, the two sites appear to overlap (30–34). Interestingly, corepressors also affect several biological properties of steroid receptor-agonist complexes such as the total amount of induced activity and the position of the dose-response curve (and the steroid concentration required for half-maximal induction, or EC50) (28, 29, 35–37). This suggests that whereas corepressors may not bind to agonist complexes of the nuclear receptors, there is at least a functional interaction with agonist complexes of the classical steroid receptors. The interactions of corepressors with steroid receptors are of additional importance because they may provide a mechanism for differentiating between the activated complexes of various steroid receptors (androgen, glucocorticoid, mineralocorticoid, and progestin) in a cell-specific manner. Whereas each receptor displays preferential binding of the naturally occurring ligands, each activated receptor-steroid complex is able to bind to the same hormone response element (HRE). Thus the specificity of ligand binding is lost due to the commonality of potential HREs. Nevertheless, overexpressed NCoR and SMRT produce diametrically opposite effects on both the dose-response curve of agonists and the partial agonist activity of antagonists bound to PRs vs. GRs for induction of the same gene in the same cells (37). Thus, corepressor interactions with DNA-bound steroid receptor complexes can restore some of the specificity that appears to be lost upon binding to common HREs. Furthermore, the effects of added corepressor on GR complexes are different in CV-1 vs. 1470.2 cells (36, 37). These results suggest that the observed effects of corepressors on GR transcriptional properties can be modified by tissue-specific factors. Finally, the corepressor SMRT can antagonize the ability of the coactivator TIF2 to modulate the position of the dose-response curve of GR-agonist complexes and the amount of partial agonist activity of GR-antagonist complexes (36). These findings led us to propose an equilibrium model in which coactivators and corepressors each interact with both agonist- and antagonist-b (...truncated)