Evidence for a common step in three different processes for modulating thekinetic properties of glucocorticoid receptor-induced gene transcription

The dose-response curve of steroid hormones and the associated EC50 value a re critical parameters both in the development of new pharmacologically act ive compounds and in the endocrine therapy of various disease states. We ha ve recently described three different variables that can reposition the dos e-response curve of agonist-bound glucocorticoid receptors (GRs): a 21-base pair sequence of the rat tyrosine aminotransferase gene called a glucocort icoid modulatory element (GME), GR concentration, and coactivator concentra tion, At the same time, each of these three components was found to influen ce the partial agonist activity of antiglucocorticoids. In an effort to det ermine whether these three processes proceed via independent pathways or a common intermediate, we have examined several mechanistic details. The effe cts of increasing concentrations of both GR and the coactivator TIF2 are fo und to be saturable. Furthermore, saturating levels of either GR or TIF2 in hibit the ability of each protein, and the GME, to affect further changes i n the dose-response curve or partial agonist activity of antisteroids. This competitive inhibition suggests that all three modulators proceed through a common step involving a titratable factor. Support for this hypothesis co mes from the observation that a fragment of the coactivator TIF2 retaining intrinsic transactivation activity is a dominant negative inhibitor of each component (GME, GR, and coactivator). This inhibition was not due to nonsp ecific effects on the general transcription machinery as the VP16 transacti vation domain was inactive. The viral protein E1A also prevented the action of each of the three components in a manner that was independent of E1A's ability to block the histone acetyltransferase activity of CBP. Collectivel y, these results suggest that three different inputs (GME, GR, and coactiva tor) for perturbing the dose-response curve, and partial agonist activity, of GR-steroid complexes act by converging at a single step that involves a limiting factor prior to transcription initiation.