Transactivation Function-2 of Estrogen Receptor α Contains Transactivation Function-1-regulating Element

Abstract ERα has a ligand dependent transactivation function in the ligand binding domain of ERα C-terminus (AF-2) and a ligand independent activation function in the N-terminus (AF-1). It is still not fully understood how AF-1 and AF-2 activities are regulated cooperatively by ligands. To evaluate the AF-1 involvement in the estrogenic activities of various compounds, we analyzed these transactivation functions using AF-1 truncated and AF-2 mutated ERα mutants. AF-2 is composed of two domains with flexible and static regions. We used an AF-2 flexible region mutant and an AF-2 static region mutant. Both mutants have been reported as non-E2 responsive, due to disruption of E2-mediated coactivator recruitment to the AF-2. The AF-2 mutants were not activated by agonists but surprisingly antagonists and SERMs activated the AF-2 mutants. This antagonist reversal activity was derived from AF-1. Furthermore, we demonstrated that the AF-2 contains an AF-1 suppression function using C-terminal truncated ERα mutants. From these findings, we hypothesized that the mutation of AF-2 disrupted its ability to suppress AF-1 causing the antagonist reversal. To assess the AF-2 mediated AF-1 suppression, we analyzed the transcription activity of physically separated AF-1 and AF-2 using a novel hybrid reporter assay. We observed that the AF-1 activity was not suppressed by the physically separated AF-2. Furthermore, SERMs did not induce the AF-1 mediated activity from the separated mutant AF-2, that differed from the intact protein. These results imply that SERM activity is dependent on a conformational change of the full-length ERα molecule which allows for AF-1 activation.