Coregulators for nuclear receptors (NR) are factors that either enhance or repress their transcriptional activity. Both coactivators and corepressors have been shown to use similar but functionally distinct NR interacting determinants containing the core motifs LxxLL and ⌽xx⌽⌽, respectively. These interactions occur through a hydrophobic cleft located on the surface of the ligand-binding domain (LBD) of the NR and are regulated by ligand-dependent activation function 2 (AF-2). In an effort to identify novel coregulators that function independently of AF-2, we used the LBD of the orphan receptor RVR (which lacks AF-2) as bait in a yeast twohybrid screen. This strategy led to the cloning of a nuclear protein referred to as CIA (coactivator independent of AF-2 function) that possesses both repressor and activator functions. Strikingly, we observed that CIA not only interacts with RVR and Rev-ErbA␣ in a ligand-independent manner but can also form complexes with estrogen receptor alpha (ER␣) and ER␤ in vitro and enhances ER␣ transcriptional activity in the presence of estradiol (E 2 ). CIA-ER␣ interactions were found to be independent of AF-2 and enhanced by the antiestrogens EM-652 and ICI 182,780 but not by 4-hydroxytamoxifen and raloxifene. We further demonstrate that CIA-ER␣ interactions require the presence within CIA of a novel bifunctional NR recognition determinant containing overlapping LxxLL and ⌽xx⌽⌽ motifs. The identification and functional characterization of CIA suggest that hormone binding can create a functional coactivator interaction interface in the absence of AF-2.Nuclear receptors belong to a superfamily of transcription factors that modulate hormone-regulated physiological pathways involved in reproduction, development, growth, and metabolism (38). Members of the nuclear receptor superfamily have been shown to possess the dual ability to activate and repress the expression of target genes through the recruitment of coactivators and corepressors (reviewed in references 21 and 41). These regulatory proteins associate mostly in a liganddependent manner with the ligand-binding domain (LBD) of the receptor. A short helical motif that is located at the C-terminal end of the LBD and is referred to as activation function 2 (AF-2) has been shown to play a central role in coregulator-receptor interaction, as its integrity is essential for ligand-dependent coactivator binding (12, 18, 31, 50) whereas its deletion favors corepressor binding (52,66). Comparative analysis of the crystal structures of several unliganded and liganded nuclear receptors has revealed that the AF-2 helix appears to take a distinct configuration in the presence of ligand, suggesting that ligand binding modified the conformation of the LBD and promotes the recruitment of coactivators through the formation of a novel interacting surface (reviewed in reference 42).A large number of coactivators have been characterized to date. These proteins generally possess multiple functional domains which cooperate to maximize receptor activity thr...