Mechanistic Principles in NR Box-Dependent Interaction between Nuclear Hormone Receptors and the Coactivator TIF2

ABSTRACT

Nuclear hormone receptors exert transcriptional activation of target genes upon hormone induction via interactions with the basal transcription machinery. This interaction is mediated by cofactors which physically bind to receptors, thereby acting as coactivators or corepressors leading to activation or repression, respectively. Here we report the screening for and cloning of a peroxisome proliferator receptor-interacting protein, the rat homolog of TIF2. By sequence comparison with the related coactivator SRC-1, we identified three short conserved motifs (NR boxes) in both proteins which are the putative binding sites of TIF2 to nuclear hormone receptors. We demonstrate here by generation of amino acid exchanges within the NR boxes that all three boxes located in the receptor interaction domain of TIF2 are necessary and sufficient for interaction. The three boxes individually can bind to hormone receptors but display preferences in binding for certain receptors. In addition, we show that the interaction domain of TIF2 can compete with other AF-2-dependent cofactors for binding to receptors. Finally, we demonstrate cooperative binding of two TIF2 molecules to a heterodimeric nuclear receptor complex even in the presence of only one cognate ligand, indicating an allosteric effect on the heterodimeric partner upon coactivator binding.

ACKNOWLEDGMENTS

We thank M. G. Parker, B. W. O’Malley, and S. Nilsson for generously providing constructs. We are grateful to Dorothee Feltkamp and Franziska Wiebel for providing material, and we thank all members of the orphan receptor group for helpful discussions, especially AnneMarie Witte for excellent technical support.

J.L. receives a grant from the European Community (MCFA). E.T. obtained a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. This work was supported by a grant from the Swedish Cancer Society.

ADDENDUM

During the process of review of this paper, similar results regarding the binding preferences of different NR boxes within TIF2 and SRC-1 to nuclear receptors were reported independently (12a, 21a, 40a).