Identification of Tamoxifen-Induced Coregulator Interaction Surfaces within the Ligand-Binding Domain of Estrogen Receptors

Abstract

Tamoxifen is a selective estrogen receptor (ER) modulator that is clinically used as an antagonist to treat estrogen-dependent breast cancers but displays unwanted agonistic effects in other tissues. Previous studies on ERα have delineated a role of the N-terminal activation function AF-1 in mediating the agonistic effects of tamoxifen, while the mechanisms for how ERβ mediates tamoxifen action remain to be elucidated. As peptides can be used to detect distinct receptor conformations and binding surfaces for coactivators and corepressors, we attempted in this study to identify previously unrecognized peptides that interact specifically with ERs in the presence of tamoxifen. We identified two distinct peptides among others that are highly selective for tamoxifen-bound ERα or ERβ. Domain mapping and mutation analysis suggest that these peptides recognize a novel tamoxifen-induced binding surface within the C-terminal ligand-binding domain that is distinct from the agonist-induced AF-2 surface. Peptide expression specifically inhibited transcriptional ER activity in response to tamoxifen, presumably by preventing the binding of endogenous coactivators. Moreover, tamoxifen-responsive and ER subtype-selective coactivators were engineered by replacing the LXXLL motifs in the coactivator TIF2 with either of the two peptides. Finally, our results indicate that related coactivators may act via the novel tamoxifen-induced binding surface, referred to as AF-T, allowing us to propose a revised model of tamoxifen agonism.

We thank P. Kushner and S. Nilsson for providing plasmids. We are grateful to members of the Receptor Biology Unit for sharing materials and stimulating discussions.

This work was supported by KaroBio AB (Huddinge, Sweden) and by the Swedish Cancer Society.