Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor α

In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ER alpha) in an agonist-dependent manner. The interaction is specific for ER alpha as ERP and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ER alpha was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ER alpha is sufficient for interaction with RAP80. RAP80 overexpression reduces ER alpha polyubiquitination, increases the level of ER alpha protein, and enhances ER alpha-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ER alpha protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UlMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UlMs does not affect the ability of RAP80 to interact with ER alpha, but eliminates the effects of RAP80 on ER alpha polyubiquitination, the level of ER alpha protein, and ER alpha-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ER alpha as a new RAP80- interacting protein and suggests that RAP80 may be an important modulator of ER alpha activity.