Fulvestrant (ICI 182,780)-dependent interacting proteins mediate immobilization and degradation of estrogen receptor-α

The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-alpha(ER alpha) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ER alpha interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)ER alpha-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ER alpha-interacting proteins, cytokeratins 8 and 18 (CK8 . CK18). We determined, using ER alpha-activating function-2 mutants, that helix 12 (H12) of ER alpha, but not its F domain, is essential for fulvestrant-induced ER alpha-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ER alpha immobilization/degradation, transient transfection assays were performed using wild type ER alpha, ER alpha with amutated H12, and ER alpha with a deleted F domain. Of those, only the ER alpha H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ER alpha degradation in CK8 . CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ER alpha degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ER alpha-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ER alpha immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ER alpha to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ER alpha turnover.