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-α (ERα) 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-ERα interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERα-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 ERα-interacting proteins, cytokeratins 8 and 18 (CK8·CK18). We determined, using ERα-activating function-2 mutants, that helix 12 (H12) of ERα, but not its F domain, is essential for fulvestrant-induced ERα-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERα immobilization/degradation, transient transfection assays were performed using wild type ERα,ERα with a mutated H12, and ERα with a deleted F domain. Of those, only the ERα H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERα 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 ERα 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 ERα-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 ERα immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERα to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERα turnover.

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