Proteomics analysis of the estrogen receptor alpha receptosome

Abstract

The estrogen receptors (ERs) are ligand-dependent transcription factors that activate transcription by binding to estrogen response elements. Estrogen-mediated effects are tissue- and cell type-specific, determined by the cofactor recruitment to the ERs among other factors. To understand these differences in estrogen action, it is important to identify the various compositions of the ER complexes (ER receptosomes). In this report, we describe a fast and efficient method for the isolation of the ERalpha receptosome for proteomics analysis. Using immobilized estrogen response element on a Sepharose column in combination with two-dimensional electrophoresis and MALDI-TOF MS, significant amounts of proteins could be isolated and identified. Differences in ERalpha complex composition with the ER ligands 17beta-estradiol, 4-hydroxytamoxifen, and ICI-182,780 could also be observed. Thus, this approach provides an easy and relevant way of identifying ERalpha cofactor and transcription factor recruitment under different conditions.

Fig. 1.

Strategy for isolation and identification of DNA-bound ERα complex. A, 90% nuclear and 10% cytosolic extract from ∼3 × 10⁹ MCF-7 cells was used for protein complex isolation. 100 nm ligand and 20 μg of recombinant human ERα were added to the cell extract. The extract was incubated at 37 °C for 45 min and split into two samples, one applied to a column containing 9×ERE-Sepharose and the other applied to a column containing 9×NF-ERE (control) run in parallel. Elution was performed with 1 m NaCl, 10 mm Tris-HCl, pH 7.4, and the eluted proteins were concentrated and desalted. The proteins were then separated both by 1-DE (e.g. for Western blot analysis) and by IEF-2-DE for better separation. Spots were compared between gels using advanced two-dimensional gel analysis software and then excised for MALDI MS identification. B, sequences immobilized onto the HiTrap NHS column: functional trimers of 3×ERE or non-functional trimers of 3×NF-ERE. The ERE sites are underlined, and the lowercase letters indicate exchanged bases in the NF-ERE.

Fig. 2.

Western blot analysis of ERα and ERβ levels and identification of known ERα interactors. A, analysis of endogenous levels of ERα and ERβ in MCF-7 cell extracts (90% nuclear, 10% cytosolic). B, 15% of the volume of eluted ERE-interacting proteins from extracts treated with 100 nm E₂, ICI-182,780, or 4-OHT. The proteins were separated by one-dimensional SDS-PAGE, electrotransferred to a nitrocellulose membrane, and immunoblotted using antibodies directed against ERα, p300, TIF2, Pol-II, and HDAC3. Control samples were treated equally but separated on 9×NF-ERE. Input represents 40 μg of cell extract prior to column chromatography. ICI, ICI-182,780.

Fig. 3.

1-DE and 2-DE separation of ERE-interacting proteins. A, Coomassie staining of proteins isolated on 9×ERE-Sepharose and separated by 2-DE (pI 3–11, 10–150 kDa) from extracts treated with 100 nm E₂, ICI-182,780 (ICI), or 4-OHT. The protein pattern of each complex is a representation of at least three independent two-dimensional gels. The box represents the Hsp70 spot and its quantification by comparing its peak intensity in the different two-dimensional gels (B). C, 1-DE separation of 15% of the volume of eluted ERE-interacting proteins from 100 nm E₂, ICI-182,780, and 4-OHT treatments as well as control sample (E₂-treated and isolated on 9×NF-ERE column). The indicated bands (a–h) correspond to the proteins listed in Table I. Control samples were treated equally but separated on 9×NF-ERE. For simplicity, only the E₂ control sample is shown (4-OHT and ICI-182,780 control samples are available as supplemental Fig. S1).

Fig. 4.

Venn diagram showing overlap of proteins found in E₂-, ICI-182,780-, and 4-OHT-induced ERα complexes. Of 108 proteins identified, 25 proteins were found enriched at the ERE under E₂ treatment and 20 were enriched under 4-OHT treatment, whereas no significant enrichment could be observed under ICI-182,780 (ICI) treatment. Additionally, seven proteins were found in all eluates, including control sample (not shown here). A 2-fold enrichment cutoff was set to discriminate between the treatments.

Fig. 5.

Western blot analysis of MS-identified ERE-interacting proteins. 15% of the volume of eluted ERE-interacting proteins from extracts treated with 100 nm E₂, ICI-182,780 (ICI), and 4-OHT was analyzed. The proteins were separated by one-dimensional SDS-PAGE, electrotransferred to a nitrocellulose membrane, and immunoblotted using antibodies against Hsp70, SFPQ, 100-kDa coactivator SND1, and PHAPI2. Control samples were treated equally but separated on 9×NF-ERE. Input represents 40 μg of cell extract prior to column chromatography.