title: Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens.
abstract: The diverse biological effects of xenoestrogens may be explained by their ability to differentially recruit co-regulatory proteins to the estrogen receptor (ER). We employed high-throughput receptor affinity binding and co-regulatory protein recruitment screening assays based on fluorescence polarization and time resolved florescence resonance energy transfer (TR-FRET), respectively, to assess xenoestrogen-specific binding and co-regulatory protein recruitment to the ER. Then we used a functional proteomic assay based on co-immunoprecipitation of ER-bound proteins to isolate and identify intact co-regulatory proteins recruited to a ligand-activated ER. Through these approaches, we revealed differential binding affinity of bisphenol-A (BPA) and genistein (GEN) to the human ERα (ESR1) and ligand-dependent recruitment of SRC-1 and SRC-3 peptides. Recruitment profiles were variable for each ligand and in some cases were distinct compared to 17β-estradiol (E2). For example, E2 and GEN recruited both SRC-1 and -3 peptides whereas BPA recruited only SRC-1 peptides. Results of the functional proteomic assay showed differential recruitment between ligands where E2 recruited the greatest number of proteins followed by BPA then GEN. A number of proteins share previously identified relationships with ESR1 as determined by STRING analysis. Although there was limited overlap in proteins identified between treatments, all ligands recruited proteins involved in cell growth as determined by subnetwork enrichment analysis (p<0.05). A comparative, in silico analysis revealed that fewer interactions exist between zebrafish (Danio rerio) esr1 and zebrafish orthologs of proteins identified in our functional proteomic analysis. Taken together these results identify recruitment of known and previously unknown co-regulatory proteins to ESR1 and highlight new methods to assay recruitment of low abundant and intact, endogenous co-regulatory proteins to ESR1 or other nuclear receptors, in both human and aquatic species.
authors.affiliation: Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, USA.
authors.forename: L Cody
authors.initials: LC
authors.lastname: Smith
authors.affiliation: Department of Civil and Environmental Engineering and Nicholas School of the Environment, Duke University, Durham, NC 27708, USA.
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Clark
authors.affiliation: Department of Environmental and Global Health and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Joseph H
authors.initials: JH
authors.lastname: Bisesi
authors.affiliation: Department of Civil and Environmental Engineering and Nicholas School of the Environment, Duke University, Durham, NC 27708, USA.
authors.forename: P Lee
authors.initials: PL
authors.lastname: Ferguson
authors.affiliation: Department of Environmental and Global Health and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, USA. Electronic address: sabo@phhp.ufl.edu.
authors.forename: Tara
authors.initials: T
authors.lastname: Sabo-Attwood
mesh_terms: D000818:Animals; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D061986:MCF-7 Cells / Q000187:drug effects; D056918:Nuclear Receptor Coactivators / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D060066:Protein Interaction Maps / Q000187:drug effects; D015262:Xenobiotics / Q000494:pharmacology*; D015027:Zebrafish / Q000378:metabolism*
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D004967:Estrogens; D056918:Nuclear Receptor Coactivators; D010446:Peptide Fragments; D015262:Xenobiotics
keywords: Estrogen; Estrogen receptor; Fluorescence polarization; Steroid receptor co-regulator, co-immunoprecipitation, proteomics; Time resolved fluorescence resonance energy transfer; Xenoestrogens