Measurement of estrogen receptor alpha homodimerization caused by xenoestrogens using bimolecular fluorescence complementation
P. Tarnow, D. Hunecke & A. Luch
Introduction: After binding its ligand, a conformational change in estrogen receptor alpha (ERα) occurs, leading to subsequent homodimerization of two monomeric ERs. The dimer then binds to specific DNA-elements or to other transcription factors already bound on gene promoters to regulate target gene expression. Protein-fragment complementation assays in general are simple tools to monitor proteinprotein interactions and are useful to visualize the subcellular sites of interactions in living cells. In case of bimolecular fluorescence complementation (BiFC), two fragments of a fluorescent protein (e.g. YFP) are genetically fused to proteins of interest. The single fragments show no fluorescence, but when the proteins mutually interact, the fluorescent protein refolds and gives a fluorescence signal. Here we aimed to use BiFC to monitor ERα homodimerization after stimulation with several estrogenic substances present in human environment.
Methods: To monitor ERα dimerization we fused full length ERα to the carboxyterminal and aminoterminal fragments of the citrine variant of YFP and cotransfected these constructs transiently into COS-7 cells. After stimulation with estrogens, fluorescence was measured in a photometer or visualized by confocal microscopy.
Results and Conclusions: Compared to mock transfected cells cotransfection caused a visible fluorescence signal in the nuclei of transfected cells. Stimulation of the cells with the ERα agonists 17β-estradiol (E2), bisphenol A, genistein and butylparabene but also with the antagonist fulvestrant (ICI) and the selective estrogen receptor modulator 4-hydroxytamoxifen (4-OHT) dose dependently increased fluorescence signals. Interestingly, ICI and 4-OHT stimulation caused higher fluorescence signals than E2. If this is caused by a higher amount of dimers or by conformational differences remains to be shown. Except stimulation with ICI, which translocated ERα-dimers into cytoplasma, fluorescence signals were only seen in the nuclei. The partial ERα agonist 4-bethylbenzylidene camphor as well as the heavy metal cadmium caused no increase of the fluorescence signal.
Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.