Development of a bioluminescence resonance energy transfer (BRET) for monitoring estrogen receptor α activation

Estrogen receptor (ER) is a ligand-activated transcriptional factor, able to dimerize after activation and to bind specific DNA sequences (estrogen response elements). thus activating gene target transcription. Since ER homo- and heterodimerization (giving alpha-alpha and alpha-beta isoforms) is a fundamental step for receptor activation, we developed an assay for detecting compounds that induce human ERalpha homo-dimerization based on bioluminescence resonance energy transfer (BRET). BRET is a non-radiative energy transfer, occurring between a luminescent donor and a fluorescent acceptor, that strictly depends on the closeness between the two proteins and can therefore be used for studying protein-protein interactions. We cloned ERalpha coding sequence in frame with either a variant of the green fluorescent protein (enhanced yellow fluorescent protein. EYFP) or Renilla luciferase (RLuc). Upon ERalpha homo-dimerization, BRET process takes place in the presence of the RLuc substrate coelenterazine resulting in EYFP emission at its characteristic wavelength. The ER alpha-Rluc and ER alpha-EYFP fusion proteins were cloned. then the occurrence of BRET in the presence of ER alpha activators was assayed both in vivo, within cells, and in vitro, with purified fusion proteins.