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AstraZeneca Pharmaceuticals, Wilmington, Delaware 19850 (J.L., K.S.K., C.W.S.); 431 83 Mölndal, Sweden (H.K., G.A., E.N.); and 151 85 Södertälje, Sweden (C.D.)
Address all correspondence and requests for reprints to: Jianwei Liu, AstraZeneca Pharmaceuticals, 1800 Concord Pike, LW207D, Wilmington, Delaware 19850. E-mail: jianwei.liu{at}astrazeneca.com.
Estrogen receptor (ER)-mediated gene transcription occurs via the formation of a multimeric complex including ligand-activated receptors and nuclear coactivators. We have developed a homogeneous in vitro functional assay to help study the ligand-dependent interaction of ERs with various nuclear coactivators. The assay consists of FLAG-tagged ER
or ERß ligand binding domain (LBD), a biotinylated coactivator peptide, europium-labeled anti-FLAG antibody, and streptavidin-conjugated allophycocyanin. Upon agonist binding, the biotinylated coactivator peptide is recruited to FLAG-tagged ER LBD to form a complex and thus allow fluorescence resonance energy transfer (FRET) to occur between europium and allophycocyanin. Compounds with estrogen antagonism block the agonist-mediated recruitment of a coactivator and prevent FRET. The assay was used to evaluate the preference of ERs for various coactivators and ligands. Both ER and ERß exhibited strong preferences for coactivator peptides corresponding to steroid receptor coactivator-1 and PPAR
coactivor-1 vs. peroxisome proliferator-activated receptor-interacting protein and cAMP response element binding protein-binding protein. 17ß-Estradiol acted as a nonselective agonist for ER and ERß. Genistein showed full agonism for ER and only partial agonism for ERß, but with higher potency for ERß than ER. Both raloxifene and tamoxifen behaved as full antagonists in this functional assay. The results obtained using compounds with a wide range of potency correlated well with those from a cell-based reporter gene assay. Therefore, this simple in vitro functional assay is predictive of ligand-dependent transactivation function of the receptor and, as such, is useful in nuclear receptor applications including mechanistic studies.
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