title: A homogeneous in vitro functional assay for estrogen receptors: coactivator recruitment.
abstract: 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 ERalpha or ERbeta 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 ERalpha and ERbeta exhibited strong preferences for coactivator peptides corresponding to steroid receptor coactivator-1 and PPARgamma coactivor-1 vs. peroxisome proliferator-activated receptor-interacting protein and cAMP response element binding protein-binding protein. 17beta-Estradiol acted as a nonselective agonist for ERalpha and ERbeta. Genistein showed full agonism for ERalpha and only partial agonism for ERbeta, but with higher potency for ERbeta than ERalpha. 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.
authors.affiliation: AstraZeneca Pharmaceuticals, Wilmington, Delaware 19850, USA. jianwei.liu@astrazeneca.com
authors.forename: Jianwei
authors.initials: J
authors.lastname: Liu
authors.forename: Katharine S
authors.initials: KS
authors.lastname: Knappenberger
authors.forename: Helena
authors.initials: H
authors.lastname: Kack
authors.forename: Gunilla
authors.initials: G
authors.lastname: Andersson
authors.forename: Ewa
authors.initials: E
authors.lastname: Nilsson
authors.forename: Christine
authors.initials: C
authors.lastname: Dartsch
authors.forename: Clay W
authors.initials: CW
authors.lastname: Scott
mesh_terms: D000595:Amino Acid Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D004965:Estrogen Antagonists / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D005063:Europium / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D051548:Histone Acetyltransferases; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D056920:Nuclear Receptor Coactivator 1; D009842:Oligopeptides; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D020849:Raloxifene Hydrochloride / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011960:Receptors, Estrogen / Q000819:agonists / Q000378:metabolism*; D013629:Tamoxifen / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D018160:Receptors, Cytoplasmic and Nuclear; D011960:Receptors, Estrogen; D014157:Transcription Factors; D013629:Tamoxifen; D005063:Europium; D020849:Raloxifene Hydrochloride; D004958:Estradiol; C085706:FLAG peptide; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1