A Genetic Screening for Ligand Binding by the Human Estrogen Receptor.
Final rept. 15 Aug 94-14 Aug 98,
EUROPEAN MOLECULAR BIOLOGY LAB HEIDELBERG (GERMANY)
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FLP recombinase-steroid receptor fusion proteins convert ligand binding into DNA recombination in yeast and mammalian cells. We describe a ligand responsive FLP-estrogen receptor binding domain FLP-EBD in yeast to follow ligand binding interactions. Shortening the distance between FLP and the EBD in the fusion protein specifically blocks induction only by antagonists, presenting an assay for them in yeast. We show using this assay for ligand binding that correlates with transcriptional responsiveness, that agonists and antagonists differently position the C-terminus of the ligand binding domain helix 12 and the F domain. Upon antagonist binding, the F domain interferes with the fusion protein activity. Mutational disruption of helix 12 alters the position of the F domain, imposing interference after agonist or antagonist binding. Numerous genetically selected inversion mutations where now agonists, but not antagonists, induce interference are similarly reliant on helix 12 and F domain positioning. Our results demonstrate that agonists and antagonists differently position helix 12 and implicate the F domain in mechanisms of antagonist action. Mutagenesis of the EBD has generated numerous mutations with altered ligand specificity. This study is consistent with the conclusions of the recent x-ray structures of a static EBD to define specific amino acid contacts in the ligand binding pocket, namely at E353, R394, G521, H524, L540. The structures clarify and aid new drug design.
- Medicine and Medical Research