A Genetic Screen for Ligand Binding by the Human Estrogen Receptor.
Annual rept. 15 Aug 95-14 Aug 96,
EUROPEAN MOLECULAR BIOLOGY ORGANIZATION HEIDELBERG (GERMANY)
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FLP recombinase-steroid receptor fusion proteins convert ligand binding into DNA recombination. We describe a ligand responsive FLP - estrogen receptor binding domain FLP-EBD in yeast that accurately reflects known estrogen receptor agonist affinities. All tested estrogens, whether agonists or antagonists, induce FLP-EBD 251-595 recombination, indicating that all induce EBD release from the Hsp9O complex. Altering the distance between FLP and the EBD domains in the fusion protein affects ligand inducibility. A FLP-EBD 3O4-595, with 53 fewer amino acids, shows reduced inducibility by agonists, and unexpectedly, complete insensitivity to induction by all antagonists tested. Thus we observe a tethered interference between FLP and the EBD domains that differs depending on the distance between the two domains and the conformations induced by agonists or antagonists, presenting a distinction between estrogen agonists and antagonists in yeast. Combining this distinction with mutagenesis of the EBD has generated numerous mutations with altered ligand specificity, sometimes inverting the activation effects of hormones and antihormones. Further study will define the specific mechanisms leading to antihormone action, especially with respect to the therapeutically important antihormones, tamoxifen and raloxifene.
- Medicine and Medical Research