A genetic, non-transcriptional assay for nuclear receptor ligand binding in yeast.
We describe the development of a genetic, non-transcriptional assay for the detection of ligand binding to nuclear receptors based on the ligand-dependent reconstitution of the defective Ras/cAMP viability pathway of the Saccharomyces cerevisiae strain CDC25-2. We have characterized the assay using the estrogen receptor (ER) alpha as an example and found it to be extremely sensitive, stringent, rapid and selective. We applied this assay to different ligands and ligand-binding domains (LBDs) and analyzed co-stimulation with 17beta-estradiol (E2) and the synthetic ligand 4-hydroxytamoxifen (4-OHT) in vivo. This simple and inexpensive assay may be useful for the study of steroid hormone receptor (SHR) actions at the plasma membrane and for the analysis of ligand binding in vivo. Furthermore, it may allow for the selection of novel ligands and ligand-binding domains and has significant potential for application in compound screening.
Institut für Biologie III/Genetik, Albert-Ludwigs-Universität, Schänzlestrasse 1, D-79104 Freiburg, Germany. fabian.koehler@biologie.uni-freiburg.de
Fabian
F
Köhler
André
A
Zimmermann
Martin
M
Hager
Albrecht E
AE
Sippel
D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014170:Transformation, Genetic; D018631:ras Proteins / Q000235:genetics
D047628:Estrogen Receptor alpha; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D011960:Receptors, Estrogen; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; D018631:ras Proteins