Random mutagenesis of human estrogen receptor ligand binding domain identifies mutations that decrease sensitivity to estradiol and increase sensitivity to a diphenol indene-ol compound: basis for a regulatable expression system.
We have used low fidelity polymerase chain reaction amplification to generate mutations in the human estrogen receptor ligand binding domain (LBD). Screening of libraries of mutants in yeast revealed a variety of phenotypic changes including decreased responsiveness to estradiol and increased responsiveness to synthetic compounds. Identification of the mutations responsible for these phenotypic changes indicated discrete regions of the LBD that are important for human estrogen receptor function. Cumulative rounds of mutagenesis and screening allowed us to produce a mutant estrogen receptor that was of reversed specificity as compared with the wild type LBD, in that it was more responsive to a diphenol indene-ol than to estradiol. This mutant may form the basis of a useful regulatable expression system in mammalian cells.
Miller
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N
Geneva Biomedical Research Institute, Plan-les-Ouates, Switzerland.
Whelan
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D004958:Estradiol / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D007192:Indenes / Q000494:pharmacology; D008024:Ligands; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010636:Phenols / Q000494:pharmacology; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011960:Receptors, Estrogen / Q000819:agonists / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D007192:Indenes; D008024:Ligands; D010636:Phenols; D011960:Receptors, Estrogen; D004958:Estradiol