Use of the yeast one-hybrid system to screen for mutations in the ligand-binding domain of the estrogen receptor.
This report describes a novel yeast one-hybrid system which easily allows for the detection of mutations in the ligand-binding domain of the estrogen receptor. This screen is based on the observation that a fusion protein consisting of the GAL4 DNA-binding domain and the estrogen receptor can interact with a GAL4 upstream activating sequence and induce the expression of an integrated GAL1-lacZ gene only in the presence of estradiol. Various deletion mutants of the estrogen receptor were tested in this assay and activating function 1 which is present in the N-terminus of the estrogen receptor was found to be responsible for the transactivation produced in the assay. To test if the screen could be used to detect random mutants in the ligand-binding domain of the estrogen receptor the region of the human receptor between amino acids 381 to 403 was mutated by oligonucleotide saturation mutagenesis. Two of the mutants generated by this mutagenesis were characterized to demonstrate that the results obtained from the screen in the yeast screen are relevant to mammalian systems. One of the mutants which has a valine at position number 388 instead of a glycine was able to transactivate in both the yeast and a mammalian system. This mutant was a more potent activator of transcription and also appeared to have a higher affinity for [3H]estradiol in vivo than the wild type receptor. The other mutant which was characterized has five amino acid changes from amino acids 390 through 400. This mutant was nonfunctional in the yeast and mammalian transcription assays and did not bind [3H]estradiol in vivo or in vitro.
Bush
S M
SM
Department of Pharmacology, University of Vermont, Burlington 05405, USA.
Folta
S
S
Lannigan
D A
DA
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004274:DNA, Recombinant; D004958:Estradiol / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D015533:Transcriptional Activation
D004274:DNA, Recombinant; D011960:Receptors, Estrogen; D004958:Estradiol