A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins.
A number of proteins have been rendered functionally oestrogen-dependent by fusion with the hormone-binding domain of the oestrogen receptor. There are, however, several significant disadvantages with such fusion proteins. First, their use in cells in vitro requires phenol red-free medium and laborious stripping of steroid hormones from serum in order to avoid constitutive activation. Secondly, control of oestrogen receptor fusion proteins in vivo is precluded by high endogenous levels of circulating oestrogens. Thirdly, the hormone-binding domain of the oestrogen receptor functions as a hormone-dependent transcriptional activation domain making interpretation of fusions with transcription factors problematical. In order to overcome these drawbacks we have used a transcriptionally inactive mutant of the murine oestrogen receptor which is unable to bind oestrogen yet retains normal affinity for the synthetic ligand, 4-hydroxytamoxifen. When the hormone-binding domain of this mutant oestrogen receptor is fused to the C-terminus of the c-Myc protein, Myc-induced proliferation and apoptosis in fibroblasts becomes dependent on 4-hydroxytamoxifen, but remains refractory to 17 beta-oestradiol.
Littlewood
T D
TD
Molecular Endocrinology Laboratories, Imperial Cancer Research Fund, London, UK.
Hancock
D C
DC
Danielian
P S
PS
Parker
M G
MG
Evan
G I
GI
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D004958:Estradiol / Q000494:pharmacology; D015870:Gene Expression; D005786:Gene Expression Regulation; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D011960:Receptors, Estrogen / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013629:Tamoxifen / Q000494:pharmacology; D014162:Transfection
D017931:DNA Primers; D004268:DNA-Binding Proteins; D016271:Proto-Oncogene Proteins c-myc; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; D004958:Estradiol