Cloning and characterization of a 77-kDa oestrogen receptor isolated from a human breast cancer cell line.
We have cloned and characterized a 77-kDa oestrogen receptor (ER) from an oestrogen-independent subclone of the MCF-7 human breast cancer cell line. This receptor contains an in-frame, tandem duplication of exons 6 and 7, located in the steroid-binding domain of the ER. This mutation has abrogated ligand binding, but not DNA binding, in this mutant ER. We previously described the partial structure of a unique oestrogen receptor (ER) that is expressed in an oestrogen-independent MCF-7:2A subclone of the breast cancer cell line MCF-7 (Pink JJ, Wu SQ, Wolf DM, Bilimoria MM, Jordan VC 1996a, Nucleic Acids Res 24 962-969). Sequence analyses determined the molecular weight of this 80-kDa ER to be 77 kDa, and hereafter this protein will be designated as ER77. Examination of the entire coding sequence of the ER77 mRNA indicates that it contains a tandem duplication of exons 6 and 7. Using a coupled transcription/translation system, a 77-kDa ER, which corresponds to the protein observed in the MCF-7:2A cells, was expressed. The ER77 protein does not bind the ligands [3H] oestradiol or [3H]tamoxifen aziridine. In DNA binding gel shift assays, the in vitro synthesized ER77 binds to a consensus vitellogenin A2 oestrogen-response element. In transient transfection experiments, the mutant ER, alone or in combination with the wild-type ER, does not induce expression of an oestrogen-responsive luciferase reporter construct. In fact, expression of the ER77 in the ER-positive T47D:A18 cell line inhibits E2-induced luciferase expression. Overexpression of wild-type ER in T47D:A18 cells leads to elevated constitutive expression of the luciferase reporter, which was inhibited by co-transfection with ER77. These data suggest that the ER77 can interfere with normal ER activity and does not act as a constitutive activator of oestrogen-independent growth in MCF-7:2A cells. Consequently, the constitutive growth observed in MCF-7:2A cells is probably the result of other ER-mediated pathways.
Pink
J J
JJ
Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison 53792, USA.
Fritsch
M
M
Bilimoria
M M
MM
Assikis
V J
VJ
Jordan
V C
VC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001943:Breast Neoplasms / Q000737:chemistry; D002999:Clone Cells / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D005091:Exons; D005260:Female; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011960:Receptors, Estrogen / Q000737:chemistry / Q000302:isolation & purification*; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D008024:Ligands; D011960:Receptors, Estrogen