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. 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.