Yeast Two-hybrid System Demonstrates That Estrogen Receptor Dimerization Is Ligand-dependent in Vivo(*)

Previous studies using in vitro procedures have not clearly established whether the estrogen receptor (ER) acts as a monomer or dimer in the cell. We have used the yeast two-hybrid system as an in vivo approach to investigate the dimerization of the estrogen receptor in the absence and presence of estrogen and anti-estrogens. This system is independent of ER binding to the estrogen response element. Two vectors, expressing GAL4 DNA binding domain-human ER and GAL4 transactivation domain-human ER, were constructed. Control experiments showed that each fusion protein had a high affinity binding site for estradiol-17β and could transactivate an ERE-LacZ reporter gene in yeast similar to the wild type ER. The two fusion proteins, GAL4 DB-hER and GAL 4 TA-hER, were expressed in the yeast strain, PCY2, which carries a GAL1 promoter-lacZ reporter. ER dimerization was measured via reconstitution of GAL4 through interaction of the fusion proteins, which transactivates LacZ through the GAL1 promoter. When both ER fusion proteins were expressed, β-galactosidase activity was estradiol-17β-inducible. Furthermore, we showed that both tamoxifen and ICI 182,780 also induced β-galactosidase activity, albeit lower than that induced by estradiol-17β. These results strongly argue that ER dimerization is ligand-dependent and the dimer can be induced by estradiol-17β, tamoxifen, or ICI 182,780. We also treated the yeast containing the two fusion proteins with estradiol-17β and tamoxifen or ICI 182,780 simultaneously to determine the effects on ER dimerization. β-Galactosidase activity was lower when the yeast was treated with a higher ratio of tamoxifen or ICI 182,780 to estrogen than estradiol-17β alone. Taken together, we conclude that ER dimerization is ligand (estradiol-17β, tamoxifen, or ICI 182, 780)-dependent, and we suggest that estradiol-17β-induced dimers are destabilized when estradiol-17β is used with tamoxifen or ICI 182,780 simultaneously.

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