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

1995; Elsevier BV; Volume: 270; Issue: 40 Linguagem: Inglês

10.1074/jbc.270.40.23322

1083-351X

Hong Wang, Gregory A. Peters, Xin Zeng, Moli Tang, Wallace Ip, SA Khan,

Fungal Plant Pathogen Control

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- lac Z 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.