Abstract

Functional analyses, performed with the estrogen receptor (ER) isolated from different sources or produced with various expression systems, led to contradictory results concerning the role of estrogen (E2) and antiestrogens in ER DNA binding. Here we report the DNA-binding properties of the human ER and show that the wild type ER (HEG0) binds in vitro to an estrogen response element (ERE) as a dimer, irrespective of the presence or absence of estrogen. We also show that the two antihormones, 4-hydroxytamoxifen (OHT, a partial ER agonist) and ICI 164,384 (a pure antagonist) do not impair HEG0 dimerization and DNA binding in vitro. Exposure of HEG0 to elevated temperature (37 C) in vitro results in a much faster reduction of its binding capacity to an ERE in the absence of ligand or in the presence of ICI 164,384 than in the presence of either E2 or OHT. The Gly to Val mutation at amino acid 400 present in the human ER that we initially cloned (HE0), is responsible for an even faster heat inactivation of unliganded receptor compared with HEG0 and largely accounts for the previously observed in vitro ligand-dependent DNA binding of ER. We also show that, as previously observed for OHT, ICI 164,384 does not prevent ER binding to an ERE in vivo, even though ICI 164,384 acts as a pure antagonist for transcriptional activation by ER. We discuss these results in the context of a ligand-dependent interaction between the C-terminal region E, which contains the ligand-binding domain, and the N-terminal A/B region, which contains the activation function AF-1.