Differential DNA-binding abilities of estrogen receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells.

We have developed a transient transfection system using the Cytomegalovirus (CMV) promoter to express the human estrogen receptor (ER) at very high levels in COS-1 cells and have used it to study the interaction of agonist and antagonist receptor complexes with estrogen response element (ERE) DNA. ER can be expressed to levels of 20-40 pmol/mg or 0.2-0.3% of total soluble protein and all of the soluble receptor is capable of binding hormone. The ER binds estradiol with high affinity (Kd 0.2 nM), and is indistinguishable from native ER in that the receptor is capable of recognizing its cognate DNA response element with high affinity, and of transactivating a transgene in an estradiol-dependent manner. Gel mobility shift assays reveal interesting ligand-dependent differences in the binding of receptor complexes to ERE DNA. Receptors occupied by estradiol or the type I antiestrogen transhydroxytamoxifen bind to DNA response elements when exposed to the ligand in vitro or in vivo. Likewise, receptors exposed to the type II antiestrogen ICI 164,384 in vitro bind to ERE DNA. However, when receptor exposure to ICI 164,384 is carried out in vivo, the ER-ICI 164,384 complexes do not bind to ERE DNA, or do so only weakly. This effect is not reversed by subsequent incubation with estradiol in vitro, but is rapidly reversible by in vivo estradiol exposure of intact COS-1 cells. This suggests there may be some cellular process involved in the mechanism of antagonism by the pure antiestrogen ICI 164,384, which is not observed in cell-free extracts.