Antiestrogen-liganded estrogen receptor interaction with estrogen responsive element DNA in vitro.

The mechanism whereby antiestrogens alter the ability of the estrogen receptor (ER) to enhance transcription of estrogen-regulated genes is largely unknown. The effect that selected estrogenic and antiestrogenic ligands have on binding of ER to specific DNA sequences, estrogen responsive elements (EREs) has been quantitated. No differences in purification properties of calf uterine ER liganded with 4-hydroxytamoxifen (4-OHT-ER), ICI 164,384 (ICI 164,384-ER) or estradiol (E2-ER) were detected. A microtiter well plate assay was employed in which liganded ER bound to plasmid DNA is preferentially retained compared to free liganded ER. Binding of E2-ER, 4-OHT-ER, or ICI 164,384-ER was measured to plasmids containing or lacking a 38bp consensus ERE in vitro. The EREs tested contain an inverted repeat (5'-CAGGTCAGAGTGACCTG-3'). Both E2-ER and 4-OHT-ER showed similar high affinity specific binding (Kd = 0.24 and 0.16 nM, respectively) to one copy of the ERE. ICI 164,384-ER did not bind to plasmids containing one ERE. At saturation, however, 4-OHT-ER binding was about 50% of that observed for E2-ER. When the plasmid contained 3 or 4 tandem copies of the ERE, binding of E2-ER, 4-OHT-ER, and ICI 164,384-ER binding was measurable. E2-ER bound in a cooperative manner as suggested by convex Scatchard plots and Hill coefficients > 1.5. In contrast, 4-OHT-ER binding displayed much reduced cooperativity, and ICI 164,384-ER did not display cooperative binding. From these results, we propose that the conformation of ER induced by 4-OHT reduces its binding capacity to this consensus ERE without altering its affinity of binding. Furthermore, higher order protein-protein interactions between antiestrogen-liganded ER bound to DNA differ from those of E2-ER bound to ERE.