The inhibition of the estrogen receptor's positive cooperative [³H]estradiol binding by the antagonist, clomiphene

The calf uterine estrogen receptor showed positive cooperativity of [³H]estradiol equilibrium binding; the Scatchard plot was convex and the Hill coefficient was 1.69 ± 0.021 (n = 14). The effects of the estrogenic antagonists, zuclomiphene (cis-2-(p-[2-chloro-1,2-diphenylvinyl]phenoxyl)triethylamine citrate) and enclomiphene (trans-2-(p-[2-chloro-1,2-diphenylvinyl]phenoxy)triethylamine citrate), on the positive cooperativity of [³H]estradiol binding were measured by titrating the receptor with a variable concentration of [³H]estradiol and antagonist while maintaining a constant excess in a specific ratio of the antagonist to the [³H]estradiol. With a 45- to 55-fold molar excess of zuclomiphene or an 820- to 900-fold molar excess of enclomiphene above the [³H]estradiol concentration, the receptor's positive cooperative [³H]estradiol binding was inhibited. A transition from a convex to a linear Scatchard plot and a decrease in the Hill coefficient from 1.69 to 1.10 ± 0.02 (n = 6) were induced. The specifically bound [³H]estradiol was inhibited 43 to 50% by the zuclomiphene and enclomiphene. The addition of unlabeled estradiol in a 1- or 2.3-fold molar excess above that of the [³H]estradiol concentration produced a 50 to 75% competitive displacement of the specifically bound [³H]estradiol; nevertheless, the Scatchard plot remained convex and the Hill coefficient was 1.74 an 1.80, respectively. Thus, inhibition of the positive cooperativity of [³H]estradiol binding by the clomiphene isomers was not due to dilution of the specifically bound [³H]estradiol by the antagonist. These data demonstrate that there are two molecular mechanisms by which an estrogen antagonist interferes with the function of the receptor: as a competitor, thus blocking the estrogen receptor's binding site to an agonist, and second by inducing conformational changes that inhibit site:site interactions and receptor activation.