The present paper shows that the antiestrogen tamoxifen and different tamoxifen derivatives bind to two distinct cytoplasmic binding sites in the fetal uterus of guinea pig. The first one (Site A) corresponds to the estrogen receptor and binds tamoxifen with a dissociation constant (Kd) of 1.8 ± 0.4 nm. The dissociation rate constant (k-1) of the Site A:tamoxifen complex at 4° is 8.3 ± 2 × 10-4 sec-1 and at 26° is 123 ± 26 × 10-4 sec-1. The binding ability of Site A appears to be thermolabile, being destroyed by heating at 37°. The hydroxylated derivatives of tamoxifen (Metabolites B and D) have a higher affinity for Site A as compared to tamoxifen. The second binding site for tamoxifen (Site B) appears to be specific for the triphenylethylene class of antiestrogens (nafoxidine and several tamoxifen metabolites), the hydroxylation of tamoxifen decreasing the affinity for Site B. In contrast, natural and synthetic estrogens as well as cortisol, testosterone, and progesterone do not compete for Site B. Site B shows a higher affinity for tamoxifen (Kd 0.39 ± 0.01 nm) as compared to Site A and a higher stability of the complex at both 4 and 26° (k-1 0.81 ± 0.14 and 3.0 ± 0.4 × 10-4 sec-1, respectively). The binding ability of Site B appears to be resistant to heating at 37°. Both Sites A and B are destroyed by proteolytic treatment and are precipitated by 36% saturated ammonium sulfate. The tamoxifen:Site A complex translocates into the uterine nuclei in a “cell free” system by a temperature-dependent process, but the tamoxifen:Site B complex does not. Nevertheless, a site with a Kd similar to cytoplasmic Site B (0.47 ± 0.1 nm) is spontaneously present in untreated fetal uterine nuclei. In other fetal organs which contain no estrogen receptor (heart) or very low levels (lung), the concentration of Site B is found to be significantly lower than in the fetal uterus (5 to 6 times). Furthermore, progressively lower levels of Site B are found in neonatal, immature, and mature uteri (as compared to fetal uterus) which contain also decreasing amounts of estrogen receptors.

It is concluded that, besides binding to the estrogen receptor, the triphenylethylene antiestrogens bind to a specific site distinct from the estrogen receptor, that appears to be localized mainly in estrogen target cells.

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Part of the expenses of this work was supported by the Centre National de la Recherche Scientifique (Equipe de Recherche du C.N.R.S. 187) and by the Recherche Médicale Française.

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