Binding characteristics of 5-androstene-3β, 17β-diol and estradiol-17β to the cytoplasmic estrogen receptor of the immature rat uterus

Abstract

In earlier studies we have shown that the androgen 5-androstene-3β, 17β-diol (Adiol) acts as a weak but fully potent estrogen in the immature rat uterus. In vivo administration of 1 mg Adiol to the immature female rat causes the nuclear translocation of the estrogen receptor by Adiol itself without aromatization. In order to elucidate in more detail the mechanism underlying these effects, we have compared the interactions of [ 3H]-Adiol and [ 3H]-estradiol-17β (E 2) with the cytoplasmic estrogen receptor, using the protamine-precipitation technique. This technique which allows a very rapid separation of free and bound ligand appears to be very handy and useful especially for the study of relatively rapid dissociating hormone-receptor complexes. Adiol binds to the estrogen receptor with a rather high affinity ( K d ⋍ 8 nM), measured at 0°C from protamine-sulphate-precipitated estrogen receptor. The low amount of androgen receptor sites in immature rat uterus and the stereospecificity of the binding of Adiol which was identical to that of E 2 exclude that binding to the androgen receptor was involved. The lower affinity of Adiol than E 2 for the estrogen receptor was caused by its more rapid dissociation from the estrogen receptor ( t 1 of the Adiol-receptor complex was about 3 1 2 hr, while dissociation of the E 2-receptor complex was hardly demonstrable in the first 24 h at 0°C). The association rate of Adiol with the receptor was somewhat higher than that of E 2. Analysis of the interactions between Adiol and E 2 at the receptor binding site revealed competitive inhibition of each others' binding. Adiol binding to the estrogen receptor appeared to protect the latter against thermo-inactivation. These data suggest a common binding site for Adiol and E 2 on the estrogen receptor and present further evidence in favor of the concept that Adiol itself binds to the cytoplasmic estrogen receptor, causes its activation and nuclear translocation, followed by the expression of estrogenic effects in a way almost identical to that of the classical phenolic estrogens.