Characterization of a Unique Population of Unfilled Estrogen-Binding Sites Associated with the Nuclear Fraction of Immature Rat Uteri*

Abstract

Studies were initiated to determine whether resident nuclear estrogen receptors with unfilled binding sites existed in the nuclear fraction of uteri from untreated immature rats. These studies revealed a small population (mean ± sem, 0.087 ± 0.017 pmol/uterus) of unfilled estrogen-binding sites in the uterine nuclear fraction from untreated immature rats, which bound [³H]estradiol in the absence of added cytoplasmic receptor. These nuclear estrogen-binding sites occurred in addition to the 0.60–1.2 pmol estrogen-binding activity/uterus in the uterine cytoplasmic fraction of these animals. [³H]Estradiol bound reversibly with high affinity (apparent K_d = 1.4 × 10⁻¹⁰m) to these binding sites, and only estrogenic compounds competed for this binding. Binding studies done at a variety of temperatures (0–37 C) showed that there were no estradiol-filled receptor sites associated with the nuclear fraction. In addition, these unfilled nuclear estrogen-binding sites remained unfilled 15 min after injections of either 1.0 or 0.1 μg estradiol/rat. Extraction of these sites was achieved with 0.4 m KCl, and when this extract was centrifuged on a 5–20% linear 0.010 m Tris-HCl, 0.0015 m Na₂EDTA, and 0.40, m KCl sucrose gradient, the binding activity exhibited a sedimentation coefficient of 3.6S. These unfilled nuclear estrogen-binding sites did not appear to have arisen as a contaminant from the estrogen-binding proteins present in either uterine cytoplasm or serum during tissue homogenization. The existence of these unfilled nuclear estrogen-binding sites does not represent a major exception to the classical two-step theory of estrogen action; instead, they seem to be novel forms of the estrogen receptor whose physiological role has not yet been determined. (Endocrinology106: 1776, 1980)