Characterization of a unique population of unfilled estrogen-binding sites associated with the nuclear fraction of immature rat uteri.

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 Kd = 1.4 x 10(-10)M 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 micrograms 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 been determined.