Heterogeneity of estrogen binding sites in the cytosol of the rat uterus

Abstract

Saturation analysis over a wide range of [3H]estradiol concentrations (0.05 to 80 IBM) yields data indicative of two distinct types of binding sites in cytosol prepared from uteri of mature or immature rats. Type I sites have a dissociation constant (&) of 0.8 nM and constitute approximately 1.0 pmol of the sites present in an immature rat uterus (wet weight, 35 mg; DNA, 300 pg). The affinity of type II sites for estradiol is considerably lower than type I sites (Kd, 30 IBM); however, the numbers of sites are 4-fold greater than type I. Type I sites represent the classical estrogen receptor which has a sedimentation coefficient of 8 S on low salt sucrose density gradients and will undergo translocation from the cytoplasm to the nucleus. In contrast, type II sites do not translocate to the nucleus and [3H]estradiol dissociates from these sites when cytosol is labeled prior to sucrose density gradient centrifugation. However, type II sites are readily measured as a 4 S macromolecule by the hydroxylapatite adsorption assay of postlabeled gradient fractions. This technique permits the measurement of both 8 and 4 S sites under equilibrium conditions and thus avoids underestimates due to dissociation of C3H]estradiol from both sites during the centrifugation period. This postlabeled hydroxylapatite-gradient method should be of value in the accurate determination of the quantities of both 8 and 4 S binding sites in all receptor systems and should be particularly valuable for measuring these sites in biopsy samples of mammary cancer tissue. Type II sites are found predominantly in estrogen-sensitive tissues and only low levels occur in nontarget tissues. Estradiol, estrone, estriol, and diethylstilbestrol compete equally well for binding to type II sites while progesterone and testosterone have no effect on [3H]estradiol binding. These characteristics, plus the presence of these sites in adult tissues, exclude the possibility that they represent a-fetoprotein. The presence of type II sites complicates the accurate measurement and differentiation of type I sites. Valid estimates of binding parameters of both sites can be obtained by saturation analysis over a wide range of [3H]estradiol concentrations and resolution of the two binding components by the methods of Scatchard and Rosenthal (Scatchard, G. (1949) Ann. N. Y. Acud Sci. 51, 660-672; Rosenthal, H. E. (1967) Anal. Biochem. 48,317-338). The function of this second