Susan Upchurch

Heterogeneity of estrogen receptors in the cytosol and nuclear fractions of the rat uterus.

Multiple species of estrogen binding sites have been demonstrated in cytosol and nuclear fractions of uteri from immature and adult ovariectomized female rats. Equilibrium binding analyses of uterine cytosol yielded two binding sites, I and II, with dissociation constants of 0.8 and 33 nM respectively. The high affinity cytosol site (0.8 nM - Type I) translocated to the nuclear compartment following estrogen treatment in vivo and appears to represent the classical estrogen receptor which can be measured by 3H-estradiol exchange. Type II sites remain in the cytosol after estrogen injection. A third binding component was observed in the nuclear compartment (nuclear Type II) which binds 3H-estradiol at 4° and displayed cooperative binding characteristics. The presence of these sites in both cytosol and nuclear compartments complicates the accurate measurement and differentiation of these sites. Valid estimates of binding parameters for cytosol Type I and II sites may be obtained by saturation analyses over a wide range of 3H-estradiol concentrations (0.05–40 nM). Nuclear Type I can be differentiated from nuclear Type II by performing saturation analysis under exchange conditions which measure both Type I and II sites and comparing the values obtained when the assay is performed at 4°C which measures only Type II sites.

Progesterone and dexamethasone antagonism of uterine growth: A role for a second nuclear binding site for estradiol in estrogen action

Abstract Administration of estradiol to mature-ovariectomized rats caused nuclear binding of the estrogen receptor and elevated the number of secondary estradiol binding sites (type II sites) in uterine nuclei. These events are correlated with the stimulation of uterine growth. Administration of dexamethasone or progesterone blocks the stimulation of nuclear type II sites and inhibits uterine growth. The inhibition of growth occurs even though the estrogen receptor undergoes normal nuclear translocation, nuclear retention and cytoplasmic replenishment. It is speculated that the stimulation of nuclear type II sites may be an intermediate step in the mechanism by which estradiol causes uterine growth.

Effects of estradiol-17α on nuclear occupancy of the estrogen receptor, stimulation of nuclear type II sites and uterine growth

Estriol and estradiol-17 alpha (E2-17 alpha) have classically been described as weak or impeded estrogens since they are incapable of stimulating true uterine growth when administered acutely by single injection. We have demonstrated [16] that estriol is capable of stimulating true uterine growth when the hormone is administered by paraffin implant. The possibility that E2-17 alpha is similar to estriol was examined. A single injection of E2-17 alpha causes a rapid accumulation of the estrogen receptor in uterine nuclei and this is correlated with the stimulation of early uterotropic responses. The nuclear receptor content declines rapidly and no stimulation of nuclear type II sites or true uterine growth is observed. E2-17 alpha does however stimulate the replenishment of cytoplasmic estrogen receptor. This receptor-response profile is typical of a short acting estrogen such as estriol. Chronic exposure (96 h) of mature-ovariectomized rats to estradiol-17 alpha (4 mg) by beeswax implant results in continual nuclear occupancy by estrogen receptors, dramatic stimulation of nuclear type II sites and true uterine growth. It is not possible to determine whether the uterotropic stimulation was due to direct effects of E2-17 alpha since this isomer was partially metabolized to E2-17 beta and both isomers were found in uterine nuclei after an implant of E2-17 alpha. We conclude that E2-17 alpha is capable of acting as an estrogen, either by its inherent estrogenicity or by its conversion to E2-17 beta, and that it may be dangerous to consider this steroid to be an ineffective or inadequate estrogen.

Heterogeneity of Estrogen Binding Sites: Relationship to Estrogen Receptors and Estrogen Responses

Publisher Summary This chapter discusses the significance of the various forms of estrogen binding sites. Estrogen receptors are macromolecules that bind estrogen to form receptor hormone complexes. These complexes are generally involved with the stimulation of cellular growth and metabolism. This concept of steroid hormone action is widely known. Saturation analysis of uterine cytosol over a wide range of [ 3 H] estradiol concentrations reveals a curve for specifically bound [ 3 H] estradiol. Two types of macro molecule exist that bind estrogen in a stereospecific fashion: (1) type I, which has the properties of the classical cytosol estrogen receptor, and (2) type II, which has a lower affinity and a higher capacity for estradiol than type I. Type II sites also differ from type I as they do not undergo translocation from the cytoplasm to the nucleus after an estrogen injection. The implications of the existence of type II sites are far-reaching. Their presence interferes with the measurement of type I sites, producing an overestimate of type I and an incorrect identification of type II as type I.

Estrogen binding in the rat uterus: heterogeneity of sites and relation to uterotrophic response.

Abstract Multiple species of estrogen binding sites have been demonstrated in cytosol and nuclear fractions of uterine cells from immature and adult ovariectomized rats. Cytosol . Type I sites show properties identical to the classical cytoplasmic estrogen receptor. Type II binding sites have lower affinity for the ligand, exist in higher concentrations and do not undergo nuclear translocation after estrogen stimulation. These secondary sites may be involved in the retention of estrogens within the uterus, by creating an estrogen rich environment for binding to type I sites which in turn translocate estrogen as a receptor-hormone complex to the nucleus. Since it is not known whether type II sites are intra- or extracellular, they may act in this buffer capacity in either or both cellular compartments. Another possibility is that type II sites are precursors of type I sites. Nucleus . In addition to type I sites, a second site was also observed in the nuclear compartment. This second nuclear binder does not appear to be related to the cytosol type II site; however they have similar dissociation constant and share hormone and tissue specificity. Estradiol administration causes an increase in both specific binding components in uterine nuclei of mature ovariectomized rats. The second component (type II) binds estradiol with a higher capacity than type I sites, and displays a saturation curve which is sigmoidal. The stimulation of type II sites is a specific estrogenic response and is highly correlated with uterine growth. A single injection of estradiol results in long term retention of type I sites (6h), rapid and sustained elevations of type II sites (1–72 h) and true uterine growth. In contrast, estriol injection caused a rapid increase in type I sites which was not accompanied by an increase in type II sites and no true uterine growth occurred. Conversely the administration of estriol by paraffin implant sustained elevated nuclear levels of type I sites, increased nuclear type II sites 2–3 fold above controls and stimulated true uterine growth 48 h following hormone administration. These data suggest that estrogen stimulation of true uterine growth may require long term (6–24 h) nuclear retention of type I sites and sustained elevation in type II sites. These secondary sites do not appear to be translocated to the nucleus but instead may be chromosomal proteins which are present in the nucleus of uterine cells at alt times. We conclude that elevated levels of nuclear type II sites correlate well with the biosynthetic events associated with the long term uterotrophic response to estrogenic hormones.

Nuclear binding of the estrogen receptor: heterogeneity of sites and uterotropic response.

Two kinds of estradiol binding sites are present in purified nuclei from the rat uterus following estradiol injection. One of these sites (type I) corresponds to the well-known estrogen receptor which undergoes translocation from the cytoplasm to the nucleus. The second site (type II) is not translocated from the cytoplasm to the nucleus, however, estradiol treatment does stimulate an increased number of these sites. Type II sites are observed in purified nuclei and chromatin isolated from the uterus but not from non-target tissues such as the spleen and diaphragm. Thus an elevation in the levels of type II sites appear to be a specific nuclear response of the rat uterus to estradiol. Saturation analysis over a wide range of [3H]-estradiol concentrations produces a binding curve for type II sites which is sigmoidal and hence no accurate estimation of the dissociation constant is possible. The binding of [3H]-estradiol to nuclear type II sites is inhibited by estradiol and diethylstilbestrol but not by progesterone, testosterone, or corticosterone. Extraction of nuclei isolated from estrogen treated rat uteri with KCl provides a complex picture. Direct labeling of nuclear estrogen receptors either by in vivo injection or in vitro incubation of intact uteri with [3H]-estradiol measures only a fraction of the specific estrogen binding sites associated with the nuclear pellet following 0.4 M KCl extraction. These sites are more accurately determined by performing saturation analysis over a wide range of [3H]-estradiol concentrations by exchange which measures specific estrogen binding sites, not [3H]-steroid. Saturation analysis of estradiol binding to KCl extracted nuclei when performed by exchange, with appropriate corrections for type II binding, reveals that approximately 1000--2000 receptors per nucleus are resistant to KCl extraction 1 hr after administration. The same numbers of type I sites display long-term nuclear retention. A single injection of estradiol results in long term (greater than 6 h) retention of type I sites, rapid and sustained elevations (1--72h) in type II sites and true uterine growth (uterine wet weight at 24--43 h). Estriol injections caused a rapid increase in nuclear type I sites which was not accompanied by an increase in type II sites and no true uterine growth occurred. Administration of estriol or estradiol as a pellet implant, which causes continuous occupancy of type I sites, increases the quantity of nuclear type II sites and stimulates true uterine growth. Therefore, we conclude that elevated levels of nuclear type II sites correlate with the long term uterotropic response to estrogenic hormones. Although we do not understand the function of this second class of binding sites it is possible that the type II sites represent a major component in the mechanism by which estrogens stimulate growth of the uterus.

Estrogen Receptor Heterogeneity and Uterotropic Response

For several years investigators have suggested that at least two forms of estrogen binding sites exist in the uterus (Best-Belpomme et al. 1970; Ellis and Ringold 1971 ; Erdos et al. 1969; Michel et al. 1974; Puca et al. 1971 ; Sanborn et al. 1971; Steggles and King 1970). One of these sites, the estrogen receptor, has been intensively investigated (Baulieu et al. 1975; Clark et al. 1978a; Gorski and Gannon 1976; Jensen et al. 1974; O’Malley and Means 1974). This receptor is a protein macromolecule that binds estrogens in a stereospe- cific manner and is found in the cytosol of estrogen-sensitive cells. It has a very high affinity for estradiol (K d ~ 10-9 M) and is generally considered to exist in uterine cells at a concentration of about 20,000 sites per cell or ~0.5 pmol/100 μg DNA (Anderson et al. 1972; Clark and Gorski 1969; Katzenellenbogen et al. 1973). The other binding site(s) has received little attention and is often ignored or considered to result from serum albumin or α-fetoprotein (Katzenellenbogen et al. 1973). Early work indicated that dissociation of estrogen from a single binding site could not easily account for this multiplicity, and so two or more sites were proposed (Sanborn et al. 1971; Best-Belpomme et al. 1970; Erdos et al. 1969; Ellis and Ringold 1971; Puca et al. 1971). Rochefort and Baulieu (1969) had noted previously the presence of a secondary site in the uterus that bound estradiol with low affinity but very high capacity.