A two-step mechanism for the interaction of estradiol with rat uterus.

Abstract

During the past ten years much information has been accumulated concerning the interaction of tritiated estrogens with hormone-dependent tissues. Their striking affinity for estradiol, both in vivol-5 and in vitro,6-9 first suggested that such target tissues as uterus, vagina, and anterior pituitary possess unique components called "estrogen receptors." Strong but reversible association of hormone with receptor, without chemical transformation of the steroid molecule, appears to be a primary step in the uterotrophic process, not affected by such inhibitors of early estrogen response as puromycin or actinomycin D.10 The interaction of estradiol with target tissues involves two distinct phenomenauptake and retention; the latter process, but not the former, becomes saturated in vivo if the hormone administered exceeds the physiological level.1' Centrifugal fractionation experiments'0-'4 demonstrate two sites of estrogen binding in uterine cells. As confirmed by autoradiography,"5 16 most estradiol, both in endometrium and in myometrium, resides in the nuclei, but a certain amount is bound to a macromolecular substance appearing in the supernatant fraction. Toft and Gorski"7 made the important observation that the radioactive estradiol-receptor complex in the supernatant fraction can be characterized by ultracentrifugation in sucrose density gradients where it migrates with a sedimentation coefficient of 9.5S; disruption of the complex by proteases but not by nucleases suggests that the receptor substance is a protein. We have confirmed these findings and have observed further8-20 that a different estradiol-receptor complex, sedimenting at about 5S, can be extracted from uterine nuclei by cold 0.3 M KC1, which solubilizes little uterine DNA.21 Although the 9.5S complex does not appear to be a simple aggregate of the 5S, the two receptor substances show many similar characteristics, discussed in more detail elsewhere.22 An important difference is the ability of the 9.5S complex to form spontaneously on addition of tritiated estradiol to supernatant fraction;9' 20 23 the 5S complex is not produced by adding estradiol to nuclear extract,24 although it can be formed in the whole homogenate. This paper presents evidence that the 9.5S receptor is extranuclear and involved in estrogen uptake, whereas the nuclear 5S complex, probably responsible for hormone retention and growth initiation, is formed from the 9.5S complex by a process which consumes 9.5S receptor and which is retarded at low temperature. Materials and Methods.-These investigations used estradiol-6, 7-H' (spec. act. 57.4 c/mmole), KRH buffer (pH 7.3, Krebs-Ringer-Henseleit-glucose, containing in gm/liter: NaCI, 8.0; KC1, 0.20; Na2HPO4-7H20, 1.73; KH2PO4, 0.20; CaCl2, 0.10; MgCl2, 0.048; and glucose, 1.0), and Tris-EDTA buffer (pH 7.4, 0.01 M 2-amino-2-hydroxymethyl-1,3-propanediol plus 0.0015 M ethylenediaminetetraacetic acid, disodium salt).

Changes in the concentration of high-affinity oestradiol receptors in rat uterine supernatant preparations during the oestrous cycle, pseudopregnancy, pregnancy, maturation and after ovariectomy

“…All the Scatchard plots obtained by this method were linear, indicating that the receptors measured were homogeneous in The fluctuations in cytoplasmic binding capacity may reflect changes in the distribution of receptors between cytoplasm and nucleus. This might be explained in terms of the two-step mechanism for oestrogen action proposed by Jensen et al (1968), who showed that transfer of oestradiol from cytoplasm to nucleus was accompanied by a decrease in the supernatant binding capacity. Alternatively the receptors may be localized in a particular type of cell of which the number is subject to hormonal influence.…”

“…Little is known at present about the interaction between these two or their precise role in mediating hormone action. It has been observed that the binding capacity of the cytoplasm decreases as oestradiol enters the nucleus (Jensen et al 1968), which may imply a transport function for the cytoplasmic receptor.…”

Transformation of Adrenergic Receptors in the Myocardium

“…Similar1y, the temperature sensitive transformation of an estrogen binding protein in the cytoso1 of rat uterus is thought to involve allosteric changes that permit migration of the receptor protein to the cell nucleus (Jensen et ~., 1968;Shyamala and Gorski, 1969 Kunos and Szentivanyi (1968) also noted the difference between winter and summer frog hearts and reported that it required combination of a metabolic inhibitor (2,4-dinitrophenol) and cooling to al10w block by a-adrenergic antagonists in the latter.…”

Spatiotemporal dynamics of the expression of estrogen receptors in the postnatal mouse brain

“…40,41 Offspring of the pregnant mice were decapitated on P7. The brain was removed and frozen using dry ice powder, and stored at À80 1C until the time of assay.…”