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).

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The steroid hormone receptors are characterized by binding to relatively rigid, inflexible endogenous steroid ligands. Other members of the nuclear receptor superfamily bind to conformationally flexible lipids and show a corresponding degree of elasticity in the ligand-binding pocket. Here, we report the X-ray crystal structure of the oestrogen receptor a (ERa) bound to an oestradiol derivative with a prosthetic group, ortho-trifluoromethlyphenylvinyl, which binds in a novel extended pocket in the ligand-binding domain. Unlike ER antagonists with bulky side groups, this derivative is enclosed in the ligand-binding pocket, and acts as a potent agonist. This work shows that steroid hormone receptors can interact with a wider array of pharmacophores than previously thought through structural plasticity in the ligand-binding pocket.

The interaction of estradiol with uterine cells involves the association of the hormone with an extranuclear receptor protein, followed by temperature dependent translocation of the resulting complex to the nucleus. During this process, the steroid binding unit of the protein undergoes an alteration, called "receptor transformation," that can be recognized by an increase in its sedimentation rate from 3.8S to 5.2S, and by its acquisition of the ability to bind to isolated uterine nuclei and to alleviate a tissue specific deficiency in the RNA synthesizing capacity of such nuclei. Receptor transformation can be effected in the absence of nuclei by warming uterine cytosol with estradiol. This preparation of transformed complex resembles that extracted from nuclei both in its sedimentation rate (5.3S) and in its ability to bind to uterine nuclei and augment RNA synthesis, properties that are not shown by the native complex. It is proposed that receptor transformation is an important step in estrogen action and that a principal role of the hormone is to induce conversion of the receptor protein to a biochemically functional form.

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