Adele J. Wolfson

Non-activated form of the progesterone receptor from chick oviduct: characterization.

Summary A non-activated form of the progesterone receptor from chick oviduct cytosol can be stabilized and prepared in the presence of sodium molybdate. This form of the receptor sediments at 8–9 S on 0.15 M KCl salt-containing sucrose gradients. Its Stokes radius, as determined on Ultrogel AcA-22, is 7.9 nm. The data permit calculation of a molecular weight for the non-activated receptor form of ∼ 290,000 d and a frictional ratio of 1.7. The non-activated receptor is eluted from DEAE-cellulose as a single peak, at an ionic strength of 0.1. Studies of dissociation kinetics of the hormone-receptor complex demonstrate that the non-activated complex dissociates 2.5 times faster than the activated complex (t 1/2 at 0°: 19 and 48 h, respectively).

Non-activated progesterone receptor extracted from nuclei of hen oviduct

When the progesterone receptor was extracted from nuclei of laying hen oviduct with 0.5 M sodium molybdate, a large, 7-8 S, form of the receptor was observed. This receptor form resembled non-activated cytoplasmic receptor not only in displaying the same sedimentation coefficient, but also in rapid dissociation rate of the hormone-receptor complex. This finding suggests that either activation may occur within the nuclear compartment, or that activation may be reversed under certain conditions.

Cyanoketone competition with estradiol for binding to the cytosolic estrogen receptor

Cyanoketone, an inhibitor of many steroidogenic processes, has been found to inhibit binding of estradiol to its receptor in a competitive manner. The Ki observed was 1.2 X 10(-6)M. This action may explain some of cyanoketones effects in vivo.

Binding of activated progesterone receptor to microsomes

Specific binding of steroid hormones to microsomes has been reported for several tissues. In the hen oviduct, this receptor appears to be very similar to activated cytosolic receptor. The microsomal receptor is readily solubilized, and resembles the cytosolic receptor in all physico-chemical characteristics: sedimentation coefficient approximately 4 S, Stokes radius 5.5 nm, slow dissociation rate of the complex, adsorption to polyanions. It is precipitated by an antibody to the cytosolic receptor. Microsomes display saturable binding of cytosolic receptor, with a Bmax of approximately 300 fmol/mg protein. This binding is also observed using microsomes from non-target tissues, and is decreased by treatment with RNase. It seems likely that microsomal binding is due to the high affinity of activated cytosolic receptor for RNA.

Characterization of ‘native’ and ‘activated’ progesterone and oestrogen receptors from chick oviduct cytosol

Soon after the discovery, in the cytosol of mammalian uterus, of specific oestrogen-binding proteins termed ‘receptors’ (Talwar et al., 1964, Toft et al., 1967; Baulieu et al., 1967), it was established that exposure of the tissue to oestradiol in vivo or at 25–30°C in vitro leads to a rapid shift of the receptor-hormone complexes to the nuclear compartment (Jensen et al., 1968; Gorski et al., 1968). It was also found that, in cell-free systems, this ‘translocation’ of the receptor-oestradiol complexes from cytosol to the nucleus also can be obtained by incubation at 25–30°C, and that upon warming at this temperature of cytosol oestradiol-receptor complexes, a change of the sedimentation coefficient takes place (initially ~ 4S in 0.3 M KCl containing buffer, it becomes ~ 5S), while they acquire the ability to bind to nuclei (Jensen et al., 1971). Detailed studies with other receptor-steroid complexes have shown that this ‘transformation’ or ‘activation’ can be obtained in all cases, and that it results in increased binding not only to nuclei but also to DNA and to certain other polyanions (Higgins et al., 1973; Milgrom et al., 1973), although the changes in observable physico-chemical parameters due to activation vary with the hormone and with the species (see, for example, reviews by Baulieu et al., 1975; Gorski and Gannon, 1976; Katzenellenbogen, 1980, Simmons, 1980).

Non-activated Form of the Progesterone Receptor in the Chick Oviduct

According to current ideas, in order to exert their biological effects, steroid hormones must first bind to their cytoplasmic receptors, and the complex then moves into the nucleus of the target cell. The process by which the receptor-hormone complex acquires the ability to bind to nuclei and to polyanions, such as DNA, is termed “activation”, the mechanism of which is unknown. It is important to clarify what structural changes in the receptor accompany the activation process, for a better understanding of both activation itself, and the mechanism of hormone action as a whole. Our approach to this problem has been to study the physical and biological properties of the progesterone receptor before and after activation has occurred.

INTERACTION OF OESTROGENS, ANTIOESTROGENS AND PROGESTERONE WITH RECEPTORS AND REGULATION OF GENE EXPRESSION IN THE CHICK OVIDUCT

Publisher Summary This chapter presents the recent results concerning the receptor activation step in the chick oviduct, and discusses these in relation to later events in the control of specific gene expression influenced by oestrogens and progesterone. Experiments were carried out with oestrogen stimulated and with withdrawn chicks. Steroid hormones regulate the expression of specific genes in target tissues often switching these genes “on” and “off” in an all or non fashion. It has been well established that the first step in this process is binding of the hormone to an intracellular cytoplasmic protein called the “receptor.” The “native” receptor-ligand complex subsequently undergoes “activation,” a process by which it acquires the capacity to bind to polyanions such as phosphocellulose or DNA, and to whole nuclei. The activated receptor-ligand complex is translocated to the nucleus, where it presumably interacts with the chromatin in an as yet unknown way, probably inducing changes in the rate of transcription of the hormonally controlled genes.