Liselotte E. Closs

Receptors Reconsidered: A 20-Year Perspective

Publisher Summary The original discovery of steroid hormone receptors and essentially all information concerning their interaction and function in target cells have depended on experiments in which a radioactive steroid serves as a marker for the receptor protein to which it binds. The female reproductive tissues, such as uterus, vagina, and anterior pituitary, contain a characteristic estrogen-binding component which was first indicated by their striking ability to take up and retain tritiated hexestrol and estradiol after the administration of physiological doses of these substances to immature animals. This chapter discusses the biochemical mechanism by which the reaction of estradiol and other estrogenic hormones with receptor substances elicits hormonal response has been the subject of extensive investigation. The activated steroid-receptor complex is translocated to the nucleus where it binds to chromatin and in some way modulates RNA synthesis which appears to be characteristically restricted in hormone-dependent tissues.

Antibodies to estrophilin: comparison between rabbit and goat antisera.

Abstract The immunochemical similarity of mammalian estrophilins has been demonstrated by the ability of immunoglobulin (Ig-i) obtained from rabbits and a goat immunized with purified estradiol-receptor complex of calf uterine nuclei to cross-react with all radioactive estradiol-receptor complexes (E∗R) tested to date, as determined by sucrose gradient, gel filtration and double antibody precipitation techniques. Rabbit antibodies to the nuclear receptor of calf uterus react with 5.2 S nuclear E∗R from calf, rat, rabbit and sheep uterus, as well as from rat endometrial and pituitary tumors to give two more rapidly sedimenting E∗R-Ig-i complexes (7–8 S and 10–12 S). Extranuclear E∗R (4 S) from uteri and tumors of several mammalian species, and from rat and human mammary cancers, as well as nuclear and extranuclear E∗R (3.5 S) from MCF-7 human breast tumor cells, give a single major sedimentation peak (7–8 S) with rabbit Ig-i. The hormone specificity of rabbit Ig-i has been demonstrated by the absence of any interaction between Ig-i and androgen receptors of rat prostate, or mammalian and non-mammalian progesterone receptors. Also, there is no reaction with free estradiol, with non-specific binding components in human breast cancer cytosol, or with 125I-labeled mouse or rat a-fetoprotein. Goat antibodies to calf uterine E∗R react with nuclear and extranuclear E∗R of all tested tissues, including uterus, tumor and human breast cancer, to give a major new entity sedimenting at 11–14 S. The titer of antibodies to estrophilin in goat Ig-i is considerably higher than in rabbit Ig-i. Unlike rabbit antibody, interaction of goat Ig-i with extranuclear estrophilin causes a decrease in affinity and a reduction in the number of binding sites for estradiol. Fab fragments from both rabbit and goat Ig-i react with nuclear E∗R from calf uterus to give a product sedimenting slightly ahead of the original E∗R complex.

Estrophilin: Pro and anti

Abstract Antibodies to the estrogen-receptor protein (estrophilin) of calf uterus have been prepared in the rabbit, the goat and two strains of rat. Because these antibodies react with the receptor without preventing or displacing estradiol binding, antibody-antigen interaction is demonstrated and studied conveniently by its effect on the gel filtration and sedimentation properties of estrophilin, using radioactive estradiol as a marker. Antibodies to the nuclear form of calf uterine estrophilin produced in the rabbit and the goat cross react with nuclear as well as extranuclear estradiol-receptor complexes from reproductive tissues of every species tested; antiestrophilin obtained recently in the ACI rat appears similarly cross reactive. In contrast, antiestrophilin generated in the Lewis rat reacted only with receptor from calf uterus. Polyethylene glycol-mediated fusion of spleen cells from the immunized Lewis rat with three different mouse myeloma lines produced viable hybridoma cultures yielding monoclonal hybridoma cell lines secreting either rat IgG or IgM. which react specifically with calf uterine estrophilin. Production and cloning of hybridomas derived from spleen cells of the ACI rat, in an attempt to obtain cross reacting monoclonal antibody, are in progress. This assortment of either cross reacting or species-specific antibodies to estrophilin, especially those produced by monoclonal hybridomas, offers immunochemical approaches to receptor assay and purification as well as novel probes of receptor structure and function, including the question of whether estrophilin is synthesized in target cells by way of a proestrophilin incapable of binding steroid.

The immunoendocrinology of estrophilin.

Immunoglobulin from the serum of rabbits immunized with highly purified estradiol-receptor complex from calf uterine nuclei has been shown to contain specific antibodies to estrophilin by five criteria. Antibodies to calf nuclear estrophilin cross react with nuclear estradiol-receptor complexes of rat, rabbit and sheep uterus, rat endometrial and pituitary tumor, and MCF-7 human breast cancer cell line. They also react with extranuclear receptor of calf, rat, mouse, rabbit, guinea pig, monkey and sheep uterus, rat mammary, endometrial and pituitary tumor, and human breast cancer. There is no interaction of the antibody with estradiol itself. The nuclear form of estrophilin appears to bind more immunoglobulin molecules than does the cytosol form. The antibodies do not react with either the nuclear or extranuclear dihydrotestosterone-receptor complexes of rat prostate, with the extranuclear progesterone-receptor complexes of rabbit uterus, chick oviduct or rat endometrial tumor, or with rat and mouse alpha-fetoprotein. These findings indicate an immunochemical similarity among estrophilins from several mammalian species, as well as between nuclear and extranuclear forms of the receptor, but not among receptor proteins for different steroid hormones. Immunoglobulin from the serum of a goat immunized with similar antigen shows a considerably higher titer of antibodies to estrophilin. These react with nuclear and extranuclear estradiol-receptor complexes of calf uterus to produce somewhat larger entities than those formed with the rabbit antibody. Unlike the rabbit antibody, interaction with the goat antibody causes a noticeable decrease in estradiol-binding affinity of the extranuclear estrophilin as well as an apparent decrease in the total hormone-binding capacity. Specific antibodies to estrophilin offer promise as valuable reagents for receptor analysis and purification, as well as for the elucidation of many still unresolved questions concerning receptor synthesis, localization and function.