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FIGURE 1-28 (A and B) Demonstration that unoccupied steroid hormone receptors may be associated with the cell nucleus. In this study the unoccupied estrogen receptor in MCF-7 human breast cancer cells was visualized through application of monoclonal antibodies which are specific for the estrogen receptor; the cells shown in B were treated identically except that a nonimmune monoclonal antibody was substituted. [Reproduced with permission from Figures 3A and B of King, W. J., and Greene, G. L. (1984). Localization of estrogen receptor in MCF-7 human breast cancer cells: Monoclonal antibodies localize estrogen receptor in the nuclei of target cells. Nature 307, 745-747. (C and D) Demonstration that occupied steroid hormone receptors are localized in the cell nucleus. This panel is an autoradiogram of MCF-7 human breast cancer

DNA-binding proteins, where they regulate the expression of genes related to the biological response of the hormone in question (described in detail later in this chapter).

Determination of the exact site of the subcellular location of the unliganded steroid receptors has been a subject of extensive research. While all of the steroid receptors have a high degree of structural homology there are still some key differences in biochemical properties, which result in differing proportions of the unoccupied receptor being present in the cytosol or nuclear compartments; some of the properties of the members of the family of steroid receptors are summarized in Table 1-5.

The unliganded forms of many of the receptors form hetero-oligomers that include one or more receptor molecules and a dimer of the 90-kDa heat shock protein (HSP), as well as other proteins (e.g., see Chapter 10). The function of the interaction of the unoccupied receptors with the HSP may be to occlude the DNA-binding domain of the unoccupied receptor and prevent inappropriate interaction with the DNA. Interaction with other proteins through HSP-90 may locate the receptor to the cytoskeleton. After the steroid receptor binds ligand, the receptor dissociates from the HSP and undergoes some form of conformational change that will optimize specific interaction with regions of DNA (see Figure 1-26).

Figure 1-26 presents a generic model of a typical steroid receptor. The protein consists of a single polypeptide chain divided into six domains. Starting at the N-terminus, the domains are, respectively, (i, ii) the A/B or variable (sequence and length) domain; (iii) the C or DNA binding domain which contains two zinc fingers; (iv) the D or variable hinge region; (v) the E or ligand binding domain; and (vi) the F or variable domain. The functioning of these domains will be presented later under the topic of mechanism of steroid action.

As shown in Fig. 1-27, there is a striking structural homology between all of the domains of the cell of the members of the steroid receptor gene superfamily; clearly, all of these proteins are related evolutionarily.

FIGURE 1-28—Continued cells incubated for 2 hr with high specific activity tritiated estradiol at 37°C for 2 hr. (C) 1 X 10~9 M) at 0°C and (D) at 37°C. The predominant localization is nuclear at both 0 and 37°C. Large black dots seen in the nuclei of unlabeled cells are nucleoli. Stained with methylgreen-pyronin, 2-week exposure, 4-/im sections, X900. [Reproduced by permission from Figure 4A of Sheridan, P. J., Buchanan, J. M., Anselmo, F. C., and Martin, P. M. (1979). Equilibrium: The intracellular distribution of steroid receptors. Nature 282, 579-582.

FIGURE 1-29 Progress curve for determining specific hormone binding to a receptor.

The ancestor to these proteins may be the oncogene product v-erbA or c-erbA. v-erbA is an oncogene product that binds to DNA but has no ligand-binding domain. Thus, v-erbA can constitutively activate a subset of genes that would normally be dependent upon the presence of hormone to occupy the ligand-binding domain. It is interesting that the thyroid hormone and retinoic acid receptors, as well as the hormonally active form of vitamin D3, l,25(OH)2D3, are members of this family of hormone receptors, since the structures of their ligands are not classically steroidal in nature.

2. Peptide-Protein Hormone Receptor

A number of protein hormones and, in some instances, their receptors, such as nerve growth factor (NGF), epidermal growth factor (EGF), and insulin, have been found by autoradiography experiments (employment of radioactive preparations of the hormone) to be localized in the nucleus or nuclear membrane of their target cells. It is not yet clear whether these hormone-receptor complexes are functional; they could have arrived at their intracellular location by the process of endocytosis, as described in Figure 1-20.

D. Measurement of Hormone-Receptor Interactions

Historically there have been two different techniques employed for studying the interaction of hormones with their receptors: autoradiography, which determines localization of the hormone under both in vivo and in vitro conditions, and saturation analysis, which is carried out under in vitro conditions. Both techniques are dependent upon access to radioactive preparations of the hormone under study with a high specific activity.

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