Estrogen Binding Component of Mouse Leydig Cell Tumor: Anin VitroConversion from Nonreceptor to Receptor-Like Molecule*

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Abstract
The estorgen-binding components in the cytosols prepared from estrogen-dependent (tumor code 22137) and -independent (tumor code 124958) mouse Leydig cell tumors were examined. The estrogen-dependent tumor was found to contain the typical estrogen receptor. The estrogen-independent tumor has a unique estrogen-binding component with low affinity for 17β-estradiol (E2; Kd = 10-7 M) and no affinity for diethylstilbesterol (DES). However, removal of small molecules from the cytosol by dialysis, gel filtration, or ammonium sulfate fractionation resulted in a marked increase in affinity for E2 (Kd = 1O-9-10-10 M) and DES. Similar changes in the dissociation constant and steroid specificity were induced by simply allowing the cytosol to remain at 0 C for more than 18 h or exposing the cytosol to 0.4 M NaSCN for 30 min. These treatments, which induced the tight binding of [3H]E2, made it possible to determine the sedimentation constant in a sucrose gradient. Gel filtration, dialysis, or allowing the cytosol to stand for 24 h at 0 C resulted in the appearance of 7–8S and 3–4S peaks in a low salt medium but a 4–5S peak in a high salt medium. A NaSCNinduced high affinity binder migrated in the 2.8S region in a gradient containing 0.4 M NaSCN. The sedimentation constant of the estrogen receptor from tumor 22137 was 4S in a 0.4-M NaSCN sucrose gradient. The Stokes radii of estrogen-binding components treated with NaSCN were estimated by Sephadex G-200 chromatography in high salt buffer (0.4 M KCI). The components from tumor 124958 and tumor 22137 had the Stokes radii of 31.5 and 40.5 Å, respectively. The binding behavior of this estrogen-binding component from tumor 124958 to isolated nuclei or DNA was also examined. When the cytosol was labeled with [3H]E2 and then incubated with nuclei or DNA cellulose, no appreciable binding of [3H]E2 with nuclei or DNA was observed, even though a conventional heat activation procedure was employed. However, dialysis of the cytosol caused a marked increase in nuclear or DNA binding of the estrogen-binding component. This binding was also found to be ionic strength dependent. On the other hand, a NaSCNinduced high affinity component did not bind to nuclei, while the estrogen receptor of tumor 22137 showed an increase in nuclear binding after treatment with NaSCN. These results suggest that the cytosol in the estrogen-independent tumor contains a small-sized estrogen-binding component with a low affinity for E2 and no affinity for DES, which can be converted to an estrogen receptor-like molecule by removal of a dialyzable compound(s).

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