Estrone receptor formation during the processing of estradiol-receptor complex in MCF-7 cells.

Human breast cancer cells (MCF-7, maintained in long-term culture) contain separate estrogen receptors specific for either 17 beta-estradiol or estrone. Utilizing optimum conditions for the protamine sulfate assay, it has been possible to demonstrate both receptors in the 0.6 M KCl extract of nuclei and in the cytosol. Similarly, in the exchange assay, high-affinity low-capacity binding sites for 17 beta-estradiol and estrone have been found in the salt-extracted nuclear residue. Dissociation constants and binding capacities were determined for either receptor in the absence of the other [e.g., estrone receptor (E1R) in the cytosol or nuclear residue from 17 beta-estradiol-stimulated cells] or, when both receptors were present, a saturating amount of the other estrogen (unlabeled) was added to the assay mixture (e.g., the salt-extractable nuclear receptors). Specificity was demonstrated by the inability of estrone to compete with 17 beta-[2,4,6,7-3H]estradiol for the 17 beta-estradiol receptor (E2R) at molar excesses less than 10-fold. Likewise, there was no inhibition of [6,7-3H]estrone binding to its receptor by molar excesses of 17 beta-estradiol below 100-fold. Other steroid hormones were very weak competitors of [6,7-3H]estrone, even at 1000-fold molar excesses. The quantitative relationships of these two estrogen receptors were shown to fluctuate in the various cellular compartments following incubation (37 degrees) of MCF-7 cells with 10(-8) M 17 beta-estradiol. This level of 17 beta-estradiol elicited the translocation of all detectable cytosolic E2R to the nucleus, where, after an incubation of 1 hr, the salt-resistant 17 beta-estradiol disappeared and 40% of the extractable 17 beta-estradiol-binding capacity was lost (processed). Simultaneously, the E1R which remained in the nuclear residue appeared in the nuclear extract, and ultimately this receptor accumulated in the cytosol. The estrone-binding capacity (0.78 pmol/mg DNA) which appeared following the processing of E2R nearly equalled the loss of 17 beta-estradiol binding sites per cell (0.85 pmol/mg DNA). Concentrations of 17 beta-estradiol which elicited the greatest processing of E2R in these incubations brought about the appearance of maximum levels of E1R in MCF-7 cells. Considering these results in the light of data previously reported from this laboratory concerning the metabolic and ligand fate of 17 beta-[3H]estradiol in MCF-7 cells, processing would appear to involve the formation of E1R in the salt-resistant nuclear compartment followed by the accumulation of E1R in the cytosol.