Differences between estrogen- and antiestrogen-estrogen receptor complexes from human breast tumors identified with an antibody raised against the estrogen receptor.

Radiolabeled estrogens 17 beta-[3H]estradiol and diethylstilbestrol ( [3H]DES) and the antiestrogen [3H]monohydroxytamoxifen ( [3H]MHT) all bind with high affinity to the extranuclear estrogen receptor (ER) from the MCF-7 human breast tumor cell line (Kd = 3 X 10(-10), 2 X 10(-10), and 0.63 X 10(-10) M, respectively). A polyclonal antibody raised in a goat to the calf nuclear ER selectively decreased the binding affinity and number of binding sites for 17 beta-[3H]estradiol, but did not appear to affect these binding parameters for [3H]MHT. In the presence of goat antibody, the binding of the nonsteroidal estrogen DES was so perturbed that it was not possible to quantitate the decreased number of binding sites or affinity of this compound as assessed by Scatchard saturation analysis. These results were confirmed in human breast tumor cytosols by sucrose density gradient analysis. The binding of 17 beta-[3H]-estradiol and [3H]DES to the ER was significantly reduced by preincubation with the polyclonal antibody, whereas the binding of [3H]MHT was reduced only when the tumor cytosol was preincubated with a very high concentration of antibody. At these concentrations of antibody, the binding of 17 beta-[3H]estradiol and [3H]DES to the receptor was prevented completely. In contrast, when the antibody was added to the tumor cytosol after the 3H-ligand had bound to the receptor, the binding properties of all 3H-ligands were unaffected. The [3H]MHT-ER antibody complex consistently sedimented as a higher-molecular-weight complex on sucrose density gradients than did the corresponding estrogenic complexes. The decrease in the affinity of estrogenic ligands can be explained in part by an increase in the dissociation rate at 4 degrees of these compounds from the ER. The dissociation rate of MHT was unaffected by the goat antibody. These results imply that there are important differences in the binding of antiestrogen and estrogens to the tumor cytosol ER. A ligand-binding model is proposed that may aid in the understanding of antiestrogen action.