Antibodies to the estrogen receptor-α modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors

Antibodies (Abs) raised against the estrogen receptor-alpha (ER alpha) were used to investigate the role of ER alpha proteins located at the plasma membrane in mediating the rapid, estrogen-stimulated secretion of prolactin (PRL) from rat pituitary GH(3)/B6/F10 cells. Exposure of the cells to 1 nM 17 beta-estradiol (E-2) significantly increased PRL release after 3 or 6 min. When ER alpha Abs that bind specifically to ER alpha but are too large to diffuse into cells were tested for activity at the cell membrane, Ab R4, targeted to an ER alpha hinge region sequence, increased PRL release in a time-, and concentration-dependent fashion. Ab H151, directed against a different hinge region epitope, decreased PRL release and blocked the stimulatory action of E-2. Abs raised against the DNA binding domain (H226) or the carboxyl terminus (C542) were not biologically active. When each Ab was examined for recognition of ER alpha on the cell surface by immunocytochemistry, all except H151 generated immunostaining in aldehyde-fixed cells. In live cells, however, Ab H151 but not Ab R4 blocked the membrane binding of fluorescently tagged E-2-BSA. Overall, the data indicate that plasma membrane ER alpha proteins mediate estrogen-stimulated PRL release from GH(3)/B6/F10 cells. These results may also convey information about conformationally sensitive areas of the membrane form of ER alpha involved in rapid, nongenomic responses to estrogens,-Norfleet, A. M., Clarke, C. H., Gametchu, B., Watson, C. S. Antibodies to the estrogen receptor-alpha modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors.