Membrane estrogen receptor-enriched GH₃/B6 cells have an enhanced non-genomic response to estrogen

We immunoselected GH₃/B6 cells for a membrane estrogen receptor (mER) using antibodies generated against the rat intracellular ER (iER). Immunocytochemistry with anti-ER antibodies revealed bright fluorescence distributed in patches over the surface of mER-enriched cells, while cells immuno-depleted for mER showed only low-level membrane immunofluorescence. Quantitation via digital image analysis confirmed that immunoenriched populations show increases in both stained cell numbers and intensity of staining. Short-term culturing with serum reversibly decreased the intensity of immunostaining in mER-enriched cells to immuno-depleted cell levels. The mER-enriched populations initially contained ∼85% immunopositive cells in defined medium, but when cultured continuously with serum gradually decline to ∼22% immunopositive cells by 10 weeks. Cells enriched for mER showed a significant increase in rapid (after 2 or 5 min) prolactin release when treated with 17β-estradiol, while mER-depleted cells lacked this response. Immunoprecipitabie membrane proteins isolated from mER-enriched cells were 60,000, 74,000 and ∼ 200,000 MW, compared to an iER size of 67,000. Therefore, the presence and level of an mER that is antigenically related to iER is correlated with the ability of GH₃/B6 cells to mediate a rapid action of estrogen.