A comparison of membrane vs. intracellular estrogen receptor-α in GH₃/B6 pituitary tumor cells using a quantitative plate immunoassay

The plasma membrane form of the estrogen receptor-α (mER-α) is involved in rapid estrogen-induced prolactin release from GH₃/B6 rat pituitary tumor cells and can be detected immunocytochemically using several estrogen receptor-α (ER-α) antibodies. We recently described staining of fixed cells via a biotin-avidin-alkaline phosphatase sandwich assay. From this protocol, we have developed a rapid, quantifiable 96-well plate immunoassay for mER-α, using a different alkaline phosphatase substrate, para-nitrophenylphosphate, which generates a soluble yellow product, para-nitrophenol. We also permeabilized cells with detergent during fixation to measure intracellular ER-α (iER-α) with the same assay and then compared intracellular versus membrane ER-α levels in two GH₃/B6 cell subclones originally selected for high and absent mER-α expression by immunocytochemistry. While the F10 subclone expresses plentiful amounts of the mER-α, the D9 subclone has undetectable levels of mER-α using this assay. In addition, there is a seven-fold difference in iER-α expression between the high (F10) and no (D9) mER-α expressing subclones. In the high mER-α expressing cell line, the mER-α totals approximately one third of total cellular ER-α. Neither membrane or intracellular forms of ER-β were detected with this assay. The pNp assay allows convenient and quantitative comparison of multiple parameters of mER-α and iER-α regulation and should be applicable to other antigens that are expressed on the cell surface as well as intracellularly.