A comparison of membrane vs. intracellular estrogen receptor-alpha in GH(3)/B6 pituitary tumor cells using a...

The plasma membrane form of the estrogen receptor-alpha (mER-alpha) is involved in rapid estrogen-induced prolactin release from GH(3)/B6 rat pituitary tumor cells and can be detected immunocytochemically using several estrogen receptor-alpha (ER-alpha) 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-alpha, 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-alpha (iER-alpha) with the same assay and then compared intracellular versus membrane ER-alpha levels in two GH(3)/B6 cell subclones originally selected for high and absent mER-alpha expression by immunocytochemistry. While the F10 subclone expresses plentiful amounts of the mER-alpha, the D9 subclone has undetectable levels of mER-alpha using this assay. In addition, there is a seven-fold difference in iER-alpha expression between the high (F10) and no (D9) mER-alpha expressing subclones. In the high mER-alpha expressing cell line, the mER-alpha totals approximately one third of total cellular ER-alpha. Neither membrane or intracellular forms of ER-beta were detected with this assay. The pNp assay allows convenient and quantitative comparison of multiple parameters of mER-alpha and iER-alpha regulation and should be applicable to other antigens that are expressed on the cell surface as well as intracellularly.