Abstract
We used modified immunocytochemical conditions to quantify a membrane form of estrogen receptor-alpha (mERalpha) in a rat pituitary tumor cell line, GH3/B6/F10. We studied the regulation of mERalpha vs. levels of intracellular ERalpha (iERalpha) using our 96-well plate immunoassay. The anti-ERalpha antibody C542 was used to label the ERalpha (via conjugated alkaline phosphatase) in fixed permeabilized (for iERalpha) vs. nonpermeabilized cells (for mERalpha). Expression of mERalpha was highest at low cell densities (<1000 cells/well) and decreased significantly at densities where cellular processes touched, whereas the more abundant iERalpha increased with increasing cell density over the same range. Serum starvation for 48 h caused increases in mERalpha, whereas iERalpha levels showed no significant changes. A large decline in mERalpha and iERalpha levels with cell passage number was observed. Minutes after nM 17beta-estradiol (E2) treatment, a portion of the cells rounded up and detached from the culture plate, whereas nM cholesterol had no such effect. Although E2 treatment did not change mERalpha levels, the antigen was reorganized from a fine particulate to aggregation into asymmetric large granules of staining. That common culturing conditions favor down-regulation of mERalpha may explain the relatively few reports of this protein in other experimental systems.