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Regulation of the membrane estrogen receptor-: role of cell density, serum, cell passage number, and estradiol

Human Biological Chemistry and Genetics Department, University of Texas, Medical Branch, Galveston Texas, USA; and
^* Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

¹Correspondence: Department of Human Biological Chemistry and Genetics, University of Texas, Medical Branch, Galveston, TX 77555-0645, USA. E-mail: cswatson{at}utmb.edu

We used modified immunocytochemical conditions to quantify a membrane form of estrogen receptor- (mER) in a rat pituitary tumor cell line, GH₃/B6/F10. We studied the regulation of mER vs. levels of intracellular ER (iER) using our 96-well plate immunoassay. The anti-ER antibody C542 was used to label the ER (via conjugated alkaline phosphatase) in fixed permeabilized (for iER) vs. nonpermeabilized cells (for mER). Expression of mER was highest at low cell densities (<1000 cells/well) and decreased significantly at densities where cellular processes touched, whereas the more abundant iER increased with increasing cell density over the same range. Serum starvation for 48 h caused increases in mER, whereas iER levels showed no significant changes. A large decline in mER and iER levels with cell passage number was observed. Minutes after nM 17ß-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 mER 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 mER may explain the relatively few reports of this protein in other experimental systems.—Campbell, C. H., Bulayeva, N., Brown, D. B., Gametchu, B., Watson, C. S. Regulation of the membrane estrogen receptor-: role of cell density, serum, cell passage number, and estradiol.

Key Words: estrogens • nongenomic effects of steroids • rapid • nuclear receptors

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