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

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.

Membrane Glucocorticoid Receptor Activation Induces Proteomic Changes Aligning with Classical Glucocorticoid Effects

“…In situ PLA was sensitive enough to detect the mGR in these cell lines without the need for serum starvation, known to enhance membrane expression of steroid receptors (45). As expected GC withdrawal increased mGR expression in all cell lines (except Jurkat cells) (supplemental Fig.…”

Mechanisms of membrane estrogen receptor-α-mediated rapid stimulation of Ca2+levels and prolactin release in a pituitary cell line

“…This procedure was used to normalize the PRL concentration to cell number (5). Briefly, after collection of the supernatant, cells were fixed in 2% paraformaldehyde-0.1% glutaraldehyde for 30 min at RT.…”

Nongenomic actions of low concentration estrogens and xenoestrogens on multiple tissues

“…Collectively, these receptors inhabit nuclei and cytoplasm (Mangelsdorf et al 1995), plasma membranes or perimembrane spaces (Clarke C.H., Norfleet, Clarke M.S.F., Watson, Cunningham K.A., & Thomas 2000;Thomas et al 2005;Watson & Gametchu 2003b), and endoplasmic reticulum and mitochondria (Evans, Jr. & Muldoon 1991;Revankar et al 2005;Yager & Chen 2007); they also sometimes change locations or arrangements within their locations, depending upon liganding or other circumstances (Campbell et al 2002;Song et al 2002). [Though they are other versions of ERs, the family of estrogen receptor-related (ERR) proteins are probably not potential targets because of their very small ligand binding pockets that can only accommodate ~4 carbon atoms (Kallen et al 2004). ]…”