Membrane estrogen receptors identified by multiple antibody labeling and impeded‐ligand binding

TLDR

The nongenomic estrogen response in GH3/B6 pituitary tumor cells lacks a defined protein mediator. The authors used monoclonal antibodies H226, H222, and polyclonal ER21, each targeting distinct intracellular ER epitopes, to immunohistochemically label membrane proteins in GH3/B6 cells. Rapid prolactin release occurs within 5 min of nanomolar estrogen exposure, and confocal microscopy, antibody labeling, and fluorescent estrogen‑BSA binding demonstrate a membrane‑localized ER that colocalizes with the ligand and appears structurally similar to the intracellular ER. Published in FASEB Journal 1995 (9:404–410) by Pappas et al.

Abstract

GH3/B6 rat pituitary tumor cells exhibit rapid prolactin release (within 5 min) when treated with nanomolar amounts of estrogen. However, the putative protein mediator of this nongenomic action has not been described. Using antibodies directed against a peptide representing the hinge region of the intracellular estrogen receptor (iER), we have demonstrated that these cells contain a membrane ER (mER). We now report that confocal scanning laser microscopy of cells labeled live with the anti-peptide antibody further supports a membrane localization of ER. The monoclonal antibodies H226 and H222 and a polyclonal antibody, ER21, each recognizing a unique epitope on iER (NH2 terminal to the DNA-binding region, within the steroid binding region, and the NH2-terminal end, respectively), also immunohistochemically label membrane proteins of immuno-selected GH3/B6 cells. These cells also specifically bind a fluorescent estrogen-BSA conjugate. Coincubation of cells with anti-ER antibody and the fluorescent estrogen-BSA conjugate reveals that these labels colocalize on cells. These results suggest that mER may be structurally similar to iER.—Pappas, T. C., Gametchu, B., Watson, C. S. Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J. 9, 404–410 (1995)