Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding.
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
TC
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0645.
Gametchu
B
B
Watson
C S
CS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001666:Binding Sites, Antibody; D002462:Cell Membrane / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D007150:Immunohistochemistry; D008024:Ligands; D018613:Microscopy, Confocal; D011485:Protein Binding; D051381:Rats; D011960:Receptors, Estrogen / Q000276:immunology / Q000378:metabolism*; D012710:Serum Albumin, Bovine; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004967:Estrogens; D008024:Ligands; D011960:Receptors, Estrogen; D012710:Serum Albumin, Bovine; D016650:Fluorescein-5-isothiocyanate