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

T. C. Pappas, B. Gametchu, C. S. Watson

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)404-410
Number of pages7
JournalFASEB Journal
Volume9
Issue number5
StatePublished - Jan 1 1995

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Keywords

  • E
  • GH/B6
  • confocal microscopy
  • monoclonal antibody

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Pappas, T. C., Gametchu, B., & Watson, C. S. (1995). Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB Journal, 9(5), 404-410.