Estrogen receptor-α detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry

Andrea M. Norfleet, Mary L. Thomas, Bahiru Gametchu, Cheryl S. Watson

    Research output: Contribution to journalArticle

    150 Citations (Scopus)

    Abstract

    A population of estrogen receptor-α (ERα) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ERα at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2% paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ERα. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ERα Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity of staining for ERα was confirmed by three approaches: first, treatment with an antisense oligodeoxynucleotide to nuclear ERα mRNA reduced immunolabeling of both membrane and nuclear ERα; second, labeling by two Abs raised against different ERα oligopeptides was neutralized by competing peptide; third, six Abs (ER21, H226, R4, H222, MC20, and C542) that recognize unique epitopes on rodent ERα produced immunolabeling, but neither primate-specific ERα Ab nor Ab to ERβ caused staining. In addition to demonstrating the plasma membrane ERα in GH3/B6/F10 cells, this method should be applicable to other cell types that exhibit nongenomic responses to estrogen or other steroid hormones.

    Original languageEnglish (US)
    Pages (from-to)3805-3814
    Number of pages10
    JournalEndocrinology
    Volume140
    Issue number8
    StatePublished - 1999

    Fingerprint

    Pituitary Neoplasms
    Aldehydes
    Estrogen Receptors
    Immunohistochemistry
    Cell Membrane
    Enzymes
    Antibodies
    Staining and Labeling
    Detergents
    Estrogens
    Nuclear Antigens
    Oligopeptides
    Gastrin-Secreting Cells
    Avidin
    Oligodeoxyribonucleotides
    Nuclear Envelope
    Glutaral
    Surface Antigens
    Cytoplasmic and Nuclear Receptors
    Biotin

    ASJC Scopus subject areas

    • Endocrinology
    • Endocrinology, Diabetes and Metabolism

    Cite this

    Estrogen receptor-α detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry. / Norfleet, Andrea M.; Thomas, Mary L.; Gametchu, Bahiru; Watson, Cheryl S.

    In: Endocrinology, Vol. 140, No. 8, 1999, p. 3805-3814.

    Research output: Contribution to journalArticle

    Norfleet, Andrea M. ; Thomas, Mary L. ; Gametchu, Bahiru ; Watson, Cheryl S. / Estrogen receptor-α detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry. In: Endocrinology. 1999 ; Vol. 140, No. 8. pp. 3805-3814.
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    abstract = "A population of estrogen receptor-α (ERα) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ERα at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2{\%} paraformaldehyde/0.1{\%} glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ERα. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ERα Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity of staining for ERα was confirmed by three approaches: first, treatment with an antisense oligodeoxynucleotide to nuclear ERα mRNA reduced immunolabeling of both membrane and nuclear ERα; second, labeling by two Abs raised against different ERα oligopeptides was neutralized by competing peptide; third, six Abs (ER21, H226, R4, H222, MC20, and C542) that recognize unique epitopes on rodent ERα produced immunolabeling, but neither primate-specific ERα Ab nor Ab to ERβ caused staining. In addition to demonstrating the plasma membrane ERα in GH3/B6/F10 cells, this method should be applicable to other cell types that exhibit nongenomic responses to estrogen or other steroid hormones.",
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