A population of estrogen receptor-alpha (ER alpha) 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 ER alpha 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 ER alpha. 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. ER alpha 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 ER alpha was confirmed by three approaches: first, treatment with an antisense oligodeoxynucleotide to nuclear ER alpha mRNA reduced immunolabeling of both membrane and nuclear ER alpha; second, labeling by two Abs raised against different ER alpha oligopeptides was neutralized by competing peptide; third, six Abs (ER21, H226, R4, H222, MC20, and C542) that recognize unique epitopes on rodent ER alpha produced immunolabeling, but neither primate-specific ER alpha Ab nor Ab to ER beta caused staining. In addition to demonstrating the plasma membrane ER alpha 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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Antibodies (Abs) raised against the estrogen receptor-alpha (ERalpha) were used to investigate the role of ERalpha proteins located at the plasma membrane in mediating the rapid, estrogen-stimulated secretion of prolactin (PRL) from rat pituitary GH(3)/B6/F10 cells. Exposure of the cells to 1 nM 17beta-estradiol (E(2)) significantly increased PRL release after 3 or 6 min. When ERalpha Abs that bind specifically to ERalpha but are too large to diffuse into cells were tested for activity at the cell membrane, Ab R4, targeted to an ERalpha hinge region sequence, increased PRL release in a time- and concentration-dependent fashion. Ab H151, directed against a different hinge region epitope, decreased PRL release and blocked the stimulatory action of E(2). Abs raised against the DNA binding domain (H226) or the carboxyl terminus (C542) were not biologically active. When each Ab was examined for recognition of ERalpha on the cell surface by immunocytochemistry, all except H151 generated immunostaining in aldehyde-fixed cells. In live cells, however, Ab H151 but not Ab R4 blocked the membrane binding of fluorescently tagged E(2)-BSA. Overall, the data indicate that plasma membrane ERalpha proteins mediate estrogen-stimulated PRL release from GH(3)/B6/F10 cells. These results may also convey information about conformationally sensitive areas of the membrane form of ERalpha involved in rapid, nongenomic responses to estrogens.

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There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) α or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERα were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERα, rabbit polyclonal IgG antibody which does not recognize ERβ was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERα, but abundant labeling for ERα throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 µM antisense oligonucleotide directed against the translation start site of ERα reduced ERα immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERα. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERα in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERα as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.