Estrogen Receptor-  Detected on the Plasma Membrane of Aldehyde-Fixed GH3/B6/F10 Rat Pituitary Tumor Cells by Enzyme-Linked Immunocytochemistry

Abstract

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.

Membrane Oestrogen Receptors on Rat Pituitary Tumour Cells: Immuno‐Identification and Responses to Oestradiol and Xenoestrogens

“…n.d., not done. a, Norfleet et al 1999a;b, Norfleet et al 1999b;c, Pappas et al 1995a;d, Pappas et al 1995b;e, Pappas et al 1994;f, Watson et al 1995. results from these experiments are summarized in Fig. 1, which depicts recognition of seven epitopes by nine antibodies to iERá.…”

Sex differences in drug abuse

“…A number of immunocytochemical studies have demonstrated that antibodies to ERα will bind to the exterior of the cell. Watson and colleagues have shown that seven different antibodies to ERα bind to the membrane of rat pituitary tumor cells and antibodies that bind to the hormone-binding domain of ERα modulate rapid E-induced prolactin release [94]. In the hippocampus estradiol regulates the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites [143] and there is ultrastructural evidence that there are ERα .…”

Rapid mechanisms of glucocorticoid signaling in the Leydig cell☆

“…This case has been confirmed in other nuclear receptor such as estrogen receptor α (ERα). In ERα-expressing pituitary tumor cells (GH3 cells), ERα is located in the plasma membrane and mediates the estrogen-induced rapid release of prolactin, because it is blocked or stimulated directly by antibodies directed to different amino acid sequences of ERα [32].…”