title: Antibodies to the estrogen receptor-alpha modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors.
abstract: 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.
authors.affiliation: Department of Human Biological Chemistry, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Norfleet
authors.forename: C H
authors.initials: CH
authors.lastname: Clarke
authors.forename: B
authors.initials: B
authors.lastname: Gametchu
authors.forename: C S
authors.initials: CS
authors.lastname: Watson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D047628:Estrogen Receptor alpha; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D010911:Pituitary Neoplasms / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism*; D051381:Rats; D011960:Receptors, Estrogen / Q000276:immunology*
chemical_list: D000906:Antibodies; D047628:Estrogen Receptor alpha; D008565:Membrane Proteins; D011960:Receptors, Estrogen; D011388:Prolactin