title: Characterization of a membrane-associated estrogen receptor in a rat hypothalamic cell line (D12).
abstract: The ability of estrogens to produce rapid changes in cellular function has been firmly established. The question remains whether these changes are mediated by a modified form of the nuclear estrogen receptor (ER) that is associated with the plasma membrane (mER) or by a completely novel membrane receptor. Therefore, we characterized the biochemical properties of the nuclear and membrane-associated ERs expressed endogenously in a rat hypothalamic endothelial cell line (D12). Radioligand binding experiments using D12 membrane fractions showed that these cells exhibit properties consistent with a binding site specific for estrogens (mER). Equilibrium binding assays using [125I]16-alpha-iodo-3,17- beta-estradiol revealed saturable binding to mER, an affinity value similar to nuclear ER, with differing receptor expression levels. Competition assays revealed that 9 of 12 ER ligands tested had comparable affinities for mER and ER. For example, 17-alpha-estradiol and estrone had similar binding characteristics for both receptors while differences were noted for raloxifene, 17beta-estradiol (E2), and genistein. Western blot and immunocytochemical analyses using antibodies specific for ERalpha confirmed that D12 cells expressed a membrane-associated protein with a molecular mass (67 kDa) similar to that of ERalpha that colocalized with caveolae-enriched membranes. A rapid increase in intracellar Ca2+ levels in the presence of E2 suggests that mER can mediate physiologic changes through calcium mobilization. These data support the expression of mER in these brain-derived endothelial cells that is similar to, but biochemically distinguishable from, nuclear ERalpha.
authors.affiliation: Women's Health Research Institute, Wyeth Research, Collegeville, PA 19426, USA. Deeched@Wyeth.com
authors.forename: Darlene C
authors.initials: DC
authors.lastname: Deecher
authors.forename: Pamela
authors.initials: P
authors.lastname: Swiggard
authors.forename: Donald E
authors.initials: DE
authors.lastname: Frail
authors.forename: Lawrence T
authors.initials: LT
authors.lastname: O'Connor
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D051242:Caveolin 1; D022461:Caveolins / Q000502:physiology; D042783:Endothelial Cells; D004958:Estradiol / Q000378:metabolism / Q000502:physiology*; D004970:Estrone / Q000502:physiology; D005470:Fluorometry; D019833:Genistein / Q000494:pharmacology; D007031:Hypothalamus / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008970:Molecular Weight; D020849:Raloxifene Hydrochloride / Q000494:pharmacology; D051381:Rats; D011960:Receptors, Estrogen / Q000378:metabolism / Q000502:physiology*; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology
chemical_list: D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D011960:Receptors, Estrogen; D020845:Selective Estrogen Receptor Modulators; D004970:Estrone; D020849:Raloxifene Hydrochloride; D004958:Estradiol; D019833:Genistein; D002118:Calcium