Characterization of a membrane-associated estrogen receptor in a rat hypothalamic cell line (D12) |
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Authors: | Deecher Darlene C Swiggard Pamela Frail Donald E O'Connor Lawrence T |
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Institution: | (1) Pfizer, St. Louis, MO;(2) American Medical Association, Chicago, IL;(3) Women’s Health Research Institute, Wyeth Research N3114, 500 Arcola Road, 19426 Collegeville, PA |
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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-α-iodo-3,17-β-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-α-estradiol and estrone had similar binding characteristics for both receptors while differences were noted for
raloxifene, 17β-estradiol (E2), and genistein. Western blot and immunocytochemical analyses using antibodies specific for
ERα confirmed that D12 cells expressed a membrane-associated protein with a molecular mass (67 kDa) similar to that of ERα
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 ERα. |
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Keywords: | Membrane estrogen receptor estradiol estrone raloxifene radioligands MC20 |
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