“Estradiol induces the expression of an estrogen receptor alpha variant at the cell- surface of hypothalamic neurons”

S.V. GOROSITO, A. G. LORENZO AND M.J. CAMBIASSO

Buzios, RJ, Brasil

Congreso; ”. I Congreso IBRO/LARC de Neurociencias de América Latina, Caribe y Península Ibérica; 2008

Estradiol induces the expression of an estrogen receptor α variant at the cell- surface of hypothalamic neurons. S.V. Gorosito,  A. G. Lorenzo and M.J. Cambiasso Instituto Ferreyra (INIMEC-CONICET) Córdoba, Argentina sgorosito@immf.uncor.edu  Previous studies from our laboratory have demonstrated that the axogenic effect of 17-b-estradiol (E2) on male hypothalamic neurons is not exerted through the classical intracellular estrogen receptor (ER) but depends on a membrane mechanism involving Ca2+, PKC and ERK signaling. Recently, we found that a truncated ERa was localized in membrane fractions of hypothalamic tissue from E16 embryos. Moreover, our experiments extend these results to hypothalamic neurons in vitro showing that ERα can be detected from the cell exterior as a biotinylated cell-surface protein. In the present study we investigate the effect of E2 treatment on ERα localization. To this end, we used the membrane impermeant sulfo-NHS-biotin to label cell-surface proteins of hypothalamic neurons grown for 48h with or without 10 nM E2. The labeled proteins were collected with avidin-agarose beads to obtain 2 fractions: the pellet fraction, with the cell-surface biotinylated proteins; and the supernatant fraction, with the intracellular un-biotinylated proteins. In E2 treated cultures, Western blot analysis revealed the presence of ERα in both fractions; in the supernatant there appeared the full-length (66 kDa) and the truncated (~ 55 kDa) ERα, whereas in the pellet there appeared only the truncated isoform of ERα. In control cultures, ERα was not evident in the pellet fraction. Using different antibodies against ERβ (ZYMED and SCBT) and GPR30 (MBL), no immunoreactive bands were detected in the pellet fraction. Immunocytochemical analysis showed the expression of ERα in the cell- surface of non-permeabilized neurons.  A strong staining at the cytoplasmic and nuclear levels was observed in permeabilized neurons. In summary, these results confirm the presence of an ERα on the plasma membrane of hypothalamic neurons in vitro. We have also shown that E2 treatment induces the expression of the truncated isoform of ERa at neuronal cell-surface. Although the mechanisms of E2 translocation to the neuronal cell-surface will have to be addressed, the evidence presented in this report indicates the need to re-evaluate the current estrogen signaling models. Supported by CONICET and FONCyT.