Estradiol induces the expression of an estrogen receptor ¨¢ variant at the cell- surface of hypothalamic neurons.

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

Buzios, Brasil

Congreso; 1st IBRO/LARC Congress of Neurosciences; 2008

Estradiol induces the expression of an estrogen receptor ¦Á variant at the cell- surface of hypothalamic neurons. S.V. Gorosito,  A. G. Lorenzo and M.J. Cambiasso Instituto Ferreyra (INIMEC-CONICET) C¨®rdoba, 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 ER¦Á 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 ER¦Á 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 ER¦Á in both fractions; in the supernatant there appeared the full-length (66 kDa) and the truncated (~ 55 kDa) ER¦Á, whereas in the pellet there appeared only the truncated isoform of ER¦Á. In control cultures, ER¦Á was not evident in the pellet fraction. Using different antibodies against ER¦Â (ZYMED and SCBT) and GPR30 (MBL), no immunoreactive bands were detected in the pellet fraction. Immunocytochemical analysis showed the expression of ER¦Á 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 ER¦Á 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.