AN ESTROGEN RECEPTOR Á VARIANT LOCATED AT THE CELL- SURFACE OF HYPOTHALAMIC NEURONS IS REGULATED BY ESTRADIOL

CAMBIASSO, M.J., GOROSITO S.V

Villa Gualino, Torino, Italia

Congreso; V Meeting Steroids and Nervous System; 2009

Consejo superior de investigaciones cientificas

An estrogen receptor á variant located at the cell- surface of hypothalamic neurons is regulated by estradiol Cambiasso M.J., Gorosito S.V. Instituto de Investigación Médica M y M Ferreyra (INIMEC-CONICET), Friuli 2434 (5016) Córdoba, Argentina. Fax: +54-351-4695163. E-mail: jcambiasso@immf.uncor.edu   Although the biological activity of estrogen is generally mediated through nuclear estrogen receptors, a large body of evidence indicates that estrogen may also affect target cells upon binding to putative membrane estrogen receptors (mER) coupled to intracellular signaling cascades. 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 [1, 2]. 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 ERá can be detected from the cell exterior as a biotinylated cell-surface protein [3]. In the present study we investigate the effect of E2 treatment on ERá membrane 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 ERá in both fractions; in the supernatant there appeared the full-length (66 kDa) and the truncated (~ 55 kDa) ERá, whereas in the pellet there appeared only the truncated isoform of ERá. In control cultures, ERá was not evident in the pellet fraction. Using different antibodies against ERâ (ZYMED and SCBT) and GPR30 (MBL), no immunoreactive bands were detected in the pellet fraction. Immunocytochemical analysis showed the expression of ERá 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 ERá 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. These evidences indicate the need to address the mechanism of ER translocation to the neuronal cell-surface.   Research is supported by CONICET (PIP6238) and FONCyT (PICT26331).   Reference List   1. Carrer, H.F., Cambiasso, M.J., Gorosito, S., 2005. Effects of estrogen on neuronal growth and differentiation. J.Steroid Biochem. Mol. Biol., 93, 319-323. 2. Gorosito, S., Cambiasso, M.J., 2008. Axogenic effect of estrogen in male hypothalamic neurons involves Ca2+, PKC and ERK signaling. J Neurosci Res., 86(1), 145-157. 3. Gorosito, S., Lorenzo, A., Cambiasso, M.J., 2008. Estrogen receptor a is expressed on the cell-surface of embryonic hypothalamic neurons. Neuroscience 154(4), 1173-7.