Distribution and Posttranslational Modification of Synaptic ERα in the Adult Female Rat Hippocampus

Acute 17 beta-estradiol (E2) signaling in the brain is mediated by extranuclear estrogen receptors. Here we used biochemical methods to investigate the distribution, posttranslational modification, and E2 regulation of estrogen receptor-alpha (ER alpha) in synaptosomal fractions isolated by differential centrifugation from the adult female rat hippocampus. We find that ER alpha is concentrated presynaptically and is highly enriched with synaptic vesicles. Immunoisolation of vesicles using vesicle subtype-specific markers showed that ER alpha is associated with both glutamate and gamma-aminobutyric acid-containing neurotransmitter vesicles as well as with some large dense core vesicles. Experiments using broad spectrum and residue-specific phosphatases indicated that a portion of ER alpha in synaptosomal fractions is phosphorylated at serine/threonine residues leading to a mobility shift in SDS-PAGE and creating a double band on Western blots. The phosphorylated form of ER alpha runs in the upper of the two bands and is particularly concentrated with synaptic vesicles. Finally, we used E2 with or without the acyl protein thioesterase 1 inhibitor, Palmostatin B, to show that 20 min of E2 treatment of hippocampal slices depletes ER alpha from the synaptosomal membrane by depalmitoylation. We found no evidence that E2 regulates phosphorylation of synaptosomal ER alpha on this time scale. These studies begin to fill the gap between detailed molecular characterization of extranuclear ER alpha in previous in vitro studies and acute E2 modulation of hippocampal synapses in the adult brain. (Endocrinology 154: 819-830, 2013)