Estradiol-Induced Estrogen Receptor-α Trafficking

Estradiol has rapid actions in the CNS that are mediated by membrane estrogen receptors (ERs) and activate cell signaling pathways through interaction with metabotropic glutamate receptors (mGluRs). Membrane-initiated estradiol signaling increases the free cytoplasmic calcium concentration ([Ca2+](i)) that stimulates the synthesis of neuroprogesterone in astrocytes. Weused surface biotinylation to demonstrate that ER alpha has an extracellular portion. In addition to the full-length ER alpha [apparent molecular weight (MW), 66 kDa], surface biotinylation labeled an ER alpha-immunoreactive protein (MW, similar to 52 kDa) identified by both COOH-and NH2-directed antibodies. Estradiol treatment regulated membrane levels of both proteins in parallel: within 5 min, estradiol significantly increased membrane levels of the 66 and 52 kDa ER alpha. Internalization, a measure of membrane receptor activation, was also increased by estradiol with a similar time course. Continuous treatment with estradiol for 24-48 h reduced ER alpha levels, suggesting receptor downregulation. Estradiol also increased mGluR1a trafficking and internalization, consistent with the proposed ER alpha-mGluR1a interaction. Blocking ER with ICI 182,780 or mGluR1a with LY 367385 prevented ER alpha trafficking to and from the membrane. Estradiol-induced [Ca2+](i) flux was also significantly increased at the time of peak ER alpha activation/internalization. These results demonstrate that ER alpha is present in the membrane and has an extracellular portion. Furthermore, membrane levels and internalization of ER alpha are regulated by estradiol and mGluR1a ligands. The pattern of trafficking into and out of the membrane suggests that the changing concentration of estradiol during the estrous cycle regulates ER alpha to augment and then terminate membrane-initiated signaling.