Bondar Galyna, Kuo John, Hamid Naheed, Micevych Paul
Department of Neurobiology, Laboratory of Neuroendocrinology and Brian Research Institute, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
J Neurosci. 2009 Dec 2;29(48):15323-30. doi: 10.1523/JNEUROSCI.2107-09.2009.
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 (Ca(2+)) that stimulates the synthesis of neuroprogesterone in astrocytes. We used surface biotinylation to demonstrate that ERalpha has an extracellular portion. In addition to the full-length ERalpha [apparent molecular weight (MW), 66 kDa], surface biotinylation labeled an ERalpha-immunoreactive protein (MW, approximately 52 kDa) identified by both COOH- and NH(2)-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 ERalpha. 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 ERalpha levels, suggesting receptor downregulation. Estradiol also increased mGluR1a trafficking and internalization, consistent with the proposed ERalpha-mGluR1a interaction. Blocking ER with ICI 182,780 or mGluR1a with LY 367385 prevented ERalpha trafficking to and from the membrane. Estradiol-induced Ca(2+) flux was also significantly increased at the time of peak ERalpha activation/internalization. These results demonstrate that ERalpha is present in the membrane and has an extracellular portion. Furthermore, membrane levels and internalization of ERalpha 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 ERalpha to augment and then terminate membrane-initiated signaling.
雌二醇在中枢神经系统中具有快速作用，这些作用由膜雌激素受体（ERs）介导，并通过与代谢型谷氨酸受体（mGluRs）相互作用激活细胞信号通路。膜启动的雌二醇信号传导增加了游离细胞质钙浓度（Ca(2+)），从而刺激星形胶质细胞中神经孕酮的合成。我们使用表面生物素化来证明ERα具有细胞外部分。除了全长ERα[表观分子量（MW），66 kDa]外，表面生物素化还标记了一种由COOH和NH(2)导向抗体鉴定的ERα免疫反应蛋白（MW，约52 kDa）。雌二醇处理同时调节这两种蛋白的膜水平：在5分钟内，雌二醇显著增加了66 kDa和52 kDa ERα的膜水平。内化是膜受体激活的一种测量指标，雌二醇也以类似的时间进程增加了内化。用雌二醇连续处理24 - 48小时会降低ERα水平，提示受体下调。雌二醇还增加了mGluR1a的转运和内化，这与所提出的ERα - mGluR1a相互作用一致。用ICI 182,780阻断ER或用LY 367385阻断mGluR1a可阻止ERα往返膜的转运。在ERα激活/内化峰值时，雌二醇诱导的Ca(2+)通量也显著增加。这些结果表明ERα存在于膜中且具有细胞外部分。此外，ERα的膜水平和内化受雌二醇和mGluR1a配体调节。进出膜的转运模式表明，发情周期中雌二醇浓度的变化调节ERα以增强然后终止膜启动的信号传导。
