Laboratory of Neuroendocrinology of the Brain Research Institute and Department of Neurobiology, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90095-1763, USA.
J Neurosci. 2010 Sep 22;30(38):12589-96. doi: 10.1523/JNEUROSCI.1038-10.2010.
Estrogen receptors (ERs) and estrogen-binding proteins have been localized intracellularly and on the cell surface. The membrane-associated proteins initiate signaling that activates a myriad of cellular responses including the modulation of ion channels and ultimately transcription. Although many of the downstream actions of membrane ERs, including ERα and ERβ, have been characterized, the mechanisms regulating membrane ER levels have remained elusive in the nervous system. In the present study, we used surface biotinylation to identify and study the estradiol regulation of membrane ERα in mixed-sex, cultured hypothalamic neurons from rat. Following surface biotinylation, Western blot analysis revealed full-length 66 kDa ERα and several ERα splice variants, most notably a biotinylated 52 kDa ERα-immunoreactive protein. Treatment of the neurons with estradiol caused a rapid and transient increase of the biotinylated 52 kDa and 66 kDa ERα proteins in the plasma membrane. Exposure of the neurons to estradiol also significantly increased internalization of 52 kDa and 66 kDa ERα membrane proteins, a measure of receptor activation. In the hypothalamus, membrane ERα signaling depends on transactivation of metabotropic glutamate receptor-1a (mGluR1a). Estradiol treatment increased the internalization of mGluR1a in parallel with ERα, a finding consistent with the hypothesis of an ERα-mGluR1a signaling unit. These results demonstrate that estradiol regulates the amount of ERα in the membrane, suggesting estradiol can regulate its own membrane signaling.
雌激素受体 (ERs) 和雌激素结合蛋白已在细胞内和细胞表面定位。膜相关蛋白启动信号转导，激活包括离子通道调节在内的多种细胞反应，最终激活转录。尽管已经对膜 ER（包括 ERα 和 ERβ）的许多下游作用进行了描述，但在神经系统中，调节膜 ER 水平的机制仍然难以捉摸。在本研究中，我们使用表面生物素化来鉴定和研究混合性别培养的大鼠下丘脑神经元中膜 ERα 的雌二醇调节。表面生物素化后，Western blot 分析显示全长 66 kDa ERα 和几种 ERα 剪接变体，特别是生物素化的 52 kDa ERα-免疫反应性蛋白。用雌二醇处理神经元会导致质膜中生物素化的 52 kDa 和 66 kDa ERα 蛋白快速且短暂增加。神经元暴露于雌二醇也显著增加了 52 kDa 和 66 kDa ERα 膜蛋白的内化，这是受体激活的一种衡量标准。在下丘脑，膜 ERα 信号取决于代谢型谷氨酸受体 1a（mGluR1a）的转激活。雌二醇处理会增加 mGluR1a 的内化，与 ERα 平行，这一发现与 ERα-mGluR1a 信号单元的假说一致。这些结果表明，雌二醇调节膜中 ERα 的数量，表明雌二醇可以调节其自身的膜信号。
