The Regulation of Plasma Membrane Estrogen Receptor-a Levels by Endocytosis

说明(申请人提供)：雌二醇(17-雌二醇)是一种性类固醇激素，被认为可以防止缺血性中风和神经退行性变引起的神经元丢失。随着美国人口老龄化的预期，保护老化的大脑将变得越来越重要。雌二醇通过一种鲜为人知的机制激活ER？(雌二醇受体？)，从而防止神经元丢失。数据有力地表明，定位于质膜(ER最初被确定为细胞内蛋白)的ER亚群的激活和ERK(细胞外信号调节激酶)途径的激活起着关键作用。我们以前的文献表明：(1)雌激素治疗原代下丘脑神经元导致生物素化ER迅速进入和流出质膜，我们的发现证实了这一发现，因为我们发现皮层神经元通过内小体内化了膜不通透的雌二醇。(2)雌二醇治疗原代培养的海马神经元和皮质神经元，(A)迅速启动G蛋白偶联受体激酶-2(GRK2)的激活，(B)促进ER/α-arrestin-1免疫复合体的形成。(3)siRNA下调α-arrestin-1可阻止雌激素诱导的ERK磷酸化。我们的发现支持雌激素通过触发内吞作用来下调质膜ER水平的概念。他们还强烈地表明，膜ER的调节类似于视紫红质受体和其他GPCRs的调节。到目前为止，大多数研究膜启动的雌二醇信号的实验室都严重依赖于容易受到固定和组织破坏伪影影响的技术。应该可以使用活细胞以及成像和生物物理技术来更深入地了解膜内质网的调节，使我们能够检查实时的、空间上可分辨的事件。为了扩展我们之前的工作，我们提出了以下假设：雌激素通过与受体介导的内吞作用相关的机制来调节ER？质膜水平。这项拟议的研究建立在我们之前关于雌二醇作用的工作的基础上，它引入了该领域的新方法，包括实时成像(全内反射荧光成像显微镜 和荧光蛋白-受体融合构建)和生物物理(质膜电容测量)技术。这项工作将研究膜内质网调节和视紫红质受体调节之间的强烈相似性。此外，这项工作开始为研究雌二醇的神经保护作用铺设一个新的框架。这项研究将得益于单细胞成像、生物物理测量和膜运输方面的专家Robert Chow博士和感光细胞生理学专家Jeannie Chen博士的指导，特别是与视紫红质和arrestin信号有关的感光细胞生理学，以及感光细胞退化。 公共卫生相关性：由缺血性中风和神经退行性变引起的神经元丢失可通过雌二醇治疗而减轻。雌激素的这种保护作用的机制尚不完全清楚，但数据表明，定位于质膜的雌激素受体的激活是至关重要的。这些研究试图极大地扩展我们对质膜ER激活与神经系统保护之间联系的机制的认识。好了！

DESCRIPTION (provided by applicant): Estradiol (17¿-estradiol) is a sex steroid hormone that is recognized to protect against neuronal loss caused by ischemic stroke and neurodegeneration. Protection of the aging brain will assume increasing significance with the projected graying of America's population. The prevention of neuronal loss by estradiol involves the activation of ER¿ (estradiol receptor-¿) by a mechanism that is poorly understood. Data strongly suggest that activation of a sub-population of ER¿ localized to the plasma membrane (ER¿ was originally identified as a intracellular protein) and of the ERK (extracellular-signal regulated kinase) pathway play critical roles. Our previous publications show that (1) estradiol treatment of primary hypothalamic neurons causes the rapid trafficking of biotinylated ER¿ into and out of the plasma membrane, a finding we corroborated in cortical neurons by showing internalization of membrane-impermeable estradiol via endosomes. (2) Estradiol treatment of primary hippocampal and cortical neurons (a) rapidly initiates GRK2 (G protein-coupled receptor kinase-2) activation, and (b) promotes the formation of ER¿/¿-arrestin-1 immuno-complexes. (3) The knockdown of ¿- arrestin-1 using siRNA prevents estradiol-induced ERK phosphorylation. Our findings support the concept that estradiol down-regulates plasma membrane ER¿ levels by triggering endocytosis. They also strongly suggest that the regulation of membrane ER¿ is analogous to the regulation of rhodopsin receptor and other GPCRs. To date, the majority of laboratories studying membrane-initiated estradiol signaling have relied heavily on techniques that are subject to artifacts of fixation and tissue disruption. Deeper insight into the regulationof membrane ER¿ should be accessible using live cells along with imaging and biophysical techniques, enabling us to examine real-time, spatially resolved events. To extend our previous work, we propose the following HYPOTHESIS: Estradiol regulates ER¿ plasma membrane levels by a mechanism associated with receptor-mediated endocytosis. The proposed investigation builds on our previous work on estradiol action, and it introduces novel approaches to the field including real-time imaging (total internal reflection fluorescence imaging microscopy and fluorescent protein-receptor fusion constructs) and biophysical (plasma membrane capacitance measurements) techniques. The work will investigate the strong analogy between the regulation of membrane ER¿ and regulation of rhodopsin receptors. Furthermore, the work begins to lay out a new framework for investigation of the neuroprotective effects of estradiol. The investigation will benefit from guidance by Dr. Robert Chow, an expert in single-cell imaging, biophysical measurements and membrane trafficking, and Dr. Jeannie Chen, an expert in photoreceptor physiology, especially pertaining to rhodopsin and arrestin signaling, and photoreceptor degeneration. PUBLIC HEALTH RELEVANCE: Neuronal loss, due to ischemic stroke and neurodegeneration, is attenuated by estradiol treatment. The mechanism involved in this protective action of estradiol is not completely understood but data shows the activation of ER¿ (estrogen receptor) localized to the plasma membrane is critical. These studies seek to significantly expand our knowledge of the mechanism linking the activation of plasma membrane ER¿ to the protection of the nervous system. !