TRAFFICKING OF A NEURONAL GLUTAMATE TRANSPORTER, EAAC1

描述：(申请人摘要) 谷氨酸和天冬氨酸是主要的快速兴奋性神经递质 在哺乳动物的中枢神经系统(CNS)中，并参与了几个 发育中的和成人神经系统的可塑性形式。过份 EAA受体的激活与脑损伤有关 对中枢神经系统的严重侮辱，包括中风和头部创伤。胞外 这些兴奋性氨基酸(EaS)的浓度受 依赖Na+的高亲和力转运蛋白家族。我们最近开发了 血小板衍生生长因子(PDGF)增加细胞表面的证据 神经元谷氨酸转运蛋白EAAC1的表达我们还有 发现磷脂酰肌醇3-K(PI3-K)的抑制剂Wortmannin， 减少这种转运蛋白在细胞表面的表达。的总目标是 本项目旨在研究控制这一规则的机制，并 定义这种改变的细胞表面表达的功能后果。 根据我们的初步数据，细胞表面表达的这些变化是 由于细胞内小泡和质膜之间的运输。我们 假设PDGF通过PI3-K和a蛋白增加细胞表面表达 丝氨酸-苏氨酸激酶又称Akt(也称为蛋白激酶B)。我们 假设... 陷阱参与了向细胞表面的运输，而动力素，一种 GTP酶在转运蛋白的内吞作用中起重要作用。我们假设 这项规定是针对EAAC1亚类型的转运体的，并且 嵌合体将有助于描述这两种调节机制 以及EAAC1管理这种受管制的贩运的部分。最后，我们 假设PDGF介导的细胞表面表达的增加可能 有助于其先前记载的神经保护活性。这些 假设将通过C6胶质瘤进行检验，C6胶质瘤是一个只表达 转运蛋白的EAAC1亚型和原代神经元富集型培养。我们 建议使用各种免费的生化、分子生物学、 解决这些假说的药理学和细胞生物学方法。 由于EAAC1富含皮质和海马体，因此这两个区域 特别容易受到兴奋毒性侮辱，拟议的研究可能 为制定限制兴奋性中毒性大脑的新策略提供机会 损坏。

DESCRIPTION: (Applicant's Abstract) Glutamate and aspartate are the predominant rapid excitatory neurotransmitters in the mammalian central nervous system (CNS), and are involved in several forms of plasticity in the developing and adult nervous system. Excessive activation of EAA receptors contributes to brain damage observed in several acute insults to the CNS, including stroke and head trauma. The extracellular concentrations of these excitatory amino acids (EAAs) are controlled by a family of NA+-dependent high-affinity transporters. We have recently developed evidence that platelet-derived growth factor (PDGF) increases cell surface expression of EAAC1, one of the neuronal glutamate transporters. We have also found that wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), decreases cell surface expression of this same transporter. The overall goal of this project is to study the mechanisms that control this regulation, and to define the functional consequences of this altered cell surface expression. Based on our preliminary data, these alterations in cell surface expression are due to trafficking between intracellular vesicles and the plasma membrane. We hypothesize that PDGF increases cell surface expression through PI3-K and a serine-threonine kinase called Akt (also known as protein kinase B). We hypothesize that SNAREs are involved in the trafficking to the cell surface, and that dynamin, a GTPase, is important for endocytosis of the transporter. We hypothesize that this regulation is specific for the EAAC1 subtype of transporter, and that chimeras will help to delineate both the mechanisms involved in this regulation and the portions of EAAC1 that govern this regulated trafficking. Finally, we hypothesize that this PDGF-mediated increase in cell surface expression may contribute to its previously documented neuroprotective activity. These hypotheses will be tested using C6 glioma, a model system that only expresses the EAAC1 subtype of transporter and primary neuron-enriched cultures. We propose using a variety of complimentary biochemical, molecular biological, pharmacological, and cell biologic approaches to address these hypotheses. Since EAAC1 is enriched in cortex and hippocampus, two areas that are particularly vulnerable to excitotoxic insults, the proposed studies may provide opportunities to develop new strategies to limit excitotoxic brain damage.