PGDF-Dependent Regulation of EAAC1

描述（由申请人提供）：兴奋性氨基酸，谷氨酸，由谷氨酸转运蛋白家族从突触中清除。EAAC1是一种神经元亚型，在海马体和皮质中富集，这些区域对兴奋性毒性损伤非常敏感。EAAC1亚细胞定位在C6胶质瘤细胞中受刺激调节。PDGF已被证明具有神经保护作用，EAAC1的调节可能是PDGF发挥其神经保护作用的一种方式。本提案的目的是了解pdgf依赖性EAAC1调控的机制。目的一是确定是否需要通过与Cbl适配蛋白（CAP）的相互作用，对原癌基因产物Cbl进行pdgf依赖性磷酸化。这将通过表达显性干扰型CAP结构和测量PDGF处理后细胞表面EAAC1的表达来检验。目的II是通过用肌动蛋白解聚剂预处理细胞并测量谷氨酸摄取和细胞表面EAAC1，来测试刺激运输是否需要完整的肌动蛋白丝。目的二是利用EAAC1的嵌合体和突变变体，鉴定pdgf依赖性运输所需的EAAC1的结构基序。

DESCRIPTION (provided by applicant): The excitatory amino acid, glutamate, is cleared from the synapse by a family of glutamate transporters. EAAC1, a neuronal subtype, is enriched in the hippocampus and cortex, areas that are extremely sensitive to excitotoxic insults. EAAC1 subcellular localization is regulated by stimulation in C6 glioma cells. PDGF has been shown to be neuroprotective, and regulation of EAAC1 may represent one way in which PDGF exerts its neuroprotective effects. The goal of this proposal is to understand the mechanisms underlying PDGF-dependent regulation of EAAC1. Aim I is to determine if PDGF-dependent phosphorylation of the proto-oncogene product, Cbl, via an interaction with the Cbl adapter protein (CAP) is required. This will be examined by expressing a dominant interfering CAP construct and measuring cell surface EAAC1 expression after PDGF treatment. Aim II is to test whether intact actin filaments are required for stimulated trafficking, by pre-treating cells with an actin depolymerizer and measuring glutamate uptake and cell surface EAAC1. Aim Ill is to identify structural motifs of EAAC1 required for PDGF-dependent trafficking, using chimeras and mutant variants of EAAC1.