PGDF-Dependent Regulation of EAAC1

描述(申请人提供)：兴奋性氨基酸，谷氨酸，由谷氨酸转运体家族从突触中清除。EAAC1是一种神经元亚型，富含在海马区和皮质中，这两个区域对兴奋性毒性侮辱极其敏感。在C6胶质瘤细胞中，EAAC1的亚细胞定位受刺激的调节。PDGF被证明具有神经保护作用，调节EAAC1可能是PDGF发挥神经保护作用的一种方式。这项建议的目的是了解依赖PDGF调控EAAC1的机制。目的I确定原癌基因产物Cb1是否需要依赖PDGF通过与Cb1适配蛋白(CAP)相互作用而磷酸化。这将通过表达显性干扰CAP结构和测量PDGF处理后细胞表面EAAC1的表达来检验。目的二是通过用肌动蛋白解聚器对细胞进行预处理，并测量谷氨酸摄取和细胞表面EAAC1，来测试完整的肌动蛋白细丝是否需要刺激运输。目的利用EAAC1的嵌合体和突变体，鉴定依赖PDGF运输所需的EAAC1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.