PGDF-Dependent Regulation of EAAC1

描述（申请人提供）：兴奋性氨基酸谷氨酸通过谷氨酸转运蛋白家族从突触中清除。EAAC1是一种神经元亚型，在海马和皮质中富集，这些区域对兴奋性毒性损伤非常敏感。C6胶质瘤细胞中EAAC1亚细胞定位受刺激调节PDGF已被证明是神经保护性的，并且EAAC 1的调节可能代表PDGF发挥其神经保护作用的一种方式。该提案的目标是了解EAAC1的PDGF依赖性调节机制。目的是确定是否需要原癌基因产物Cbl通过与Cbl衔接蛋白（CAP）的相互作用进行PDGF依赖性磷酸化。这将通过表达显性干扰CAP构建体并在PDGF处理后测量细胞表面EAAC 1表达来检查。目的II是测试是否需要完整的肌动蛋白丝刺激贩运，通过用肌动蛋白解聚剂预处理细胞和测量谷氨酸摄取和细胞表面EAAC 1。目的III是使用EAAC 1的嵌合体和突变体来鉴定PDGF依赖性运输所需的EAAC 1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.