MOLECULAR ORGANIZATION OF CNS SYNAPSES

描述：（改编自申请人的摘要）突触功能和 突触可塑性对于信息传递和存储至关重要， 但是哺乳动物中枢神经系统的突触结构 神经系统（CNS）的了解很少，特别是在分子水平上。 水平 本提案的广泛长期目标是了解 突触后组织和多样性的分子基础， 中枢神经系统，特别是关于蛋白质-蛋白质相互作用， 突触膜 具体目标是定义和描述 介导两种不同分子的聚集和锚定的分子 神经递质受体（NMDA受体和GABAA受体） 分别在兴奋性和抑制性突触后位点。 的 实验方法是鉴定细胞内蛋白质， 特别是NMDA和GABAA受体，通过使用主要的 这些受体亚基的胞内结构域作为酵母中的诱饵 脑cDNA文库的双杂交筛选。 在初步研究中，A 已知的肌动蛋白结合蛋白（α-辅肌动蛋白）和一种新的延伸卷曲螺旋 蛋白质（称为NIP-1），已被确定为相互作用的分子 直接与单独的结构域与细胞质C-末端尾 NMDA受体亚基NR 1。 这些结果证明 双杂交方法检测潜在相互作用的可行性 整合突触膜蛋白和突触膜的组成部分之间的联系 突触后皮质细胞骨架。 通过双杂交鉴定蛋白质相互作用的真实性 将通过重组蛋白的结合在体外证实系统， 通过免疫组织化学共定位在体内（大鼠脑）， 免疫沉淀。 介导相互作用的蛋白质结构域将是 将通过缺失突变来定义，希望能识别出 用于蛋白质-蛋白质相互作用的新序列基序或模块， 细胞膜 受体结合的功能意义 通过共转染实验研究其结合蛋白 在培养的细胞中。 发育和细胞表达模式 蛋白质与NMDA受体和GABAA受体的相互作用将是 以大鼠脑为特征。 确定突触后神经元中受体的分子结构 膜将进一步我们了解的机制， 突触发生和突触结构可塑性，从而学习 以及大脑的发育和衰老过程。 从 从临床的角度来看，组织的异常 突触后膜和细胞骨架在中枢神经系统可能会导致 神经系统疾病，就像肌肉膜的缺陷 细胞骨架蛋白肌营养不良蛋白可引起肌营养不良。 在 在背景下，有趣的是，我们的初步研究发现 肌营养不良蛋白（α-辅肌动蛋白）的亲戚，作为一种假定的锚定蛋白， NMDA受体。

DESCRIPTION: (adapted from Applicant's Abstract) Synaptic functions and synaptic plasticity are crucial for information transfer and storage in the brain, but the structure of synapses in the mammalian central nervous system (CNS) is poorly understood, especially at the molecular level. The broad long term objective of this proposal is to understand the molecular basis of postsynaptic organization and diversity in the CNS, with particular reference to protein-protein interactions at the synaptic membrane. The specific aim is to define and characterize the molecules which mediate the clustering and anchoring of two different classes of neurotransmitter receptor (NMDA receptors and GABAA receptors) at excitatory and inhibitory postsynaptic sites, respectively. The experimental approach is to identify intracellular proteins which bind specifically to NMDA and GABAA receptors, by using the major intracellular domain of subunits of these receptors as baits in a yeast two-hybrid screen of a brain cDNA library. In Preliminary studies, a known actin binding protein (a-actinin) and a novel extended coiled-coil protein (termed NIP-1), have been identified as molecules that interact directly with separate domains with the cytoplasmic C-terminal tail of the NMDA receptor subunit NR1. These results demonstrate the feasibility of the two-hybrid approach to detect potential interaction between integral synaptic membrane proteins and components of the postsynaptic cortical cytoskeleton. The authenticity of the protein interactions identified by the two-hybrid system will be confirmed in vitro by binding of recombinant proteins, and in vivo (rat brain) by immunohistochemical co-localization and immunoprecipitation. Protein domains mediating the interaction will be will be defined by deletional mutagenesis, with the hope of identifying novel sequence motifs or modules for protein-protein interactions at the cell membrane. The functional significance of receptor association with its binding protein will be investigated by co-transfection experiments in cultured cells. Developmental and cellular expression patterns of the proteins interaction with NMDA receptor and GABAA receptors will be characterized in rat brain. Determining the molecular organization of receptors in postsynaptic membranes will further our understanding of the mechanisms of synaptogenesis and synaptic structural plasticity, and hence of learning and memory, and developmental and aging processes in the brain. From a clinical point of view, abnormalities in the organization of the postsynaptic membrane and cytoskeleton in the CNS could results in neurological disease, just as defects in the muscle membrane cytoskeletal protein dystrophin can cause muscular dystrophy. In the context, it is of interest that our preliminary studies have identified a relative of dystrophin (a-actinin) as a putative anchoring protein for the NMDA receptor.