MOLECULAR ORGANIZATION OF CNS SYNAPSES

中枢神经系统突触的分子组织

• 批准号: 2416406
• 负责人: MORGAN H. SHENG
• 金额: $ 12.16万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2416406
• 关键词: GABA receptor; NMDA receptors; alpha actinin; cytoskeleton; immunocytochemistry; laboratory rat; neural plasticity; receptor binding; receptor expression; recombinant proteins; site directed mutagenesis; synaptogenesis

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.

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 大脑，但哺乳动物中枢的突触结构 人们对神经系统（CNS）知之甚少，尤其是在分子层面 等级。 该提案的广泛长期目标是了解 突触后组织的分子基础和多样性 CNS，特别是蛋白质与蛋白质的相互作用 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 神经递质受体的类别（NMDA 受体和 GABAA 受体） 分别位于兴奋性和抑制性突触后位点。 这 实验方法是鉴定结合的细胞内蛋白质 专门针对 NMDA 和 GABAA 受体，通过使用主要 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， 已知的肌动蛋白结合蛋白（α-肌动蛋白）和新型延伸卷曲螺旋 蛋白质（称为 NIP-1）已被确定为相互作用的分子 直接与具有细胞质C末端尾部的单独结构域 NMDA 受体亚基 NR1。 这些结果表明 双混合方法检测潜在相互作用的可行性 完整的突触膜蛋白和突触膜的成分之间 突触后皮质细胞骨架。 双杂交鉴定的蛋白质相互作用的真实性 系统将通过重组蛋白的结合在体外得到证实，并且 体内（大鼠脑）通过免疫组织化学共定位和 免疫沉淀。 介导相互作用的蛋白质结构域将是 将通过删除突变来定义，希望能够识别 用于蛋白质-蛋白质相互作用的新序列基序或模块 细胞膜。 受体关联的功能意义 其结合蛋白将通过共转染实验进行研究 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 大鼠大脑中的特征。 确定突触后受体的分子组织 膜将进一步加深我们对机制的理解 突触发生和突触结构可塑性，以及学习的影响 和记忆，以及大脑的发育和衰老过程。 从 从临床角度来看，组织异常 中枢神经系统中的突触后膜和细胞骨架可能导致 神经系统疾病，就像肌肉膜缺陷一样 细胞骨架蛋白肌营养不良蛋白可引起肌营养不良。 在 有趣的是，我们的初步研究发现 肌营养不良蛋白（a-肌动蛋白）的亲戚，作为推定的锚定蛋白 NMDA 受体。