MOLECULAR ORGANIZATION OF CNS SYNAPSES

中枢神经系统突触的分子组织

• 批准号: 2892053
• 负责人: MORGAN H. SHENG
• 金额: $ 13.98万
• 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/2892053
• 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输和存储至关重要 大脑，但是哺乳动物中央突触的结构 神经系统（CNS）的理解很少，尤其是在分子上 等级。 该提议的长期目标是了解 突触后组织和多样性的分子基础 中枢神经系统，特别是指蛋白质 - 蛋白质相互作用 突触膜。 具体目的是定义和表征 介导两个不同的聚类和锚定的分子 神经递质受体（NMDA受体和GABAA受体）的类别 在兴奋性和抑制性突触后部位。 这 实验方法是鉴定结合的细胞内蛋白 专门针对NMDA和GABAA受体，通过使用专业 这些受体亚基的细胞内结构域，作为酵母中的诱饵 脑cDNA库的两个杂交屏幕。 在初步研究中 已知的肌动蛋白结合蛋白（a-肌动蛋白）和新型的延伸盘绕螺旋 蛋白质（称为NIP-1）已被鉴定为相互作用的分子 直接带有单独的域，带有细胞质C末端尾 NMDA受体亚基NR1。 这些结果表明 两种杂交方法检测潜在相互作用的可行性 在整体突触膜蛋白和成分之间 突触后皮质细胞骨架。 两杂交鉴定的蛋白质相互作用的真实性 通过重组蛋白的结合，将在体外确认系统，并且 通过免疫组织化学共定位和体内体内（大鼠脑）和 免疫沉淀。 介导相互作用的蛋白质结构域将是 将通过缺失诱变来定义，希望识别 新型序列基序或模块用于蛋白质 - 蛋白质相互作用的模块 细胞膜。 受体关联与 它的结合蛋白将通过共转染实验研究 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 在大鼠大脑中表征。 确定突触后受体的分子组织 膜将进一步了解我们对机制的理解 突触发生和突触结构可塑性，因此学习 和记忆，以及大脑中的发展和衰老过程。 从 临床观点，组织的异常 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肉营养不良。 在 上下文，我们的初步研究很感兴趣 肌营养不良蛋白（a-肌动蛋白）的亲戚作为假定的锚定蛋白 NMDA受体。