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：(改编自申请者摘要)突触功能和 突触可塑性是脑内信息传递和存储的关键 大脑，但哺乳动物中枢的突触结构 人们对神经系统(CNS)了解甚少，尤其是在分子水平 水平。这项建议的广泛长期目标是理解 突触后组织和多样性的分子基础 CNS，特别是关于蛋白质之间的相互作用 突触膜。其具体目标是定义和描述 调节两个不同分子的聚集和锚定的分子 神经递质受体的分类(NMDA受体和GABAA受体) 分别位于兴奋性和抑制性突触后部位。这个 实验方法是识别结合在细胞内的蛋白质 针对NMDA和GABAA受体，通过使用主要的 这些受体亚单位的胞内结构域在酵母中作为诱饵 脑cdna文库的双杂交筛选。在初步研究中，一个 已知的肌动蛋白结合蛋白(α-肌动蛋白)和一种新的延伸螺旋线圈 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和细胞质C-末端尾部 NMDA受体亚基NR1。这些结果证明了 两种混合方法检测潜在相互作用的可行性 在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 该系统将通过与重组蛋白的结合在体外得到确认，以及 在体内(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。受体与肌动蛋白相互作用的功能意义 它的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 与NMDA受体和GABAA受体相互作用的蛋白质将是 以大鼠大脑为特征的。 突触后受体分子组成的测定 膜将进一步加深我们对其机制的理解 突触发生和突触结构可塑性，从而学习 和记忆，以及大脑的发育和衰老过程。从… 从临床的角度来看，组织中的异常 中枢神经系统中的突触后膜和细胞骨架可能导致 神经性疾病，就像肌肉膜的缺陷 细胞骨架蛋白营养不良蛋白可导致肌肉营养不良。在 令人感兴趣的是，我们的初步研究已经确定 肌营养不良蛋白的近亲(α-肌动蛋白)，作为推测的锚定蛋白 NMDA受体。