SUBUNIT ASSEMBLY DOMAINS--TARGETS TO LIMIT NEUROTOXICITY

与许多配体门控离子通道一样，谷氨酸受体(GluRs)是由多个跨膜亚单位组成的寡聚糖蛋白复合体。GluRs的功能特性在很大程度上由组成受体的亚基决定。要形成具有适当功能性质的GluRs，必须存在调节亚基组装的机制。因此，GluR亚基似乎包含高度特异的相互作用和识别结构域，其功能是调节受体的亚单位组装和可能的亚单位化学计量。本申请中提出的实验旨在鉴定NMDA和非NMDA类型的GluR亚基的关联和组装结构域。烟碱型乙酰胆碱受体的亚单位，抑制性甘氨酸和γ-氨基丁酸受体包含决定亚单位化学计量比的结构域，这些结构域是组装受体所必需的。对于组成这些通道的亚基，“结合域”位于亚基多肽的胞外氨基末端。本申请中提出的研究的总体目标是确定NMDA和非NMDA GluR亚基的结构域，这些结构域相互作用，是准确组装功能性同聚体和异构体受体所必需的。一旦确定了NMDA和AMPA受体亚单位的组装结构域，就将测试组装域肽在EAA激活的培养小脑颗粒细胞神经元死亡期间的神经保护能力，这是一个研究EAA诱导的神经毒性的成熟模型系统。细胞外和细胞内递送的GluR结合域肽的神经保护特性将被确定。通过将结合域肽与触角虫同源域第三螺旋上的16个氨基酸的多肽连接起来，将促进多肽在细胞内的传递，该多肽可被神经元迅速内化。识别抑制兴奋性毒性的神经保护相关域肽，可能是开发一类针对单个GluRs亚型的新型临床重要神经保护剂的第一步。

Like many ligand gated ion channels, glutamate receptors (GluRs) are oligomeric glycoprotein complexes composed of multiple membrane spanning subunits. The functional properties of GluRs are largely determined by the subunits that compose the receptor. For GluRs with the appropriate functional properties to form, there must exist mechanisms to regulate subunit assembly. It seems likely therefore, that GluR subunits contain highly specific interaction and recognition domains that function to regulate subunit assembly and possibly subunit stoichiometry of the receptor. The experiments proposed with in this application are aimed at identifying the association and assembly domains of both the NMDA and non-NMDA types of GluR subunits. Subunits of the nicotinic acetylcholine receptor, the inhibitory glycine and gamma-aminobutyric acid receptors contain structural domains that determine subunit stoichiometry and that are necessary for assembly of the receptor. For the subunits composing those channels, the "association domains" are located within the extracellular amino-terminus of the subunit polypeptides. The overall goal of the research proposed in this application is to identify structural domains of NMDA and non-NMDA GluR subunits that interact and that are required for the accurate assembly of functional homomeric and heteromeric receptors. Once assembly domains for the NMDA and AMPA receptor subunits are defined, assembly domain peptides will be tested for their ability to be neuroprotective during periods of EAA activated neuronal death in cultured cerebellar granule cells, a well established model system to study EAA induced neurotoxicity. The neuroprotective properties of both extracellular and intracellularly delivered GluR association domain peptides will be determined. The intracellular delivery of peptides will be facilitated by linking the association domain peptides to a 16 amino acid peptide from the third helix of the antennapedia homeodomain that is rapidly internalized by neurons. The identification of neuroprotective association domain peptides that inhibit excitotoxicity, may be the first steps toward the development of a new class of clinically important neuroprotective agents that target individual subtypes of GluRs.