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（GluR）是由多个跨膜亚基组成的寡聚糖蛋白复合物。 GluRs的功能特性在很大程度上由组成受体的亚基决定。为了形成具有适当功能特性的GluRs，必须存在调节亚基组装的机制。 因此，GluR亚基似乎含有高度特异性的相互作用和识别结构域，其功能是调节亚基组装和可能的受体亚基化学计量。 本申请中提出的实验旨在鉴定NMDA和非NMDA类型的GluR亚基的缔合和组装结构域。 烟碱乙酰胆碱受体、抑制性甘氨酸和γ-氨基丁酸受体的亚基含有决定亚基化学计量的结构域，并且是受体组装所必需的。 对于组成这些通道的亚基，“缔合结构域”位于亚基多肽的胞外氨基末端内。 本申请中提出的研究的总体目标是确定相互作用的NMDA和非NMDA GluR亚基的结构域，以及功能性同源和异源受体的准确组装所需的结构域。一旦确定了NMDA和AMPA受体亚基的组装结构域，将在培养的小脑颗粒细胞中测试组装结构域肽在EAA激活的神经元死亡期间的神经保护能力，所述小脑颗粒细胞是研究EAA诱导的神经毒性的良好建立的模型系统。 将确定细胞外和细胞内递送的GluR缔合结构域肽的神经保护性质。 肽的细胞内递送将通过将缔合结构域肽连接到来自被神经元快速内化的触角足同源结构域的第三螺旋的16个氨基酸的肽来促进。 识别神经保护性结合域肽抑制兴奋性毒性，可能是第一步的一类新的临床上重要的神经保护剂的发展目标个别亚型的GluRs。

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.