Polarized Targeting of Metabotropic Glutamate Receptors

代谢型谷氨酸受体的极化靶向

• 批准号: 6720312
• 负责人: ANNA FRANCESCONI
• 金额: $ 19.31万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-25 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6720312
• 关键词: MDCK cell; alpha actinin; binding proteins; caveolins; embryo /fetus tissue /cell culture; glutamate receptor; immunoprecipitation; laboratory rat; mass spectrometry; nerve /myelin protein; protein localization; protein protein interaction; protein purification; protein signal sequence; protein transport; proteomics; receptor binding; vesicle /vacuole; vimentin; yeast two hybrid system

DESCRIPTION (provided by applicant): Neurotransmitter receptor targeting to postsynaptic and presynaptic sites requires correct sorting, insertion/retention at the plasma membrane, and clustering. To date, the molecular mechanisms underlying targeting of receptors to dendrites vs. axons remain largely unknown. The underlying hypothesis of this proposal is that neurotransmitter receptors may interact via targeting signals with adaptor proteins mediating recruitment to specialized cargo vesicles or, alternatively, with scaffolding proteins, which may cause compartmentalized retention and/or stabilization. The experimental approach is to identify proteins that bind to targeting signals contained within the different splice variants of the metabotropic glutamate receptor 1 (mGluR1). Preliminary studies have identified a signal sequence in the intracellular tail of the mGluRlb isoform required for exclusion from distal dendrites and for axonal targeting. This signal is also required for apical targeting in Madin-Darby canine kidney (MDCK) cells. Aim 1 will identify proteins involved in mGluRlb targeting using the yeast two-hybrid system. A brain cDNA library will be screened with the intracellular tail of mGluRlb as bait. Binding partners will be tested for interaction with a mutant bait lacking the targeting signal. Preliminary studies, have identified a novel protein, termed mGRAB, attesting to the feasibility of this approach. In Aim 2, a novel technology, Tandem Affinity Purification (TAP), will be used to isolate native complexes of proteins interacting with mGluRlb in MDCK cells. The receptor-interacting protein complex will be analyzed by mass spectrometry to identify individual proteins. Preliminary results have identified three proteins, alpha-actinin-4, myosin IC and vimentin as potential components of the mGluRlb associated complex. Understanding the molecular mechanisms underlying regulation of mGluR trafficking has important implications for the understanding of the molecular mechanisms underlying synaptic activity in normal and pathological conditions. Moreover, mGluRs are an important therapeutic target for the treatment of neurological disorders, such as Parkinson's disease, and of psychotic symptoms and dysfunctions of cognitive processes associated with schizophrenia. This application is ideally suited to the R21 format in that it employs high-risk experiments based on new technologies that may lead to major advances in this field.

描述（由申请人提供）： 靶向突触后和突触前位点的神经递质受体需要正确的排序、在质膜上的插入/保留和聚类。迄今为止，受体靶向树突与轴突的分子机制仍然很大程度上未知。该提议的基本假设是，神经递质受体可能通过靶向信号与介导招募到专门货物囊泡的衔接蛋白相互作用，或者与支架蛋白相互作用，这可能导致区室化保留和/或稳定。实验方法是鉴定与代谢型谷氨酸受体 1 (mGluR1) 不同剪接变体中包含的靶向信号结合的蛋白质。初步研究已经确定了 mGluRlb 亚型细胞内尾部的信号序列，该信号序列是从远端树突中排除和轴突靶向所需的。 Madin-Darby 犬肾 (MDCK) 细胞的顶端靶向也需要该信号。目标 1 将使用酵母双杂交系统鉴定参与 mGluRlb 靶向的蛋白质。以mGluRlb的细胞内尾部作为诱饵来筛选脑cDNA文库。将测试结合伙伴与缺乏靶向信号的突变诱饵的相互作用。初步研究发现了一种名为 mGRAB 的新型蛋白质，证明了这种方法的可行性。在目标 2 中，串联亲和纯化 (TAP) 新技术将用于分离 MDCK 细胞中与 mGluRlb 相互作用的天然蛋白质复合物。受体相互作用蛋白复合物将通过质谱分析来鉴定单个蛋白。初步结果已确定三种蛋白质：α-肌动蛋白-4、肌球蛋白 IC 和波形蛋白是 mGluRlb 相关复合物的潜在成分。了解 mGluR 运输调节的分子机制对于理解正常和病理条件下突触活动的分子机制具有重要意义。此外，mGluR是治疗神经系统疾病（例如帕金森病）以及精神分裂症相关的精神病症状和认知过程功能障碍的重要治疗靶点。该应用程序非常适合 R21 格式，因为它采用基于新技术的高风险实验，可能会导致该领域取得重大进展。