Polarized Targeting of Metabotropic Glutamate Receptors

描述(由申请人提供)： 以突触后和突触前部位为靶点的神经递质受体需要正确的分类、在质膜上的插入/保持和聚集。到目前为止，受体靶向树突和轴突的分子机制在很大程度上仍不清楚。这一提议的基本假设是，神经递质受体可能通过靶向信号与接头蛋白相互作用，介导募集到特定的货物小泡，或者，替代地，与支架蛋白相互作用，这可能导致分区保留和/或稳定。实验方法是识别与代谢性谷氨酸受体1(MGluR1)的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，这是排除远端树突和轴突靶向所必需的。这一信号也是Madin-Darby犬肾(MDCK)细胞顶端靶向所必需的。目标1将使用酵母双杂交系统鉴定与mGluRlb靶向有关的蛋白质。以mGluRlb的胞内尾巴为诱饵，筛选出一个脑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。初步研究已经确定了一种新的蛋白质，称为mGRAb，证明了这种方法的可行性。在目标2中，将使用一种新的技术，串联亲和纯化(TAP)，在MDCK细胞中分离与mGluRlb相互作用的天然蛋白质复合体。受体相互作用的蛋白质复合体将通过质谱学进行分析，以识别单个蛋白质。初步结果表明，α-肌动蛋白-4、肌球蛋白IC和波形蛋白是mGluRlb相关复合体的潜在成分。了解mGluR转运调控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，mGluRs是治疗帕金森氏病等神经疾病以及与精神分裂症相关的精神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，因为它采用了基于可能导致该领域重大进步的新技术的高风险实验。

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.