Polarized Targeting of Metabotropic Glutamate Receptors

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分选、插入/保留在质膜上和成簇。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以通过靶向信号与介导募集到专门的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。实验方法是识别与代谢型谷氨酸受体1（mGluR 1）的不同剪接变体内包含的靶向信号结合的蛋白质。初步研究已经鉴定了mGluRlb同种型的细胞内尾部中的信号序列，所述信号序列是从远端树突排除和轴突靶向所需的。该信号也是Madin-Darby犬肾（MDCK）细胞中顶端靶向所需的。目的1将使用酵母双杂交系统鉴定参与mGluRlb靶向的蛋白。将用mGluRlb的细胞内尾作为诱饵筛选脑cDNA文库。将测试结合配偶体与缺乏靶向信号的突变体诱饵的相互作用。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，一种新的技术，串联亲和纯化（TAP），将用于分离天然复合物的蛋白质相互作用与mGluRlb在MDCK细胞。将通过质谱法分析受体相互作用蛋白复合物，以鉴定单个蛋白。初步结果已经确定了三种蛋白质，α-辅肌动蛋白-4，肌球蛋白IC和波形蛋白作为mGluRlb相关复合物的潜在组分。了解mGluR运输调节的分子机制对于了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是治疗神经障碍（例如帕金森病）和精神病症状以及与精神分裂症相关的认知过程功能障碍的重要治疗靶标。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.