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Proteomic mining of intra- and extracellular proteins that modulate AMPA receptor function

调节 AMPA 受体功能的细胞内和细胞外蛋白质的蛋白质组学挖掘

基本信息

批准号：
9328253
负责人：
Hana L Goldschmidt
金额：
$ 5.71万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-03-06 至 2020-03-05
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9328253
关键词：
AMPA Receptors Address Architecture Biological Assay Biotinylation Brain Brain Diseases Cell Extracts Cell Fractionation Chemicals Chemosensitization Co-Immunoprecipitations Complementary DNA Data Disease Excitatory Synapse Extracellular Domain Extracellular Protein Functional disorder Glutamate Receptor Kainic Acid Receptors Knowledge Label Laboratories Learning Length Long-Term Depression Long-Term Potentiation Mass Spectrum Analysis Mediating Memory Mental disorders Messenger RNA Mining Modeling Molecular Mus Neuraxis Neurons Neurotransmitter Receptor Play Post-Translational Protein Processing Proteins Proteomics Regulation Research Research Proposals Role Surface Synapses Synaptic Transmission Synaptic plasticity Testing Western Blotting Work density experimental study expression vector extracellular in vivo insight interest kainate knock-down mutant nervous system disorder new therapeutic target novel overexpression protein protein interaction receptor receptor density receptor expression receptor function small hairpin RNA spatiotemporal trafficking

项目摘要

PROJECT SUMMARY AMPA-type glutamate receptors (AMPARs) are the major excitatory neurotransmitter receptor in the central nervous system. Dynamic regulation of the density, subunit composition and trafficking of AMPARs into and out of excitatory synapses is a key mechanism to facilitate changes in synaptic strength important for various forms of synaptic plasticity relevant to learning and memory. Over the last twenty years, work from Huganir Laboratory and others has made significant contributions to uncovering the molecular mechanisms underlying synaptic targeting of AMPARs that strengthens (long-term potentiation, LTP) or weakens (long-term depression, LTD) synaptic transmission. These studies have contributed to the prevailing model of LTP, whereby subunit-specific protein interactions and post-translational modifications within the cytosolic, carboxy (C)-terminus of AMPARs facilitate the trafficking of AMPARs to synapses and thereby enhance synaptic transmission. Recent work challenged this model and the requirement for the C-termini of AMPARs, as well as their interacting partners, to mediate LTP. It was found that not only could AMPAR mutants that lack the C- terminal domain rescue LTP in mice deficient all AMPARs (GluA1-A3), but so could GluK1, which belongs to the distinct, Kainate subclass of ionotropic glutamate receptors not normally found at these synapses. While these data remain a topic of debate, AMPA and Kainate receptors shared conserved structural architecture in their extracellular domains, raising the intriguing and less explored possibility that the extracellular domains of specific AMPAR subunits might also play key roles in regulating the synaptic targeting of the receptors. In this research proposal we will investigate the hypothesis that synaptic targeting of AMPARs required to induce long-term changes in synaptic strength is bi-directionally regulated by coordinated protein-protein interactions with the N- and C-termini. To test this hypothesis we have developed an assay that uses proximity- labeling with APEX2-tagged to the N- or C-termini of AMPAR subunits to perform an unbiased, proteomic screen to identify proteins that endogenously interact with either the extracellular or intracellular domains of GluA1- and GluA2-containing AMPARs during synaptic plasticity. Selected candidate proteins that demonstrate validated, dynamic and specific interactions with AMPAR subunits will be further characterized using overexpression and knockdown approaches to evaluate their role in regulating the synaptic targeting and function of AMPARs during synaptic plasticity. By identifying and characterizing new, functionally-relevant interactions between synaptic proteins and AMPARs, the proposed research will provide significant insight into the molecular mechanisms underlying plasticity relevant to learning, memory and higher brain function. Further, these studies have broad implications for brain disorders involving synaptic dysfunction as they might reveal novel therapeutic targets for the treatment of neurological and psychiatric diseases.

项目总结