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The Neuronal Nicotinic Acetylcholine Receptor Interactome via a Knock-In Mouse

通过敲入小鼠的神经元烟碱乙酰胆碱受体相互作用组

基本信息

批准号：
7296157
负责人：
Edward Hawrot
金额：
$ 22.61万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-09-25 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7296157
关键词：
Address Affinity Amino Acid Substitution Antibodies Area Arts Award Behavior Behavioral Binding Binding Sites Blood capillaries Cell physiology Cervical Complex Condition Coupled Cytoplasmic Protein Cytoplasmic Tail Databases Detergents Development Drosophila acetylcholine receptor alpha-subunit Drug Addiction Drug Delivery Systems Drug abuse Exons Genes Habenula Health Human Individual Intervention Investigation Knock-in Mouse Knock-out Knowledge Maintenance Maps Mass Spectrum Analysis Medial Mediating Messenger RNA Methods Modeling Molecular Mus Muscle Nervous system structure Neurons Nicotine Nicotine Dependence Nicotinic Receptors Oocytes Peptides Phase Phosphorylation Site Physiological Play Preparation Proteins Proteomics Psychological reinforcement Public Health Regulation Research Research Personnel Research Proposals Resolution Rewards Role Scientist Seasons Signal Transduction Specificity Spectrometry Structure of superior cervical ganglion Synapses Synaptic Transmission System Technology Testing Transgenic Organisms Trypsin Ventral Tegmental Area Wild Type Mouse Work alpha Bungarotoxin base capillary concept ganglion cell genetic regulatory protein in vivo innovation insight mass spectrometer member mutant nano-electrospray novel novel strategies programs protein protein interaction receptor tandem mass spectrometry tool

项目摘要

DESCRIPTION (provided by applicant): This proposal describes an innovative research approach aimed at ultimately revealing the molecular mechanisms that produce and maintain nicotine dependence. To accomplish this, we propose to develop new and generalizable investigative tools by combining cutting-edge features in emerging transgenic and proteomic technologies, specifically by using novel pharmatope-tagged alpha-3-subunit-containing nicotinic receptors in an existing novel knock-in mouse to harness the powerful and sophisticated capabilities of modern mass spectrometry . This project is a new venture in our lab that seeks to apply the fundamental insights gained from our previous structural and functional work to outstanding problems in the drug addiction area. A detailed understanding of nicotine-induced alterations in intracellular protein-protein interactions (i.e., the interactome) involving the putative regulatory cytoplasmic domain of neuronal nicotinic acetylcholine receptors (nAChRs) promises to provide uniquely valuable insights into how behavioral manifestations of nicotine reinforcement, tolerance and sensitization develop. The core concept is that novel pharmatope-tagged knock-in mice can provide general tools to study regulatory proteins interacting with the unusually large cytoplasmic loop that is a feature common to all nAChR subtypes. Our alpha3-knock-in introduces an alpha-bungarotoxin (Bgtx) binding site into neuronal nAChRs that are normally Bgtx- insensitive. The ability to bind Bgtx will be used to biochemically isolate the receptor under mild conditions, together with associated regulatory signaling complexes. These associated proteins will be identified using tandem mass spectrometry and their phosphorylation sites functionally mapped. To critically test this approach, we will focus on the Bgtx-sensitive alphas subunit expressed in primary cultures of superior cervical ganglionic neurons whose post-synaptic alpha3-containing nAChRs are essential for synaptic transmission. This system will allow us to probe the effects of nicotine exposure on the nAChR interactome profile, and will pave the way for studies of alpha3 signaling complexes in the CNS; in particular, in the ventral tegmental area and medial habenula, regions strongly implicated in nicotine addiction. Public Health Relatedness: Knowledge of the cascade of cellular processes and protein interactions regulated by nicotine will contribute to the development of new health interventions.

描述（由申请人提供）：该提案描述了一种创新的研究方法，旨在最终揭示产生和维持尼古丁依赖的分子机制。为了实现这一目标，我们建议通过结合新兴转基因和蛋白质组学技术的前沿特征，特别是通过在现有的新型敲入小鼠中使用新型药物表位标记的α-3-亚基含烟碱受体来开发新的和可推广的调查工具，以利用现代质谱的强大和复杂的功能。这个项目是我们实验室的一个新项目，旨在将我们以前的结构和功能工作中获得的基本见解应用于药物成瘾领域的突出问题。详细了解尼古丁诱导的细胞内蛋白质-蛋白质相互作用的改变（即，涉及神经元烟碱乙酰胆碱受体（nAChR）的假定调节胞质结构域的相互作用组（interactome）有望为尼古丁强化、耐受性和致敏性的行为表现如何发展提供独特的有价值的见解。核心概念是，新的pharmatope-tagged敲入小鼠可以提供一般的工具来研究与异常大的细胞质环相互作用的调节蛋白，这是所有nAChR亚型共有的特征。我们的α 3-敲入将α-银环蛇毒素（Bgtx）结合位点引入通常对Bgtx不敏感的神经元nAChR中。结合Bgtx的能力将用于在温和条件下生物化学分离受体以及相关的调节信号复合物。这些相关的蛋白质将被确定使用串联质谱和其磷酸化位点的功能映射。要严格测试这种方法，我们将集中在Bgtx敏感的α亚基表达的上级颈神经节神经元的突触后α 3-含有nAChRs的突触传递是必不可少的原代培养。该系统将使我们能够探测尼古丁暴露对nAChR相互作用组特征的影响，并将为CNS中α 3信号复合物的研究铺平道路;特别是在腹侧被盖区和内侧缰核中，这些区域与尼古丁成瘾密切相关。公共卫生相关性：尼古丁调节的细胞过程和蛋白质相互作用级联的知识将有助于开发新的健康干预措施。