Proteomics of Nicotinic Receptor Complexes

描述（由申请方提供）：烟碱乙酰胆碱受体（AChR）介导烟草中尼古丁在中枢神经系统（CMS）中的作用。我们希望了解尼古丁对AChRs的重复激活启动和维持烟草成瘾期间发生的CMS分子和细胞变化的分子机制。我们的中心假设是，神经元乙酰胆碱受体是离子通道，与不同功能的细胞溶质蛋白的不同集合。这些蛋白质及其调节的过程可能参与改变尼古丁重复激活后AChR的密度、功能组织和性质。AChR的这些变化随后导致表达AChR的神经网络（例如中皮质边缘多巴胺系统）的下游适应性变化，从而维持对尼古丁的成瘾。这个建议的主要目的是确定直接和间接与阿尔法和阿尔法？AChR亚基，因为它们是神经元中表达的两种主要AChR亚型的代表性亚基。我们将通过结合两种方法来实现这一点：1）一种新的蛋白质组学方法，使用标记的AChR亚基胞质环的可溶性融合蛋白，在神经细胞中异源表达，以亲和纯化相关蛋白，然后通过质谱法鉴定;和2）通过进行酵母双杂交筛选细胞系衍生的，因此富集的cDNA文库。我们正在申请R21，因为拟议研究的一部分采用了一种创新但高风险的蛋白质组学方法，该方法仅用于纯化可溶性蛋白质复合物，以鉴定与整合膜蛋白相关的蛋白质复合物。我们假设，这些蛋白质或它们发挥功能的途径可以通过开发药物以高特异性另外靶向，这些药物破坏它们与该域中特定序列的相互作用，以调节AChR的生物合成，从而调节CMS中尼古丁作用的特定生理效应物。尼古丁受体已被证明在许多疾病中受到影响，包括精神分裂症和烟草成瘾。这项工作将阐明哪些其他新的蛋白质调节它们在脑神经细胞中的功能，从而为治疗这些神经疾病的治疗操作提供新的靶点。

DESCRIPTION (provided by applicant): Nicotinic acetylcholine receptors (AChRs) mediate the actions of nicotine in tobacco in the central nervous system (CMS). We wish to understand the molecular mechanisms by which the repetitive activation of AChRs by nicotine initiates and sustains the molecular and cellular changes of the CMS that occur during tobacco addiction. Our central hypothesis is that neuronal AChRs are ion channels that are associated with distinct sets of cytosolic proteins of different functions. It is likely that these proteins, and the processes they regulate, participate in the changing the density, functional organization, and properties of AChRs following their repetitive activation by nicotine. These changes in AChRs then lead to downstream adaptive changes in neural networks within which they are expressed (e.g. mesocorticolimbic dopamine system) and thus sustain addiction to nicotine. The main objective of this proposal is to identify cytosolic proteins associated both directly and indirectly with the alphas and alpha? AChR subunits, because they are representative subunits of two major AChR subtypes expressed in neurons. We will to accomplish this by combining two approaches: 1) a new proteomic approach using soluble fusion proteins of tagged AChR subunit cytoplasmic loops, heterologously expressed in neural cells, to affinity purify associated proteins, which will then be identified by mass spectrometry; and 2) by performing yeast two-hybrid screens of cell line-derived, and thus enriched, cDNA libraries. We are applying for a R21 because a part of the proposed study adapts an innovative, but high risk proteomic approach, which has only been used to purify soluble protein complexes, to identify protein complexes associated with integral membrane proteins. We hypothesize that these proteins or the pathways in which they function could be additionally targeted with high-specificity by the development of drugs that disrupt their interactions with specific sequences in this domain to modulate the biogenesis of AChRs, and thus specific physiological effectors of nicotine's action in the CMS. Nicotinic receptors have been shown to be affected in many diseases including schizophrenia and tobacco addiction. The proposed work will clarify what other novel proteins regulate their function in brain nerve cells and thus provide new targets for therapeutic manipulation for the treatment of these neurolgical diseases.