Proteomics of Nicotinic Receptor Complexes

描述(由申请人提供)：烟碱型乙酰胆碱受体(AChRs)介导烟草中尼古丁在中枢神经系统(CMS)的作用。我们希望了解尼古丁反复激活AChRs启动和维持烟草成瘾过程中CMS的分子和细胞变化的分子机制。我们的中心假设是，神经元AChRs是离子通道，与不同功能的胞浆蛋白相关。这些蛋白质及其调控的过程很可能参与了尼古丁重复激活AChRs后AChRs密度、功能组织和性质的变化。AChRs的这些变化随后导致表达AChRs的神经网络(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。这个建议的主要目标是鉴定与α和α？直接或间接相关的胞浆蛋白。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，使用在神经细胞中异源表达的标记AChR亚单位胞质环的可溶性融合蛋白来亲和纯化相关蛋白，然后通过质谱仪进行鉴定；2)通过酵母双杂交筛选来自细胞系的从而丰富的cDNA文库。我们申请R21是因为拟议研究的一部分采用了一种创新的但高风险的蛋白质组学方法，这种方法只被用于纯化可溶性蛋白质复合体，以鉴定与完整膜蛋白相关的蛋白质复合体。我们假设，这些蛋白质或它们发挥作用的途径可以通过药物的开发以高特异性的方式额外靶向，这些药物可以破坏它们与该区域中特定序列的相互作用，以调节AChRs的生物发生，从而调节尼古丁在CMS中的作用的特定生理效应。尼古丁受体已被证明在包括精神分裂症和烟草成瘾在内的许多疾病中都受到影响。这项拟议的工作将阐明哪些新的蛋白质调节它们在脑神经细胞中的功能，从而为这些神经内科疾病的治疗提供新的靶点。

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.