Proteomics of Nicotinic Receptor Complexes

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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.