McClure-Begley_2013_Genomics.Proteomics.Bioinformatics_11_207

Neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha4 and beta2 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affinity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between alpha4beta2-containing (alpha4beta2( *)) nAChRs and other proteins remains limited. In this study, we identified proteins that interact with alpha4beta2( *) nAChRs in a genedose dependent pattern by immunopurifying beta2( *) nAChRs from mice that differ in alpha4 and beta2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Reduced expression of either the alpha4 or the beta2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-immunoprecipitated with these nAChRs. Furthermore, stratified linear regression analysis indicated that levels of 17 proteins was correlated significantly with expression of alpha4beta2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a beta2( *) nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of alpha4beta2 nAChRs and their downstream signaling mechanisms.