Exploring the nicotinic acetylcholine receptor-associated proteome with iTRAQ and transgenic mice

Neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and β2 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 α4β2-containing (α4β2(∗)) nAChRs and other proteins remains limited. In this study, we identified proteins that interact with α4β2(∗) nAChRs in a genedose dependent pattern by immunopurifying β2(∗) nAChRs from mice that differ in α4 and β2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Reduced expression of either the α4 or the β2 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 α4β2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a β2(∗) nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of α4β2 nAChRs and their downstream signaling mechanisms.