Exploring the Nicotinic Acetylcholine Receptor-associated Proteome with iTRAQ and Transgenic Mice

摘要

Neuronal nicotinic acetylcholine receptors（nAChRs） containing a4 and b2 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affnity.These nAChRs are involved in nicotine dependence,mood disorders,neurodegeneration and neuroprotection.However,our understanding of the interactions between a4b2-containing（a4b2*） nAChRs and other proteins remains limited.In this study,we identifed proteins that interact with a4b2*nAChRs in a gene-dose dependent pattern by immunopurifying b2*nAChRs from mice that differ in a4 and b2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation（iTRAQ）.Reduced expression of either the a4 or the b2 subunit results in a correlated decline in the expression of a number of putative interacting proteins.We identifed 208 proteins co-immunoprecipitated with these nAChRs.Furthermore,stratifed linear regression analysis indicated that levels of 17 proteins was correlated signifcantly with expression of a4b2 nAChRs,including proteins involved in cytoskeletal rearrangement and calcium signaling.These fndings represent the frst application of quantitative proteomics to produce a b2* nAChR interactome and describe a novel technique used to discover potential targets for pharmacological manipulation of a4b2 nAChRs and their downstream signaling mechanisms.