Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Nicotine acetylcholine receptors (nAChRs) comprise a family of ligand-gated channels widely expressed in the mammalian brain. The beta 2 subunit is an abundant protein subunit critically involved in the cognitive and behavioral properties of nicotine as well as in the mechanisms of nicotine addiction. In this work, we used matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS) to uncover protein interactions of the intracellular loop of the beta 2 subunit and components of immunoprecipitated beta 2-nAChR complexes from mouse brain. Using the beta 2-knockout mouse to exclude nonspecific binding to the beta 2 antibody, we identify 21 nAChR-interacting proteins (NIPs) expressed in brain. Western blot analysis confirmed the association between the beta 2 subunit and candidate NIPs. Based on their functional profiles, the hypothesis is suggested that the identified NIPs can regulate the trafficking and signaling of the beta 2-nAChR. Interactions of the 132 subunit with NIPs such as G protein a, G protein-regulated inducer of neurite outgrowth 1, and G protein-activated K+ channel 1 suggest a link between nAChRs and cellular G protein pathways. These findings reveal intracellular interactions of the beta 2 subunit and may contribute to the understanding of the mechanisms of nAChR signaling and trafficking in neurons.