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Identifying the α7‐nicotinic acetylcholine receptor interactome from a human neuroblastoma cell line
Author(s) -
Mulcahy Matthew John,
Barrantes Francisco,
Hawrot Edward
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.884.2
Subject(s) - interactome , acetylcholine receptor , chemistry , nicotinic agonist , cytisine , nicotinic acetylcholine receptor , receptor , cell culture , hek 293 cells , biochemistry , microbiology and biotechnology , biology , gene , genetics
The α7‐nicotinic acetylcholine receptor (α7‐nAChR) is a ligand‐gated ion channel widely expressed in the mammalian CNS that is associated with numerous physiological functions. α7‐nAChRs are also the principal high‐affinity α‐bungarotoxin (bgtx) binding proteins in the mammalian brain. There is growing evidence that α7‐nAChRs are involved in a number of protein‐protein signaling cascades. The SH‐EP1‐hα7‐Ric‐3 cell line (Valles et al., 2009) is derived from the SH‐EP1 human neuroblastoma cell line which has been transfected with human α7‐nAChR (hα7) and human Resistance to Inhibitors of Cholinesterase 3 (Ric‐3). Bgtx‐sensitive protein complexes were isolated from SH‐EP1‐hα7‐Ric‐3 cells using bgtx affinity immobilization. Total bound protein was eluted, reduced and alkylated prior to digestion with trypsin in‐solution. The resulting peptides were analyzed with an LTQ‐Orbitrap mass spectrometer and data analyzed using ProteoIQ™. More than 20 proteins (1% peptide FDR; 90–100% probability) that were not present in SH‐EP1 controls were identified from multiple preparations of SH‐EP1‐hα7‐Ric‐3 cells. These proteins, which may be involved in receptor function and biogenesis, will serve as a foundation for further investigations of SH‐EP1‐hα7‐Ric‐3 and other human‐derived cells lines as well as primary human tissue. This research is supported by NIH 1R21AG038774, 1S10RR027027, and NSF EPS‐1004057.