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Proteomic investigation of human α7‐nicotinic acetylcholine receptor interacting proteins (1147.5)
Author(s) -
Mulcahy Matthew,
Lukas Ronald,
Barrantes Francisco,
Hawrot Edward
Publication year - 2014
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.28.1_supplement.1147.5
Subject(s) - acetylcholine receptor , nicotinic agonist , chemistry , interactome , cell culture , orbitrap , cytisine , receptor , trypsin , nicotinic acetylcholine receptor , biochemistry , microbiology and biotechnology , biology , enzyme , mass spectrometry , genetics , chromatography , gene
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 evidence that α7‐nAChRs are involved in a number of protein‐protein signaling cascades. SH‐SY5Y and SH‐EP1 human clonal cell lines have been utilized extensively to characterize nAChRs. SH‐SY5Y cells naturally express α7‐nAChRs. SH‐EP1‐hα7 and SH‐EP1‐hα7‐Ric‐3 cells have been engineered to stably and heterologously express human α7‐nAChRs alone or along with human Resistance to Inhibitors of Cholinesterase 3 (Ric‐3). Bgtx‐sensitive protein complexes were isolated from each cell line using bgtx affinity immobilization. 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™. 216 total proteins (1% peptide FDR; 90‐100% probability) that were not present in controls were identified from multiple preparations of the three cell lines. Identified interacting proteins will serve as a foundation for further investigations in human‐derived cells lines and primary human tissue. Grant Funding Source : Supported by NIH 1R21AG038774, 1S10RR027027, NSF EPS‐1004057 (EH); PIP 2012 and PICT 2011‐0604 (FJB)