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Proteomes 2018, 6(4), 42; https://doi.org/10.3390/proteomes6040042

Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo

1
Department of Psychiatry, Yale University School of Medicine, 34 Park Street, 3rd Floor Research, New Haven, CT 06508, USA
2
Yale/NIDA Neuroproteomics Center, 300 George Street, New Haven, CT 06509, USA
3
W.M. Keck Biotechnology Resource Laboratory, Yale University School of Medicine, 300 George Street, New Haven, CT 06509, USA
4
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editors: Angus C. Nairn and Kenneth R. Williams
Received: 12 September 2018 / Revised: 10 October 2018 / Accepted: 11 October 2018 / Published: 15 October 2018
(This article belongs to the Special Issue Neuroproteomics)
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Abstract

Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following co-expression of α4/β2 nAChR subunits with CaMKII in HEK cells, mass spectrometry identified 8 phosphorylation sites in the α4 subunit. One of these sites and an additional site were identified when isolated α4/β2* nAChRs were dephosphorylated and subsequently incubated with CaMKII in vitro, while 3 phosphorylation sites were identified following incubation with protein kinase A (PKA) in vitro. We then isolated native α4/β2* nAChRs from mouse brain following acute or chronic exposure to nicotine. Two CaMKII sites identified in HEK cells were phosphorylated, and 1 PKA site was dephosphorylated following acute nicotine administration in vivo, whereas phosphorylation of the PKA site was increased back to baseline levels following repeated nicotine exposure. Significant changes in β2 nAChR subunit phosphorylation were not observed under these conditions, but 2 novel sites were identified on this subunit, 1 in HEK cells and 1 in vitro. These experiments identified putative CaMKII and PKA sites on α4/β2* nAChRs and novel nicotine-induced phosphorylation sites in mouse brain that can be explored for their consequences on receptor function. View Full-Text
Keywords: nicotinic receptor; CaMKII; PKA; quantitative phosphoproteomics; mouse; phosphorylation; nicotine nicotinic receptor; CaMKII; PKA; quantitative phosphoproteomics; mouse; phosphorylation; nicotine
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Miller, M.B.; Wilson, R.S.; Lam, T.T.; Nairn, A.C.; Picciotto, M.R. Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo. Proteomes 2018, 6, 42.

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