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

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.

Nicotinic Receptors Underlying Nicotine Dependence: Evidence from Transgenic Mouse Models

“…For instance, estradiol has been shown to bind to the Cterminal tail of the a4 subunit to potentiate the activation of a4* nAChRs in the presence of acetylcholine, an effect that was selective for a4 as differences were not found with the a3 subunit (Curtis et al 2002). More recently, phosphorylation sites have been identified on a4b2* nAChRs, suggesting a direct role for the receptor in mediating calcium/calmodulin-dependent protein kinase II and protein kinase A intracellular signaling (Miller et al 2018). Together, these nAChR subtype specific interactions, along with cell type-specific expression patterns, may allow for selective modulation of various aspects of cholinergic signaling, thereby permitting each endogenous modulator to differentially regulate neural processes.…”

From Synapse to Function: A Perspective on the Role of Neuroproteomics in Elucidating Mechanisms of Drug Addiction

“…Subsequent use of this approach in post-mortem cortical tissue from nicotine-dependent mice and human subjects revealed 17 dose-dependent nicotine-induced changes in protein interactions in mice, with eight of these, including CaMKIIα, recapitulated in tissue from human smokers. The molecular relevance of the proposed interaction was recently elucidated by the Picciotto group using both in vitro and in vivo preparations [43]. These studies provide an excellent example of the utility of interactome studies in identifying novel protein interactions that may serve as the basis for developing precision medicine.…”

Editorial for Special Issue: Neuroproteomics

“…Just as protein phosphorylation plays a key role in the molecular mechanisms underlying drug addiction, the articles by Bertholomey et al [31] and Miller et al [32] indicate that this PTM also plays an important role in alcohol use disorders (AUDS) and nicotine addiction, respectively. Bertholomey et al [31] describe how early life stress is associated with an increased risk of developing AUDs.…”

“…As described by Miller et al [32], high-affinity nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs, where * denotes other, potentially unidentified subunits) are essential for the rewarding and reinforcing properties of nicotine. α4/β2* nAChRs are ion channel-containing proteins that flux positive ions, including calcium, in response to nicotine or the endogenous neurotransmitter acetylcholine.…”

“…Interactions have previously been identified between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brains [33,34]. Following co-expression of α4/β2 nAChR subunits with CaMKII in human embryonic kidney (HEK) cells, MS/proteomic analyses described by Miller et al [32] identified eight phosphorylation sites in the α4 subunit. One of these sites and an additional site were identified when α4/β2* nAChRs were dephosphorylated and then incubated with CaMKII in vitro, while three phosphorylation sites were identified following incubation with protein kinase A (PKA) in vitro.…”