Review
Proteins interacting with nicotinic acetylcholine receptors: expanding functional and therapeutic horizons

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Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that carry out the fast actions of the neurotransmitter acetylcholine (ACh). Over the past 30 years, it has become clear that the activity of nAChRs is dependent on their interaction with a host of proteins, and the number of these that have been identified has increased considerably with recent large-scale proteomic analyses. This review focuses on these interacting proteins, discussing how they regulate a wide range of functions including receptor assembly, and trafficking to and from the cell surface, as well as how they directly modulate functional characteristics such as sensitivity and the degree of response to ACh. Mutations giving rise to disease states highlight the importance of these interacting proteins. Here, we consider their potential as future therapeutic targets for treating diseases associated with altered nAChR function.

Section snippets

Protein–protein networking is crucial for all levels of cellular function

The function of many proteins can only be understood in the context of their interactions with other proteins, and in turn, such interactions must be understood in the context of the complex network of all protein–protein interactions, the ‘interactome’ [1]. In the realm of signalling in the nervous system, for example, our appreciation of the complexity of ligand–receptor interactions has moved to a new level by the finding that the N-methyl-D-aspartate receptors (NMDARs) for the

Nicotinic acetylcholine receptors

The nAChRs, like NMDARs, are excitatory receptors, mediating cholinergic transmission involving the neurotransmitter acetylcholine (ACh). However, they belong to a separate receptor class: the cys-loop ligand-gated ion channel (LGIC) superfamily, which also includes receptors for serotonin (5-hydroxytryptamine), γ-aminobutyric acid and glycine. nAChR family members play key roles in the brain, autonomic ganglia and at the neuromuscular junction (NMJ).

Each nAChR consists of five subunits forming

A protein complex that clusters nAChRs at the neuromuscular junction

Just over 30 years ago, rapsyn was co-purified with muscle-type nAChRs [3]. This was the first nAChR interactor to be identified, and it was found to promote clustering of nAChRs at the postsynapse of the NMJ. Rapsyn therefore plays an important role in packing nAChRs to the high density (10 000 receptors per μm2) necessary to achieve efficient cholinergic transmission. Using chimeric constructs and immunoprecipitation, it was shown that rapsyn interacts with the β1 subunit to mediate

Interacting proteins also cluster neuronal nAChRs

As in muscle, neuronal nAChRs are clustered, most likely by a different complex of proteins ([3] and references therein). Rapsyn, which has been detected in the nervous system, is incapable of clustering α3β2 or α4β2 nAChRs at the cell surface [3]. In addition, it was observed that the clustering of α5- and β2-containing nAChRs is unaffected in mice lacking rapsyn, indicating that rapsyn is not necessary for neuronal nAChR clustering. However, a more recent study found that rapsyn was present

Kinases affect nAChR function

Despite being very variable in amino acid sequence, the TM3–TM4 intracellular loop of nAChRs usually contains one or more phosphorylation sites. Phosphorylation, which is important for the clustering and stability of muscle nAChRs, can also directly affect nAChR function. The Src family kinases (SFKs) Src, Fyn and Lyn bind to the TM3–TM4 intracellular loop of α7, phosphorylating tyrosine residues, which reduces the response to ACh of heterologously expressed α7 and of native receptors in the

Assembly and trafficking

nAChR subunits are folded and assembled into oligomers in the endoplasmic reticulum (ER) before being trafficked to the cell surface. Two proteins, RIC-3 (resistance to inhibitors of cholinesterase), and UNC-50 (uncoordinated), first identified in the nematode Caenorhabditis elegans, were also found to perform in mammals roles in nAChR assembly and maturation, thereby influencing the number of receptors present at the cell surface [3]. Heterologous expression studies using Xenopus oocytes have

Extracellular interactors

Most proteins found to interact with nAChRs are intracellular, but there are examples of extracellular protein interactors (Figure 1b). These include lynx1, lynx2, secreted Ly-6/urokinase plasminogen activator receptor-related protein (SLURP)-1 and SLURP-2, which are members of the Ly-6/neurotoxin superfamily with a three-finger motif bearing structural similarity to snake α-neurotoxins. Lynx1 and lynx2 are GPI-anchored proteins expressed in neuronal populations that are both distinct and

Proteomics in large-scale identification of nAChR associated proteins

Two proteomics studies have considerably expanded the number of proteins predicted to be associated with nAChRs 32, 33. Researchers in the first study used matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS) to identify 21 proteins (Table 1) that were either pulled down with the TM3–TM4 loop of mouse β2 or co-immunoprecipitated with β2 [32]. The results provide insights into β2 trafficking, suggesting that it might be dependent on dynamin

Future prospects: an expanded horizon in drug design?

Although we have provided several examples of nAChR-interacting proteins, it is beyond the scope of this review to consider each in detail. However, Table 1 lists a number of nAChR-interacting proteins to illustrate the currently known extent of interactor diversity. Knowledge of these interactions enhance our understanding of protein complexes that play a role in all aspects of nAChR function, and should help to identify factors underlying nAChR-related diseases, such as congenital myasthenic

Conclusion

This review highlights the requirement of different techniques, such as immunoprecipitation, pull down and yeast two-hybrid (see [46] for a review of techniques used to study protein–protein interactions), to build a picture of interactome components linked to nAChRs. The picture is far from finished, and we anticipate that the continuation of these studies in addition to the application of large-scale proteomics and high-throughput approaches will reveal large protein complexes associated with

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