Trends in Pharmacological Sciences
ReviewProteins interacting with nicotinic acetylcholine receptors: expanding functional and therapeutic horizons
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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2016, Journal of Functional FoodsCitation Excerpt :In this study, we found that the α9-nAchRs were significantly induced in primary cultured monocytes isolated from the heavy smokers upon treatment with a low concentration (1 µL) of nicotine (Fig. 5C). Similar results were observed for other types of nAchRs, in which protein–protein interactions enhanced either the function or the stability of the receptors (Jones, Buckingham, & Sattelle, 2010). We demonstrated that monocytes overexpressing α9-nAchR in heavy smokers were more likely to form foam cells than those in non-smokers exposed to the same concentration of ox-LDL and nicotine (Fig. 6B).
Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor: Spinosyn interaction
2015, Insect Biochemistry and Molecular BiologyCitation Excerpt :These differences could either decrease the amount of receptor translated, reduce the capacity of spinosad to bind to the receptor, make the receptor less responsive even if spinosad does bind or alter the binding sites of accessory proteins that are required for wildtype α6 containing receptors to be formed. Loss of binding interactions with modulatory proteins or chaperones could lead to poorer function or impaired assembly of a correct channel with other Drosophila nAChR subunits (Jones et al., 2010). Differences in the Bovα6 and the Pxα6 protein sequence compared with other dipteran orthologues are concentrated in the C-terminal region and in the TM3-TM4 cytoplasmic loop.
Looking below the surface of nicotinic acetylcholine receptors
2015, Trends in Pharmacological SciencesExpression of cloned α6∗nicotinic acetylcholine receptors
2015, NeuropharmacologyCitation Excerpt :For example, expressing α7 AChRs in mammalian cells is extremely hard without the help of the chaperone RIC-3 (resistance to inhibitors of cholinesterase-3) (Castillo et al., 2005; Lansdell et al., 2005; Williams et al., 2005). Although many chaperones have been identified for various AChR subtypes to facilitate subunit folding and assembly, enhance trafficking, or prevent proteasome degradation (as reviewed in Jones et al., 2010; Colombo et al., 2013), none of them has been reported positive for α6* AChRs. This may be partially due to lack of a good way to express recombinant α6 in vitro to fish out the right chaperones.
The use of α-conotoxin ImI to actualize the targeted delivery of paclitaxel micelles to α7 nAChR-overexpressing breast cancer
2015, BiomaterialsCitation Excerpt :These effects of nAChRs are mediated by multiple signaling cascades, such as ERK and PI3K/mTOR signaling pathways. Alpha 7 subunit is the main nAChR subunit that mediates the proliferative effects of nicotine in cancer cells, making homomeric α7 nAChR to be a valuable molecular target for the therapy of cancers [20]. α7 nAChR antagonists (d-tubocurarine/α-cobratoxin [α-CbT]) have been considered as potential anticancer agents for non-small cell lung cancer and malignant pleural mesothelioma [21].