RIC-3 differentially modulates α4β2 and α7 nicotinic receptor assembly, expression, and nicotine-induced receptor upregulation

Abstract

BackgroundRecent work has shown that the chaperone resistant to inhibitors of acetylcholinesterase (RIC-3) is critical for the folding, maturation and functional expression of a variety of neuronal nicotinic acetylcholine receptors. α7 nicotinic receptors can only assemble and functionally express in select lines of cells, provided that RIC-3 is present. In contrast, α4β2 nicotinic receptors can functionally express in many cell lines even without the presence of RIC-3. Depending on the cell line, RIC-3 has differential effects on α4β2 receptor function – enhancement in mammalian cells but inhibition in Xenopus oocytes. Other differences between the two receptor types include nicotine-induced upregulation. When expressed in cell lines, α4β2 receptors readily and robustly upregulate with chronic nicotine exposure. However, α7 nicotinic receptors appear more resistant and require higher concentrations of nicotine to induce upregulation. Could the coexpression of RIC-3 modulate the extent of nicotine-induced upregulation not only for α7 receptors but also α4β2 receptors? We compared and contrasted the effects of RIC-3 on assembly, trafficking, protein expression and nicotine-induced upregulation on both α7 and α4β2 receptors using fluorescent protein tagged nicotinic receptors and Förster resonance energy transfer (FRET) microscopy imaging.ResultsRIC-3 increases assembly and cell surface trafficking of α7 receptors but does not alter α7 protein expression in transfected HEK293T cells. In contrast, RIC-3 does not affect assembly of α4β2 receptors but increases α4 and β2 subunit protein expression. Acute nicotine (30 min exposure) was sufficient to upregulate FRET between α4 and β2 subunits. Surprisingly, when RIC-3 was coexpressed with α4β2 receptors nicotine-induced upregulation was prevented. α7 receptors did not upregulate with acute nicotine in the presence or absence of RIC-3.ConclusionsThese results provide interesting novel data that RIC-3 differentially regulates assembly and expression of different nicotinic receptor subunits. These results also show that nicotine-mediated upregulation of α4β2 receptors can be dynamically regulated by the presence of the chaperone, RIC-3. This could explain a novel mechanism why high affinity α4β2 receptors are upregulated in specific neuronal subtypes in the brain and not others.

Identification and Characterization of a G Protein-binding Cluster in α7 Nicotinic Acetylcholine Receptors

“…N2a cells were co-transfected with Ric-3, which has been shown to promote the trafficking of ␣7 nAChRs to the cell surface (32)(33)(34). Visualization of fBgtx labeling in non-permeablized N2a cells transfected with ␣7 or ␣7 345-348A suggest that a mutation of the GPBC does not interfere with ␣7 nAChR cell surface expression in a heterologous system (Fig.…”

Regulation of nicotinic acetylcholine receptors in Alzheimer׳s disease: A possible role of chaperones

“…At its low levels, it promotes α7subunit assembly in the ER and surface delivery, but at its higher levels, RIC-3 suppresses α7 surface delivery and maintains it in the ER (Alexander et al, 2010b). Although some studies have shown that RIC-3 does not change protein levels of individual α7-nAChR subunits and it just enhances their assembly and forward trafficking to the cell surface (Dau et al, 2013), many others have claimed that it modestly elevates the expression of α7-nAChR subunits (Wang et al, 2009;Williams et al, 2005, Vallés et al, 2009. Moreover, RIC-3 does not alter the transport of α7-nAChRs from the ER to the cell membrane; rather, it promotes the correct assembly at the ER and increases the expression of functional α7-nAChRs on the cell surface (Vallés et al, 2009).…”

“…RIC-3 is an essential chaperone protein for folding, assembly and surface trafficking of α7-nAChRs (Dau et al, 2013;Rezvani et al, 2009) and its co-expression is required for proper function and surface expression of these receptors (Shteingauz et al, 2009;Treinin, 2008). This chaperone seems to make cysteine residues in α7-nAChRs accessible to palmitoylation at the ER that is a necessary step for subsequent functional expression of this receptor at the plasma membrane of the neurons (Alexander et al, 2010a;Drisdel et al, 2004).…”

RIC-3, a potential target for regulating cholinergic signaling and inflammation

“…Second, RIC-3 was shown to affect different aspects of nAChR maturation depending on identity of the receptor's subunits. Specifically, RIC-3 increased assembly and trafficking to the plasma membrane of α7 receptors, but not the quantity (likely to indicate stability) of α7 nAChR subunits; in contrast, RIC-3 increased quantity of α4 and β2 subunits while having no effect on their assembly into α4β2 receptors 29 . Thus, RIC-3 may interact with and affect different nAChRs differently.…”

“…Additionally, brain expression patterns of mouse ric3 mRNA and CHRNA7 overlap, a result consistent with the suggestion that mammalian RIC-3 is required for α7 nAChR's maturation in vivo 19 . RIC-3 was shown to affect stability of unassembled nAChR subunits, assembly of subunits to form a nAChR, and trafficking of nAChRs [24][25][26][27][28][29] ( Figure 1 and below). In C. elegans loss of ric-3 gene function led to reduced plasma membrane expression of multiple nAChRs, demonstrating a positive, nAChR expression promoting function for RIC-3 22 .…”