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EMBO J. Jul 20, 2005; 24(14): 2566–2578.
Published online Jun 30, 2005. doi:  10.1038/sj.emboj.7600741
PMCID: PMC1176467

Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans

Abstract

Nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory neurotransmission in neurons and muscles. To identify nAChR accessory proteins, which may regulate their expression or function, we performed tandem affinity purification of the levamisole-sensitive nAChR from Caenorhabditis elegans, mass spectrometry of associated components, and RNAi-based screening for effects on in vivo nicotine sensitivity. Among the proteins identified was the calcineurin A subunit TAX-6, which appeared to function as a negative regulator of nAChR activity. We also identified five proteins not previously linked to nAChR function, whose inactivation conferred nicotine resistance, implicating them as positive regulators of nAChR activity. Of these, the copine NRA-1 colocalized with the levamisole receptor at neuronal and muscle plasma membranes, and, when mutated, caused reduced synaptic nAChR expression. Loss of SOC-1, which acts in receptor tyrosine kinase (RTK) signaling, also reduced synaptic levamisole receptor levels, as did mutations in the fibroblast growth factor receptor EGL-15, and another RTK, CAM-1. Thus, tandem affinity purification is a viable approach to identify novel proteins regulating neurotransmitter receptor activity or expression in model systems like C. elegans.

Keywords: copine, levamisole receptor, SOC-1, tandem affinity purification, TAX-6 calcineurin

Introduction

Nicotinic acetylcholine receptors are pentameric, ligand-gated ion channels (LGICs) that mediate excitatory neurotransmission in neurons and muscles (Changeux and Edelstein, 1998; Unwin, 2005). During biogenesis, synaptic function, and turnover, nAChRs need to interact with a plethora of proteins (Sanes and Lichtman, 2001). A limited number of such proteins is known; among them are the endoplasmic reticulum (ER) chaperones BiP (Blount and Merlie, 1991), calnexin (Keller et al, 1996), and the 14-3-3β ER forward transport protein (O'Kelly et al, 2002). At the cell surface, rapsyn stabilizes nAChRs at postsynaptic sites (Gautam et al, 1995), and a receptor tyrosine kinase, MuSK, induces clustering of nAChRs in response to neuron-secreted agrin (DeChiara et al, 1996; Gautam et al, 1996; Sanes and Lichtman, 2001). On cultured Xenopus muscle, also the FGF receptor can induce nAChR clustering (Peng et al, 1991). nAChR functional properties are regulated by kinases (Swope et al, 1999), and protein phosphatase 2B (PP2B; calcineurin) affects the rate at which nAChRs recover from desensitization (Khiroug et al, 1998; Liu and Berg, 1999).

Additional proteins that may interact with nAChRs have been identified by genetic analysis in Caenorhabditis elegans. The best studied nAChR in C. elegans is the levamisole-sensitive nAChR (levamisole receptor), which is expressed in muscles and (motor-) neurons (Fleming et al, 1997; Culetto et al, 2004; Towers et al, 2005). Five putative subunits, UNC-29, LEV-1 (non-α-subunits), UNC-38, UNC-63, and LEV-8 (α-subunits), were identified in genetic screens for resistance to the nematocidal cholinergic agonist levamisole (Lewis et al, 1980). However, in vivo interaction in C. elegans of those subunits in a single nAChR has not been shown, and the exact subunit composition of the levamisole receptor is not clear. By electrophysiology, UNC-29 and UNC-38 are required for levamisole receptor function at the neuromuscular junction (NMJ), but not for function of another, pharmacologically different nAChR (Richmond and Jorgensen, 1999). Three additional C. elegans genes have recently been shown to encode potential nAChR accessory proteins: (1) RIC-3 enhances the expression of multiple nAChRs in the cell periphery and on the plasma membrane of heterologous cells (Halevi et al, 2002). (2) LEV-10 facilitates clustering of levamisole receptors at the NMJ (Gally et al, 2004). (3) EAT-18 affects expression of pharyngeal nAChRs containing the subunit EAT-2 (McKay et al, 2004). It can be expected that other accessory proteins with important functional roles remain to be identified.

Evidence for physical association of accessory proteins with nAChRs is often indirect. For example, rapsyn was first identified simply as a major protein present in nAChR-rich membrane preparations (LaRochelle and Froehner, 1986). Ligand affinity chromatography of nAChRs and conventional peptide sequencing, however, allowed identification of only few accessory proteins (Briley and Changeux, 1977). Thus, such proteins may interact with nAChRs only with low affinity, and more advanced methods for isolation and identification, like tandem affinity purification (Rigaut et al, 1999) and mass spectrometry, may be required to identify them. However, generic purification methods, in particular for integral membrane proteins of low abundance, like nAChRs, yet have to be established for multicellular organisms expressing them, including C. elegans. Though inherently more difficult, this approach is preferable over the use of heterologous expression systems, since it ensures that protein–protein interactions identified are as close as possible to the in vivo situation.

Here we report identification and characterization of proteins associated with the levamisole receptor. We used, for the first time in C. elegans, the tandem affinity purification to isolate the levamisole receptor, and identified copurified proteins by mass spectrometry. Potential roles in nAChR function were determined after RNAi and/or in mutants, revealing proteins whose inactivation conferred altered nicotine sensitivity in vivo. For three of those proteins, including the fibroblast growth factor receptor (FGFR) substrate adaptor SOC-1, the copine NRA-1, and the calcineurin TAX-6, we provide additional results showing functions in cell surface expression and regulation of nAChRs, as well as colocalization with the levamisole receptor in vivo.

Results

Purification of the levamisole receptor

To biochemically isolate the levamisole receptor and associated proteins, we employed the tandem affinity purification (TAP) (Rigaut et al, 1999). This method involves two genetically encoded affinity tags (Protein A attached to the tobacco etch virus (TEV) protease cleavage site and calmodulin-binding peptide (CBP)) to purify a transgenic protein and associated molecules through successive IgG- and calmodulin-chromatography steps. UNC-29, C-terminally fused with the TAP tag, could be detected in the detergent extract from whole animals and pulled down with IgG agarose (Figure 1A and B). The UNC-29::TAP protein was functional, since it restored normal nicotine sensitivity in unc-29(x29) loss-of-function mutants (Figure 1C). To avoid purification of proteins that associate only with UNC-29 monomers, we varied the TAP purification, using a ‘split' TAP tag. We fused the Protein A/TEV tag to UNC-29 and the CBP tag to LEV-1; thus, only assembled complexes containing both subunits would be purified. Alternatively, we generated a strain that coexpressed UNC-29::TAP with hexa-histidine-tagged versions of LEV-1, UNC-38, and UNC-63, allowing Ni2+ chromatography as a third purification step. In total, four purifications were performed: two with the simple TAP tag on UNC-29, one with the third Ni2+ chromatography, and one with the split TAP tag. As a control, we also performed a mock purification from a wild-type (WT) extract containing no tagged protein (Figure 1D; Supplementary Figure 7 (for stronger contrast)).

Figure 1
TAP of the levamisole receptor. (A) Immunodetection of the ProtA portion of UNC-29::TAP (lane 1, *) in detergent extracts of strain AQ748 and N2 (WT, lane 2). (B) UNC-29::TAP and UNC-29::TEV-ProtA were precipitated with IgG ...

We identified the purified proteins by analyzing tryptic peptides obtained from the whole sample using multi-dimensional protein identification technology (MudPIT; Link et al, 1999; Washburn et al, 2001) and searching the C. elegans proteome database (Tabb et al, 2002). Multiple peptides of each of the levamisole receptor subunits, UNC-29, UNC-38, UNC-63, LEV-8, and LEV-1, were identified in four different purifications (Table I and Supplementary data), verifying that the purification was successful, and showing that these subunits physically associate in vivo. Many other proteins were reproducibly represented by multiple peptides. For example, the BiP ortholog HSP-3 (Supplementary Table 1), which was shown previously to associate with nAChR subunits in vertebrates (Blount and Merlie, 1991), was identified all the four times. Together, these data indicate that our procedure indeed purified the levamisole receptor.

Table 1
nAChR subunits and proteins copurified with the levamisole receptor, that cause altered sensitivity to nicotine, when depleted by RNAi

Novel nAChR subunits copurified with the levamisole receptor

Two of the purified proteins were additional nAChR α-subunits not previously implicated in levamisole receptor function: ACR-8, and ACR-12 (Table I). We investigated whether these proteins, as well as ACR-13, that had just recently been identified as LEV-8 (Towers et al, 2005), functionally resemble levamisole receptor subunits, mutations which cause resistance to paralysis by cholinergic agonists (i.e. unc-29(x29); Figure 2A and B). Mutants in acr-8 (a Tc1 transposon insertion, cxP821, as well as a genomic deletion, ok1240; not shown), acr-12(ok367), and acr-13/lev-8(x15) were tested for sensitivity to nicotine and levamisole (Figure 2A and B). Interestingly, while acr-8 and acr-13 mutations conferred nicotine resistance, acr-12(ok367) did not. Moreover, only acr-13/lev-8(x15) animals were levamisole resistant. Thus, not all of the nAChR subunits copurified equally contribute to levamisole receptor function.

Figure 2
Analysis of novel nAChR subunits copurified with the levamisole receptor. (A, B) Paralysis of mutants lacking additional nAChR α-subunits that copurified with the levamisole receptor, acr-8(cxP821), acr-12(ok367), and acr-13/lev-8(x15), was either ...

The finding that five nAChR α-subunits were copurified with the two non-α-subunits LEV-1 and UNC-29 could indicate that multiple distinct classes of levamisole receptors with differing α-subunit composition may exist. To address this possibility, we studied the expression patterns of these proteins. C-terminal GFP fusions of ACR-13 (truncated in the large cytoplasmic loop) and full-length ACR-8 were expressed in body muscles, few head and tail neurons, and nervecord synapses, but apparently not in ventral cord motor neurons (Supplementary Figure 1A and C). In contrast, full-length ACR-12::GFP was exclusively expressed in neurons, including ventral cord motor neurons (Supplementary Figure 1B). Thus, the expression domains of ACR-8, -12, and -13 collectively overlap with that of known levamisole receptor subunits in body muscles and motor neurons (Fleming et al, 1997; Culetto et al, 2004; Towers et al, 2005; data not shown).

To test for colocalization of ACR-8 and ACR-12 with the levamisole receptor subunit UNC-38 in individual postsynaptic receptor clusters, we developed a new method for in vivo labeling of cell-surface epitopes. We expressed nAChR subunits with epitope tags at the extracellular C-termini and detected them with fluorescent antibodies injected into the body cavity (Supplementary Figure 2). Thus, we could detect cMYC-tagged UNC-38 in puncta along nervecords. These were most likely postsynaptic receptor clusters, since they were juxtaposed to, but did not overlap with, presynaptic synaptobrevin (SNB-1::GFP; Figure 2C; Supplementary Figure 6C). UNC-38::3xMYC showed complete colocalization in the nervecord puncta with UNC-29::GFP (Figure 2D; Supplementary Figure 6D), as did HA-epitope-tagged LEV-1 (Supplementary Figure 5). However, HA-tagged ACR-8 or ACR-12 was present only in some of the clusters containing UNC-38::3xMYC (Figure 2E and F; Supplementary Figure 6E and F). Thus, ACR-8 and ACR-12 may contribute to levamisole receptors in only a subset of postsynaptic clusters.

RNAi reveals proteins affecting in vivo nicotine sensitivity

In addition to the nAChR subunits described above, more than 200 proteins were detected in the fractions containing the purified levamisole receptor. Though some of them were represented by multiple peptides, indicating high relative abundance, many others were identified by single peptides only. While the high sensitivity of our protein identification method facilitates discovery of low-affinity interactors, it probably also led to detection of nonspecific contaminants. Thus, we needed to identify those proteins likely to be genuine functional interactors. We included for further analysis all proteins isolated with the split TAP tag (i.e. the sample containing only assembled receptors), as well as proteins that were detected several times in the other three purifications (225 proteins; Table I and Supplementary Tables 1 and 2). As a means of judging the specific enrichment of proteins by our affinity purification over abundance in an extract, we compared our list of proteins with those found in a recent mass spectrometric analysis of the C. elegans proteome, which identified 1616 abundant proteins (including membrane proteins) in crude extracts by comparable identification methods (Mawuenyega et al, 2003). Based on this comparison, 69 of the 225 proteins were not clearly enriched by our TAP purification (Supplementary Tables 1 and 2). Since at least some of these proteins were probably nonspecific contaminants, we assigned low priority for further analysis to these proteins.

Next, we functionally assessed the identified proteins for effects on cholinergic neurotransmission. We depleted each protein by feeding the appropriate strain from a bacterial RNAi library (Kamath et al, 2003) to rrf-3(pk1426) mutant animals, which exhibit enhanced sensitivity to RNAi (Simmer et al, 2002), but show normal responses to nicotine (Figure 3A). Since levamisole receptor function is a major determinant of nicotine sensitivity in the paralysis assay (Figure 2A and B) and depletion of known levamisole receptor subunits causes strong nicotine resistance (Figure 3A and Table I), loss of a protein functionally associated with the levamisole receptor may confer altered sensitivity to nicotine. We could test 157 proteins this way; the remaining proteins were either not represented in the library (40 proteins), or RNAi caused lethal phenotypes or paralysis (28 proteins).

Figure 3
RNAi of proteins copurified with the levamisole receptor and mutation of TAX-6 calcineurin affect nicotine responses. (A) RNAi-sensitized animals of genotype rrf-3(pk1426) were depleted of either UNC-63, UNC-38, or a protein copurified with the levamisole ...

Through this RNAi screening, we identified 11 proteins whose depletion caused resistance to nicotine, including 6 nAChR subunits (Table I and Figure 3B). Depletion of 44 other proteins caused nicotine hypersensitivity (Table I; Supplementary Table 1 and Figure 3A); of these, 28 were more abundant in the purified sample than in a whole-proteome analysis (Mawuenyega et al, 2003) and therefore enriched by our purification. Thus, our results suggested potential functional interactions between 33 proteins and the seven nAChR subunits purified as part of the levamisole receptor (Table I).

Calcineurin sets the nicotine sensitivity of neurons and muscles

The most abundant protein whose depletion caused nicotine hypersensitivity was the calcineurin A subunit TAX-6 (Kuhara et al, 2002). Active calcineurin contains also the regulatory subunits calcineurin B (CNB-1) and calmodulin (also copurified; Supplementary Table 2). The regulatory subunits bind TAX-6 in a Ca2+-dependent manner to replace its auto-inhibitory C-terminus from the active site (Hashimoto et al, 1990). We found that loss-of-function mutations in both tax-6(p675) and cnb-1(jh103) (Bandyopadhyay et al, 2002) conferred hypersensitivity to nicotine (Figure 3C). Further, we prepared a synthetic gain-of-function (g.o.f.) allele of tax-6 by expressing constitutively active TAX-6 (lacking the auto-inhibitory C-terminus) in tax-6(p675) animals (Figure 3C; Supplementary Figure 3A). Significantly, these animals were nicotine resistant. Thus, calcineurin appears to negatively regulate levamisole receptor function.

TAX-6 is coexpressed with UNC-38 in muscles and neurons (Kuhara et al, 2002) (Supplementary Figure 3B). To study whether TAX-6 expression in muscle is sufficient to control nicotine sensitivity, we expressed TAX-6 in p675 mutants either from its endogenous promoter or from the muscle-specific promoter pmyo-3 (Supplementary Figure 3A). TAX-6 expressed in muscle rescued the nicotine sensitivity phenotype only partially, while expression from the tax-6 promoter fully restored WT sensitivity (Figure 3C). Expression of the tax-6 g.o.f. allele in muscles restored nicotine sensitivity to a level comparable to WT, but did not cause resistance (Figure 3C). These results suggest that calcineurin negatively regulates nicotine sensitivity to a certain degree by affecting nAChRs in body muscles; however, it is most likely required in neurons as well. Our findings parallel observations in rat chromaffin cells, where calcineurin inhibitors increased the rate at which nAChRs recover from nicotine-induced desensitization (Khiroug et al, 1998), while they increased the rundown of nicotine-induced peak currents mediated by chick ciliary ganglion α7 nAChRs (Liu and Berg, 1999). Our results suggest that a direct or indirect physical interaction, which (given the abundance of TAX-6 in the purified sample) may extend beyond the transient interaction expected for a phosphatase and its substrate, could mediate nAChR regulation by calcineurin in C. elegans.

Novel non-nAChR proteins reduce levamisole receptor function when depleted

Depletion or mutation of five non-nAChR proteins that had not previously been implicated in nAChR function conferred moderate resistance to nicotine (Figures 3B and and4A;4A; Table I). These are: (1) a homolog of vertebrate Nicalin (encoded by T05F1.1), a putative type I ER-membrane protein that antagonizes Nodal signaling (Haffner et al, 2004); (2) a putative Ca2+-dependent phospholipid-binding protein (T28F3.1) of the copine family (Creutz et al, 1998); (3) a protein containing a PHD Zn-finger motif and putative transmembrane domains (C17G1.4); (4) a POLO box-like serine/threonine kinase, PLK-2, whose mammalian homologs Fnk and Snk affect synaptic plasticity (Kauselmann et al, 1999; Pak and Sheng, 2003); and (5) SOC-1, which functions as a multisubstrate adaptor protein in FGF signaling (Schutzman et al, 2001). All five proteins are conserved in vertebrates (Table I). To further test whether these proteins are required for nicotinic receptor function, we obtained loss-of-function alleles for all of them: soc-1(n1789), plk-2(tm1395), T28F3.1(ok1025), T05F1.1(tm1453), and C17G1.4(tm1649). In all cases, nicotine resistance paralleling the RNAi phenotype was observed (Figure 4A). On the basis of these mutant phenotypes, the latter three genes were designated nra-1–3 (for nicotinic receptor associated).

Figure 4
Pharmacological analysis of genomic mutants of levamisole receptor-associated proteins. (A) Genomic deletion or mutation of plk-2(tm1395), nra-1(ok1025), soc-1(n1789), nra-2(tm1453), and nra-3(tm1649) cause significant resistance to 31 mM nicotine, verifying ...

To investigate the specificity of these genes' effects on nicotine sensitivity, we assayed sensitivity to other drugs, namely levamisole (a specific levamisole receptor agonist) and muscimol, an agonist of the inhibitory γ-aminobutyric acid receptor (GABAR) encoded by unc-49. We observed moderate levamisole resistance in soc-1(n1789), nra-1(ok1025), nra-2(tm1453), and nra-3(tm1649) mutants, indicating that their nicotine resistance phenotype was due, at least in part, to effects on levamisole receptor function (Figure 4B). Second, of all of the mutants tested, only soc-1(n1789) showed resistance to muscimol in our assay (Figure 4C and D). These results indicated that the effects of plk-2(tm1395), nra-1(ok1025), nra-2(tm1453), and nra-3(tm1649) were specific for nAChR function, while SOC-1 may have more general effects on the function of the body muscle NMJ and/or its receptors. Finally, all of the mutants except nra-1(ok1025) showed moderate to strong uncoordinated (unc) phenotypes in thrashing assays, further indicating effects on NMJ function (Figure 4C).

Loss of the copine NRA-1 causes reduced synaptic levamisole receptor expression

Copines possess two C2 domains, which can bind phospholipids in a Ca2+-dependent manner, and a protein interaction domain (Tomsig et al, 2003), by which they may recruit other proteins to membranes. The levamisole resistance of nra-1 mutants may, in principle, be due to reduction of either activity or (synaptic) expression of the levamisole receptor. We thus examined if loss of NRA-1 affected levamisole receptor expression, either throughout the cell or specifically at synaptic sites. We expressed LEV-1::GFP and UNC-38::3xMYC in rrf-3(pk1426) mutants and depleted them of NRA-1 by RNAi. LEV-1::GFP, as a marker for total cellular levamisole receptor expression, was not reduced after RNAi of nra-1 (Figure 5A). However, when we probed synaptic expression of UNC-38::3xMYC with injected anti-MYC-antibodies, we found a significant reduction in synaptic UNC-38 expression levels (28% reduction, P<0.0002; Figure 5B). As a control for our assay's ability to detect changes in synaptic levamisole receptor levels, we tested ric-3(md158) mutants, which are defective for nAChR expression in the cell periphery (Halevi et al, 2002). Significantly, in md158 animals expressing LEV-1::4xHA (Figure 5C), levels of surface levamisole receptor were largely reduced (by 65%, P<2.3 × 10−9; comparable results were obtained for the UNC-38::3xMYC transgene; data not shown). To verify the RNAi-based effects of NRA-1 depletion, we expressed and measured in vivo antibody-labeled LEV-1::4xHA in nra-1 mutants. Significantly, these showed a reduction of synaptic levamisole receptor expression by 23%, P<0.001 (Figure 5C). In contrast, a MYC-epitope-tagged UNC-49 GABAR subunit showed normal synaptic expression (Figure 5D). Thus, the copine NRA-1 may, directly or indirectly, control expression levels of synaptic levamisole receptors, but not of GABARs.

Figure 5
Reduced synaptic levamisole receptor expression in nra-1 and soc-1 mutants. (A, B) Muscular expression levels of LEV-1::GFP (A), and synaptic protein levels of UNC-38::3xMYC (B) in animals of strain AQ1019 depleted by RNAi of either NRA-1 ...

To study the subcellular site of action of NRA-1, we looked at its expression pattern. Full-length NRA-1::GFP was expressed from its endogenous promoter in many cell types (Figure 6A–E), including neurons in the head and tail, ventral cord motor neurons, and body muscles. Importantly, NRA-1::GFP localized largely to plasma membranes in muscles and neurons (Figure 6B and D) and colocalized with antibody-labeled LEV-1::4xHA (Figure 6E), though it was also observed outside LEV-1 puncta. Thus, NRA-1 may function as a general membrane organizer that, however, specifically affects cell surface expression of levamisole receptors, but not of the GABAR UNC-49.

Figure 6
Coexpression of the copine NRA-1::GFP and LEV-1 at plasma membranes of neurons and muscles. (A) Expression of NRA-1::GFP in head neurons, in body wall muscles (B) (note the preferential expression at the plasma membrane), in hypodermal cells ...

The FGFR pathway specifically affects synaptic levamisole receptor expression

Another levamisole receptor-associated protein, SOC-1, acts in RTK signaling, specifically downstream of the FGF receptor EGL-15 (Borland et al, 2001; Schutzman et al, 2001). Among its many biological activities, EGL-15 affects muscle protein degradation in starved animals (Szewczyk and Jacobson, 2003). This indicates that EGL-15 (and thus probably SOC-1) is expressed in body muscle, where also the antagonistic RTK phosphatase CLR-1 is expressed (Kokel et al, 1998), while EGL-15 is also found in other tissues (Bulow et al, 2004; Huang and Stern, 2004). SOC-1 is a homolog of the mammalian GAB multisubstrate adaptor proteins that, after phosphorylation by FGFR, recruits additional targets to the activated RTK; however, GAB proteins act downstream also of other RTKs (Liu and Rohrschneider, 2002). A second, parallel signaling pathway downstream of egl-15 utilizes the adaptor protein SEM-5 (homologous to human GRB2), and activates the GTPase ras via the guanine nucleotide exchange factor SOS-1, assisted by the leucine-rich repeat protein SOC-2. Signaling through both pathways, either dependent on SOC-1 or SEM-5, is thought to activate a MAP kinase cascade (Schutzman et al, 2001).

Since loss of soc-1 caused resistance to nicotine and levamisole (Figure 4A and B), we tested whether soc-1(n1789) mutants showed altered synaptic expression of levamisole receptors. This was the case: Synaptic LEV-1::4xHA expression was significantly reduced in n1789 animals (by 36%; P=3.6 × 10−8; Figure 5C). Consistently, this was also found for epitope-tagged UNC-38::3xMYC, in rrf-3(pk1426) mutants depleted of SOC-1 by RNAi (reduced by 29%, P=0.0005; Figure 5B), while LEV-1::GFP expression was not altered (Figure 5A). Thus, synaptic targeting of levamisole receptors, rather than their general expression, appears to be specifically affected by SOC-1. Interestingly, soc-1 mutants also exhibited reduced synaptic expression of the UNC-49 GABAR (51% reduction, P<1e−6; Figure 5D), consistent with the observed resistance of soc-1 mutants to muscimol (Figure 4C and D). The loss of both GABARs and nAChRs from synaptic sites in soc-1 mutants suggests a more general role of SOC-1 in assisting expression of LGICs at the NMJ, possibly acting downstream of EGL-15, and maybe also of other RTKs.

Of the proteins known to act in EGL-15 signaling, only SOC-1 was copurified with the levamisole receptor. Thus, to test if its involvement in synaptic nAChR expression correlated with its role in RTK signaling, as opposed to a secondary function not related to the FGF receptor, we studied nicotine sensitivity of other mutants affecting FGF signaling. Significantly, two partial loss-of-function alleles of egl-15(n484 and n1477) (DeVore et al, 1995; Goodman et al, 2003), as well as mutations in three additional positive regulators of FGF signaling, soc-2(ku167), sos-1(c548), and sem-5(n1779), caused moderate nicotine resistance, while mutation of the antagonistic phosphatase clr-1(n1745) caused hypersensitivity to nicotine (Figure 7A). egl-15(n484) mutants were also levamisole resistant (Figure 7B). Importantly, the drug response phenotypes observed for the mutants in egl-15 and other positive FGF signaling regulators correlated with a reduction of synaptic LEV-1::4xHA expression, just like in soc-1(n1789) (Figure 7C; Supplementary Figure 4B). Our findings implicate SOC-1 and the FGFR cascade in levamisole receptor cell surface expression and/or clustering.

Figure 7
Mutations in soc-1, egl-15, and cam-1 affect levamisole receptor function and synaptic expression. (A) Responses to 31 mM nicotine in paralysis assays were compared in WT (N2), soc-1(n1789), egl-15(n484 and n1477), and cam-1(ks52 and ak37) mutant animals, ...

In vertebrates, such functions have been demonstrated for another RTK, namely MuSK (see introduction). We thus analyzed two mutations in CAM-1, which shows the highest homology to MuSK, and found that cam-1(ak37), containing a premature stop before the TM domain (M Francis and A Maricq, personal communication), significantly reduced synaptic LEV-1::4xHA expression (by 22%, P<0.0006; Figure 5C) and conferred resistance to levamisole (Figure 5B). Also, deletion of the kinase domain alone (allele ks52; Koga et al, 1999) caused levamisole resistance (Figure 5B); however, ks52 did not reduce synaptic levamisole receptor levels (Figure 5C), and neither cam-1 allele was nicotine resistant (Figure 5A). These results indicate a complex involvement of cam-1 also in levamisole receptor expression. To study whether CAM-1 and EGL-15 may cooperatively regulate synaptic expression of levamisole receptors, we depleted each RTK by RNAi either in egl-15(n1477) or cam-1(ak37) animals, respectively, and analyzed synaptic LEV-1::4xHA expression with injected antibodies (Figure 7D). Significantly, synaptic LEV-1 was reduced for both combinations of mutation and RNAi depletion relative to either mutant alone. Thus, EGL-15 and CAM-1 may act in parallel to govern synaptic levamisole receptor expression. Reduction of synaptic LEV-1 in both egl-15 and cam-1 mutants was further enhanced by SOC-1 RNAi, suggesting that loss of SOC-1 in cam-1 mutants might reduce signaling also through EGL-15, and vice versa (Figure 7D). Like soc-1(n1789) (Figure 4C), egl-15 and cam-1 mutants exhibited uncoordinated phenotypes (Supplementary Figure 4C). However, in contrast to SOC-1, they did not affect muscimol sensitivity, indicating that they do not influence GABAR expression. Though many details of these putative signaling pathways remain to be investigated, our findings suggest that the functional interaction between nAChRs and SOC-1 may represent a mechanism for regulation of synaptic nAChR clustering and/or maintenance through RTK signaling.

Discussion

We have shown (to our knowledge for the first time) that tandem affinity purification can be used to isolate an integral membrane protein complex of low abundance, the C. elegans levamisole-sensitive nAChR, from its native environment in a multicellular organism. Even though the yields of purified receptor complex were low, we could identify novel proteins that functionally interact with this nAChR by combining proteomic analysis with genetic and behavioural assays. Most of these proteins have not previously been implicated in affecting the expression or modulation of nAChRs. Others, like calcineurin and BiP, were known to affect nAChR functional properties or expression (Blount and Merlie, 1991; Khiroug et al, 1998; Liu and Berg, 1999). Copurification of these proteins with the levamisole receptor may indicate direct interactions. Alternatively, some of the proteins identified may interact indirectly, perhaps through a protein scaffold containing this nAChR. Since the high sensitivity of mass spectrometry certainly led to the identification also of unspecific contaminants, especially among abundant proteins in C. elegans extracts (Mawuenyega et al, 2003), the use of RNAi to identify nicotine-resistance genes was an invaluable secondary screen that allowed us to focus on proteins that affect either the functional properties of nAChRs (like calcineurin), or their synaptic expression (like the copine NRA-1).

We identified five novel non-nAChR proteins that cause nicotine resistance when depleted. For two of those, the copine NRA-1 and the multisubstrate adaptor SOC-1, we could show a requirement for maintenance of normal levels of nAChRs at synaptic sites. Our findings indicate that RTK signaling, utilizing the multisubstrate adaptor SOC-1, promotes clustering and/or maintenance of both nAChRs and GABARs at synaptic sites in an intact animal, expanding previous observations made in cell culture (Peng et al, 1991). Our data further imply that SOC-1-like adaptor proteins may do so via direct or indirect physical interaction with neurotransmitter receptor proteins.

The association of the copine NRA-1 with the levamisole receptor gives new insight into the functions of this novel, widely occurring family of proteins (Creutz et al, 1998). Since copines, after binding Ca2+, have the potential to direct other proteins to phospholipid membranes (Tomsig et al, 2003), NRA-1 may recruit proteins that interact with the levamisole receptor, possibly in an activity-dependent manner. Deletion of nra-1 caused resistance to cholinergic agonists and reduced synaptic levamisole receptor levels, but did not affect synaptic levels of GABARs; thus, NRA-1 may play a relatively specific role in targeting or stabilizing the levamisole receptor at the plasma membrane.

Among the remaining proteins that we copurified are many whose known or inferred function may suggest a possible role in nAChR biology, for example, a casein kinase I homolog (F46F2.2), depletion of which caused nicotine hypersensitivity. Possibly even some of the copurified proteins lacking nicotine sensitivity phenotypes (Supplementary Table 2) may function in nAChR biology and have escaped detection due to ineffective RNAi or subtle phenotypes. Many of the proteins copurified with the levamisole receptor bear membrane-spanning domains, and may thus interact with it in the membrane environment. Future experiments, including coimmunoprecipitation, membrane-specific protein interaction assays, and electrophysiology, will allow us to define the exact functions of the novel nAChR interactors identified in our study.

Most of the proteins identified have vertebrate homologs; thus, their functions may be conserved in humans and could provide new insight into the molecular mechanisms underlying nicotine addiction. Our combined proteomic and genetic approach to define functional protein complexes in the multicellular organism C. elegans holds great potential for future applications, not only for the study of nAChRs, but also for analysis of other protein complexes in the nervous system and other tissues.

Materials and methods

Extract preparation, Western analysis, and TAP

Nematodes were resuspended in buffer D (20 mM Tris–HCl, pH 7.9; 150 mM NaCl; 10% glycerol). Then we added, adjusted to the total volume: 0.5 mM DTT; 0.5 mM PMSF; 1.0 mM EDTA; 1.0 μg/ml Nα-p-tosyl-L-arginin-methyl-ester; 2.0 μg/ml of each pepstatin, leupeptin, and chymostatin (Sigma). The suspension was dripped into liquid N2 and stored at −80°C. Frozen suspension, corresponding to 40 g of worms, was ground under liquid N2 to a fine powder, which was slowly thawed on ice (as all subsequent steps). After dilution with buffer D and additives (see above) to 160 ml and homogenization in a glass tissue homogenizer (Kontes, New Jersey), the suspension was centrifuged at 22 500 g for 30′. The supernatant was centrifuged for 1 h at 125 000 g, the pellets of both centrifugations combined and resuspended in 120 ml buffer D with additives and 1% Triton-X 100. The suspension was homogenized and gently stirred for 2 h. After centrifugation at 22 500 g for 30′, the supernatant was centrifuged for 1 h at 125 000 g. The clear interphase was collected and dialyzed twice for 2 h against buffer D with 0.05% Triton-X 100. The TAP tag was immunochemically detected using peroxidase antiperoxidase soluble complex (PAP, Sigma), on blots of either total nematode lysate or IgG agarose immunoprecipitates from ca. 200 μl of extract. Tandem affinity purification was performed as described, substituting NP-40 by 0.05% Triton-X 100 in all buffers (Rigaut et al, 1999). Purification with a TAP tag on UNC-29 (strain AQ748) yielded all levamisole receptor subunits; however, UNC-29 was enriched (14 individual peptides identified, compared to 3–5 for the other subunits), indicating copurification of UNC-29 monomers. Purification using the split TAP tag (strain AQ839) yielded all subunits in similar abundance of 3–8 individual peptides.

RNAi screen

For RNAi, we used rrf-3(pk1426) mutants (Simmer et al, 2002), either by feeding bacterial strains from the Ahringer library (Kamath et al, 2003), or (in few cases) by soaking with in vitro transcribed double-stranded RNA (Maeda et al, 2001). For feeding, bacteria were grown on NGM plates containing 25 μg/ml carbenicillin for 48 h, then transcription was induced by spotting 100 μl of 100 mM IPTG onto the bacterial lawn. Three L4 animals were placed onto the dried plate, and transferred to a freshly induced plate after 24 h. Nicotine sensitivity of the progeny was scored 96 h later (see below). For candidates causing nicotine sensitivity phenotypes, the RNAi experiment was repeated to verify the finding.

Drug response and thrashing assays

A total of 30 young adult animals were transferred to NGM plates containing 0.425 or 0.5% (v/v) (−)-nicotine, or 1 or 0.2 mM levamisole. Paralysis was followed by visual inspection every 15′ and defined as lack of movement in response to prodding. For thrashing assays, animals were placed into M9 salt solution in 96-well plates, with a pad of NGM at the bottom of the wells, either without or with 1.5 mM muscimol for 1 h. Then, animals were filmed and body thrashes per minute were counted for 20 or more animals per genotype and drug condition and averaged.

In vivo antibody-binding assay

Animals expressing C-terminally epitope-tagged versions of nAChR subunits, or N-terminally tagged UNC-49 GABAR subunits, were mounted on dry agarose pads under halocarbon oil. They were injected into the pseudocoelom with fluorescently labeled monoclonal antibodies (anti-HA, clone 16B12, coupled to Alexa488, Molecular Probes; or anti-cMYC, clone 9E10, coupled to Cy3, Sigma) in a 1:200 dilution in injection buffer (20 mM K3PO4, 3 mM K citrate, 2% PEG 6000, pH 7.5). We injected until a few eggs were pushed out, assuming that this happens once a certain internal pressure is reached, and thus ensuring roughly equivalent concentration of antibody from animal to animal. Animals were recovered from the pads in M9 salt solution, and transferred to NGM plates seeded with OP50. Animals that moved normally, fed and laid eggs, were imaged after ca. 6 h (during this time, the coelomocytes took up excess antibody from the body fluid). Quantitative analysis of nervecord fluorescence was performed using ImageJ. Line scans were traced along fluorescent puncta of the ventral nervecord, which we analyzed in the midbody of the worm, within ca. 1/4 of the length of the animal either anterior or posterior of the vulva. After background correction, fluorescence values were averaged for individual line scans; then, the averaged values of line scans from 15–111 different animals of the same genotype and experimental conditions were averaged.

Note added in proof

While this paper was undergoing final revisions, Francis et al (2005) Neuron 46: 581 reported that mutation of cam-1 affects synaptic currents at the NMJ, specifically for a C. elegans nicotine-sensitive nAChR comprising the ACR-16 subunit.

Supplementary Material

Supplementary Information

Acknowledgments

We thank I Mori, B Séraphin, and A DeAntoni for providing plasmids, J Liewald for comments on the manuscript, C Schultheis, A Kruse, and Y Martinez-Fernandez for technical assistance, L Ségalat for acr-8(cxP821), J Ahnn for cnb-1(jh103), M Francis and V Maricq for cam-1(ak37), M Nonet for NM670, B Bamber and A Benham for FY386, the C. elegans knockout consortium and the Japanese National Bioresource for the nematode for deletion strains. Some nematode strains used in this work were provided by the CGC, which is funded by the NIH NCRR. AG was a Long-Term Fellow of the Human Frontier Science Program. This work was funded by NIH grants to WRS and JRY, and by grants from the DFG, BMBF, and HMWK to AG.

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