SEARCH

SEARCH BY CITATION

Keywords:

  • nicotinic acetylcholine receptor;
  • receptor-associated protein;
  • resistance to inhibitors of cholinesterase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

RIC-3 is a transmembrane protein which enhances maturation (folding and assembly) of neuronal nicotinic acetylcholine receptors (nAChRs). In this study, we report the cloning and characterisation of 11 alternatively spliced isoforms of Drosophila melanogaster RIC-3 (DmRIC-3). Heterologous expression studies of alternatively spliced DmRIC-3 isoforms demonstrate that nAChR chaperone activity does not require a predicted coiled-coil domain which is located entirely within exon 7. In contrast, isoforms containing an additional exon (exon 2), which is located within a proline-rich N-terminal region, have a greatly reduced ability to enhance nAChR maturation. The ability of DmRIC-3 to influence nAChR maturation was examined in co-expression studies with human α7 nAChRs and with hybrid nAChRs containing both Drosophila and rat nAChR subunits. When expressed in a Drosophila cell line, several of the DmRIC-3 splice variants enhanced nAChR maturation to a significantly greater extent than observed with human RIC-3. In contrast, when expressed in a human cell line, human RIC-3 enhanced nAChR maturation more efficiently than DmRIC-3. The cloning and characterisation of 11 alternatively spliced DmRIC-3 isoforms has helped to identify domains influencing RIC-3 chaperone activity. In addition, studies conducted in different expression systems suggest that additional host cell factors may modulate the chaperone activity of RIC-3.

Abbreviations used
5HT3R

5-hydroxytryptamine type 3 receptor

αBTX

α-bungarotoxin

CeRIC

Caenorhabditis  elegans RIC-3

DmRIC

Drosophila melanogaster RIC-3

FCS

foetal calf serum

hRIC-3

human RIC-3

nAChR

nicotinic acetylcholine receptor

RIC

resistance to inhibitors of cholinesterase

Neuronal nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in both vertebrates and invertebrates. They have been implicated in several human neurological disorders (Gotti et al. 2006) and are targets for commercially important insecticides used in crop protection and animal health applications (Raymond Delpech et al. 2005; Millar and Denholm 2007). Nicotinic receptors, in common with other members of the Cys-loop family of ligand-gated ion channels, are oligomeric cell-surface proteins in which five subunits co-assemble to form a central cation-selective pore. Seventeen nAChR subunits (α1–α10, β1–β4, γ, δ and ε) have been identified in vertebrate species which generate a diverse family of heteromeric and homomeric nAChR subtypes (Le Novère et al. 2002; Millar 2003). In the model insect species Drosophila melanogaster, 10 nAChR subunits (Dα1–Dα7 and Dβ1–Dβ3) have been identified (Jones et al. 2007; Millar and Denholm 2007). Heterologous expression of cloned nAChR subunits has provided a powerful approach to the characterisation of these receptors, but difficulties have been encountered with the efficient expression of some nAChR subtypes. Most notable have been difficulties in the expression in cultured cell lines of the vertebrate α7 nAChR subunit (Cooper and Millar 1997; Kassner and Berg 1997; Rangwala et al. 1997) and of almost all insect nAChR subunits (Lansdell et al. 1997; Millar 1999).

RIC-3 is a transmembrane protein which is able to enhance nAChR maturation (folding and assembly). The gene encoding RIC-3 (ric-3) was first identified as one of several genes in the nematode Caenorhabditis  elegans which, when mutated, conferred RIC such as aldicarb (Nguyen et al. 1995; Miller et al. 1996). The nomenclature ‘RIC’ is derived from the phenotype resistance to inhibitors of cholinesterase. Evidence indicating that the protein encoded by ric-3 is required for the maturation of nAChRs in C. elegans was obtained from genetic studies aimed at identifying mutations able to suppress neuronal degeneration caused by a gain of function mutation in the nAChR subunit DEG-3 (Halevi et al. 2002). Further evidence to indicate that RIC-3 is able to enhance nAChR maturation has been obtained from studies in which RIC-3 was co-expressed with recombinant nAChRs (Halevi et al. 2002, 2003). Unlike many other chaperone proteins which interact with a diverse selection of target proteins, RIC-3 appears to have a highly restricted target protein specificity. Other than nAChRs, the only other protein RIC-3 has been shown to interact with or modulate is a closely related member of the Cys-loop family, the 5-hydroxytryptamine type 3 receptor (5HT3R) (Halevi et al. 2002; Castillo et al. 2005; Cheng et al. 2005, 2007). No evidence of interaction with glutamate- or GABA-gated ion channels has been observed (Halevi et al. 2002; Lansdell et al. 2005).

A clear example of the ability of RIC-3 to facilitate nAChR maturation and functional expression has come from co-expression studies conducted with the vertebrate nAChR α7 subunit. The α7 nAChR subunit is capable of generating functional homomeric receptors when expressed in Xenopus oocytes (Couturier et al. 1990), but fails to do so when expressed in most non-neuronal cultured cell lines (Cooper and Millar 1997). Studies conducted in Xenopus oocytes demonstrated that co-expression of the human or C. elegans RIC-3 (hRIC-3 or CeRIC-3) protein results in more efficient functional expression of α7 nAChRs (Halevi et al. 2002, 2003). Significantly, co-expression of RIC-3 also facilitates the functional expression of α7 nAChRs in otherwise non-permissive cultured cell lines (Castillo et al. 2005; Lansdell et al. 2005; Williams et al. 2005).

In this study, we have identified and cloned a family of 11 alternatively spliced RIC-3 isoforms (D. melanogaster RIC-3; DmRIC-3) from the model insect species D. melanogaster. Heterologous expression studies of DmRIC-3 and hRIC-3 in both Drosophila and human cell lines has revealed that the nAChR-chaperone activity of RIC-3 is influenced by the host cell environment. Furthermore, comparison of alternatively spliced DmRIC-3 isoforms has identified the importance of DmRIC-3 domains in modulating nAChR maturation.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Plasmids, cDNAs and antibodies

The molecular cloning of the hRIC-3 cDNA and its subcloning into the mammalian expression vector pRK5 has been described previously (Lansdell et al. 2005). Human RIC-3 was subcloned into the Drosophila expression vector pRmHa3 (Bunch et al. 1988) with EcoRI. The human α7 nAChR subunit was subcloned into pRmHa3 with KpnI and SalI. The subcloning of other nAChR subunits into pRmHa3 has been described previously (Lansdell et al. 1997; Lansdell and Millar 2000a,b, 2002), as has the introduction of FLAG epitope tags into Drosophila subunits (Lansdell and Millar 2002). Monoclonal antibody mAb319, raised against the α7 nAChR subunit, and mAbFLAG-M2, raised against the recombinant FLAG epitope, were obtained from Sigma, Poole, UK.

Molecular cloning and sequence analysis of Drosophila RIC-3

Synthetic oligonucleotide primers corresponding to the predicted 5′ and 3′ untranslated regions of the predicted Drosophila gene sequence CG30296 were designed and used for PCR amplification with a Drosophila head λZAPII cDNA library (provided by Dr Ron Davis, Baylor College of Medicine, Houston, TX, USA). Amplified fragments were cloned into the cloning vector pCRII (Invitrogen, Paisley, UK) and verified by nucleotide sequencing. Identification of coiled-coil motifs (Lupas et al. 1991) was performed with the program coils (http://www.ch.embnet.org/software/COILS_form.html). Cloned DNA encoding alternatively spliced DmRIC-3 isoforms were subcloned into the Drosophila expression vector pRmHa3 and the mammalian expression vector pRK5. Sequences of the 11 DmRIC-3 isoforms have been deposited in the sequence databases. Database accession numbers are: DmRIC-32,7 (AM902266); DmRIC-32,7a (AM902267); DmRIC-32,7a,9 (AM902268); DmRIC-36,7 (AM902269); DmRIC-36,7a (AM902270); DmRIC-36,7,9 (AM902271); DmRIC-36,7a,9 (AM902272); DmRIC-37 (AM902273); DmRIC-37a (AM902274); DmRIC-37,9 (AM902275); and DmRIC-37a,9 (AM902276).

Expression in cultured cell lines

Schneider’s Drosophila S2 cells (Schneider 1972) were grown in Shields and Sang M3 medium (Sigma) containing 12.5% heat-inactivated FCS (Sigma), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Paisley, UK) at 25°C. Exponentially growing S2 cells were transfected by a modified calcium phosphate method as described previously (Lansdell et al. 1997). Cells were transiently transfected with plasmid expression vector pRmHa3 containing appropriate nAChR subunit cDNAs. Expression of nAChR subunit cDNAs from the metallothionein promoter of pRmHa3 was induced by the addition of CuSO4 (0.6 mM) for 24 h. Human embryonic kidney tsA201 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen) containing 10% FCS (Sigma), 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies). Cells were maintained in a humidified incubator containing 5% CO2 at 37°C. Mammalian cells were transfected using the Effectene reagent (Qiagen, Crawley, UK) according to the manufacturer’s instructions. After overnight incubation in Effectene, cells were incubated at 37°C or 25°C for 24 h before being assayed for radioligand binding. For low temperature experiments, tsA201 cells were transfected overnight at 37°C (as above) and then transferred to 25°C in a cooled incubator containing 5% CO2 for 24 h before assaying by radioligand binding.

Radioligand binding

[3H]epibatidine (48 Ci/mmol) was purchased from Perkin Elmer, Seer Green, UK. [125I]α-bungarotoxin ([125I]αBTX; 150–200 Ci/mmol) was purchased from GE Healthcare, Amersham, UK. Radioligand binding with [3H]epibatidine (3 nM) to disrupted cells was performed as described previously (Lansdell and Millar 2000a). Samples were assayed by filtration onto Whatman GF/B filters (Brandel, Gaithersburg, MD, USA) pre-soaked in 0.5% polyethylenimine followed by rapid washing using a Brandel cell harvester (Brandel, Gaithersburg, MD, USA) (Lansdell and Millar 2000a). Cell-surface binding with [125I]αBTX (3 nM) was performed as described previously (Lansdell and Millar 2004). Samples were in Hank's buffered saline solution buffer containing 0.5% bovine serum albumin and harvested onto Whatman GF/A filters pre-soaked in 0.5% polyethylenimine using a Brandel cell harvester (Lansdell and Millar 2004). Amounts of total cellular protein were determined by a Bio-Rad DC protein assay (Bio-Rad, Hemel Hempsted, UK) using bovine serum albumin standards.

Metabolic labelling and immunoprecipitation

Drosophila S2 cells were metabolically labelled by growth in methionine-free Shields and Sang M3 medium for 15 min, cells were labelled with 250 μCi ‘Pro-mix’ (GE Healthcare), a mixture of [35S]-methionine and [35S]-cysteine, in 1.5 mL methionine-free medium for 3 h. Medium containing 30 mg/L methionine and 10% heat-inactivated foetal calf serum (FCS) was then added and the cells incubated for a further 90 min. Cells were washed three times with 10 mL phosphate-buffered saline and harvested into 300 μL ice-cold lysis buffer (150 mM NaCl, 50 mM Tris/Cl, pH 8.0, 5 mM EDTA and 1% Triton X-100) containing protease inhibitors (0.2 mM phenylmethylsulphonyl fluoride, 2 mM N-ethylmaleimide and 10 μg/mL, each, of leupeptin, apoprotinin and pepstatin). Solubilisation and all subsequent steps were performed at 4°C. After 1 h solubilisation the cell lysate was pre-cleared by incubation overnight with 30 μL pre-washed protein G-sepharose (GE Healthcare) in a 1 : 1 mixture with lysis buffer. Non-solubilised material was pelleted by centrifugation at 14 000 g for 15 min. Cell lysates were incubated with mAbFLAG-M2 or mAb319 for 3.5 h. The antibody–receptor complex was immunoprecipitated by the addition of 35 μL protein G-sepharose, incubated for a further 3.5 h and isolated by centrifugation at 16 000 g. Samples were washed with 4 × 1 mL lysis buffer. Samples were examined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis followed by autoradiography as described previously (Lansdell et al. 1997).

Electrophysiology

Human kidney tsA201 cells, grown on glass coverslips coated in collagen and polylysine (both 10 μg/mL), were cotransfected with plasmids encoding the human nAChR α7 subunit and RIC-3 isoforms, together with pEGFP-C2 (Clontech, Mountain View, CA, USA), encoding green fluorescent protein. Whole-cell recordings were performed at 22°C, 36–48 h after transfection, as described previously (Lansdell et al. 2005).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Molecular cloning of Drosophila RIC-3

As had been reported previously (Halevi et al. 2002), the CeRIC-3 protein shows weak sequence similarity to a predicted protein (CG9349) identified by the Drosophila genome sequencing project. As a consequence of more recent annotations of the Drosophila genome, CG9349 has been assigned the name CG30296, but does not, as yet, have a defined biological function. Synthetic oligonucleotide primers corresponding to the predicted 5′ and 3′ untranslated regions of CG30296 were synthesised and used for PCR amplification from a Drosophila head λZAPII cDNA library. Several alternatively spliced cDNA clones have been isolated and sequenced. Comparison of these cDNA sequences with genomic sequence data indicates that the gene encoding CG30296 contains 11 exons. As is illustrated in Fig. 1, 11 alternative cDNA isoforms have been identified by PCR amplification, all of which maintain the putative open reading frame and will be referred to as DmRIC-3 isoforms (Fig. 1). DmRIC-3 isoforms were identified which either contain or lack exon 2 (40 amino acids), exon 6 (7 amino acids) and exon 9 (30 amino acids). In addition, two alternative versions of exon 7 (which we have referred to as exon 7 and exon 7A) were identified. Exons 7 and 7A encode 83 and 34 amino acids respectively. We consider exons 7 and 7A to be genuine alternative exons (rather than exons which may be either present or absent), as the presence or absence of both exons would result in disruption of the open reading frame. The alternatively spliced DmRIC-3 isoforms have been assigned names on the basis of the presence or absence of exons 2, 6, 7, 7A and 9 (Fig. 1), thus DmRIC-32,7a,9 contains exons 2, 7A and 9 but lacks exon 6 and 7.

image

Figure 1.  Alternatively spliced variants of Drosophila RIC-3. Eleven alternatively spliced variants of RIC-3 have been cloned from Drosophila melanogaster (DmRIC-3). Nomenclature of the alternatively spliced variants of DmRIC-3 reflects the presence or absence of exons 2, 6, 7, 7A and 9. Numbers and letters in parenthesis refer to exons present in the alternatively spliced cDNA clones. The position of hydrophobic, presumed transmembrane, domains (M1 and M2) and predicted coiled-coil domain are indicated.

Download figure to PowerPoint

The influence of RIC-3 on the maturation of human α7 nAChRs

A robust assay of RIC-3's chaperone activity is its ability to facilitate the maturation of the nAChR α7 subunit into a conformation which can be detected by the high affinity nAChR antagonist αBTX (Lansdell et al. 2005). In the absence of co-expressed RIC-3, the α7 nAChR subunit fails to adopt a conformation which can be detected by [125I]αBTX in the human embryonic kidney cell line tsA201 (Fig. 2). However, when α7 nAChR subunit is co-expressed with hRIC-3, high levels of specific cell-surface [125I]αBTX binding are detected (307 ± 51 fmol/106 cells, = 4; Fig. 2b).

image

Figure 2.  The influence of hRIC-3 and DmRIC-3 on the expression of human α7 nAChRs. The human nAChR α7 subunit was co-expressed with either DmRIC-3 splice variants or hRIC-3 by transient transfection in human tsA201 cells maintained at 37°C (a), tsA201 cells maintained at 25°C (b) or Drosophila S2 cells maintained at 25°C (c). Specific cell-surface binding of [125I]αBTX (3 nM) was determined. Data are presented as specific radioligand binding (fmol/106 cells). Data are mean of at least four independent experiments performed in triplicate.

Download figure to PowerPoint

The alternatively spliced DmRIC-3 cDNAs were subcloned into the mammalian expression vector pRK5 and co-expressed by transient transfection with the human nAChR α7 subunit in tsA201 cells. Co-expression of some, but not all, isoforms of DmRIC-3 resulted in specific cell-surface binding of [125I]αBTX (Fig. 2a). The most notable difference between DmRIC-3 isoforms is that higher levels of [125I]αBTX binding are detected with isoforms lacking exon 2 (Fig. 2a). All DmRIC-3 isoforms lacking exon 2 facilitated cell-surface [125I]αBTX binding (∼25–120 fmol/106 cells, = 4). However, the levels of [125I]αBTX binding detected were significantly lower than the level of binding detected when the α7 nAChR subunit was co-expressed with hRIC-3 (< 0.01; Fig. 2a).

A further series of experiments was performed in which the influence of culturing transfected tsA201 cells at a lower temperature was examined. In tsA201 cells which were maintained at 25°C, rather than 37°C, co-expression of the human α7 nAChR subunit with either hRIC-3 or DmRIC-3 isoforms lacking exon 2 gave similar levels of [125I]αBTX binding (Fig. 2b). As had been found in cells maintained at 37°C, no specific [125I]αBTX binding was detected when the α7 nAChR subunit was expressed in the absence of RIC-3 or with DmRIC-3 isoforms which contain exon 2 (Fig. 2b). For DmRIC-3 isoforms lacking exon 2, the level of [125I]αBTX binding was not significantly different to that observed with hRIC-3.

To examine whether RIC-3 proteins might facilitate nAChR maturation more efficiently in a different host cell type, co-expression studies were performed in Drosophila S2 cells (which are maintained routinely at 25°C). In the absence of co-expressed RIC-3, no specific binding of [125I]αBTX to α7 nAChRs was detected in S2 cells (Fig. 2c). The hRIC-3 cDNA and alternatively spliced DmRIC-3 cDNAs were subcloned into the Drosophila expression vector pRmHa3 (Bunch et al. 1988) and expressed by transient transfection with the nAChR α7 subunit in S2 cells. As was observed in tsA201 cells, no significant binding of [125I]αBTX binding was detected with DmRIC-3 isoforms containing exon 2 (Fig. 2c). High levels of [125I]αBTX binding were, however, detected when the α7 subunit was co-expressed with DmRIC-3 isoforms which lack exon 2 (∼90–150 fmol/106 cells, = 4–9). Furthermore, in contrast to the situation in tsA201 cells, significantly higher levels of cell-surface [125I]αBTX binding were detected with DmRIC-3 isoforms lacking exon 2 than with hRIC-3 (28 ± 6 fmol/106 cells, = 6, < 0.01; Fig. 2a).

The influence of RIC-3 on nAChRs containing Drosophila subunits

Considerable difficulties have been encountered with the expression of insect recombinant nAChRs in heterologous expression systems (Millar 1999, 2003). Indeed, we are unaware of any reports of the successful expression of insect recombinant nAChRs in cultured cell lines. Some success has been achieved by using a Drosophila cell line, rather than mammalian cell line, but only by the co-expression of insect nAChR α subunits with a mammalian non-α subunit (Lansdell et al. 1997; Huang et al. 1999; Lansdell and Millar 2000a). For example, co-expression of the Drosophila nAChR Dα2 subunit with a mammalian β2 subunit in Drosophila S2 cells results in high levels of specific binding of the nicotinic radioligand [3H]epibatidine (Lansdell et al. 1997). In addition, co-expression of Dα2 and mammalian β2 subunits has been shown to generate functional nAChRs (Bertrand et al. 1994; Lansdell et al. 1997). Therefore, to examine the influence of RIC-3 on nAChRs containing insect nAChR subunits, hRIC-3 and DmRIC-3 isoforms were co-expressed with a hybrid nAChR containing the Drosophila Dα2 subunit and the rat β2 subunit (Dα2 + β2) in Drosophila S2 cells. Compared with the level of radioligand binding detected in the absence of RIC-3, significantly higher levels of [3H]epibatidine binding were detected when Dα2 + β2 was co-expressed with DmRIC-3 isoforms which lack exon 2 (∼2.5- to 3.5-fold higher = 3–10; < 0.01; Fig. 3c). No significant increase in levels of [3H]epibatidine binding was detected when Dα2 + β2 was co-expressed with either hRIC-3 or with DmRIC-3 isoforms which contain exon 2 (Fig. 3c).

image

Figure 3.  The influence of hRIC-3 and DmRIC-3 on the expression of Drosophila Dα2-containing nAChRs. Drosophila nAChR Dα2 subunit, together with the rat β2 subunit, was co-expressed with either DmRIC-3 splice variants or hRIC-3 by transient transfection in human tsA201 cells maintained at 37°C (a), tsA201 cells maintained at 25°C (b) or Drosophila S2 cells maintained at 25°C (c). Specific binding of [3H]epibatidine (3 nM) to cell membranes was determined. To normalise for differences in transfection efficiency between experiments and to provide a clearer indication of the effect of co-expression of RIC-3, data are presented as the fold increase in specific radioligand binding compared with Dα2β2 expressed in the absence of RIC-3. The level of [3H]epibatidine binding detected in the absence of RIC-3 for each of the expression systems examined was 3.3 ± 0.4 fmol/mg protein (= 7) in human tsA201 cells at 37°C, 10.0 ± 0.5 fmol/mg protein (= 7) in human tsA201 cells at 25°C and 103 ± 20 fmol/mg protein (= 9) in Drosophila S2 cells. Fold increase data are mean of at least four independent experiments performed in triplicate.

Download figure to PowerPoint

Hybrid nAChR subunit combinations such as Dα2 + β2 generate [3H]epibatidine binding sites very inefficiently in mammalian cells cultured at 37°C (Lansdell et al. 1997). In contrast to the situation in S2 cells (Fig. 3c), none of the DmRIC-3 isoforms significantly enhanced levels of [3H]epibatidine binding to Dα2 + β2 nAChRs when expressed in tsA201 cells at 37°C (Fig. 3a). However, co-expression of hRIC-3 with Dα2 + β2 resulted a substantial enhancement in levels of [3H]epibatidine binding (3.4 ± 0.4 fold higher, = 4; Fig. 3a), significantly higher (< 0.01) than the levels detected with DmRIC-3 or in the absence of RIC-3.

Previous studies have shown that when insect nAChR subunits are expressed in mammalian cells, higher levels of radioligand binding are detected in cells maintained at temperatures lower than 37°C (Lansdell et al. 1997). To examine the influence of lower temperature, transfected tsA201 cells were maintained at 25°C prior to radioligand binding. Whereas none of the DmRIC-3 isoforms caused a significant increase in levels of [3H]epibatidine binding to Dα2 + β2 nAChRs in tsA201 cells maintained at 37°C (Fig. 3a), several DmRIC-3 isoforms caused enhanced levels of binding in tsA201 cells maintained at 25°C (∼2.2- to 4.1-fold higher, = 4; Fig. 3b). However, the facilitatory effect of DmRIC-3 isoforms in tsA201 cells at 25°C was lower than that observed with hRIC-3 in tsA201 cells at 25°C (5.5 ± 1.0 fold increase, = 4; Fig. 3b).

A motivation for cloning DmRIC-3 was the hope that it might facilitate the maturation and heterologous expression of recombinant receptors assembled exclusively from insect nAChR subunits. We have examined an extensive series of RIC-3 isoforms and Drosophila nAChR subunit combinations by heterologous expression in S2 cells. To date, no combination of RIC-3 and insect nAChR subunits has generated significant levels of nicotinic radioligand binding. Clearly there is a huge number of possible combinations of 11 DmRIC-3 isoforms and 10 Drosophila nAChR subunits (Dα1-Dα7 and Dβ1-Dβ3), particularly since the appropriate combinations of nAChR subunits are unknown. Consequently, we have not performed an exhaustive study of all such combinations but, to date, we have examined > 40 combinations of DmRIC-3 isoforms and Drosophila nAChR subunit combinations and have detected no specific binding of radioligands such as [3H]epibatidine and [125I]αBTX.

Co-precipitation of DmRIC-3 with nAChR subunits

A consistent difference between DmRIC-3 isoforms is that those containing exon 2 (DmRIC-32,7, DmRIC-32,7a and DmRIC-32,7a,9) appear to act much less efficiently as a nAChR chaperone than those lacking exon 2 (Figs 2 and 3). Previous studies employing co-immunoprecipitation have demonstrated that hRIC-3 associates with nAChR subunits (Lansdell et al. 2005; Williams et al. 2005). We have therefore performed co-immunoprecipitation studies to examine whether differences in chaperone activity of exon 2-containing and exon 2-lacking DmRIC-3 isoforms could be attributed to their ability to co-assemble with nAChR subunits. Two DmRIC-3 isoforms were selected, one containing exon 2 (DmRIC-32,7a,9) and one lacking exon 2 (DmRIC-37a,9). These two DmRIC-3 isoforms were co-expressed with several epitope (FLAG)-tagged Drosophila nAChR subunits. Using a monoclonal antibody specific for the FLAG epitope (mAbFLAG-M2), clear evidence for co-precipitation of both DmRIC-3 isoforms was obtained. A representative experiment in which DmRIC-32,7a,9 and DmRIC-37a,9 were co-precipitated with Dα2FLAG is illustrated in Fig. 4a. Similar data were obtained with all other Drosophila subunits which were examined (Dα3FLAG, Dα6FLAG and Dβ3FLAG; not shown). A similar experiment was performed in which DmRIC-3 isoforms were co-expressed with the human nAChR α7 subunit. As with the Drosophila subunits, clear evidence of co-precipitation was observed for DmRIC-3 isoforms containing and lacking exon 2. Initially, experiments were performed with the same pair of DmRIC-3 isoforms (DmRIC-32,7a,9 and DmRIC-37a,9) as examined with the Drosophila subunits. Although clear evidence for co-precipitation of the exon 2-containing DmRIC-3 isoform (DmRIC-37a,9) was obtained (Fig. 4), the exon 2-lacking DmRIC-3 isoform (DmRIC-37a,9) appeared to co-migrate with the α7 nAChR subunit, precluding clear identification (not shown). For this reason, the experiment was repeated with another exon 2-lacking DmRIC-3 isoform (DmRIC-37,9) and evidence of co-precipitation of this isoform with the α7 nAChR subunit was detected (Fig. 4b).

image

Figure 4.  Co-immunoprecipitation of DmRIC-3. Two alternatively spliced DmRIC-3 variants, one containing exon 2 and one lacking exon 2, were co-expressed in Drosophila S2 cells together with either a FLAG epitope-tagged Dα2 nAChR subunit (a) or the α7 nAChR subunit (b). Cells were metabolically labelled and the ability of DmRIC-3 to co-assemble with Dα2FLAG or α7 subunits was demonstrated by immunoprecipitation with mAbFLAG-M2 or mAb319 respectively (lanes 5 and 6). The absence of cross-reactivity of DmRIC-3 isoforms with either mAbFLAG-M2 or mAb319 was confirmed (lanes 1 and 2). The experiment was performed on four occasions. On all occasions the Dα2FLAG subunit migrated as a doublet. The more intense labelling of the Dα2FLAG bands in lane 6 (a) was not consistent between experiments and is presumably because of differences in gel loading. The position of molecular weight markers is shown. The apparent molecular weights of the bands are 49 kDa (human α7), 59 kDa and 61 kDa (Dα2FLAG), 48 kDa (DmRIC-37a,9), 51 kDa (DmRIC-32,7a,9) and 55 kDa (DmRIC-37,9).

Download figure to PowerPoint

A predicted coiled-coil domain is not required for DmRIC-3 function

In common with RIC-3 proteins from other species, the DmRIC-3 protein contains a predicted coiled-coil domain. Interestingly, this coiled-coil domain is located entirely within exon 7 (Fig. 1). As DmRIC-3 isoforms containing exon 7 or the alternative exon 7A do not differ significantly in their chaperone activity, it appears that such activity does not require the coiled-coil domain. In order to examine the role of the C-terminal domain further, a truncated DmRIC-3 construct (DmRIC-3Δ) was generated by PCR amplification with specific oligonucleotide primers. DmRIC-3Δ contains only exons 1, 3, 4 and 5 (Fig. 5). Specific cell-surface binding of [125I]αBTX was detected when DmRIC-3Δ was co-expressed with the α7 nAChR subunit in either Drosophila S2 cells (31.9 ± 5.8 fmol/106 cells) or human tsA201 cells at 25°C (39.0 ± 4.4 fmol/106 cells). As we have demonstrated previously that full-length RIC-3 facilitates the functional expression of α7 nAChRs (Lansdell et al. 2005), we examined the ability of DmRIC-3Δ to do so. Rapidly desensitising agonist-induced responses, characteristic of α7 nAChRs, were detected when the α7 subunit was co-expressed with DmRIC-3Δ which were blocked by the α7 nAChR antagonist methyllycaconitine (Fig. 5). As reported previously (Lansdell et al. 2005), no functional expression of α7 nAChRs was observed in the absence of RIC-3.

image

Figure 5.  Functional co-expression of α7 nAChRs with a truncated DmRIC-3. A truncated DmRIC-3 construct (a) containing only exons 1, 3, 4 and 5 (DmRIC-3Δ) was co-expressed with the nAChR α7 subunit in tsA201 cells cultured at 37°C. Representative whole-cell recordings are shown (b). Rapidly desensitising responses, characteristic of α7 nAChRs, were observed in response to application of 200 μM ACh. Block and recovery of whole-cell responses after bath application of 100 nM MLA is illustrated in the middle and right hand traces respectively.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

RIC-3 is a molecular chaperone which enhances nAChR maturation

RIC-3 is a nAChR-associated transmembrane protein which acts as a molecular chaperone to enhance nAChR maturation (Halevi et al. 2002, 2003). A clear illustration of the influence of RIC-3 upon nAChR maturation is its ability to facilitate the functional expression of vertebrate nAChR subunits such as α7 in non-neuronal mammalian cell lines (Castillo et al. 2005; Lansdell et al. 2005; Williams et al. 2005). In the absence of RIC-3, the α7 subunit fails to generate functional nAChRs and also fails to fold into a conformation which can be detected by nicotinic radioligands (Cooper and Millar 1997; Kassner and Berg 1997; Rangwala et al. 1997). Whereas previous studies of RIC-3 have characterised the chaperone activity of hRIC-3 and CeRIC-3, here we describe a family of alternatively spliced RIC-3 proteins from the model insect species D. melanogaster.

In contrast to many chaperone proteins, which have relatively broad specificity for target proteins, RIC-3 appears to act almost exclusively as a chaperone of nAChRs and of the closely related 5HT3R (Halevi et al. 2002; Castillo et al. 2005; Cheng et al. 2005, 2007). RIC-3 is reported to have no affect on maturation of glutamate-gated ion channels (Halevi et al. 2002, 2003). In addition, whereas RIC-3 has been shown to co-precipitate with nAChR and 5HT3R subunits (Cheng et al. 2005; Lansdell et al. 2005), no co-precipitation has been detected with GABA-gated ion channel subunits (Lansdell et al. 2005).

In both vertebrate and invertebrate species, RIC-3 has been detected in muscle and nerve cells (Halevi et al. 2002, 2003). It is concentrated in cell bodies (Halevi et al. 2002) and appears to be located predominantly within the endoplasmic reticulum (Halevi et al. 2002; Castillo et al. 2005; Cheng et al. 2007), which is the site of nAChR subunit folding and assembly (Green and Millar 1995). Indeed, there is evidence that RIC-3 is able to interact with unassembled nAChR subunits (Lansdell et al. 2005) which are thought to be located exclusively within intracellular compartments such as the endoplasmic reticulum (Green and Millar 1995).

The first reported molecular cloning of RIC-3 was from C. elegans (Halevi et al. 2002). Analysis of the predicted CeRIC-3 amino acid sequence revealed the presence of two hydrophobic putative transmembrane domains. Consequently, the CeRIC-3 protein has been predicted to be a transmembrane protein containing two transmembrane domains with intracellular N- and C-termini (Halevi et al. 2002). Human RIC-3 also contains two hydrophobic domains (Halevi et al. 2003), but analysis of its primary amino acid sequence suggests that its N-terminal hydrophobic domain is likely to be a cleavable signal sequence (Castillo et al. 2005; Cheng et al. 2005), a conclusion which is supported by experimental evidence (Cheng et al. 2007). Thus, the hRIC-3 protein would be expected to have a single transmembrane-spanning domain (Castillo et al. 2005; Cheng et al. 2005). Analysis of the DmRIC-3 amino acid sequence reveals two strongly hydrophobic domains, located within exons 1 and 4 (Fig. 1) but, like CeRIC-3, is not predicted to contain a cleavable signal sequence. It appears, therefore, the DmRIC-3 is a transmembrane protein with a similar membrane topology to that which has been predicted for CeRIC-3 (Halevi et al. 2002).

The influence of DmRIC-3 alternative splicing

A clear difference between the 11 alternatively spliced isoforms of DmRIC-3 examined in the present study is the finding that those which contain exon 2 display little, if any, ability to enhance maturation of either α7 or Dα2β2 nAChRs. In contrast DmRIC-3 isoforms lacking exon 2 enhance nAChR maturation to a dramatic extent (Figs 2 and 3). Exon 2 encodes 40 amino acids and lies between the two predicted transmembrane domains of DmRIC-3 (Fig. 1). In DmRIC-3 isoforms lacking exon 2, the region between the two predicted transmembrane domains is highly proline rich (containing about 20% proline residues), a feature which is common to RIC-3 proteins from other species (Halevi et al. 2002, 2003). Although the role of this proline-rich domain is unclear, it may be significant that the 40 amino acids encoded by exon 2 contains only 2 proline residues (equivalent to just 5%). The presence of exon 2 may, therefore, be disrupting the correct functioning of this protein domain. Interestingly, amino acid sequence alignments indicate that hRIC-3 and CeRIC-3 proteins do not contain a region homologous to exon 2 of DmRIC-3. We have demonstrated co-precipitation with nAChRs subunits of DmRIC-3 isoforms lacking exon 2, as well as those containing exon 2 (Fig. 4). This indicates that the influence of exon 2 upon the chaperone activity of DmRIC-3 is not because of exon 2 disrupting the interaction of RIC-3 with nAChR subunits.

In contrast to the influence of exon 2, the presence or absence of exons 6, 7/7A or 9 does not appear to exert a significant effect on the ability of DmRIC-3 to act as a nAChR molecular chaperone (Figs 2 and 3). Amino acid sequence analysis of DmRIC-3 has identified the presence of a predicted coiled-coil domain (Lupas et al. 1991), a motif which has been implicated in protein–protein interactions (Burkhard et al. 2001) and which is a feature of previously characterised RIC-3 proteins from other species (Halevi et al. 2002, 2003). Interestingly, however, the coiled-coil domain of DmRIC-3 is located exclusively within alternative exon 7 and is lacking from exon 7A (Fig. 1). As there is no significant difference in the ability of DmRIC-3 isoforms containing either of these two alternative exons, we can conclude that the predicted coiled-coil domain is not required for DmRIC-3 to act as a nAChR chaperone. This conclusion is supported by evidence that a truncated DmRIC-3 construct retains chaperone activity (Fig. 5) and is consistent with studies conducted with truncated versions of CeRIC-3 which lack both of its coiled-coil domains but retain nAChR chaperone activity (Ben-Ami et al. 2005).

DmRIC-3 as a chaperone of insect nAChRs

We have examined whether the co-expression of DmRIC-3 can circumvent the considerable problems which have been encountered in the heterologous expression of insect nAChRs (Millar 1999, 2003). Although we have obtained evidence that DmRIC-3 can enhance maturation of hybrid nAChRs containing insect and mammalian nAChR subunits, we have been unable, as yet, to successfully express recombinant receptors assembled exclusively from insect nAChR subunits. It appears, therefore, that the successful heterologous expression of insect nAChRs may require the identification of additional chaperone or accessory proteins.

RIC-3 function is influenced by the host cell

Differences have been observed in the chaperone activity of DmRIC-3 and hRIC-3 when expressed in either a Drosophila or a human cell line. In part, this may be attributable to differences in the temperature at which these cells are maintained (37°C for the human cell line and 25°C for the Drosophila cell line). To an extent, this possibility is supported by experiments in which the human cell line was maintained at a lower temperature (25°C). As has been suggested previously (Lansdell et al. 1997), this may be a consequence of more efficient folding of insect proteins at lower temperatures. It seems likely, however, that the differences observed in RIC-3 activity in the two cell line may also be influenced by other host-cell specific factors.

In addition to evidence that RIC-3 can enhance maturation of nAChRs such as α7, there is evidence that co-expression of RIC-3 in cultured cell lines can enhance maturation levels of heteromeric nAChRs such as α4β2 and α3β4 (Lansdell et al. 2005). Surprisingly, it has also been reported that co-expression of RIC-3 can cause a reduction in the expression levels of nAChRs such as α4β2 in Xenopus oocytes (Halevi et al. 2003). These apparently conflicting findings have been difficult to explain. An interesting aspect of the present study is the finding that hRIC-3 acts more efficiently than DmRIC-3 as a nAChR chaperone in a human cell line, whereas DmRIC-3 acts more efficiently as a nAChR chaperone than hRIC-3 in a Drosophila cell line (Figs 2 and 3). This finding applies to maturation of both α7 and Dα2β2 nAChRs and suggests that the ability of RIC-3 to act as a nAChR chaperone is influenced by the host cell environment. This may provide an explanation for the apparently contradictory data which has been reported for the influence of RIC-3 on nAChR subtypes such as α4β2 (Halevi et al. 2003; Lansdell et al. 2005). The differences observed when RIC-3 is expressed in different cell types, together with the continuing difficulty in functional expression of some nAChRs, suggests that additional host-cell specific proteins may modulate the chaperone activity of RIC-3.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Financial support for this study was provided by Syngenta. TC is supported by a BBSRC doctoral training Grant PhD studentship. AY was supported by a Wellcome Trust Vacation Scholarship. VJG was supported by a Wellcome Trust PhD studentship.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Ben-Ami H. C., Yassin L., Farah H., Michaeli A., Eshel M. and Treinin M. (2005) RIC-3 affects properties and quantity of nicotinic acetylcholine receptors via a mechanism that does not require the coiled-coil domain. J. Biol. Chem. 280, 2805328060.
  • Bertrand D., Ballivet M., Gomez M., Bertrand S., Phannavong B. and Gundelfinger E. D. (1994) Physiological properties of neuronal nicotinic receptors reconstituted from the vertebrate β2 subunit and Drosophilaα subunits. Eur. J. Neurosci. 6, 869875.
  • Bunch T. A., Grinblat Y. and Goldstein L. S. B. (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucl. Acids Res. 16, 10431061.
  • Burkhard P., Stetefeld J. and Strelkov S. V. (2001) Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 11, 8288.
  • Castillo M., Mulet J., Gutiérrez L. M., Ortiz J. A., Castelán F., Gerber S., Sala S., Sala F. and Criado M. (2005) Dual role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors. J. Biol. Chem. 280, 2706227068.
  • Cheng A., McDonald N. A. and Connolly C. N. (2005) Cell surface expression of 5-hydroxytryptamine type 3 receptors is promoted by RIC-3. J. Biol. Chem. 280, 2250222507.
  • Cheng A., Bollan K. A., Greenwood S. M., Irving A. J. and Connolly C. N. (2007) Differential subcellular localization of RIC-3 isoforms and their role in determining 5-HT3 receptor composition. J. Biol. Chem. 282, 2515826166.
  • Cooper S. T. and Millar N. S. (1997) Host cell-specific folding and assembly of the neuronal nicotinic acetylcholine receptor α7 subunit. J. Neurochem. 68, 21402151.
  • Couturier S., Bertrand D., Matter J. M., Hernandez M. C., Bertrand S., Millar N., Valera S., Barkas T. and Ballivet M. (1990) A neuronal nicotinic acetylcholine receptor subunit (α7) is developmentally regulated and forms a homo-oligomeric channel blocked by α-BTX. Neuron 5, 847856.
  • Gotti C., Zoli M. and Clementi F. (2006) Brain nicotinic acetylcholine receptors: native subtypes and their relevance. Trends Pharmacol. Sci. 27, 482491.
  • Green W. N. and Millar N. S. (1995) Ion-channel assembly. Trends Neurosci. 18, 280287.
  • Halevi S., McKay J., Palfreyman M., Yassin L., Eshel M., Jorgensen E. M. and Treinin M. (2002) The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors. EMBO J. 21, 10121020.
  • Halevi S., Yassin L., Eshel M., Sala F., Sala S., Criado M. and Treinin M. (2003) Conservation within the RIC-3 gene family: effectors of mammalian nicotinic acetylcholine receptor expression. J. Biol. Chem. 278, 3441134417.
  • Huang Y., Williamson M. S., Devonshire A. L., Windass J. D., Lansdell S. J. and Millar N. S. (1999) Molecular characterization and imidacloprid selectivity of nicotinic acetylcholine receptor subunits from the peach-potato aphid Myzus  persicae. J. Neurochem. 73, 380389.
  • Jones A. K., Brown A. M. and Sattelle D. B. (2007) Insect nicotinic acetylcholine receptor gene familes: from genetic model organisms to vector, pest and beneficial species. Invert. Neurosci. 7, 6773.
  • Kassner P. D. and Berg D. K. (1997) Differences in the fate of neuronal acetylcholine receptor protein expressed in neurons and stably transfected cells. J. Neurobiol. 33, 968982.
  • Lansdell S. J. and Millar N. S. (2000a) The influence of nicotinic receptor subunit composition upon agonist, α-bungarotoxin and insecticide (imidacloprid) binding affinity. Neuropharmacol. 39, 671679.
  • Lansdell S. J. and Millar N. S. (2000b) Cloning and heterologous expression of Dα4, a Drosophila neuronal nicotinic acetylcholine receptor subunit: identification of an alternative exon influencing the efficiency of subunit assembly. Neuropharmacol. 39, 26042614.
  • Lansdell S. J. and Millar N. S. (2002) Dβ3, an atypical nicotinic acetylcholine receptor subunit from Drosophila: molecular cloning, heterologous expression and coassembly. J. Neurochem. 80, 10091018.
  • Lansdell S. J. and Millar N. S. (2004) Molecular characterisation of Dα6 and Dα7 nicotinic acetylcholine receptor subunits from Drosophila: formation of a high-affinity α-bungarotoxin binding site revealed by expression of subunit chimeras. J. Neurochem. 90, 479489.
  • Lansdell S. J., Schmitt B., Betz H., Sattelle D. B. and Millar N. S. (1997) Temperature-sensitive expression of Drosophila neuronal nicotinic acetylcholine receptors. J. Neurochem. 68, 18121819.
  • Lansdell S. J., Gee V. J., Harkness P. C., Doward A. I., Baker E. R., Gibb A. J. and Millar N. S. (2005) RIC-3 enhances functional expression of multiple nicotinic acetylcholine receptor subtypes in mammalian cells. Mol. Pharmacol. 68, 14311438.
  • Le Novère N., Corringer P.-J. and Changeux J.-P. (2002) The diversity of subunit composition in nAChRs: evolutionary origins, physiologic and pharmacologic consequences. J. Neurobiol. 53, 447456.
  • Lupas A., Van Dyke M. and Stock J. (1991) Predicting coiled coils from protein sequences. Science 252, 11621164.
  • Millar N. S. (1999) Heterologous expression of mammalian and insect neuronal nicotinic acetylcholine receptors in cultured cell lines. Biochem. Soc. Trans. 27, 944950.
  • Millar N. S. (2003) Assembly and subunit diversity of nicotinic acetylcholine receptors. Biochem. Soc. Trans. 31, 869874.
  • Millar N. S. and Denholm I. (2007) Nicotinic acetylcholine receptors: targets for commercially important insecticides. Invert. Neurosci. 7, 5366.
  • Miller K. G., Alfonso A., Nguyen M., Crowell J. A., Johnson C. D. and Rand J. B. (1996) A genetic selection for Caenorhabditis elegans synaptic transmission mutants. Proc. Natl Acad. Sci. USA 93, 1259312598.
  • Nguyen M., Alfonso A., Johnson C. D. and Rand J. B. (1995) Caenorhabditis elegans mutants resistant to inhibitors of aetylcholineasterase. Genetics 140, 527535.
  • Rangwala F., Drisdel R. C., Rakhilin S., Ko E., Atluri P., Harkins A. B., Fox A. P., Salman S. B. and Green W. N. (1997) Neuronal α-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors. J. Neurosci. 17, 82018212.
  • Raymond Delpech V., Matsuda K., Sattelle B. M., Rauh J. J. and Sattelle D. B. (2005) Ion channels: molecular targets of neuroactive insecticides. Invert. Neurosci. 5, 119133.
  • Schneider I. (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morph. 27, 353365.
  • Williams M. E., Burton B., Urrutia A., Shcherbatko A., Chavez-Noriega L. E., Cohen C. J. and Aiyar J. (2005) Ric-3 promotes functional expression of the nicotinic acetylcholine receptor α7 subunit in mammalian cells. J. Biol. Chem. 280, 12571263.