The conserved RIC-3 coiled-coil domain mediates receptor-specific interactions with nicotinic acetylcholine receptors

Abstract

RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation.

Figure 1.

L-AChR maturation requires the RIC-3 C-terminal domains. (A) Levamisole-sensitivity assays (1 mM) on transgenics expressing the different RIC-3 deletion mutants. Given is the percent per plate of paralyzed animals. n = 9–14 N = 3–6, 10 animals per plate. Levamisole sensitivity of ric-3(md1181) animals expressing RIC-3 TM or RIC-3 ΔC′ is not significantly different from the sensitivity of ric-3(md1181) animals and is significantly different from wild-type (WT) animals and from ric-3(md1181) animals expressing full-length (wild-type) RIC-3, RIC-3 minimal (min), or RIC-3 ΔCC-II. Significant differences are also seen between ric-3(md1181) animals expressing RIC-3 ΔCC-I and all other strains examined (p < 0.01, one-way Anova). (B) Schematic representation of the domain organization in RIC-3 and RIC-3 deletion mutants. Arrowheads point to the position of the GFP tag; two arrowheads are seen for RIC-3 constructs having two different tagged versions.

Figure 2.

Effects of RIC-3 TM on synaptic UNC-29 expression. Immunohistochemical analysis of synaptic UNC-29 (a L-AChR subunit) expression levels in wild-type, ric-3(md1181), ric-3(md1181) expressing RIC-3 TM from an integrated transgene, and unc-29(x29); acr-16(ok789) double mutants. Top, representative images of UNC-29 staining in the respective strains. Arrowheads indicate the nerve ring and arrows the dorsal nerve cord, where L-AChR-containing synapses are found. Bottom, quantification of images (number of animals examined is indicated in each bar). Intensity of the staining is normalized to staining of UNC-17, a presynaptic marker. Significant differences are indicated (***p < 0.001, **p < 0.01, *p < 0.05; t test). Scale bar, 10 μm.

Figure 3.

Distribution and quantity of RIC-3 deletion mutants. (A) Representative pictures showing distribution of RIC-3 deletion mutants in the body-wall muscles. Arrows point to representative puncta. (B) Expression level of each deletion mutant was quantified based on fluorescence intensity of the GFP tag; 10–15 animals for each transgenic strain. Scale bar, 5 μm.

Figure 4.

Effects and distribution of the CC-I. (A) Levamisole dose response for wild-type (WT, ▴) and wild-type overexpressing CC-I fused to GFP (▩). Given is the fraction per plate of animals moving in the presence of different concentrations of levamisole. n = 4–14, 10 animals per plate for each point. Differences are significant (p < 0.01) for levamisole concentrations 0.2 mM and 0.4 using the Student's t test for two samples. (B) Representative image showing distribution of CC-I when expressed under the RIC-3 promoter in wild type. Arrows indicate ventral cord neurons.

Figure 5.

Rescue of DEG-3(u662) and EAT-2 nAChR maturation by RIC-3 TM. Different RIC-3 derivates were examined for their ability to rescue function of a ric-3(md1181) mutant. (A) Number of DEG-3(u662)-dependent degenerations in deg-3(u662) mutants, ric-3(md1181)deg-3(u662) mutants expressing RIC-3 TM, and ric-3(md1181)deg-3(u662) mutants. Numbers are given for tail (black), head (gray), and total (light gray), in early L1 larva. N = 2–3 and n = 20–30 animals each. Rescue by RIC-3 TM is significant relative to deg-3(u662)ric-3(md1181) for total, head, and tail numbers of degenerating cells, p < 0.01. Rescue by RIC-3 TM is not significantly different from control [deg-3(u662) having wild-type copies of ric-3] for total and tail degenerations; however, for head degenerations a significant difference is seen, suggesting incomplete rescue (p < 0.01). One-way Anova was done on each group (head, tail, and total) separately. (B) Pharyngeal pumping rate in ric-3(md1181), ric-3(md1181) expressing: RIC-3 TM, RIC-3 ΔCC-I, RIC-3 minimal, or wild-type RIC-3, and wild-type animals (WT). Animals pumping at a rate less than one pump per second are considered pumping-defective. N = 3–4 and n = 12–15, 10 animals per plate. Rescue of pumping by expression of all RIC-3 derivatives is significant at p < 0.01. No significant differences were found between wild-type animals or any of the RIC-3–expressing strains; one-way Anova.

Figure 6.

RIC-3 TM does not rescue functional expression of body-wall muscle receptors. Inward currents were recorded in patch-clamped body-wall muscle cells in the whole-cell voltage-clamp mode. Agonists levamisole (A), and nicotine (B) were applied as indicated. Above-average peak currents, normalized to the currents in wild type from the indicated number of individual animals (levamisole: wild-type (WT) n = 11, ric-3(md1181) n = 7, RIC-3 TM n = 6, and RIC-3 minimal n = 7; nicotine: wild-type (WT) n = 11, ric-3(md1181) n = 7, RIC-3 TM n = 7, and RIC-3 minimal n = 7). Currents in RIC-3 minimal are not significantly different then currents in wild type but are significantly different from currents in RIC-3 TM transgenics (p < 0.01, t test). A significant difference is also seen between RIC-3 TM and ric-3(md1181); p < 0.001, t test; for levamisole-dependent responses but not for nicotine-dependent responses. Below representative original current traces for mutants, transgenics, and wild type.

Figure 7.

Effects of wild-type RIC-3 and RIC-3 deletion mutants on ACR-16–dependent currents in X. laevis oocytes. (A) Average normalized current amplitudes elicited by 1 mM acetylcholine from oocytes expressing ACR-16, ACR-16 + RIC-3 TM, ACR-16 + RIC-3 minimal, ACR-16 + RIC-3 ΔCC-I, or ACR-16 + wild-type RIC-3 (0.1 μg/μl each). Peak current amplitude for each oocyte was normalized to the average current amplitude for ACR-16-expressing oocytes from the same experiment. N = 2–4 and n = 20–33 each. Effects of wild-type RIC-3 and RIC-3 TM on ACR-16 current amplitudes and the difference between them are significant; p < 0.01. No significant difference is seen between effects of wild-type RIC-3, RIC-3 minimal, and RIC-3 ΔCC-I; one-way Anova. (B) Representative Western analysis showing the relative expression of RIC-3 and RIC-3 deletion mutants. Anti-GFP antibodies were used to detect RIC-3, RIC-3 TM, and RIC-3 minimal, or anti-RIC-3 antibodies (directed to CC-II) were used to detect RIC-3 and RIC-3 ΔCC-I. On average expression of RIC-3 deletion mutants is higher than expression of wild-type RIC-3 2.74 ± 0.63- (N = 3), 6.07 ± 1.95- (N = 2). and 2.79 ± 0.11-fold (N = 2) difference for the TM, minimal, or ΔCC-I proteins, respectively. (C) Representative current traces from oocytes expressing the indicated proteins.

Figure 8.

Interactions of RIC-3 and RIC-3 TM with ACR-16. Coimmunoprecipitation from control oocytes and oocytes expressing ACR-16; RIC-3; RIC-3 TM; ACR-16 and RIC-3, ACR-16; and RIC-3 TM (10 oocytes each). Precipitation was done using anti-GFP antibodies directed at RIC-3:GFP and RIC-3 TM:GFP, and precipitates were analyzed using antibodies against myc to detect myc tagged ACR-16 or using GFP to quantify levels of RIC-3 and RIC-3 TM in the precipitate. Also shown is the level of ACR-16, RIC-3, and RIC-3 TM expression in the same oocytes (total). Numbers on right indicate protein size in kDa.