Yeast two-hybrid screening identifies MPZ-1 and PTP-1 as candidate scaffolding proteins of metabotropic glutamate receptors in Caenorhabditis elegans
The metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptor that undergo extensive interactions with scaffolding proteins, and this is intrinsic to their function as an important group of neuromodulators at glutamatergic synapses. The Caenorhabditis elegans nervous system expresses three metabotropic glutamate receptors, MGL-1, MGL-2 and MGL-3. Relatively little is known about how the function and signalling of these receptors is organised in C. elegans. To identify proteins that scaffold the MGL-1 receptor, we have conducted a yeast two-hybrid screen. Three of the interacting proteins, MPZ-1, NRFL-1 and PTP-1, displayed motifs characteristic of mammalian mGluR scaffolding proteins. Using cellular co-expression criterion, we show mpz-1 and ptp-1 exhibited overlapping expression patterns with subsets of mgl-1 neurons. This included neurones in the pharyngeal nervous system that control the feeding organ of the worm. The mGluR agonist L-CCG-I inhibits the activity of this network in wild-type worms, in an MGL-1 and dose-dependent manner. We utilised L-CCG-I to identify if MGL-1 function was disrupted in mutants with deletions in the mpz-1 gene. The mpz-1 mutants displayed a largely wild-type response to L-CCG-I, suggesting MGL-1 signalling is not overtly disrupted consistent with a non-obligatory modulatory function in receptor scaffolding. The selectivity of the protein interactions and overlapping expression identified here warrant further investigation of the functional significance of scaffolding of metabotropic glutamate receptor function.
KeywordsCaenorhabditis elegans Electropharyngeogram Scaffolding mGluR Glutamate
Glutamate is the major excitatory neurotransmitter of the mammalian central nervous system, and it signals through two classes of receptor, ionotropic receptors (ligand-gated ion channels) and metabotropic glutamate receptors (G-protein-coupled receptors). There are eight mammalian metabotropic glutamate receptors that are divided into three subgroups (Group I-III) based upon sequence homology, pharmacology and signalling (Conn and Pin 1997). The mGluRs are non-essential, but perform pivotal modulatory roles in health and disease (Riedel et al. 1996; Riedel and Reymann 1996). The mGluR subtypes are specifically targeted either pre- or post-synaptically, and they are localised to discrete subcellular domains. Group I receptors (mGluR1/5) tend to be post-synaptic and localised to the peri-synaptic compartment (Luján et al. 1997; Nusser et al. 1994), Group II receptors (mGluR2/3) can be localised pre-and post-synaptically and are localised in pre-terminal membranes (Lujan et al. 1996; Azkue et al. 2000) and Group III receptors (for example mGluR7) tend to be localised pre-synaptically, close to the sight of neurotransmitter release (Shigemoto et al. 1996; Blümcke et al. 1996). In addition, it has been shown that mGluRs can engage in signalling crosstalk with plasma membrane ion channels, in a manner that is independent of G-protein signalling (Perroy et al. 2001).
The mGluRs have distinct domain architecture, consisting of a large extracellular N-terminal domain containing the agonist binding site and a cysteine-rich region, intracellular loops that direct G-protein interactions and an intracellular C-terminal domain (Conn and Pin 1997). The targeting, discrete sublocalisation and the signalling cascades of mGluRs can be specified by protein–protein interactions between intracellular proteins and the intracellular C-terminal domain of the receptor. A number of these intracellular proteins have been identified as interacting with specific motifs of the receptor C-terminal and are described as scaffolding proteins. Perhaps, the most conserved motif across the mGluR family is the PDZ motif and a number of proteins that perform roles in the scaffolding of receptor function interact via a PDZ-binding domain (Perroy et al. 2001). Alternative splicing of the C-terminal can create isoforms containing different combinations of scaffolding motifs, generating further functional diversity amongst the mGluR family (Perroy et al. 2001). A number of proteins that interact with the intracellular C-terminal of the mammalian receptors were identified using biochemical protein-interaction approaches, which have included the yeast two-hybrid system (Perroy et al. 2001).
The Caenorhabditis elegans genome encodes three metabotropic glutamate receptors, named MGL-1, MGL-2 and MGL-3. Sequence analysis suggests the three receptors each representing orthologues of one of the mammalian mGluR subfamilies, and the functional diversity within the mammalian family is conserved in C. elegans (Dillon et al. 2015). There is currently a limited understanding of this class of receptor, compared to the ionotropic families of glutamate receptors (viz NMDA, AMPA and Ivermectin channels) in C. elegans. We have previously shown that the C. elegans mGluR orthologue, MGL-1, is expressed in the pharyngeal nervous system. We have probed its function using the mGluR agonist L-CCG-I to show that it is a negative regulator of pharyngeal network activity. MGL-1 is required for coordinating plasticity in feeding behaviour and reproduction upon the removal of food (Dillon et al. 2015; Jeong and Paik 2017). To build upon this, we have identified candidate MGL scaffolding proteins and screened for their potential to modulate the activity of the pharyngeal network. We performed a yeast two-hybrid screen using the C-terminal of the MGL-1 receptor and used it to define potential selectivity of three candidate scaffolding partners. The molecules PTP-1, NRFL-1 and MPZ-1 contain PDZ-binding domains that robustly bound to the C-terminal domain of the receptor in the yeast two-hybrid system. We characterised these candidates and showed that PTP-1 did not selectively bind to MGL-1. NRFL-1 and MPZ-1 did bind selectively to MGL-1, and other data have highlighted that MPZ-1 also binds to targets outside of the panel of targets used in this study (Xiao et al. 2006). Our subsequent expression studies show that ptp-1 and mpz-1 but not nrfl-1 are co-expressed with mgl-1 in discrete subsets of neurones. Overall, our data identify the potential for the scaffolding of MGL-1 receptor function, which will provide additional routes to characterise the function of MGL-1 in C. elegans and provide a model to investigate dysfunction and function associated with this class of receptor.
All C. elegans strains were cultured at 20 °C under conditions previously described (Brenner 1974). Bristol N2 worms were used as the wild-type strain. Mutant alleles used in this study were as follows: mgl-1(tm1181), mpz-1(tm1136) and the strain VC559 carrying the allele mpz-1(gk273)/mIn1[dpy-10(e128) mIs14(myo-2::GFP)]. The strain carrying allele mgl-1(tm1811) and the strain VC559 were both outcrossed three and one times, respectively. The strain mpz-1(tm1136/gk273) was generated by mating mpz-1(tm1136) males with mpz-1(gk273)/mIn1[dpy-10(e128) mIs14(myo-2::GFP)] hermaphrodites. mpz-1(tm1136/gk273) hermaphrodites were selected in the F1 generation by the absence of pharyngeal GFP (marking the mIn1 inversion). The integrated strain utIs35(mgl-1::GFP) was kindly provided by Professor Katsura [previously described (Dillon et al. 2015)]. The strain pha-1(e2123) was kindly provided by R Schnabbel and used to generate the mgl-1::GFP;pha-1(e2123) selectable marker strain for the co-expression analysis of yeast two-hybrid candidates.
Yeast two-hybrid screen
The intracellular C-terminal domain of MGL-1, NMR-1, GLR-1 and LET-23 was amplified by PCR from a C. elegans cDNA (Origene Technologies Inc.). PCR products were subcloned EcoRI-SalI into pGilda containing the LexA DNA-binding domain. To test for nuclear targeting and expression, the bait plasmid was transformed into EGY48 yeast cells with the plasmid pJK101, which contains 2xOlexA-lacZ downstream of the inducible GAL-1 promoter. Transformants were plated onto GAL-H-U plates containing X-GAL (80 mg/L), and white colonies after 3 days at 30 °C indicated BD-MGL-1 was being nuclear targeted and expressed due to the suppression of lacZ expression. To test for the auto-activation of the 8xOlexA-lacZ reporter gene, each of the bait plasmids was co-transformed into EGY48 cells with the plasmid pSH18-34 (8xOlexA-lacZ reporter), plated onto GAL-His-Ura + X-GAL media and placed at 30 °C. Blue colour after 20 days indicated auto-activation. To test for auto-activation of 6xOlexA-LEU2 reporter genes, each of the bait plasmids was co-transformed into EGY48 cells with pSH18-34 and pJG4-5 (the empty prey vector), plated onto GAL–His–Ura–Trp–Leu media. Growth after 20 days at 30 °C indicated auto-activation of the LEU2 reporter gene by the bait in the absence of a prey clone.
After confirming the MGL-1 bait construct was nuclear targeted, expressed and did not auto-activate the reporter genes, we proceeded to perform a yeast two-hybrid screen to identify MGL-1 interacting proteins. The screen was performed with the DupLex-A™ yeast two-hybrid system (Origene). The cDNA library was constructed using an oligo d(T) primer and cloned into the EcoRI-XhoI sites of the prey vector pJG4-5. EGY48 cells were transformed with the plasmids pSH18-34 and selected for on GLU–Ura media; these cells were then transformed with the MGL-1 C-terminal bait (BD-MGL-1) and selected for on GLU–His–Ura media. These cells were then transformed with the C. elegans cDNA library pJG4-5 and selected on GLU–Trp–His–Ura. The number of transformants screened was titred to 400 × 106 corresponding to approximately three times the titre of viable transformants. The transformants were plated onto media containing galactose (GAL) and –Trp–His–Ura–Leu at a density of 2 × 106/plate and incubated at 30 °C. On days 4, 5 and 7, post-plating colonies were transferred to GAL–Trp–His–Ura–Leu + X-Gal. On day 10, β-galactosidase filter lift assays were performed (as described in (Breeden and Nasmyth 1985)) on the screening plates, and colonies positive for blue colour were identified and transferred onto media GAL–Trp–His–Ura–Leu + X-Gal. Yeast colonies that grew on GAL–Trp–His–Ura–Leu + X-Gal and were blue in colour after 5 days at 30 °C were re-streaked onto GAL–Trp–His–Ura–Leu + X-Gal. Those colonies that grew on GAL–Trp–His–Ura–Leu + X-Gal and were blue in colour after 4 rounds of selection were further characterised.
Prey clones prioritised for further characterisation
Number of clones identified
Number of independent clones
NRFL-1 (C01F6.6a) (2 PDZ domain protein)
MPZ-1 (10 PDZ domain protein)
PTP-1a (C48D5.2a) (FERM, PDZ, tyrosine phosphatase domain)
The 5′RACE products were obtained from two rounds of amplification using a 5′RACE primer in the first reaction and a nested 5′RACE reaction in the second reaction, which was performed using a nested 5′RACE primer and either an SL-1 primer or RACE adaptor primer. cDNA for RACE analysis was synthesised using the SMART RACE cDNA amplification kit from Clontech™. 1 µg of total RNA extracted from a mixed stage population of worms was used, and the RACE adaptor fused first-strand cDNA served as the template for the RACE PCR. The RACE primers were designed with the available sequence information from Wormbase (WS253). The RACE products were gel-purified and TOPO-cloned (Invitrogen) and then sequenced. Isoforms are named according to the annotation in Wormbase WS253.
Promoter fusion constructs were generated from genomic DNA fragments cloned into the vector backbone pHAB (provided by Dr Howard Baylis) and GFP replaced by either dsRED2 or mRFP-1. The mpz-1::mrfp-1 construct included 2315 bp of 5′UTR, the nrfl-1::dsRed2 construct included 3258 bp of 5′UTR and the ptp-1::mRFP-1 construct contained 2652 bp of 5′UTR. mRFP-1 was obtained from Dr. M. Hannah. The promoter constructs were microinjected into the selectable marker strain mgl-1::GFP;pha-1(e2123) at 30 ng/μl with the transformation plasmid marker pha-1 at 50 ng/μl. The transformants were selected by their growth at 25 °C, and three transgenic lines were analysed for co-expression.
For the mpz-1 transcript analysis, total RNA was extracted from a mixed stage population of worms and 5 μg was used to perform the first-strand cDNA synthesis (Superscript II, Invitrogen). The first-strand cDNA synthesis was performed using an mpz-1 gene-specific primer, and 2 μl of the first-strand cDNA synthesis mix was used in the PCR reaction.
Worms were mounted on 2% agarose pads and immobilised for imaging using 20 mM Na-Azide. mpz-1::mRFP-1;mgl-1::GFP confocal images were recorded using Zeiss LSM510 laser scanning confocal microscope. The settings were Objective 63 × Oil DIC, GFP excitation 488 nm, band pass filter 505–530 nm, mRFP-1 excitation 543 nm, band pass filter 560–615 nm. Optical sections were collected with a step size of 0.98 μM. ptp-1::mRFP-1;mgl-1::GFP, and nrfl-1::mRFP-1 images were captured using a Hamamatsu ORCA camera attached to a Nikon Eclipse E800 microscope, Objective × 60 Oil DIC and × 20 DIC, respectively. Images were analysed using ImageJ (NIH).
Electropharyngeograms were performed on L4 plus two- or three-day-old adult worms. The recordings were made from a semi-intact pharyngeal preparation as previously described (Dillon et al. 2015) in a modified Dent’s saline solution (144 mM NaCl, 10 mM MgCl2, 1 mM CaCl2, 6 mM KCl, 10 mM Glucose and 5 mM HEPES; pH 7.4). L-CCG-I dose response curves were generated by adding increasing concentrations to individual worms, with a 5 min wash period between each application of the drug. The wash period immediately prior to drug application was used to calculate the percentage change in the rate of pumping. L-CCG-I was supplied by TOCRIS Biosciences and diluted to the required concentration in Dent’s saline. During pharmacological experiments, solutions were added to and removed from the recording chamber using a 1 ml pipette. Dose response curves were plotted using GraphPad Prism 7, and the EC50 for each strain was calculated. The variation in the dose response curves was investigated using a two-way ANOVA test.
Yeast two-hybrid screen
Probing the specificity of the interaction between the MGL-1 C-terminal and MPZ-1, NRFL-1 and PTP-1
To assess the selectivity of the three prey clones that exhibit the in vitro interaction with the intracellular C-terminal domain of MGL-1, we compared their interaction with NMR-1, GLR-1 and LET-23. Each has a C-terminal PDZ-binding motif, and it has previously been shown that the intracellular C-terminal is important in directing the function of the receptor. The intracellular C-terminal of the ionotropic glutamate receptor subunits NMR-1 and GLR-1 and the tyrosine kinase receptor LET-23 was fused in-frame with the LexA-binding domain to create the bait plasmid constructs BD-NMR-1, BD-GLR-1 and BD-LET-23. We confirmed that each of the bait constructs was being expressed, nuclear targeted and did not auto-activate either of the reporter genes. NRFL-1 and MPZ-1 prey clones did not interact with any of the C-terminal bait constructs, and PTP-1 interacted only with the C-terminal of the GLR-1 receptor (Fig. 2). This in vitro interaction of PTP-1 with MGL-1 and GLR-1 indicates that it may perform a role in scaffolding distinct classes of glutamate receptor.
5′ RACE characterisation of mpz-1, nrfl-1 and ptp-1 and analysis of co-expression with mgl-1
Analysis of the desheathed pharynx identified co-expression of mgl-1::GFP and ptp-1::mRFP-1 in the pharyngeal nervous system (Fig. 4b). mpz-1 was identified as being expressed in the pharyngeal neurones (Fig. 4c) (including M4 and NSM based on their size and relative position), extrapharyngeal neurones, body wall muscle and vulval muscle. Co-expression of mpz-1::mRFP-1 with mgl-1::GFP was identified in neurones of the nerve ring and the pharyngeal nervous system in the desheathed pharynx preparation (Fig. 4c). Hence, in the case of ptp-1 and mpz-1, widespread expression was identified, overlapping expression with mgl-1 was only identified in a subpopulation of these cells and mgl-1 was expressed in cells that did not express mpz-1 nor ptp-1. This suggests that MPZ-1 and PTP-1 are not obligatory for MGL-1 receptor function but well placed to interact with the receptor in some of the circuits in which it might modulate function.
We identified nrfl-1 expression in the intestine and pharyngeal muscle (Fig. 4d). This cellular expression is inconsistent with the specific neuronal expression of mgl-1. Furthermore, despite some expression in neurones in the pharynx and the tail, it was not co-expressed in any of the same neurones as mgl-1::GFP. Such a mismatch in expression would argue against a functional role for NRFL-1 scaffolding of MGL-1.
Electrophysiological analysis of MGL-1 interaction with MPZ-1
Interestingly, the gk273 allele is homozygous lethal, and it is a 1183 bp genomic deletion and 8 bp insertion. The strain VC559 used in this study has the genotype mpz-1(gk273)/mIn1[dpy-10(e128) mIs14(myo-2::GFP)]; hence, it carries the gk273 allele, and it is chromosome balanced by the GFP and dpy-10 marked inversion mIn1. For this reason, it is heterozygous for the gk273 deletion allele. The same transcript analysis described for the tm1136 allele was performed on cDNA generated from mpz-1(gk273)/mIn1[dpy-10(e128) mIs14(myo-2::GFP)] worms to characterise the effect of the gk273. PCR amplification yielded two bands, corresponding to the wild type and the mutant transcript (Fig. 5). Sequencing of the mutant fragment amplified identified a 152 bp deletion at the 5′ end of mpz-1, corresponding to exon 6 of the isoform C52A11.4a. The deletion is predicted to introduce a premature stop codon into only one of the mpz-1 SL-1 trans-spliced variants identified in our study. In the case of the other two SL-1 trans-splice variants, the deletion removes an untranslated exon that resides within the 5′UTR and upstream of the predicted start codon defined by these shorter isoforms. Thus, the more profound consequence of this genomic disruption is unclear, but analysis of the phenotypic segregation of mpz-1(gk273/+) progeny identified gk273 homozygotes has an early developmental phenotype in which embryos arrest 3–6 h after being laid, corresponding to early gastrulation (data not shown).
Scaffolding proteins perform an important role in coordinating GPCR function (Romero et al. 2011). This can be in terms of subcellular targeting, localisation, immobilisation and specifying downstream signalling cascades through multi-meric protein networks. The metabotropic glutamate receptors are a class of GPCR whose function is scaffolded through interactions between the intracellular C-terminal and a variety of proteins (Enz 2012). Our current understanding of these scaffolding interactions suggests that they are important for modulating synaptic signalling and synaptic plasticity within neural networks, and their disruption can contribute to the mechanisms underlying CNS disorders such as Fragile-X (Ronesi and Huber 2008; Ronesi et al. 2012). In C. elegans, comparatively less is understood in terms of the identity and function of proteins that are involved in the scaffolding of GPCR function. This is surprising considering the worm has the largest number of genes (~ 1100) encoding GPCRs compared to humans and Drosophila (Bargmann 1998). Proteins that have emerged as scaffolding receptor function in C. elegans have been identified through either forwards genetic approaches (Byrd et al. 2001) or predictive approaches based upon known function in other organisms to screen candidates with the potential to participate in protein–protein interactions (Emtage et al. 2009; Danielson et al. 2012). In view of this limited understanding and the significant role that such mechanisms play, we used a yeast two-hybrid screen to identify protein–protein interactions that scaffold the C. elegans mGluR orthologue MGL-1.
The screen yielded three candidates that were prioritised for further analysis, MPZ-1, NRFL-1 and PTP-1. Each of the three contains one or more tandem PDZ-binding domains; this was particularly intriguing since the MGL receptor C-terminal contains a PDZ-binding motif. Based on the criteria of shared cellular expression, our observations would suggest that the NRFL-1 interaction is unlikely to have physiological relevance. Our reporters were designed to ape physiological expression, and in the case of NRFL-1, we seem to have a similar pattern to previously described (Hagiwara et al. 2012).
The mammalian orthologues of MPZ-1 and PTP-1 (MUPP-1 and PTPMEG, respectively) have each been identified as scaffolding receptor function. MUPP-1 was first identified as interacting with the 5-HT2C receptor. It has since been shown to interact with a range of different proteins, including ion channels and adhesion molecules. In general, it is considered that MUPP-1 serves to organise diverse signalling complexes (Jang et al. 2014; Fujita et al. 2012; Baumgart et al. 2014; Sindic et al. 2009), and it is has been shown that the disruption of MUPP-1 interactions can lead to the destabilisation of receptor coupling to G-proteins (Guillaume et al. 2008). In terms of the physiological function of MUPP-1, these are diverse, but perhaps the largest single body of work relates to its role in alcohol responses (Kruse et al. 2014; Milner et al. 2015; Metten et al. 2014; Buck et al. 2012; Karpyak et al. 2009) and the regulation of excitability during withdrawal. MPZ-1 has previously been shown to scaffold the function of distinct non-glutamatergic receptor types in C. elegans. MPZ-1 has been described as scaffolding the function of the 5-HT GPCR SER-1 (Xiao et al. 2006), and a molecular interaction has been identified with the insulin DAF-2 receptor (Palmitessa and Benovic 2010).
The other candidate, PTP-1, is particularly interesting because of the functional domains it contains, FERM, PDZ and PTP. PTP-1 has not been previously described as a scaffolding protein, and there is relatively little known about its function in C. elegans other than the FERM domain required for localisation in nerve processes (Uchida et al. 2002). Intriguingly, our study has identified that PTP-1 can interact with the C-terminal of the AMPA like receptor subunit GLR-1, which like MGL-1 contains a Type I PDZ motif. Indeed the mammalian orthologue of PTP-1, PTPMEG has been identified as binding to the intracellular C-terminal of the Gluδ2 ionotropic subunit, and it has been suggested that this interaction underlies mechanisms of synaptic plasticity in the cerebellum (Kohda et al. 2013). This modulation extends to the mammalian family of mGluRs, where protein phosphatase 1 (PP1) has been shown to interact with the intracellular C-termini of mGluR1a, mGluR5a/b and 7a (Croci et al. 2003; Meiselbach et al. 2006).
MPZ-1 and PTP-1 were both co-expressed with MGL-1 in neurones; in particular, we identified a substantial overlap between MPZ-1 and MGL-1 in the pharyngeal nervous system. Amongst other cells, co-expression in pharyngeal neurons is likely to include NSM and M4. NSM is serotonergic/glutamatergic and has been identified as performing a role in modulating dwelling and roaming behaviour, whereas M4 is cholinergic and controls isthmus peristalsis during feeding, respectively. Our analysis of strains carrying mutations in the mpz-1 gene identified no gross changes in pharyngeal responses to L-CCG-I, which provides a pharmacological readout for MGL-1 function. This may be a reflection of the limited contribution of the cells co-expressing MGL-1 and MPZ-1 to the network effect of L-CCG-I. We identified that MGL-1 is expressed in pharyngeal neurones independently of MPZ-1 and the function of MGL-1 in these cells may have a significant contribution to the L-CCG-I effect. Alternatively, a more discrete analysis is required to determine how MPZ-1 scaffolds the function of MGL-1 in the cells identified. Our transcript analysis of the mutants carrying the gk273 or tm1136 allele identified products corresponding to the mutant transcripts. MPZ-1 is a highly complex gene with a number of different 5′ isoforms, and 10 are currently annotated on Wormbase (Version WS253) (C52A11.4a-j). The possibility remains that protein translation could be initiated from the mutant transcripts at an alternative start codon downstream of the regions deleted. This would generate a 5′ truncated MPZ-1 protein with residual activity. Indeed, the isoform C52A11.4i is a 5′ truncated isoform that contains the 3′ region of MPZ-1 defined by the two-hybrid prey clone and could potentially interact with MGL-1.
The data from this study indicate a potent in vitro ability of the MGL-1 receptor to recruit scaffolding proteins. These can be multi-domain proteins that are involved in cross-linking and signal complex assembly, subcellular organisation (FERM domain) and impart discrete signalling (phosphatase activity). These findings hint at the integrative ability of the MGL receptors, evidenced from functional studies in higher organisms. The functional scaffolding of this class of receptor will play out in C. elegans, and the experimental tractability of this model organism will facilitate further investigation of such interactions for the modulation of neural networks and behaviour.
Strains were provided by either the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) or provided by the MITANI Laboratory through the National Bio-Resource Project of the MEXT, Japan.
Compliance with ethical standards
Conflict of interest
The author(s) declare that they have no competing interests.
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