Identification of an Ascaris G protein-coupled acetylcholine receptor with atypical muscarinic pharmacology

Acetylcholine (ACh) is a neurotransmitter/neuromodulator in the nematode nervous system and induces its effects through interaction with both ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs). The structure, pharmacology and physiological importance of LGICs have been appreciably elucidated in model nematodes, including parasitic species where they are targets for anthelmintic drugs. Significantly less, however, is understood about nematode ACh GPCRs, termed GARs (G protein-linked ACh receptors). What is known comes from the free-livingCaenorhabditis elegans as no GARs have been characterized from parasitic species. Here we clone a putative GAR from the pig gastrointestinal nematode Ascaris suum with high structural homology to the C. elegans receptor GAR-1. Our GPCR, dubbed AsGAR-1, is alternatively spliced and expressed in the head and tail of adult worms but not in dorsal or ventral body wall muscle, or the ovijector. ACh activated AsGAR-1 in a concentration-dependent manner but the receptor was not activated by other small neurotransmitters. The classical muscarinic agonists carbachol, arecoline, oxotremorine M and bethanechol were also AsGAR-1 agonists but pilocarpine was ineffective. AsGAR-1 activation by ACh was partially antagonized by the muscarinic blocker atropine but pirenzepine and scopolamine were largely ineffective. Certain biogenic amine GPCR antagonists were also found to block AsGAR-1. Our conclusion is that Ascaris possesses G protein-coupled ACh receptors that are homologous in structure to those present in C. elegans, and that although they have some sequence homology to vertebrate muscarinic receptors, their pharmacology is atypically muscarinic

A Novel G Protein-Coupled Receptor of Schistosoma mansoni (SmGPR-3) Is Activated by Dopamine and Is Widely Expressed in the Nervous System

Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3) that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of “orphan” amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs

Characterization of novel biogenic amine receptors in the human bloodfluke «Schistosoma mansoni»

The genome of the human bloodfluke Schistosoma mansoni encodes 18 putative biogenic amine-like G-protein-coupled receptors (GPCRs). These receptors are potential targets for the development of antischistosomal drugs. One of these sequences, SmGPR-1 (formerly SmGPCR), was previously cloned and was identified as a histamine receptor. In this study, we expanded the functional analysis of SmGPR-1 by studying its expression and tissue distribution both at the RNA and protein levels in different developmental stages of the parasite. In the second part of the study, we cloned and characterized two structurally related receptors, named SmGPR-2 and SmGPR-3. Bioinformatics analyses showed that the three receptors are members of a new clade of biogenic amine GPCRs and are characterized in part by the absence of a highly conserved aspartate (Asp3.32) of the third transmembrane domain. Like SmGPR-1, our first cloned receptor, SmGPR-2, was activated by histamine and its developmental expression at the mRNA level was similar to that of SmGPR-1, both receptors being upregulated in young schistosomula. However, their tissue localization was different. SmGPR-1 was enriched in the tegument, subtegumental musculature and the suckers, whereas SmGPR-2 was associated with neurons of the subtegumental plexuses. The distribution of these receptors correlated with that of histaminergic neurons, which were also detected in the subtegumental neuronal plexuses, the innervation of the suckers, elements of the central nervous system and transverse commissures. These studies suggest that histamine is an important neurotransmitter system in schistosomes. The third receptor investigated in this study, SmGPR-3, was not responsive to histamine but rather was found to have broad specificity for catecholamines, particularly dopamine and related metabolites. In vitro assays of cultured schistosomula revealed that many of the ligands that interact with SmGPR-3 also have strong effects on larval motilitAu génome de Schistosoma mansoni, un parasite sanguin de l'homme, on retrouve 18 récepteurs putatifs à amine biogène couplés aux protéines G (RCPG). Ces récepteurs ont un potentiel thérapeutique contre les infections aux schistosomes. La séquence SmGPR-1 (anciennement SmGPCR) a déjà été clonée et identifiée comme un récepteur à l'histamine. Une analyse fonctionnelle plus poussée de SmGPR-1 est l'objet de cette thèse. L'analyse de taux d'ARNm et de protéines à différents stades de développement du parasite a servi à l'étude de l'expression et la répartition tissulaire de SmGPR-1. Deux récepteurs similaires, de par leur structure, le SmGPR-2 et le SmGPR-3 ont été identifiés, clonés et caractérisés lors de cette étude. Suite à des analyses bioinformatiques, ces trois récepteurs ont révélé leur appartenance à une nouvelle variante de récepteurs à amine biogène couplés aux protéines G caractérisés par l'absence d'aspartate conservé (Asp3.32) dans le troisième domaine transmembranaire. Tout comme SmGPR-1, le récepteur SmGPR-2 est activé par l'histamine, et l'expression de l'ARNm est similaire à celle de SmGPR-1, les deux récepteurs étant régulés à la hausse chez les jeunes schistosomes. Toutefois, ils sont localisés à différents endroits, SmGPR-1 se retrouve dans le tégument, la musculature subtégumentaire et les ventouses, tandis que SmGPR-2 est associé aux plexus nerveux subtégumentaires. La localisation de ces récepteurs est similaire à celle des neurones histaminergiques que l'on retrouve dans les plexus nerveux subtégumentaires, l'innervation des ventouses, dans certains éléments du système nerveux central et les commissures transversales. Il semblerait que l'histamine soit un important système neurotransmetteur du schistosome. Le troisième récepteur identifié, SmGPR-3, n'est pas activé par l'histamine, mais semble démontrer une spécificité étendue aux catécholamines et tout particul

Immunolocalization of SmGPR-3 in larval <i>Schistosoma mansoni</i>.

<p><i>S. mansoni</i> cercaria were probed with affinity purified anti-SmGPR-3 antibody, followed by fluorescein isothiocyanate (FITC)-labelled secondary antibody. (A) Immunoreactivity (green) can be seen along the major longitudinal nerve cords (solid arrowheads) and in transverse commissures (open arrowhead), including the posterior transverse commissure near the base of the tail (open arrow). (B) No significant immunoreactivity was observed in negative controls probed with anti-SmGPR-3 antibody that was pre-adsorbed with peptide antigens or (C) controls probed with secondary antibody only. (*) non-specific labelling.</p

Effects of dopamine and related substances on schistosome motility.

<p>(A) <i>In vitro</i> transformed 3-day-old schistosomula were incubated with test drug, dopamine (DA) or epinine (EPN), each at (10⁻⁴ M) or vehicle (CT, control). Animals were treated for 5 min at room temperature, after which they were examined with a compound microscope equipped with a digital video camera and SimplePCI (Compix Inc.) for image acquisition. Images were recorded for 1 minute (∼3 frames/second) and an estimate of body length in µm was obtained for each animal in every frame. Each tracing shown is of an individual animal and is representative of 12–15 larvae per experiment and 3–4 independent experiments per treatment. (B) Experiments were repeated with various concentrations of test agonist in a range of 10⁻⁷ M–10⁻⁴ M, or in the absence of test substance (CT, control). Images were recorded as above and body length was measured for each frame. Motility is defined as the frequency of length changes (shortening and elongation) per minute of observation, as described in the Methods. The data are presented as the means and SEM of three separate experiments each with 12–15 animals. (C) Schistosomula were treated with test substances at a single concentration or in the absence of drug (CT, control) and motility was measured as above. Dopamine (DA), epinine (EPN), flupenthixol (FLPX), promethazine (PRMZ) were each tested at 50 µM. The remaining substances, adenaline (A), metanephrine (MTN) and haloperidol (HLRD) were tested at 500 µM. The data are the means and SEM of three separate experiments each with 12–15 animals. * Significantly different from the no drug control at P<0.05.</p

Functional expression of the <i>Schistosoma mansoni</i> SmGPR-3 receptor in yeast.

<p>(A) The full-length SmGPR-3 cDNA was expressed in <i>Saccharomyces cerevisae</i> strain YEX108 and grown in selective leu/histidine-deficient (leu⁻/his⁻) medium containing 2×10⁻⁴ M of each biogenic amine or vehicle (no drug control, ND). Yeast cells transformed with empty plasmid were used as a negative control (mock). Receptor activation was quantified from measurements of yeast growth in relative fluorescence units (RFU), using an Alamar blue fluorescence assay. The results are the means ± S.E.M. of 5–6 independent clones, each assayed in triplicate. The following biogenic amines were tested: adrenaline (A), noradrenaline (NA), dopamine (DA), epinine (EPN), serotonin (5-hydroxytryptamine, 5HT), octopamine (OA), tyramine (TA) and histamine (HA). (B) Functional assays were repeated with the same SmGPR-3-expressing yeast strain and variable concentrations of DA (△) or EPN (□). The mock control was tested with DA (•). EC₅₀ values for DA and EPN are 3.10×10⁻⁵ M and 2.85×10⁻⁵ M, respectively. The data are the means ± S.E.M. of two experiments, each in triplicate.</p

Sequence alignment of dopaminergic G protein-coupled receptors with <i>Schistosoma mansoni</i> SmGPR receptors.

<p>A ClustalW alignment was performed using representative examples of vertebrate dopaminergic GPCRs (D1–D5), the <i>S. mansoni</i> dopamine D2-like receptor (SmD2) and several members of the SmGPR clade. SmGPR sequences are boxed (horizontal box) and SmGPR-3 is marked by an arrow. Receptor sequences are identified by their accession numbers (brackets). The positions of the predicted seven transmembrane domains are marked by horizontal lines and the invariant residue in each TM segment <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001523#pntd.0001523-Ballesteros1" target="_blank">[37]</a> is identified by an asterisk (*) Other conserved residues of functional relevance are marked by circles (•) and conserved motifs are boxed (vertical boxes). Residues discussed in this study, R^2.64 (Arg96), D^3.32 (Asp117), S^5.42 (Ser198), T^7.39 (Thr462) and Y^7.43 (Tyr466) are identified by vertical arrows.</p

Antagonist effects on SmGPR-3 activity.

<p>(A) Yeast YEX108 auxotrophic <i>his</i> strain expressing SmGPR-3 was incubated with agonist (DA, 100 µM) and test antagonist or vehicle. Antagonists were tested at 100 µM except for flupenthixol, which was used at 10 µM. The data were normalized relative to the control sample that contained 100 µM DA but no antagonist. To test for drug induced toxicity, assays were repeated in the presence of 100 µM test antagonist in histidine-supplemented (<i>his+</i>) medium, which enables the cell to grow irrespective of receptor activation (His +ve control; see text for details). Abbreviations are as follows: SPIP, spiperone; PROP, propanolol; CLZP, clozapine; BUSP, buspirone; MINS, mianserin; CPRH, cyproheptadine; FLPX, flupenthixol; PRMZ, promethazine; HLRD, haloperidol. B–F. Dose-dependent inhibiton by haloperidol (IC₅₀ = 1.4 µM), flupenthixol (IC₅₀ = 3.9 µM), promethazine (IC₅₀ = 28.0 µM), mianserin (IC₅₀ = 45.0 µM) clozapine (IC₅₀>100 µM). The error bars are the means ± SEM for 3–4 experiments and at least 2 clones (in triplicates).</p

Dendogram analysis of biogenic amine (BA) G protein-coupled receptors (GPCR).

<p>A rooted phylogenetic tree was constructed from a ClustalW sequence alignment of vertebrate and invertebrate BA receptors, using MEGA 4 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001523#pntd.0001523-Tamura1" target="_blank">[35]</a>. Included in the alignment are 15 predicted <i>Schistosoma mansoni</i> and <i>S. japonicum</i> BA GPCR sequences, of which nine clustered together into a separate clade (SmGPR). The receptor described in this paper, SmGPR-3 is identified by an open square (□). Other <i>S. mansoni</i> receptors are marked with solid squares (▪) and <i>S. japonicum</i> receptors are marked with solid triangles (▴). Sequences are identified by their accession numbers and the species names are abbreviated as follows: A.e. (<i>Aedes aegypti</i>), A.i. (<i>Agrotis ipsilon</i>), A.m. (<i>Apis mellifera</i>), B.m. (<i>Bombyx mori</i>), B.t. (<i>Bos taurus</i>), C.e. (<i>Caenorhabditis elegans</i>), C.f. (<i>Canis familiaris</i>), C.p. (<i>Cavia porcellus</i>), D.m. (<i>Drosophila melanogaster</i>), D.j. (<i>Dugesia japonica</i>), D.r. (<i>Danio rerio</i>), H.s. (<i>Homo sapiens</i>), H.v. (<i>Heliothis virescens</i>), M.b. (<i>Mamestra brassicae</i>), M.m. (<i>Mus musculus</i>), M.mul. (<i>Macaca mulatta</i>), P.a. (<i>Periplaneta americana</i>), P.x. (<i>Papilio xuthus</i>), R.n. (<i>Rattus norvegicus</i>), S.j. (<i>S. japonicum</i>), S.med. (<i>Schmidtea mediterranea</i>), S.l. (<i>Spodoptera littoralis</i>) and S.s. (<i>Sus scrofa</i>). Predicted <i>S. mansoni</i> coding sequences are identified by their “Smp” designation obtained from the <i>S. mansoni</i> Genome database (<i>S. mansoni</i> GeneDB) and the corresponding GenBank Accession number. H1–H4, histamine type 1–4 receptors; D1–D5, dopamine type 1–5 receptors; A, adrenergic receptors; 5HT, serotonin (5-hydroxytryptamine) receptors; mACh, muscarinic acetylcholine receptors; OA/TA, octopamine/tyramine receptors.</p

Identification of an Ascaris G protein-coupled acetylcholine receptor with atypical muscarinic pharmacology

Acetylcholine (ACh) is a neurotransmitter/neuromodulator in the nematode nervous system and induces its effects through interaction with both ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs). The structure, pharmacology and physiological importance of LGICs have been appreciably elucidated in model nematodes, including parasitic species where they are targets for anthelmintic drugs. Significantly less, however, is understood about nematode ACh GPCRs, termed GARs (G protein-linked ACh receptors). What is known comes from the free-livingCaenorhabditis elegans as no GARs have been characterized from parasitic species. Here we clone a putative GAR from the pig gastrointestinal nematode Ascaris suum with high structural homology to the C. elegans receptor GAR-1. Our GPCR, dubbed AsGAR-1, is alternatively spliced and expressed in the head and tail of adult worms but not in dorsal or ventral body wall muscle, or the ovijector. ACh activated AsGAR-1 in a concentration-dependent manner but the receptor was not activated by other small neurotransmitters. The classical muscarinic agonists carbachol, arecoline, oxotremorine M and bethanechol were also AsGAR-1 agonists but pilocarpine was ineffective. AsGAR-1 activation by ACh was partially antagonized by the muscarinic blocker atropine but pirenzepine and scopolamine were largely ineffective. Certain biogenic amine GPCR antagonists were also found to block AsGAR-1. Our conclusion is that Ascaris possesses G protein-coupled ACh receptors that are homologous in structure to those present in C. elegans, and that although they have some sequence homology to vertebrate muscarinic receptors, their pharmacology is atypically muscarinic.This article is from International Journal for Parasitology 39 (2009): 1215–1222, doi:10.1016/j.ijpara.2009.03.001. Posted with permission.</p