A novel G protein-coupled receptor of Schistosoma mansoni (SmGPR-3) is activated by dopamine and is widely expressed in the nervous system

Abstract

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.

Figure 1. Dendogram analysis of biogenic amine (BA) G protein-coupled receptors (GPCR).

A rooted phylogenetic tree was constructed from a ClustalW sequence alignment of vertebrate and invertebrate BA receptors, using MEGA 4 . Included in the alignment are 15 predicted Schistosoma mansoni and S. japonicum 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 S. mansoni receptors are marked with solid squares (▪) and S. japonicum receptors are marked with solid triangles (▴). Sequences are identified by their accession numbers and the species names are abbreviated as follows: A.e. (Aedes aegypti), A.i. (Agrotis ipsilon), A.m. (Apis mellifera), B.m. (Bombyx mori), B.t. (Bos taurus), C.e. (Caenorhabditis elegans), C.f. (Canis familiaris), C.p. (Cavia porcellus), D.m. (Drosophila melanogaster), D.j. (Dugesia japonica), D.r. (Danio rerio), H.s. (Homo sapiens), H.v. (Heliothis virescens), M.b. (Mamestra brassicae), M.m. (Mus musculus), M.mul. (Macaca mulatta), P.a. (Periplaneta americana), P.x. (Papilio xuthus), R.n. (Rattus norvegicus), S.j. (S. japonicum), S.med. (Schmidtea mediterranea), S.l. (Spodoptera littoralis) and S.s. (Sus scrofa). Predicted S. mansoni coding sequences are identified by their “Smp” designation obtained from the S. mansoni Genome database (S. mansoni 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.

Figure 2. Sequence alignment of dopaminergic G protein-coupled receptors with Schistosoma mansoni SmGPR receptors.

A ClustalW alignment was performed using representative examples of vertebrate dopaminergic GPCRs (D1–D5), the S. mansoni 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 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.

Figure 3. Functional expression of the Schistosoma mansoni SmGPR-3 receptor in yeast.

(A) The full-length SmGPR-3 cDNA was expressed in Saccharomyces cerevisae 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.

Figure 4. Antagonist effects on SmGPR-3 activity.

(A) Yeast YEX108 auxotrophic his 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 (his+) 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).

Figure 5. Immunolocalization of SmGPR-3 in larval Schistosoma mansoni.

S. mansoni 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.

Figure 6. Immunolocalization of SmGPR-3 in adult Schistosoma mansoni.

Adult male worms were treated with affinity-purified anti-SmGPR-3 polyclonal IgG, followed by fluorescein isothiocyanate (FITC)-labelled secondary antibody (green). Animals were counterstained with tetramethylrhodamine B isothiocyanate (TRITC)-labelled phalloidin (red) to visualize the musculature of the body wall and digestive tract. (A) Strong SmGPR-3 immunoreactivity is visible in the region of the cerebral ganglia (cg) and along the main longitudinal nerve cords (lnc) of the CNS. (B) Green fluorescence can also be seen in peripheral nerve fibers innervating the caecum (open arrows). (C) Near the surface SmGPR-3 is strongly expressed in the peripheral innervation of the body wall muscles and the tegument. Numerous immunoreactive nerve fibers (open arrows), some varicose in appearance, are visible throughout this region. At higher magnification (D, E) we see SmGPR-3–expressing nerve fibers (open arrows) and cell bodies (solid arrowhead) innervating the body wall muscles, both circular muscle (cm) and typically spindle-shaped longitudinal muscle fibers (lm). SmGPR-3 immunoreactivity is seen in the tubercles of male worms (D, solid arrow), where it is probably associated with sensory nerve endings. (F) SmGPR-3 is expressed in the nerve plexus and small fibers of the ventral sucker. Extensive labelling of the submuscular nerve plexus can also be seen in this specimen (solid arrows). (G, H) A male worm showing strong labelling of major nerve cords (solid arrows) and the reproductive tract, including the testes (t) and associated nerves. (I) Fine SmGPR-3 immunoreactive nerve fibers (open arrows) innervate the testicular lobes of male worms. lnc, longitudinal nerve cords; cg, cerebral ganglia; c, caecum; lm, longitudinal muscle; cm, circular muscle; vs, ventral sucker; t, testes; gc, gynecophoral canal.

Figure 7. Effects of dopamine and related substances on schistosome motility.

(A) In vitro 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.

Figure 8. Homology modeling of SmGPR-3 and ligand docking.

(A) A homology model of SmGPR-3 with bound dopamine (DA) is shown. The model was generated using the β-2 adrenergic receptor (PDB Accession # 2rh1) as a structural template, as described in the Methods. The positions of the 7 predicted transmembrane (TM) helices and 3 extracellular loops (ECL) are marked. The additional intracellular helix at the C-terminal end (helix 8) is also shown. Amino acid residues that are predicted to interact with dopamine include: Arg 96 (R^2.64), Asp117 (D^3.32), Thr462 (T^7.39) and Tyr466 (Y^7.43). (B) Close-up of the predicted binding pocket showing the best docking pose of dopamine (DA) and the principal ligand binding residues. (C) Two different docking poses of dopamine (DA) are shown. Note that the position of the catechol ring in the two conformations is reversed. In the best scoring pose (yellow), the ring hydroxyl interacts with Arg96 (R^2.64) near the extracellular junction of TM2, whereas in the other docking pose (magenta) the ring interacts with Ser198 (S^5.42) of TM5 instead. In both cases the protonated amino end of dopamine is anchored to Asp117 (D^3.32) of TM3. An overlay of potential docking poses of dopamine (DA, panel D) and the structurally related ligand, epinine (EPN, panel E) shows the majority of interactions occurring with TM2, 3 and 7 for both ligands. The core interacting residues are shown and predicted H-bonds are marked by broken green lines.