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

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

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

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