Fig. 2
Phylogenetic identification and deorphanisation of an A. rubens VP/OT-type receptor. a Maximum likelihood tree showing that a VP/OT-type receptor identified by BLAST analysis of A. rubens transcriptome sequence data (boxed) is positioned within a clade comprising VP/OT-type receptors from other taxa. NPS/NG peptide/CCAP-type receptors are paralogs of VP/OT-type receptors [25]. Phylogenetic analyses of bilaterian neuropeptide receptors have shown that GnRH/AKH/ACP/CRZ-type receptors are closely related to VP/OT-type receptors and NPS/NG peptide/CCAP-type receptors [6]. Therefore, GnRH/AKH/ACP/CRZ-type receptors were included as an outgroup in the phylogenetic tree. The scale bar indicates amino acid substitutions per site, and bootstrap values are shown at nodes. Species where activation of the VP/OT-type receptor by a cognate VP/OT-type neuropeptide has been demonstrated experimentally (including A. rubens, see b) are labelled with an asterisk. Full species names and accession numbers for the receptor sequences are listed in Table S2 of Additional file 3. b Asterotocin (black circles) causes concentration-dependent activation of the A. rubens VP/OT-type receptor, demonstrated by measuring Ca2+-induced luminescence in CHO-K1 cells expressing aequorin, and transfected with the promiscuous G-protein Gα16 and the A. rubens VP/OT-type receptor (CHO-K1/G5A-ArVPOTR cells); EC50 = 5.7 × 10−8 M. CHO-K1 cells transfected with empty pcDNA vector (black square) were used as a negative control and do not exhibit luminescence when exposed to asterotocin. c Comparison of the relative luminescence responses of CHO-K1/G5A-ArVPOTR cells when exposed to asterotocin or the related peptides NGFFYamide (a paralog of asterotocin in A. rubens), human vasopressin or human oxytocin, all at a concentration of 10−4 M. Luminescence responses in the presence of vasopressin, oxytocin and NGFFYamide were not significantly higher than the basal media control (P = 0.5, P = 0.25, P = 0.25, respectively; Wilcoxon signed-rank test; n = 9)