Fig. 6

Deorphanization of C. elegans Gal/AstA-type and SST/AstC-type receptors. a Cell-based reverse pharmacology approach for receptor deorphanization. Each receptor is transiently expressed in CHO cells together with the promiscuous Gα₁₆ protein, which couples to many GPCRs and directs intracellular signaling to phospholipase Cβ (PLCβ) activity [36, 125, 126, 135]. This results in the release of calcium from intracellular storage sites, eliciting a luminescent signal from the calcium indicator aequorin. b NPR-9 is activated by three peptides encoded by the nlp-1 gene (NLP-1–1 = MDANAFRMSFamide, NLP-1–2 = MDPNAFRMSFamide, NLP-1–3 = VNLDPNAFRMSFamide) with similar potencies. EC50 for NLP-1–1 = 243.6 nM (logEC50 (M) = − 6.613, with 95% confidence interval (CI) = − 6.709 to − 6.519), for NLP-1–2 = 618.0 nM (logEC50 (M) = − 6.209, with 95% CI = − 6.266 to − 6.153), and for NLP-1–3 = 475.0 nM (logEC50 (M) = − 6.319, with 95% CI = − 6.383 to − 6.256). c Both receptor isoforms of NPR-16 are activated by the AstC-like peptide NLP-99 (GDGYGWNDCEFSPLSCLL, disulfide bridge C9-C16). EC50 for isoform a = 89.9 nM (logEC50 (M) = − 7.046, with 95% CI = − 7.113 to -6.979), and for isoform b = 65.4 nM (logEC50 (M) = − 7.184, with 95% CI = − 7.244 to − 7.125). b, c concentration-dependent activation curves are plotted as percentage of the highest peptide-evoked response (100% activation) using a nonlinear regression model. Error bars indicate Standard Error of Mean (SEM) with n ≥ 6. d Evolutionary conservation of the Gal/AstA-like receptor NPR-9 and the SST/AstC-like receptors NPR-16, NPR-24, and NPR-32, with peptides belonging to the same family. Black bars represent the number of species with mismatches in precursor-receptor conservation, meaning either only the peptide or the receptor is found. Gray and white bars indicate the number of species in which both peptide and receptor orthologs have been identified (gray), or in which none of the two were found (white). Confirmed peptide ligands of receptors are indicated in red and rank among the best scoring peptide precursors (with the lowest number of mismatches between peptide and receptor representation in nematode predicted proteomes). e Schematic representation of local search behavior assay. f Both nlp-1 and npr-9 mutants show increased turning behavior compared to wild-type animals upon removal from food. Double mutants of nlp-1 and npr-9 do not significantly differ in turning compared to the single mutants. Data are presented as mean + / − SEM including n > 6 assays with 10–15 animals tested for each strain per replicate. Statistical significance between different conditions was calculated using one-way ANOVA with Dunnett multiple comparisons test correction. * p < 0.05, ** p < 0.01. Individual data available in Additional file 9: Tables S1-S4