Functional Annotation of the Putative Orphan Caenorhabditis elegans G-protein-coupled Receptor C10C6.2 as a FLP15 Peptide Receptor

This report describes the cloning and functional annotation of a Caenorhabditis elegans orphan G-protein-coupled receptor (GPCR) (C10C6.2) as a receptor for the FMRFamide-related peptides (FaRPs) encoded on the flp15 precursor gene, leading to the receptor designation FLP15-R. A cDNA encoding C10C6.2 was obtained using PCR techniques, confirmed identical to the Worm-pep-predicted sequence, and cloned into a vector appropriate for eucaryotic expression. A [ 35 S]guanosine 5′- O -(thiotriphosphate) (GTPγS) assay with membranes prepared from Chinese hamster ovary (CHO) cells transiently transfected with FLP15-R was used as a read-out for receptor activation. FLP15-R was activated by putative FLP15 peptides, GGPQGPLRF-NH 2 (FLP15-1), RGPSGPLRF-NH 2 (FLP15-2A), its des-Arg11The abbreviations used were: FaRPs, FMRFamide-related peptides; flp, FMRFamide-like precursor protein gene; FLP15-R, FLP15 peptide receptor; FLIPR, fluorescence imaging plate reader; hRFRP, human RFamides-related peptide; GPCR, G-protein-coupled receptor; NPAF, neuropeptide AF; NPFF, neuropeptide FF; nlp, neuropeptide-like precursor protein gene; PTX, pertussis toxin; CHO, Chinese hamster ovary; GTPγS, guanosine 5′-O-(thiotriphosphate). counterpart, GPSGPLRF-NH 2 (FLP15-2B), and to a lesser extent, by a tobacco hornworm Manduca sexta FaRP, GNSFLRFNH 2 (F7G) (potency ranking FLP15-2A > FLP15-1 > FLP15-2B >> F7G). FLP15-R activation was abolished in the transfected cells pretreated with pertussis toxin, suggesting a preferential receptor coupling to G i /G o proteins. The functional expression of FLP15-R in mammalian cells was temperature-dependent. Either no stimulation or significantly lower ligand-evoked [ 35 S]GTPγS binding was observed in membranes prepared from transfected FLP15-R/CHO cells cultured at 37 °C. However, a 37 to 28 °C temperature shift implemented 24 h post-transfection consistently resulted in an improved activation signal and was essential for detectable functional expression of FLP15-R in CHO cells. To our knowledge, the FLP15 receptor is only the second deorphanized C. elegans neuropeptide GPCR reported to date.