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 [35S]guanosine 5'-O-(thiotriphosphate) (GTPgammaS) 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-NH2 (FLP15-1), RGPSGPLRF-NH2 (FLP15-2A), its des-Arg1 counterpart, GPSGPLRF-NH2 (FLP15-2B), and to a lesser extent, by a tobacco hornworm Manduca sexta FaRP, GNSFLRFNH2 (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 Gi/Go proteins. The functional expression of FLP15-R in mammalian cells was temperature-dependent. Either no stimulation or significantly lower ligand-evoked [35S]GTPgammaS binding was observed in membranes prepared from transfected FLP15-R/CHO cells cultured at 37 degrees C. However, a 37 to 28 degrees 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.
Kubiak
Teresa M
TM
Animal Health Discovery Research, Pharmacia Corporation, Kalamazoo, MI 49001, USA. teresa.m.kubiak@pfizer.com
Larsen
Martha J
MJ
Zantello
Marjorie R
MR
Bowman
Jerry W
JW
Nulf
Susan C
SC
Lowery
David E
DE
D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D009479:Neuropeptides / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D010802:Phylogeny; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D018013:Receptors, Neuropeptide / Q000096:biosynthesis / Q000235:genetics; D013696:Temperature; D014162:Transfection
D029742:Caenorhabditis elegans Proteins; D018076:DNA, Complementary; D009479:Neuropeptides; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; C499700:npr-3 protein, C elegans