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

Teresa M Kubiak,Martha J Larsen,Marjorie R Zantello,Jerry W Bowman,Susan C Nulf,David E Lowery

doi:10.1074/jbc.m304056200

Abstract

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.

Highlights

Systems of invertebrates [1,2,3,4,5,6]
A [35S]guanosine 5؅-O(thiotriphosphate) (GTP␥S) assay with membranes prepared from Chinese hamster ovary (CHO) cells transiently transfected with FLP15 receptor (FLP15-R) was used as a read-out for receptor activation
Either no stimulation or significantly lower ligand-evoked [35S]GTP␥S binding was observed in membranes prepared from transfected FLP15-R/CHO cells cultured at 37 °C

Summary

The 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; GTP␥S, guanosine 5Ј-O-(thiotriphosphate). A total of 59 distinct FaRPs, encoded on 22 flps (precursor genes), have been predicted from the Caenorhabditis elegans genome [10, 11] This number might be greater, since despite extensive bioinformatic searches, all family members have probably not been identified (10 –12). In addition to FaRPs, 23 C. elegans neuropeptide-like protein (nlp) genes have been identified defining at least 11 families of 134 unique and 151 total putative neuropeptides [13]. Despite the publication of the C. elegans genome more than 4 years ago [20], there has been only one orphan C. elegans neuropeptide GPCR matched with its cognate ligand to date [21, 22]. We propose to name this GPCR the FLP15 receptor (FLP15-R), because the receptoractivating peptides are encoded on the C. elegans precursor gene flp15 [10]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION