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

FMRFamide‐like peptides encoded on the flp‐18 precursor gene activate two isoforms of the orphan Caenorhabditis elegans G‐protein‐coupled receptor Y58G8A.4 heterologously expressed in mammalian cells

“…The mutant Chinese hamster ovary cell line CHO-10001A (referred to hereafter as CHO cells), cell culture media, transfection, and various assay reagents were as previously described. 11,12,17,18 U-73122 (phospholipase C inhibitor) and its inactive analog U-73343 were obtained from the Upjohn/Pharmacia/Pfizer compound collection. 19 Cloning and Plasmid Preparation Molecular biological techniques followed either manufacturer's recommendations or general protocols.…”

“…23 Cell Cultures, Transfection, Stable Cell Line Generation and Membrane Preparation CHO cells were cultured and transfected as described earlier. 11,12,17,18 The flp-18R1a/pCR3.1 or flp-18R1b /pCR3.1 plasmids (5 lg DNA/10 cm plate) were used for transfections. The cells were harvested 24 h after transfection and membranes were prepared as described.…”

“…The cells were harvested 24 h after transfection and membranes were prepared as described. 11,12 In cases where a 37 to 288C shift was implemented, media were supplemented with 0.1M HEPES pH 7.4 and the transfected cells were first incubated at 378C for 24 h posttransfection and then moved to a humidified 288C/3% CO 2 incubator for an additional 16-24 h before harvesting for membrane preparation or receptor signaling experiments.…”

“…Assays were run using a 96-well fluorescence imaging plate reader (FLIPR) (Molecular Devices, Sunnyvale, CA) essentially as described earlier 12,18 either with clonal cell lines or cells transiently transfected with the flp-18R1a/pCR3.1 or flp-18R1b/pCR3.1 plasmids and plated at 2 3 10 4 cells/well 24 h after transfection. In experiments incorporating a 37 to 288C temperature shift, cells plated in blackwalled 96-well plates were first incubated at 378C for 24 h and then at 288C for 16-24 h before testing in complete media supplemented with 10 mM HEPES.…”

“…[6][7][8][9] Despite intense efforts in many laboratories over the last decade, most of the C. elegans neuropeptide G-proteincoupled receptors (GPCRs) remain orphans, as only a few have been matched with their native ligands. [10][11][12][13][14][15][16] Functional expression of C. elegans neuropeptide GPCRs in mammalian expression systems has been challenging and problematic, mainly due to the apparently poor compatibility of mammalian G-proteins and other accessory proteins with heterelogously expressed nematode receptors.…”