Differential Activation of “Social” and “Solitary” Variants of the Caenorhabditis elegans G Protein-coupled Receptor NPR-1 by Its Cognate Ligand AF9

Abstract

Natural variations of wildSince neuropeptide Y shows no sequence homology to AF9 and was functionally inactive at the cloned NPR-1, we propose to rename NPR-1 and refer to it as an AF9 receptor, AF9-R1.

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.…”

“…[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.…”

Are Caenorhabditis elegans receptors useful targets for drug discovery: Pharmacological comparison of tyramine receptors with high identity from C. elegans (TYRA-2) and Brugia malayi (Bm4)

“…Unfortunately, expression only reached a maximum of ~7% in each of these lines (as visualized by immunofluorescence to the N-terminal HA tag) and isolated membranes did not exhibit significant saturable [ 3 H]LSD binding. Heat shock 24 hours after transfection, as described for the increased expression of nematode neuropeptide receptors [33], or increasing the amount of transfected DNA (maximum of 8 µg/10 cm plate) did not increase expression. Therefore, stable HEK-293 cell lines expressing Bm4 were prepared as described in methods.…”