Abstract
Here, we report the identification, cloning, and functional characterization of three Caenorhabditis elegans G protein-coupled pigment dispersing factor (PDF) receptors, which we designated as Ce_PDFR-1a, -b, and -c. They represent three splice isoforms of the same gene (C13B9.4), which share a high degree of similarity with the Drosophila PDF receptor and are distantly related to the mammalian vasoactive intestinal peptide receptors (VPAC2) and calcitonin receptors. In a reverse pharmacological screen, three bioactive C. elegans neuropeptides, which were recently identified as the Drosophila PDF orthologues, were able to activate these receptors in a dose-dependent manner with nanomolar potency (isoforms a and b). Integrated green fluorescent protein reporter constructs reveal the expression of these PDF receptors in all body wall muscle cells and many head and tail neurons involved in the integration of environmental stimuli and the control of locomotion. Using a custom data analysis system, we demonstrate the involvement of this newly discovered neuropeptide signaling system in the regulation of locomotor behavior. Overexpression of PDF-2 phenocopies the locomotor defects of a PDF-1 null mutant, suggesting that they elicit opposite effects on locomotion through the identified PDF receptors. Our findings strengthen the hypothesis that the PDF signaling system, which imposes the circadian clock rhythm on behavior in Drosophila, has been functionally conserved throughout the protostomian evolutionary lineage.
Highlights
To fortify this hypothesis, we set out to find and characterize the cognate receptors of the C. elegans PDF neuropeptides
Our functional analysis of the PDF signaling system reveals that overexpression of PDF-2 phenocopies the onic kidney; CRE, cyclic AMP response element; NLP, neuropeptide-like protein; FLP, FMRFamide-like peptide; VIP, vasoactive intestinal peptide; HPLC, high pressure liquid chromatography; GFP, green fluorescent protein
The amino-terminal extracellular region contains six conserved cysteine residues, two tryptophan residues, and one aspartate, whereas one cysteine residue appears to be conserved in extracellular loops E1 and E2 (Fig. 1b). This is typical for the class B GPCRs [12]
Summary
Strains and Media—All C. elegans strains were grown at 20 °C on nematode growth medium agar seeded with Escherichia coli OP50 bacteria. Human embryonic kidney cells (HEK293), used in the CRE-luciferase reporter assay, were maintained in Dulbecco’s modified Eagle’s medium (with L-glutamine, 4,500 mg/liter D-glucose, 110 mg/liter sodium pyruvate) supplemented with 10% fetal bovine serum and 100 IU/ml of a penicillin/streptomycin solution. Both cell lines were split every 3 days (1:10 and 1:5, respectively) and grown at 37 °C in a humidified atmosphere of 5% CO2 in air. The genomic DNA, including the predicted promoter, the open reading frame, and the 3Ј-untranslated region sequence for each gene was amplified by PCR, purified, and microinjected (100 ng/l) in wild-type C. elegans together with. The statistical significance of behavioral assays was determined using the two-tailed Student’s t test
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