Functional Characterization of Three G Protein-coupled Receptors for Pigment Dispersing Factors in Caenorhabditis elegans

Abstract

Here, we report the identification, cloning, and functional characterization of three Caenorhabditis elegans G proteincoupled 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.The neuropeptide pigment dispersing factor (PDF) 3 was initially discovered in crustaceans (as pigment-dispersing hormone), where it drives a daily rhythm of color changes (1). Thereafter, highly conserved PDF peptides were identified in many species of insects and recently also in nematodes (2). 4PDF is a crucial component of the insect circadian clock and has been characterized as a putative output factor, controlling daily rhythms in locomotor activity (3, 4). In 2005, three independent studies identified CG13758, a class B peptide G protein-coupled receptor (GPCR), as the receptor for PDF in Drosophila melanogaster (PDFR) (5-7). It is related to the mammalian VIP receptor (VPAC2) and to the calcitonin receptor, both of which are expressed in the mammalian master clock. Fly PDFR mutants and flies lacking PDF both exhibit severe deficits in free-running locomotor rhythms (4 -6). Recently, 3 endogenous PDF-like neuropeptides were discovered in the free-living nematode model organism C. elegans.4 They are expressed mainly in neurons involved in chemosensation, mechanosensation, oxygen sensing, and locomotion. Circadian analysis revealed that at least two of these peptides (e.g. PDF-1a and -b) are involved in the control of daily locomotor rhythms in C. elegans.4 Mutants lacking PDF-1 mimic the behavioral phenotype of Drosophila PDF mutants with respect to free-running locomotor rhythms. This led us to the hypothesis that the PDF signaling system, which imposes the clock rhythm on behavior, may be functionally...

PDF receptor signaling in Caenorhabditis elegans modulates locomotion and egg-laying

“…A database search based on putative transmembrane domains of the human secretin receptor family (also known as family B or II) genes predicted only six secretin receptor genes in C. elegans, one of which is the pdfr-1 (C13B9.4) gene (Harmar, 2001;Cardoso et al, 2006). Through alternative splicing, pdfr-1 produces three pigment dispersing factor (PDF) neuropeptide receptors b and c) and is expressed in all body wall muscles, in several mechanosensory neurons, chemosensory neurons, ring motor neurons and pharyngeal interneurons (Janssen et al, 2008). These three receptors are the only GPCRs of the secretin type family in C. elegans that are deorphaned and they were shown to be activated by C. elegans PDF neuropeptides (Janssen et al, 2008).…”

“…Through alternative splicing, pdfr-1 produces three pigment dispersing factor (PDF) neuropeptide receptors b and c) and is expressed in all body wall muscles, in several mechanosensory neurons, chemosensory neurons, ring motor neurons and pharyngeal interneurons (Janssen et al, 2008). These three receptors are the only GPCRs of the secretin type family in C. elegans that are deorphaned and they were shown to be activated by C. elegans PDF neuropeptides (Janssen et al, 2008). The C. elegans PDF receptors (PDFRs) are closely related to the fruit fly Drosophila melanogaster PDFR and more distantly to the mammalian VIP (VPAC 2 ) and calcitonin receptors (Janssen et al, 2008).…”

“…These three receptors are the only GPCRs of the secretin type family in C. elegans that are deorphaned and they were shown to be activated by C. elegans PDF neuropeptides (Janssen et al, 2008). The C. elegans PDF receptors (PDFRs) are closely related to the fruit fly Drosophila melanogaster PDFR and more distantly to the mammalian VIP (VPAC 2 ) and calcitonin receptors (Janssen et al, 2008).…”

“…According to their localization patterns, it is thought that PDF-1a, b and PDF-2 are mainly involved in the integration of environmental stimuli. The peptides already proved functional in locomotion as both the pdf-1 loss-of-function allele tm1996 and pdf-2 over-expression induce decreased locomotion speed, increased reversal frequency and increased number of directional changes (Janssen et al, 2008). Each of the three PDF neuropeptides is able to bind to each of the three PDF receptors but with different affinities.…”

“…Each of the three PDF neuropeptides is able to bind to each of the three PDF receptors but with different affinities. While PDFR-1a and PDFR-1b signal through Ga s and adenylate cyclase and ultimately elevate cAMP levels, PDFR-1c inhibits cAMP levels through Ga i/o (Janssen et al, 2008).…”