Regulation of PDF receptor signaling controlling daily locomotor rhythms in Drosophila

Abstract

Each day and in conjunction with ambient daylight conditions, neuropeptide PDF regulates the phase and amplitude of locomotor activity rhythms in Drosophila through its receptor, PDFR, a Family B G protein-coupled receptor (GPCR). We studied the in vivo process by which PDFR signaling turns off, by converting as many as half of the 28 potential sites of phosphorylation in its C terminal tail to a non-phosphorylatable residue (alanine). We report that many such sites are conserved evolutionarily, and their conversion creates a specific behavioral syndrome opposite to loss-of-function phenotypes previously described for pdfr. That syndrome includes increases in the amplitudes of both Morning and Evening behavioral peaks, as well as multi-hour delays of the Evening phase. The precise behavioral effects were dependent on day-length, and most effects mapped to conversion of only a few, specific serine residues near the very end of the protein and specific to its A isoform. Behavioral phase delays of the Evening activity under entraining conditions were predicted the phase of activity cycles under constant darkness. The behavioral phenotypes produced by the most severe PDFR variant were ligand-dependent in vivo, and not a consequence of changes to their pharmacological properties, nor of changes in their surface expression, as measured in vitro. The mechanisms underlying termination of PDFR signaling are complex, subject to regulation that is modified by season, and central to a better understanding of the peptidergic modulation of behavior.

The incidence of candidate binding sites for β-arrestin in Drosophila neuropeptide GPCRs

“…For the large number of GPCRs that associate with β-arrestin, such regulation is significant not only as a mechanism to decrease receptor signaling by de-sensitization and receptor endocytosis (6), but also to traffic and transition GPCRs to later stages of signaling via ERK and other signaling pathways (20, 62). Studies of β-arrestin regulation of GPCRs in Drosophila are less developed than in mammalian models, few in number and scope, and with one notable exception (63), they are limited largely to in vitro studies (34, 41, 42, 43, 44, 48). I have reported results here specifically to promote renewed consideration of β-arrestin regulation of neuropeptide GPCRs in Drosophila because β-arrestin is arguably central to understanding GPCR signaling, trafficking and turnover.…”

The incidence of candidate binding sites for β-arrestin in Drosophila neuropeptide GPCRs

“…Do the critical modifications represent phosphorylation of a specific subset of GPCR residues, or do they instead reflect an increase in the aggregate amount of phosphorylation that requires no specific pattern. There is evidence to support both possible mechanisms, for example as deduced from studies of melanopsin [OPN4–47, 48] and of neuropeptide PDF receptor [ 34 ]. A second major question asks, can phosphorylation of distinct residues on a single GPCR direct different outcomes for signaling and desensitization?…”

“…These two sub-genera are estimated to have diverged at least 60 Myr ago [32]. The demonstration that D. melanogaster and D. virilis display sequence conservation within the coding regions of a particular gene, or within the putative regulatory regions, is considered a useful indicator of functional conservation of protein or cis-regulatory sequences [e.g., [33][34][35]. As applied, this comparison reduced the occurrence of 'putatively-functional' arrestin binding sites such that only 36 of the 49 GPCR CTs contain precise BBS candidates that could be matched across the two species by position and/ or sequence.…”

“…For the large number of GPCRs that associate with β-arrestin, such regulation is significant not only as a mechanism to decrease receptor signaling by de-sensitization and receptor endocytosis [6], but also to traffic and transition GPCRs to later stages of signaling via ERK and other signaling pathways [20,63]. Studies of β-arrestin regulation of GPCRs in Drosophila are less developed than in mammalian models, few in number and scope, and with one notable exception [64], they are limited largely to in vitro studies [34,[41][42][43][44]48]. I have reported results here specifically to promote renewed consideration of β-arrestin regulation of neuropeptide GPCRs in Drosophila because β-arrestin is arguably central to understanding GPCR signaling, trafficking and turnover.…”