Jelle Caers

More than two decades of research on insect neuropeptide GPCRs: an overview

This review focuses on the state of the art on neuropeptide receptors in insects. Most of these receptors are G protein-coupled receptors (GPCRs) and are involved in the regulation of virtually all physiological processes during an insects life. More than 20 years ago a milestone in invertebrate endocrinology was achieved with the characterization of the first insect neuropeptide receptor, i.e., the Drosophila tachykinin-like receptor. However, it took until the release of the Drosophila genome in 2000 that research on neuropeptide receptors boosted. In the last decade a plethora of genomic information of other insect species also became available, leading to a better insight in the functions and evolution of the neuropeptide signaling systems and their intracellular pathways. It became clear that some of these systems are conserved among all insect species, indicating that they fulfill crucial roles in their physiological processes. Meanwhile, other signaling systems seem to be lost in several insect orders or species, suggesting that their actions were superfluous in those insects, or that other neuropeptides have taken over their functions. It is striking that the deorphanization of neuropeptide GPCRs gets much attention, but the subsequent unraveling of the intracellular pathways they elicit, or their physiological functions are often hardly examined. Especially in insects besides Drosophila this information is scarce if not absent. And although great progress made in characterizing neuropeptide signaling systems, even in Drosophila several predicted neuropeptide receptors remain orphan, awaiting for their endogenous ligand to be determined. The present review gives a précis of the insect neuropeptide receptor research of the last two decades. But it has to be emphasized that the work done so far is only the tip of the iceberg and our comprehensive understanding of these important signaling systems will still increase substantially in the coming years.

Structure-activity studies of Drosophila adipokinetic hormone (AKH) by a cellular expression system of dipteran AKH receptors.

Structure-activity studies for the adipokinetic hormone receptor of insects were for the first time performed in a cellular expression system. A series of single amino acid replacement analogues for the endogenous adipokinetic hormone of Drosophila melanogaster (pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH(2)) were screened for activity with a bioluminescence cellular assay, expressing the G-protein coupled receptor. For this series of peptide analogues, one amino acid of the N-terminal tetrapeptide was successively replaced by alanine, while those of the C-terminal tetrapeptide were successively substituted by glycine; other modifications included the blocked N- and C-termini that were replaced by an acetylated alanine and a hydroxyl group, respectively. The analogue series was tested on the AKH receptors of two dipteran species, D. melanogaster and Anopheles gambiae. The blocked termini of the AKH peptide probably play a minor role in receptor interaction and activation, but are considered functionally important elements to protect the peptide against exopeptidases. In contrast, the amino acids at positions 2, 3, 4 and 5 from the N-terminus all seem to be crucial for receptor activation. This can be explained by the potential presence of a β-strand in this part of the peptide that interacts with the receptor. The inferred β-strand is probably followed by a β-turn in which the amino acids at positions 5-8 are involved. In this β-turn, the residues at positions 6 and 8 seem to be essential, as their substitutions induce only a very low degree of receptor activation. Replacement of Asp(7), by contrast, does not influence receptor activation at all. This implies that its side chain is folded inside the β-turn so that no interaction with the receptor occurs.

Peptidomics of Neuropeptidergic Tissues of the Tsetse Fly Glossina morsitans morsitans

AbstractNeuropeptides and peptide hormones are essential signaling molecules that regulate nearly all physiological processes. The recent release of the tsetse fly genome allowed the construction of a detailed in silico neuropeptide database (International Glossina Genome Consortium, Science 344, 380–386 (2014)), as well as an in-depth mass spectrometric analysis of the most important neuropeptidergic tissues of this medically and economically important insect species. Mass spectrometric confirmation of predicted peptides is a vital step in the functional characterization of neuropeptides, as in vivo peptides can be modified, cleaved, or even mispredicted. Using a nanoscale reversed phase liquid chromatography coupled to a Q Exactive Orbitrap mass spectrometer, we detected 51 putative bioactive neuropeptides encoded by 19 precursors: adipokinetic hormone (AKH) I and II, allatostatin A and B, capability/pyrokinin (capa/PK), corazonin, calcitonin-like diuretic hormone (CT/DH), FMRFamide, hugin, leucokinin, myosuppressin, natalisin, neuropeptide-like precursor (NPLP) 1, orcokinin, pigment dispersing factor (PDF), RYamide, SIFamide, short neuropeptide F (sNPF) and tachykinin. In addition, propeptides, truncated and spacer peptides derived from seven additional precursors were found, and include the precursors of allatostatin C, crustacean cardioactive peptide, corticotropin releasing factor-like diuretic hormone (CRF/DH), ecdysis triggering hormone (ETH), ion transport peptide (ITP), neuropeptide F, and proctolin, respectively. The majority of the identified neuropeptides are present in the central nervous system, with only a limited number of peptides in the corpora cardiaca–corpora allata and midgut. Owing to the large number of identified peptides, this study can be used as a reference for comparative studies in other insects. Graphical Abstractᅟ

New genetic regulators question relevance of abundant yolk protein production in C. elegans

Vitellogenesis or maternal yolk formation is considered critical to the reproduction of egg-laying animals. In invertebrates, however, most of its regulatory genes are still unknown. Via a combined mapping and whole-genome sequencing strategy, we performed a forward genetic screen to isolate novel regulators of yolk production in the nematode model system Caenorhabditis elegans. In addition to isolating new alleles of rab-35, rab-10 and M04F3.2, we identified five mutant alleles corresponding to three novel regulatory genes potently suppressing the expression of a GFP-based yolk reporter. We confirmed that mutations in vrp-1, ceh-60 and lrp-2 disrupt endogenous yolk protein synthesis at the transcriptional and translational level. In contrast to current beliefs, our discovered set of mutants with strongly reduced yolk proteins did not show serious reproduction defects. This raises questions as to whether yolk proteins per se are needed for ultimate reproductive success.

Molecular characterization of a short neuropeptide F signaling system in the tsetse fly, Glossina morsitans morsitans

Neuropeptides of the short neuropeptide F (sNPF) family are widespread among arthropods and found in every sequenced insect genome so far. Functional studies have mainly focused on the regulatory role of sNPF in feeding behavior, although this neuropeptide family has pleiotropic effects including in the control of locomotion, osmotic homeostasis, sleep, learning and memory. Here, we set out to characterize and determine possible roles of sNPF signaling in the haematophagous tsetse fly Glossina morsitans morsitans, a vector of African Trypanosoma parasites causing human and animal African trypanosomiasis. We cloned the G. m. morsitans cDNA sequences of an sNPF-like receptor (Glomo-sNPFR) and precursor protein encoding four Glomo-sNPF neuropeptides. All four Glomo-sNPF peptides concentration-dependently activated Glomo-sNPFR in a cell-based calcium mobilization assay, with EC50 values in the nanomolar range. Gene expression profiles in adult female tsetse flies indicate that the Glomo-sNPF system is mainly restricted to the nervous system. Glomo-snpfr transcripts were also detected in the hindgut of adult females. In contrast to the Drosophila sNPF system, tsetse larvae lack expression of Glomo-snpf and Glomo-snpfr genes. While Glomo-snpf transcript levels are upregulated in pupae, the onset of Glomo-snpfr expression is delayed to adulthood. Expression profiles in adult tissues are similar to those in other insects suggesting that the tsetse sNPF system may have similar functions such as a regulatory role in feeding behavior, together with a possible involvement of sNPFR signaling in osmotic homeostasis. Our molecular data will enable further investigations into the functions of sNPF signaling in tsetse flies.

Characterization of G protein-coupled receptors by a fluorescence-based calcium mobilization assay.

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16 construct is co-transfected. Following ligand binding, activation of the Gα16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

Characterization of a neuropeptide F receptor in the tsetse fly, Glossina morsitans morsitans

Neuropeptides related to mammalian neuropeptide Y (NPY) and insect neuropeptide F (NPF) are conserved throughout Metazoa and intimately involved in a wide range of biological processes. In insects NPF is involved in regulating feeding, learning, stress and reproductive behavior. Here we identified and characterized an NPF receptor of the tsetse fly, Glossina morsitans morsitans, the sole transmitter of Trypanosoma parasites causing sleeping sickness. We isolated cDNA sequences encoding tsetse NPF (Glomo-NPF) and its receptor (Glomo-NPFR), and examined their spatial and temporal expression patterns using quantitative PCR. In tsetse flies, npfr transcripts are expressed throughout development and most abundantly in the central nervous system, whereas low expression is found in the flight muscles and posterior midgut. Expression of npf, by contrast, shows low transcript levels during development but is strongly expressed in the posterior midgut and brain of adult flies. Expression of Glomo-npf and its receptor in the brain and digestive system suggests that NPF may have conserved neuromodulatory or hormonal functions in tsetse flies, such as in the regulation of feeding behavior. Cell-based activity studies of the Glomo-NPFR showed that Glomo-NPF activates the receptor up to nanomolar concentrations. The molecular data of Glomo-NPF and Glomo-NPFR paves the way for further investigation of its functions in tsetse flies.