Neuropeptide Receptor Transcriptome Reveals Unidentified Neuroendocrine Pathways

Neuropeptides are an important class of molecules involved in diverse aspects of metazoan development and homeostasis. Insects are ideal model systems to investigate neuropeptide functions, and the major focus of insect neuropeptide research in the last decade has been on the identification of their receptors. Despite these vigorous efforts, receptors for some key neuropeptides in insect development such as prothoracicotropic hormone, eclosion hormone and allatotropin (AT), remain undefined. In this paper, we report the comprehensive cloning of neuropeptide G protein-coupled receptors from the silkworm, Bombyx mori, and systematic analyses of their expression. Based on the expression patterns of orphan receptors, we identified the long-sought receptor for AT, which is thought to stimulate juvenile hormone biosynthesis in the corpora allata (CA). Surprisingly, however, the AT receptor was not highly expressed in the CA, but instead was predominantly transcribed in the corpora cardiaca (CC), an organ adjacent to the CA. Indeed, by using a reverse-physiological approach, we purified and characterized novel allatoregulatory peptides produced in AT receptor-expressing CC cells, which may indirectly mediate AT activity on the CA. All of the above findings confirm the effectiveness of a systematic analysis of the receptor transcriptome, not only in characterizing orphan receptors, but also in identifying novel players and hidden mechanisms in important biological processes. This work illustrates how using a combinatorial approach employing bioinformatic, molecular, biochemical and physiological methods can help solve recalcitrant problems in neuropeptide research

Peptidergic control in a fruit crop pest: The spotted-wing drosophila, Drosophila suzukii

Neuropeptides play an important role in the regulation of feeding in insects and offer potential targets for the development of new chemicals to control insect pests. A pest that has attracted much recent attention is the highly invasive Drosophila suzukii, a polyphagous pest that can cause serious economic damage to soft fruits. Previously we showed by mass spectrometry the presence of the neuropeptide myosuppressin (TDVDHVFLRFamide) in the nerve bundle suggesting that this peptide is involved in regulating the function of the crop, which in adult dipteran insects has important roles in the processing of food, the storage of carbohydrates and the movement of food into the midgut for digestion. In the present study antibodies that recognise the C-terminal RFamide epitope of myosuppressin stain axons in the crop nerve bundle and reveal peptidergic fibres covering the surface of the crop. We also show using an in vitro bioassay that the neuropeptide is a potent inhibitor (EC50 of 2.3 nM) of crop contractions and that this inhibition is mimicked by the non-peptide myosuppressin agonist, benzethonium chloride (Bztc). Myosuppressin also inhibited the peristaltic contractions of the adult midgut, but was a much weaker agonist (EC50 = 5.7 μM). The oral administration of Bztc (5 mM) in a sucrose diet to adult female D. suzukii over 4 hours resulted in less feeding and longer exposure to dietary Bztc led to early mortality. We therefore suggest that myosuppressin and its cognate receptors are potential targets for disrupting feeding behaviour of adult D. suzukii

Molecular Evolution of the Neuropeptide S Receptor

The neuropeptide S receptor (NPSR) is a recently deorphanized member of the G protein-coupled receptor (GPCR) superfamily and is activated by the neuropeptide S (NPS). NPSR and NPS are widely expressed in central nervous system and are known to have crucial roles in asthma pathogenesis, locomotor activity, wakefulness, anxiety and food intake. The NPS-NPSR system was previously thought to have first evolved in the tetrapods. Here we examine the origin and the molecular evolution of the NPSR using in-silico comparative analyses and document the molecular basis of divergence of the NPSR from its closest vertebrate paralogs. In this study, NPSR-like sequences have been identified in a hemichordate and a cephalochordate, suggesting an earlier emergence of a NPSR-like sequence in the metazoan lineage. Phylogenetic analyses revealed that the NPSR is most closely related to the invertebrate cardioacceleratory peptide receptor (CCAPR) and the group of vasopressin-like receptors. Gene structure features were congruent with the phylogenetic clustering and supported the orthology of NPSR to the invertebrate NPSR-like and CCAPR. A site-specific analysis between the vertebrate NPSR and the well studied paralogous vasopressin-like receptor subtypes revealed several putative amino acid sites that may account for the observed functional divergence between them. The data can facilitate experimental studies aiming at deciphering the common features as well as those related to ligand binding and signal transduction processes specific to the NPSR

Differential Modulation of Beta-Adrenergic Receptor Signaling by Trace Amine-Associated Receptor 1 Agonists

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes

Genome Sequence of the Pea Aphid Acyrthosiphon pisum

Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems

Elucidation of signaling properties of vasopressin receptor-related receptor 1 by using the chimeric receptor approach

The identification of endogenous or surrogate ligands for orphan G protein-coupled receptors (GPCRs) represents one of the most important tasks in GPCR biology and pharmacology. The challenge lies in choosing an appropriate assay in the absence of ways to activate the receptor of interest. We investigated the signaling pathway for an orphan GPCR referred to here as vasopressin receptor-related receptor 1 (VRR1) by generating a chimeric receptor, V1a/VRR1. The engineered construct contained vasopressin V1a receptor with all three intracellular loops and C terminus replaced by those of VRR1. The chimera behaved like a typical GPCR when transiently and stably expressed in mammalian cell lines based on radioligand binding and receptor internalization studies. Upon arginine vasopressin stimulation, this chimeric receptor induced robust calcium mobilization and increase of adenylate cyclase activity. The observed signaling activities are through the activation of the chimera instead of endogenously expressed receptors, as single amino acid changes in the second transmembrane regions of the chimera drastically reduced receptor efficacy and potency. Our results suggest that VRR1 has dual signaling properties in coupling to both G(q) and G(S) pathways. Analysis of native VRR1 receptor signaling pathway by using a recently identified ligand for VRR1 confirmed this conclusion and therefore validated the utility of the chimeric receptor approach for signaling pathway identification

The effect of a selective octopamine antagonist, epinastine, on pharyngeal pumping in Caenorhabditis elegans

This paper investigates the effect of epinastine, a selective octopamine antagonist in invertebrates, in Caenorhabditis elegans. Specifically, its ability to block the inhibitory action of octopamine on C. elegans-isolated pharynx was assayed. Isolated pharynxes were stimulated to pump by the addition of 500 nM 5-hydroxytryptamine (5-HT) (113 ± 2 per 30 s, n = 15). Octopamine inhibited the 5-HT-induced pumping in a concentration-dependent manner (threshold 1-5 ?M) with a 61 ± 11% inhibition with 50 ?M (n = 5). Epinastine (0.1 ?M) antagonized the inhibitory response to octopamine (P < 0.001; n = 15). Tyramine also inhibited pharyngeal pumping induced by 5-HT but was less potent than octopamine. Tyramine, 50 ?M to 1 mM, gave a transient inhibition e.g. of 40 ± 5% at 50 ?M (n = 5). A higher (10 ?M) concentration of epinastine was required to block the tryamine response compared with octopamine. It is concluded that epinastine selectively antagonizes the effect of octopamine on C. elegans pharynx. Further studies are required to test its selectivity for octopamine in other tissues and other nematode

Human chitotriosidase CHIT1 cross reacts with mammalian-like substrates

AbstractHumans do not synthesize chitin, yet they produce a number of active and inactive chitinases. One of the active enzymes is chitotriosidase whose serum levels are elevated in a number of diseases such as Gaucher’s disease and upon fungal infection. Since the biological role of chitotriosidase in disease pathogenesis is not understood we screened a panel of mammalian GlcNAc-containing glycoconjugates as alternate substrates. LacNAc and LacdiNAc-terminating substrates are hydrolyzed, the latter with a turnover comparable to that of pNP-chitotriose. Glycolipids or glycoproteins with LacNAc and LacdiNAc represent potential chitinase substrates and the subsequent alteration of glycosylation pattern could be a factor in disease pathogenesis