Rudimentary expression of RYamide in Drosophila melanogaster relative to other Drosophila species points to a functional decline of this neuropeptide gene.

RYamides are arthropod neuropeptides with unknown function. In 2011 two RYamides were isolated from D. melanogaster as the ligands for the G-protein coupled receptor CG5811. The D. melanogaster gene encoding these neuropeptides is highly unusual, as there are four RYamide encoding exons in the current genome assembly, but an exon encoding a signal peptide is absent. Comparing the D. melanogaster gene structure with those from other species, including D. virilis, suggests that the gene is degenerating. RNAseq data from 1634 short sequence read archives at NCBI containing more than 34 billion spots yielded numerous individual spots that correspond to the RYamide encoding exons, of which a large number include the intron-exon boundary at the start of this exon. Although 72 different sequences have been spliced onto this RYamide encoding exon, none codes for the signal peptide of this gene. Thus, the RNAseq data for this gene reveal only noise and no signal. The very small quantities of peptide recovered during isolation and the absence of credible RNAseq data, indicates that the gene is very little expressed, while the RYamide gene structure in D. melanogaster suggests that it might be evolving into a pseudogene. Yet, the identification of the peptides it encodes clearly shows it is still functional. Using region specific antisera, we could localize numerous neurons and enteroendocrine cells in D. willistoni, D. virilis and D. pseudoobscura, but only two adult abdominal neurons in D. melanogaster. Those two neurons project to and innervate the rectal papillae, suggesting that RYamides may be involved in the regulation of water homeostasis.

Veenstra JA ，Khammassi H 《-》

Identification of the Drosophila and Tribolium receptors for the recently discovered insect RYamide neuropeptides.

One year ago, we discovered a new family of insect RYamide neuropeptides, which has the C-terminal consensus sequence FFXXXRYamide, and which is widely occurring in most insects, including the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum (F. Hauser et al., J. Proteome Res. 9 (2010) 5296-5310). Here, we identify a Drosophila G-protein-coupled receptor (GPCR) coded for by gene CG5811 and its Tribolium GPCR ortholog as insect RYamide receptors. The Drosophila RYamide receptor is equally well activated (EC(50), 1×10(-9)M) by the two Drosophila RYamide neuropeptides: RYamide-1 (PVFFVASRYamide) and RYamide-2 (NEHFFLGSRYamide), both contained in a preprohormone coded for by gene CG40733. The Tribolium receptor shows a somewhat higher affinity to Tribolium RYamide-2 (ADAFFLGPRYamide; EC(50), 5×10(-9)M) than to Tribolium RYamide-1 (VQNLATFKTMMRYamide; EC(50), 7×10(-8)M), which might be due to the fact that the last peptide does not completely follow the RYamide consensus sequence rule. There are other neuropeptides in insects that have similar C-terminal sequences (RWamide or RFamide), such as the FMRFamides, sulfakinins, myosuppressins, neuropeptides F, and the various short neuropeptides F. Amazingly, these neuropeptides show no cross-reactivity to the Tribolium RYamide receptor, while the Drosophila RYamide receptor is only very slightly activated by high concentrations (>10(-6)M) of neuropeptide F and short neuropeptide F-1, showing that the two RYamide receptors are quite specific for activation by insect RYamides, and that the sequence FFXXXRYamide is needed for effective insect RYamide receptor activation. Phylogenetic tree analyses and other amino acid sequence comparisons show that the insect RYamide receptors are not closely related to any other known insect or invertebrate/vertebrate receptors, including mammalian neuropeptide Y and insect neuropeptide F and short neuropeptide F receptors. Gene expression data published in Flybase (www.flybase.org) show that the Drosophila CG5811 gene is significantly expressed in the hindgut of adult flies, suggesting a role of insect RYamides in digestion or water reabsorption.

Collin C ，Hauser F ，Krogh-Meyer P ，Hansen KK ，Gonzalez de Valdivia E ，Williamson M ，Grimmelikhuijzen CJ ... -  《-》

Interspecific comparison of a Drosophila gene encoding FMRFamide-related neuropeptides.

Taghert PH ，Schneider LE 《-》

Purification and characterization of bioactive peptides RYamide and CCHamide in the kuruma shrimp Marsupenaeus japonicus.

To understand the regulation systems of appetite, bioactive peptides from the kuruma shrimp Marsupenaeus japonicus (Mj) were isolated and purified by reverse pharmacological assays using CHO cells expressing the Drosophila melanogaster G-protein-coupled receptors (GPCRs) CG5811 (a RYamide receptor) or CG14593 (a CCHamide-2 receptor). Four peptides having binding activity to GPCRs were obtained and named Mj RYamide-1, Mj RYamide-2, Mj RYamide-3, and Mj CCHamide. Genes encoding the prepropeptides of these peptides were identified using kuruma shrimp transcriptome databases. The Mj prepro-RYamide gene encodes a 130-amino acid polypeptide containing Mj RYamide-1, Mj RYamide-2, and Mj RYamide-3, whereas the Mj prepro-CCHamide gene encodes a 119-amino acid polypeptide containing a single Mj CCHamide peptide. The expression of these genes was confirmed in various neuronal organs including the brain and ventral nerve cord. In addition, prepro-RYamide gene expression is significantly reduced in the brain after starvation. RYamides may thus be associated with regulation of feeding or digestion. Changes in kayak (the c-fos ortholog in invertebrates) gene expression after administration of synthetic peptides were also investigated. Mj kayak expression levels are upregulated in hepatopancreas after treatment with Mj RYamide-3 or CCHamide. Thus, the peptides isolated in this study may have some regulatory effect on cellular metabolism in aquacultured invertebrates.

Mekata T ，Kono T ，Satoh J ，Yoshida M ，Mori K ，Sato T ，Miyazato M ，Ida T ... -  《-》

Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila.

A number of bioactive peptides are involved in regulating a wide range of animal behaviors, including food consumption. Vertebrate neuropeptide Y (NPY) is a potent stimulator of appetitive behavior. Recently, Drosophila neuropeptide F (dNPF) and short NPF (sNPF), the Drosophila homologs of the vertebrate NPY, were identified to characterize the functions of NPFs in the feeding behaviors of this insect. Dm-NPFR1 and NPFR76F are the receptors for dNPF and sNPF, respectively; both receptors are G protein-coupled receptors (GPCRs). Another GPCR (CG5811; NepYR) was indentified in Drosophila as a neuropeptide Y-like receptor. Here, we identified 2 ligands of CG5811, dRYamide-1 and dRYamide-2. Both peptides are derived from the same precursor (CG40733) and have no significant structural similarities to known bioactive peptides. The C-terminal sequence RYamide of dRYamides is identical to that of NPY family peptides; on the other hand, dNPF and sNPF have C-terminal RFamide. When administered to blowflies, dRYamide-1 suppressed feeding motivation. We propose that dRYamides are related to the NPY family in vertebrates, similar to dNPF and sNPF.

Ida T ，Takahashi T ，Tominaga H ，Sato T ，Kume K ，Ozaki M ，Hiraguchi T ，Maeda T ，Shiotani H ，Terajima S ，Sano H ，Mori K ，Yoshida M ，Miyazato M ，Kato J ，Murakami N ，Kangawa K ，Kojima M ... -  《-》