Characterization of the short neuropeptide F receptor from Drosophila melanogaster

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y(2) receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs.     

A comparative review of short and long neuropeptide F signaling in invertebrates: Any similarities to vertebrate neuropeptide Y signaling?

Neuropeptides referred to as neuropeptide F (NPF) and short neuropeptide F (sNPF) have been identified in numerous invertebrate species. Sequence information has expanded tremendously due to recent genome sequencing and EST projects. Analysis of sequences of the peptides and prepropeptides strongly suggest that NPFs and sNPFs are not closely related. However, the NPFs are likely to be ancestrally related to the vertebrate family of neuropeptide Y (NPY) peptides. Peptide diversification may have been accomplished by different mechanisms in NPFs and sNPFs; in the former by gene duplications followed by diversification and in the sNPFs by internal duplications resulting in paracopies of peptides. We discuss the distribution and functions of NPFs and their receptors in several model invertebrates. Signaling with sNPF, however, has been investigated mainly in insects, especially in Drosophila. Both in invertebrates and in mammals NPF/NPY play roles in feeding, metabolism, reproduction and stress responses. Several other NPF functions have been studied in Drosophila that may be shared with mammals. In Drosophila sNPFs are widely distributed in numerous neurons of the CNS and some gut endocrines and their functions may be truly pleiotropic. Peptide distribution and experiments suggest roles of sNPF in feeding and growth, stress responses, modulation of locomotion and olfactory inputs, hormone release, as well as learning and memory. Available data indicate that NPF and sNPF signaling systems are distinct and not likely to play redundant roles.     

The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

Neuropeptides and their receptors play vital roles in controlling the physiology and behavior of animals. Short neuropeptide F (sNPF) signaling regulates several physiological processes in insects such as feeding, locomotion, circadian rhythm and reproduction, among others. Previously, the red imported fire ant (Solenopsis invicta) sNPF receptor (S. invicta sNPFR), a G protein-coupled receptor, was immunolocalized in queen and worker brain and queen ovaries. Differential distribution patterns of S. invicta sNPFR protein in fire ant worker brain were associated both with worker subcastes and with presence or absence of brood in the colony. However, the cognate ligand for this sNPFR has not been characterized and attempts to deorphanize the receptor with sNPF peptides from other insect species which ended in the canonical sequence LRLRFamide, failed. Receptor deorphanization is an important step to understand the neuropeptide receptor downstream signaling cascade. We cloned the full length cDNA of the putative S. invicta sNPF prepropeptide and identified the putative “sNPF” ligand within its sequence. The peptide ends with an amidated Tyr residue whereas in other insect species sNPFs have an amidated Phe or Trp residue at the C-terminus. We stably expressed the HA-tagged S. invicta sNPFR in CHO-K1 cells. Two S. invicta sNPFs differing at their N-terminus were synthesized that equally activated the sNPFR, SLRSALAAGHLRYa (EC₅₀ = 3.2 nM) and SALAAGHLRYa (EC₅₀ = 8.6 nM). Both peptides decreased the intracellular cAMP concentration, indicating signaling through the G_αi-subunit. The receptor was not activated by sNPF peptides from other insect species, honey bee long NPF (NPY) or mammalian PYY. Further, a synthesized peptide otherwise identical to the fire ant sequence but in which the C-terminal amidated amino acid residue ‘Y’ was switched to ‘F’, failed to activate the sNPFR. This discovery will now allow us to investigate the function of sNPY and its cognate receptor in fire ant biology.     

Distribution of short neuropeptide F and its receptor in neuronal circuits related to feeding in larval Drosophila

Four forms of short neuropeptide F (sNPF1–4), derived from the gene snpf, have been identified in Drosophila and are known to act on a single G-protein-coupled receptor (sNPFR). Several functions have been suggested for sNPFs in Drosophila, including the regulation of feeding and growth in larvae, the control of insulin signalling and the modulation of neuronal circuits in adult flies. Furthermore, sNPF has been shown to act as a nutritional state-dependent neuromodulator in the olfactory system. The role of sNPF in the larval nervous system is less well known. To analyse sites of action of sNPF in the larva, we mapped the distribution of sNPF- and sNPFR-expressing neurons. In particular, we studied circuits associated with chemosensory inputs and systems involved in the regulation of feeding, including neurosecretory cell systems and the hypocerebral ganglion. We employed a combination of immunocytochemistry and enhancer trap and promoter Gal4 lines to drive green fluorescent protein. We found a good match between the distribution of the receptor and its ligand. However, several differences between the larval and adult systems were observed. Thus, neither sNPF nor its receptor was found in the olfactory (or other sensory) systems in the larva and cells producing insulin-like peptides did not co-express sNPFR, as opposed to results from adults. Moreover, sNPF was expressed in a subpopulation of Hugin cells (second-order gustatory neurons) only in adult flies. We propose that the differences in sNPF signalling between the developmental stages is explained by differences in their feeding behaviour.     

Functional Characterization of the Short Neuropeptide F Receptor in the Desert Locust,Schistocerca gregaria

Whereas short neuropeptide F (sNPF) has already been reported to stimulate feeding behaviour in a variety of insect species, the opposite effect was observed in the desert locust. In the present study, we cloned a G protein-coupled receptor (GPCR) cDNA from the desert locust, Schistocerca gregaria. Cell-based functional analysis of this receptor indicated that it is activated by both known isoforms of Schgr-sNPF in a concentration dependent manner, with EC₅₀ values in the nanomolar range. This Schgr-sNPF receptor constitutes the first functionally characterized peptide GPCR in locusts. The in vivo effects of the sNPF signalling pathway on the regulation of feeding in locusts were further studied by knocking down the newly identified Schgr-sNPF receptor by means of RNA interference, as well as by means of peptide injection studies. While injection of sNPF caused an inhibitory effect on food uptake in the desert locust, knocking down the corresponding peptide receptor resulted in an increase of total food uptake when compared to control animals. This is the first comprehensive study in which a clearly negative correlation is described between the sNPF signalling pathway and feeding, prompting a reconsideration of the diverse roles of sNPFs in the physiology of insects.     

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.     

Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. Here we show that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. We show that these effects are mediated through activation of extracellular signal-related kinases (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan.     

Neuropeptides of the cotton fleahopper, Pseudatomoscelis seriatus (Reuter)

The cotton fleahopper, Pseudatomoscelis seriatus (Reuter), is an economically important pest of cotton, and increasing concerns over resistance, detrimental effects on beneficial insects and safety issues associated with traditional insecticide applications have led to an interest in research on novel, alternative strategies for control. One such approach requires a more basic understanding of the neurohormonal system that regulates important physiological properties of the fleahopper; e.g. the expression of specific messenger molecules such as neuropeptides. Therefore we performed a peptidomic study of neural tissues from the fleahopper which led to the first identification of the sequences of native peptide hormones. These peptide hormones include the following neuropeptides: corazonin, short neuropeptide F (sNPF), myosuppressin, CAPA-pyrokinin and CAPA-PVK peptides. The CAPA-pyrokinin, sNPF, and CAPA-PVK peptides represent novel sequences. A comparison of fleahopper neuropeptides with those of related heteropteran species indicates that they are quite different. The sNPF of P. seriatus shows, among others, a novel substitution of Leu with Phe within the C-terminal region; a modification that sets it apart from the known sNPFs of not only other Heteroptera but of other arthropod species as well. The identity of the neuropeptides native to the fleahopper can aid in the potential development of biostable, bioavailable mimetic agonists and antagonists capable of disrupting the physiological functions that these neuropeptides regulate.     

Drosophila short neuropeptide F regulates food intake and body size

Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. Here, we report the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster.     

Functional and Genetic Characterization of Neuropeptide Y-Like Receptors in Aedes aegypti

Background

Female Aedes aegypti mosquitoes are the principal vector for dengue fever, causing 50–100 million infections per year, transmitted between human and mosquito by blood feeding. Ae. aegypti host-seeking behavior is known to be inhibited for three days following a blood meal by a hemolymph-borne humoral factor. Head Peptide-I is a candidate peptide mediating this suppression, but the mechanism by which this peptide alters mosquito behavior and the receptor through which it signals are unknown.

Methodology/Principal Findings

Head Peptide-I shows sequence similarity to short Neuropeptide-F peptides (sNPFs) that have been implicated in feeding behaviors and are known to signal through Neuropeptide Y (NPY)-Like Receptors (NPYLRs). We identified eight NPYLRs in the Ae. aegypti genome and screened each in a cell-based calcium imaging assay for sensitivity against a panel of peptides. Four of the Ae. aegypti NPYLRs responded to one or more peptide ligands, but only NYPLR1 responded to Head Peptide-I as well as sNPFs. Two NPYLR1 homologues identified in the genome of the Lyme disease vector, Ixodes scapularis, were also sensitive to Head Peptide-I. Injection of synthetic Head Peptide-I and sNPF-3 inhibited host-seeking behavior in non-blood-fed female mosquitoes, whereas control injections of buffer or inactive Head Peptide-I [Cys10] had no effect. To ask if NPYLR1 is necessary for blood-feeding-induced host-seeking inhibition, we used zinc-finger nucleases to generate five independent npylr1 null mutant strains and tested them for behavioral abnormalities. npylr1 mutants displayed normal behavior in locomotion, egg laying, sugar feeding, blood feeding, host seeking, and inhibition of host seeking after a blood meal.

Conclusions

In this work we deorphanized four Ae. aegypti NPYLRs and identified NPYLR1 as a candidate sNPF receptor that is also sensitive to Head Peptide-I. Yet npylr1 alone is not required for host-seeking inhibition and we conclude that other receptors, additional peptides, or both, regulate this important behavior.     

Minibrain/Dyrk1a Regulates Food Intake through the Sir2-FOXO-sNPF/NPY Pathway in Drosophila and Mammals

Feeding behavior is one of the most essential activities in animals, which is tightly regulated by neuroendocrine factors. Drosophila melanogaster short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding regulation. Here, we found that minibrain (mnb) and the mammalian ortholog Dyrk1a target genes of sNPF and NPY signaling and regulate food intake in Drosophila melanogaster and mice. In Drosophila melanogaster neuronal cells and mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway. Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased FOXO acetylation and increased NPY expression in the hypothalamus, as well as increased food intake. Our findings demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in Drosophila melanogaster and mammals.     

Structure and ligand specificity of the Drosophila melanogaster insulin receptor.

The insulin-binding and protein tyrosine kinase subunits of the Drosophila melanogaster insulin receptor homolog have been identified and characterized by using antipeptide antibodies elicited to the deduced amino acid sequence of the alpha and beta subunits of the human insulin receptor. In D. melanogaster embryos and cell lines, the insulin receptor contains insulin-binding alpha subunits of 110 or 120 kilodaltons (kDa), a 95-kDa beta subunit that is phosphorylated on tyrosine in response to insulin in intact cells and in vitro, and a 170-kDa protein that may be an incompletely processed receptor. All of the components are synthesized from a proreceptor, joined by disulfide bonds, and exposed on the cell surface. The beta subunit is recognized by an antipeptide antibody elicited to amino acids 1142 to 1162 of the human insulin proreceptor, and the alpha subunit is recognized by an antipeptide antibody elicited to amino acids 702 to 723 of the human proreceptor. Of the polypeptide ligands tested, only insulin reacts with the D. melanogaster receptor. Insulinlike growth factors type I and II, epidermal growth factor, and the silkworm insulinlike prothoracicotropic hormone are unable to stimulate autophosphorylation. Thus despite the evolutionary divergence of vertebrates and invertebrates, the essential features of the structure and intrinsic functions of the insulin receptor have been remarkably conserved.     

Fluorescence analysis of the size of a binding pocket of a peptide receptor at natural abundance

We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.     

Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different

Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F(1)beta pre-sequence, peptides derived from the F(1)beta pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F(1)beta pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P(1) position and small, uncharged amino acids or serine residues in the P'(1) position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the alpha-helical elements of the F(1)beta pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic alpha-helix and positively charged amino acid residues and degraded the F(1)beta pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F(1)beta pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.     

The chicken IL-1 receptor: differential evolution of the cytoplasmic and extracellular domains.

The evolutionary conservation of a sequence or part of it can help to identify the essential functional and structural domains within a protein. We have cloned and characterised a cDNA coding for the type-I interleukin-1 receptor (IL-1R) of chick (ch) embryo fibroblasts. The comparison of the amino acid (aa) sequences of the avian with that of murine (m) and human (h) IL-1Rs shows a 60% homology. The intracellular domain is the most conserved region of the chIL-1R, showing 76-79% homology to the murine and human sequences, respectively. The striking conservation of the cytoplasmic region of the receptor is confirmed by its homology with the Toll receptor protein of Drosophila melanogaster. The alignment between the chicken and D. melanogaster proteins shows the presence of four aa blocks with more than 80% homology. The possible functional significance of this homology is discussed. The extracellular binding region of the receptor has a clearly recognisable immunoglobulin-like structure although the sequence divergence is higher than in the cytoplasmic domain.     

Selective cleavage of proenkephalin-derived peptides (less than 23,300 daltons) by plasma kallikrein

The ability of human plasma kallikrein to hydrolyze several proenkephalin-derived peptides has been studied, including the synthetic peptides BAM 12P and peptides E, F, and B as well as synenkephalin-containing peptides (8.6, 18.2, and 23.3 kDa) purified from bovine adrenal medulla chromaffin granules. All the identified cleavages occurred either COOH-terminal to or between pairs of basic amino acids, with plasma kallikrein recognizing Lys-Lys, Lys-Arg, and Arg-Arg as processing signals. Moreover, plasma kallikrein was found to cleave at the COOH terminus of the basic pairs of amino acids preceding enkephalin sequences thereby releasing the biologically active form of the peptide with the free NH2-terminal Tyr needed for receptor recognition.