InteractiveFly: GeneBrief

short neuropeptide F precursor: Biological Overview | References

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. This study shows 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. These effects are mediated through activation of extracellular signal-related kinase (ERK; Rolled in Drosophila) 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 (Lee, 2008).

Neuropeptides regulate a wide range of animal behaviours related to nutrition. In particular, mammalian NPY produced in the hypothalamus of the brain controls food consumption. NPY injection in the hypothalamus of rats produces hyperphagia and obese phenotypes (Emeson, 2005; Wahlestedt, 1993). The Drosophila orthologue of NPY is sNPF. This peptide is expressed in the nervous system and it regulates food intake and body size; overexpression of sNPF produces bigger and heavier flies (Lee, 2004). Likewise, the G-protein-coupled receptor of sNPF (sNPFR1) is expressed in neurons and shows significant similarity with vertebrate NPY receptors (Feng, 2003). In mammals, however, little is known about how NPY and sNPF systemically modulate growth, metabolism and lifespan. This study shows that these neuropeptides control expression of insulin-like peptides and subsequently affect insulin signalling in target tissues (Lee, 2008).

Initially the effects of sNPF and sNPFR1 on body size were characterized by measuring the length of flies from head to abdomen. The body size of sNPF hypomorphic Drosophila mutants (sNPF^c00448) was 23% of that of the wild-type, whereas overexpressing two copies of the sNPF in the sensory neurons and sensory structures of the nervous systems by MJ94-Gal4 (MJ94>2XsNPF) increased body size by 24%. Similar changes were seen in the overall size of adult wings, which resulted from changes in both cell size and number. Effects on body size were associated with sNPF expression levels: relative to wild type, sNPF levels were 3.5-fold higher in MJ94>2XsNPF and less than half of the wild type in sNPF^c00448. In contrast to the effect of sNPF on body size, there was little effect on size from repression or overexpression of the sNPF receptor in MJ94-expressing cells (Lee, 2008).

Drosophila insulin-like peptides (Dilps) modulate growth and adult size; therefore, whether sNPF has a role in insulin-producing neurons was tested. For positive controls, Dilp2 was overexpressed in insulin-producing cells (IPCs) through Dilp2-Gal4, which increased body size, and the IPCs were ablated by expression of Dilp2>reaper to decrease body size. To investigate sNPF signalling, sNPFR1 was overexpressed in the IPCs and a 10% increase in body size was observed. Conversely, expression of the sNPFR1 dominant-negative mutant (Dilp2>sNPFR1-DN) reduced body size by 14%. Manipulation of the sNPF ligand with IPCs expressing Dilp2-Gal4, however, did not affect body size: flies overexpressing sNPF (Dilp2>2XsNPF) or in which sNPF was silenced by RNAi (Dilp2>sNPF-Ri) were of similar size to the wild type. Taken together, these results suggest that sNPF peptide may be secreted from MJ94-expressing sensory neurons and activate sNPFR1 of Dilp2-expressing IPCs (Lee, 2008).

To assess this model, the sNPF ligand and sNPFR1 receptor were visualized in the larval brain. Seven IPCs were detected in each brain hemisphere using the marker Dilp2-Gal4>nGFP. Neurons containing sNPF peptide in the axon terminal and cell body (sNPFnergic neurons) were stained adjacent to these IPCs. As expected, sNPFR1 receptors were localized in the plasma membrane of IPCs marked with Dilp2>DsRed. sNPFR1 was also localized in the neurons of the larval brain hemispheres, sub-oesophagus ganglion, ventral abdominal neurons and descending axons in the ventral ganglion (Lee, 2008).

To study genetic interactions between sNPFR1 and Dilps in IPCs, Dilp1 and Dilp2 interference mutants were generated in the sNPFR1 overexpression background. In contrast to the 10% body size increase by sNPFR1 overexpression in IPCs (Dilp2>sNPFR1), inhibition of Dilp1 and Dilp2 in IPCs (Dilp2>Dilp1-Ri and Dilp2>Dilp2-Ri) generated reduced body size by 10% and 15%, respectively. Inhibition of Dilp1 and Dilp2 with sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+Dilp1-Ri and Dilp2> sNPFR1+Dilp2-Ri) also generated a reduction in body size of 8% and 13%, indicating that Dilp1 and Dilp2 are downstream genes of sNPFR1 in IPCs for regulating body size (Lee, 2008).

To test whether sNPF regulates Dilp expression in larval IPCs, expression of Dilp1, 2, 3 and 5 were assessed in sNPF mutants. Neuronal overexpression of sNPF (MJ94>2XsNPF) markedly increased expression of Dilp2 in IPCs; it also produced novel Dilp2 expression outside of these cells. As expected, reduction of sNPF by MJ94>sNPF-Ri inhibited expression of Dilp2. In common with Dilp2, the expression of Dilp1 was positively regulated by sNPF overexpression and reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPF^c00448). Consistent with the model, expression of Dilp1 and Dilp2 was increased more than fourfold with overexpression of the receptor in IPCs (Dilp2>sNPFR1) and decreased by half with inhibition of the receptor gene in IPCs (Dilp2>sNPFR1-DN). Larval IPCs also express Dilp3 and Dilp5. Expression of Dilp3 was reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPF^c00448) but expression of Dilp5 was not regulated by any sNPF mutants. There are few functions known to distinguish these various insulin-like peptides. Nutrition-dependent growth regulation is associated with expression of Dilp3 and Dilp5, but not with that of Dilp2. Recent reports show that Dilp2 is reduced in long-lived flies expressing dFOXO or Jun-N-terminal kinase (JNK), whereas Dilp5 is uniquely upregulated upon dietary restriction that increases lifespan (Lee, 2008).

To investigate how Drosophila sNPF regulates Dilp expression, the activation of Drosophila MAP kinase signalling, which includes the action of ERK (encoded by Rolled) and JNK, was measured. sNPF overexpression with MJ94-Gal4 increased phospho-activated pERK relative to basal ERK1/2. Expression of the receptor protein sNPFR1 in IPCs also increased pERK. There were no detectable changes in phospho-activated pJNK in these sNPF and sNPFR1 mutants. Next, whether ERK activation in IPCs was sufficient to induce Dilp expression was tested. Expression of a constitutively active ERK in IPCs (Dilp2>rolled^SEM) increased expression of Dilp1 and Dilp2 more than threefold, and both transcripts were repressed less than half by the expression of an ERK inhibitory phosphatase DMKP-3 in IPCs (Dilp2>DMKP-3). In addition, the inhibition of ERK with the sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+DMKP-3) also repressed expression of Dilp1 and Dilp2 compared with that of sNPFR1 overexpression in IPCs (Dilp2>sNPFR1). These results indicate that sNPF and sNPFR1 signalling regulate ERK activation in IPCs, which in turn modulates expression of Dilp1 and Dilp2 (Lee, 2008).

To further examine the effect of sNPF on Dilp, Drosophila CNS-derived neural BG2-c6 cells, which endogenously express sNPFR1 were treated with a synthetic sNPF peptide. Dilp1 and Dilp2 were induced within 15 min, and the elevated transcript persisted for 1 h. Concomitant with this gene expression, sNPF-treated cells activated ERK. Importantly, sNPF did not induce Dilp expression significantly when cells were treated with ERK-specific kinase MEK inhibitor PD98059. To compare the functional conservation of sNPF and NPY in the regulation of insulin expression, similar tests were conduced with rat insulinoma INS-1 cells, which express NPY receptors NPYR1 and NPRY2. When treated with the human NPY peptide, expression of insulin1 and insulin2 and ERK was activated within 15 min. Furthermore, treatment with the MEK inhibitor PD98059 and NPY abolished the induction of insulin1 and insulin2. Together, these findings suggest that the regulation of insulin expression by sNPF or NPY through ERK is evolutionarily conserved in Drosophila and mammals (Lee, 2008).

To verify that sNPF induction of Dilp expression has a physiological consequence, insulin signals at a target tissue, the Drosophila fat body were examined. Fat body cells in flies with neuronal overexpression of sNPF (MJ94>2XsNP) were 42% larger than in the control, whereas inhibition of sNPF by MJ94>sNPF-Ri and sNPF^c00448 reduced cell size by 38% and 51% respectively. These differences in size correspond to changes in insulin signal transduction within the cells. Overexpression of sNPF (MJ94>sNPF and MJ94>2XsNPF) leads to phosphorylation and activation of Akt in the fat body, whereas the opposite effect was seen with neuronal inhibition of sNPF (MJ94>sNPF-Ri and sNPF^c00448). Activated Akt represses the transcription factor dFOXO by phosphorylation and subsequent cytoplasmic localization. In wild-type flies, dFOXO localized equally in the cytoplasm and nucleus. As predicted, neuronal induction of sNFP (MJ94>2XsNPF) increased the cytoplasmic localization of dFOXO, whereas inhibition of sNPF (MJ94>sNPF-Ri and sNPF^c00448) yielded fat body cells with dFOXO predominantly localized in the nucleus. Finally, dFOXO induces expression of the translational inhibitor d4E-BP, and, consistent with the current observations, expression of d4E-BP was elevated in animals where sNPF was inhibited (MJ94>sNPF-Ri and sNPF^c00448) and reduced in animals where sNPF was overexpressed (MJ94>2XsNPF) (Lee, 2008).

Besides cell growth, Drosophila insulin-like peptides modulate aspects of metabolism and ageing. For instance, ablation of the IPCs reduces animal size, elevates the level of haemolymph carbohydrates.Therefore trehalose and glucose were assessed in sNPF mutant flies. As predicted, both carbohydrates were reduced upon sNPF overexpression, and both were elevated in sNPF hypomorphs. Also the lifespan of sNPF mutants was measured. As expected, inhibition of sNPF by MJ94>sNPF-Ri increased median lifespan by 16-21%, whereas sNPF overexpression (MJ94>2XsNPF) did not affect lifespan in flies (Lee, 2008).

Overall, the effects on Dilp1 and Dilp2 expression in IPCs regulated by sNPF are associated with cellular, carbohydrate and lifespan responses that are predicted to be caused by changes in the actual level of available insulin peptides. It is concluded that sNPF ultimately regulates insulin secretion from the IPC to affect target tissue insulin/dFOXO signalling and thus modulate growth, metabolism and lifespan (Lee, 2008).

Regulation of food consumption by neuropeptides is a critical step for interventions for managing obesity and metabolic syndromes. Mammalian NPY is known to positively regulate appetite and has thus been thought to promote weight gain primarily by affecting food intake. Thus study revealed a novel physiological role for NPY that is conserved by sNPF of Drosophila. These neuropeptides can affect growth, metabolism and lifespan by modulating ERK-regulated transcription of insulin-like peptides. In Drosophila, sNPFnergic and IPC neurons are adjacent in the brain. This study found, however, that pancreatic β-cells are also responsive to NPY, which is of hypothalamic origin. Although the hypothalamic neurosecretory cells and responding pancreatic endocrine cells are spatially distinct in mammals, recent developmental analysis suggests a parallel developmental pathway for hypothalamic neurosecretory cells and the IPCs of Drosophila, raising the possibility of a common molecular mechanism for β-cell formation. This would suggest that β-cells are not only evolutionarily tied to the hypothalamic neurosecretory cells but also that they retain their functional relationship to their hypothalamic origin by regulating insulin in response to the neuropeptide NPY (Lee, 2008).

A large population of diverse neurons in the Drosophila central nervous system expresses short neuropeptide F, suggesting multiple distributed peptide functions

Insect neuropeptides are distributed in stereotypic sets of neurons that commonly constitute a small fraction of the total number of neurons. However, some neuropeptide genes are expressed in larger numbers of neurons of diverse types suggesting that they are involved in a greater diversity of functions. One of these widely expressed genes, snpf, encodes the precursor of short neuropeptide F (sNPF). To unravel possible functional diversity the distribution of transcript of the snpf gene and its peptide products wer mapped in the central nervous system (CNS) of Drosophila in relation to other neuronal markers. There are several hundreds of neurons in the larval CNS and several thousands in the adult Drosophila brain expressing snpf transcript and sNPF peptide. Most of these neurons are intrinsic interneurons of the mushroom bodies. Additionally, sNPF is expressed in numerous small interneurons of the CNS, olfactory receptor neurons (ORNs) of the antennae, and in a small set of possibly neurosecretory cells innervating the corpora cardiaca and aorta. A sNPF-Gal4 line confirms most of the expression pattern. None of the sNPF immunoreactive neurons co-express a marker for the transcription factor DIMMED, suggesting that the majority are not neurosecretory cells or large interneurons involved in episodic bulk transmission. Instead a portion of the sNPF producing neurons co-express markers for classical neurotransmitters such as acetylcholine, GABA and glutamate, suggesting that sNPF is a co-transmitter or local neuromodulator in ORNs and many interneurons. Interestingly, sNPF is coexpressed both with presumed excitatory and inhibitory neurotransmitters. A few sNPF expressing neurons in the brain colocalize the peptide corazonin and a pair of dorsal neurons in the first abdominal neuromere coexpresses sNPF and insulin-like peptide 7 (ILP7). It is likely that sNPF has multiple functions as neurohormone as well as local neuromodulator/co-transmitter in various CNS circuits, including olfactory circuits both at the level of the first synapse and at the mushroom body output level. Some of the sNPF immunoreactive axons terminate in close proximity to neurosecretory cells producing ILPs and adipokinetic hormone, indicating that sNPF also might regulate hormone production or release (Nässel, 2008).

Intrinsic neurons of Drosophila mushroom bodies express Short Neuropeptide F: Relations to extrinsic neurons expressing different neurotransmitters

Mushroom bodies constitute prominent paired neuropils in the brain of insects, known to be involved in higher olfactory processing and learning and memory. In Drosophila there are about 2,500 intrinsic mushroom body neurons, Kenyon cells, and a large number of different extrinsic neurons connecting the calyx, peduncle, and lobes to other portions of the brain. The neurotransmitter of the Kenyon cells has not been identified in any insect. This study shows expression of the gene snpf and its neuropeptide products (short neuropeptide F; sNPFs) in larval and adult Drosophila Kenyon cells by means of in situ hybridization and antisera against sequences of the precursor and two of the encoded peptides. Immunocytochemistry displays peptide in intrinsic neuronal processes in most parts of the mushroom body structures, except for a small core in the center of the peduncle and lobes and in the alpha'- and beta'-lobes. Weaker immunolabeling is seen in Kenyon cell bodies and processes in the calyx and initial peduncle and is strongest in the more distal portions of the lobes. Different antisera and Gal4-driven green fluorescent proteins were used to identify Kenyon cells and different populations of extrinsic neurons defined by their signal substances. Thus, neurotransmitter systems converging on Kenyon cells were displayed: neurons likely to utilize dopamine, tyramine/octopamine, glutamate, and acetylcholine. Attempts to identify other neurotransmitter components (including vesicular glutamate transporter) in Kenyon cells failed. However, it is likely that the Kenyon cells utilize an additional neurotransmitter, yet to be identified, and that the neuropeptides described in this study may represent cotransmitters (Johard, 2008).

What is the function of the snpf gene in Kenyon cells and is there more than one sNPF produced of the four peptides predicted by the precursor? Several peptide products from the sNPF neuropeptide precursor have been identified by mass spectrometry from different portions of the Drosophila nervous system (Predel, 2004; Baggerman, 2005; Garczynski, 2006; Wegener, 2006). Of these only sNPF-1, sNPF-14-11 and sNPF-212-19 were unequivocally identified (Predel, 2004; Wegener, 2006) and sNPF-14-11 was purified and sequenced in the present paper. Thus, the evidence for sNPF-3 and -4 (RLRWamides) is weaker because no fragmentation analysis was performed (Garczynski, 2006). However, the specific antiserum to sNPF-3 used in this study does label the same neurons as seen with that to the sNPF precursor. Therefore, unless the sNPF-3 antiserum recognizes the uncleaved peptide within the precursor, it is possible that one or both of the RWamides are also produced. In spite of their RLRWamide C-termini, both sNPF-3 and -4 can activate the sNPF receptor (Mertens, 2002; Feng, 2003; Garczynski, 2006; Johard, 2008 and references therein).

The identification of SPSLRLRFamide as the major immunoreactive product identifiable from whole fruit flies suggests that this is likely to be a predominant final product from this neuropeptide gene. In summary, this study considers the snpf-expressing neurons as producers of sNPF1 AQRSPSLRLRFamide and the sNPF1- and -2-derived peptides, SPSLRLRFamide, confirmed by peptide isolation and mass spectrometry. It is interesting in this respect to note that both vertebrate and insect types of convertases can be expected to cleave this peptide twice from the sNPF precursor (Johard, 2008).

So what are the functions of sNPFs? The first identification of an sNPF (designated Aedes head peptides) was in the mosquito Aedes aegypti and subsequently related peptides have been identified in several other insect specie. Given the widespread expression of this gene and its products in both interneurons and neurosecretory cells within the Drosophila central nervous system, it can be expected that these peptides play a variety of functional roles as central neuromodulators (or cotransmitters) and as hormones released in the circulation. Earlier studies of bioactivities of sNPF in different insects revealed that they are myostimulatory on various visceral muscles, and in locusts they stimulate ovarian growth and induce increases in vitellogeninin levels in the circulation. In female mosquitoes the sNPFs induce host seeking behavior (Johard, 2008 and references therein).

More recently, interference with snpf expression in Drosophila has shown that sNPFs are involved in regulation of feeding (Lee, 2004). In some insects sNPFs are also expressed in endocrine cells of the midgut, suggesting further regulatory roles (e.g., Veenstra, 1995). As shown in this study, one exciting role of sNPFs may be as a neuroactive compound (neuromodulator or cotransmitter) in Drosophila mushroom bodies. Thus they may play a role in higher olfactory processing or even in olfactory learning and memory or in other functions proposed for this brain center (Johard, 2008).

In summary, this study has provided a detailed mapping of the distribution of the products of the gene encoding the sNPF precursor based on in situ hybridization and immunocytochemistry. A novel finding is that snpf-derived peptides are present in a major subpopulation of intrinsic neurons of the mushroom bodies in Drosophila. Thus this study has identified the first putative neuroactive compound in intrinsic neurons of this brain center, which is known to play a major role in olfactory learning and memory. It is suspected that the sNPF-expressing Kenyon cells produce an additional fast neurotransmitter, yet to be identified (Johard, 2008).

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. This paper 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 (Lee, 2004. Full text of article).

A receptor for Short Neuropeptide F - Neuropeptide F-like Receptor 76F

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y2 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 (Mertens, 2002).

Since the Drosophila genome encodes at least 4 receptors belonging to the NPY subgroup of receptors, the Drosphila EST database was searched for the presence of EST clones, encoding one of the NPY-type receptors. PCR amplification of the EST clone (GH23382) with oligonucleotide primers specific for the predicted ORF of CG7395 (Neuropeptide F-like Receptor 76F) produced a single product of approximately 1800 bp. Sequence determination of the TA-cloned PCR product revealed a DNA insert of 1803 bp, corresponding to the sequence and size of the predicted receptor in the cDNA database of BDGP. The deduced protein encoded by the ORF of Drm-sNPF-R is 600 amino acids long. Analysis by the TMHMM program revealed that this protein is predicted to have seven transmembrane domains along with the intracellular and extracellular loops, consistent with the known G-protein coupled receptors. The N-terminal extracellular region exhibits no O-glycosylation, 2 N-glycosylation sites, along with 7 Ser/Thr phosphorylation sites (Mertens, 2002).

A phylogenetic tree based on Clustal W alignment of Drm-sNPF-R and various known NPY receptors indicates that Drm-sNPF-R is most closely related to the vertebrate neuropeptide Y2 receptors, i.e., of the domestic guinea pig (33% identity and 49% homology), humans (33% identity and 49% homology), the domestic pig (33% identity and 48% homology), and the rat (33% identity and 48% homology). An NPY-like orphan GPCR of C. elegans (C53C7.1) displays 33% identity and 47% homology. Sequence conservation among Drm-sNPF-R, the human Y2 receptor, and the C. elegans orphan receptor is depicted by similarities shown in their alignment by the AlignX program (Mertens, 2002).

Short NPFs and 'head' peptides display substantial sequence similarities and appear to belong to the same family. All NPFs have a typical R(K)–X₁–R–X₂amide motif, where the first amino acid of this motif is always a basic amino acid residue such as Arg or Lys. X₁ can be L, T or P and X₂ is always an aromatic amino acid residue such as Phe or Trp. It is proposed to (re)name all peptides with the R(K)–X₁–R–X₂amide C-terminal motif, as short NPFs; these peptides do not only occur in the head or central nervous system, but instead reach 10 times higher amounts in the abdomen and the midgut. In addition, their precursor in the Drosophila genome is annotated as the short NPF precursor. The present identification of a specific short NPF receptor in Drosophila is in favor of the presence of functional short NPFs. Several reports indicate that short NPFs have a hormonal function in insects, associated with reproduction and digestion. The expression of the short NPF receptor not only in the nervous system, but also in peripheral targets (ovaries, gut) is in agreement with a hormonal function of short NPFs. Hemolymph from sugar-fed mosquito females contains 414 fmol/μl immunoreactive short NPF. Short NPFs are also abundantly present in endocrine cells of the midgut, suggesting that they might have a function in digestion. The demonstration of the presence of the short NPF receptor transcript in the midgut favors this hypothesis (Mertens, 2002).

Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster

A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (Neuropeptide F-like Receptor 76F, NPFR76F, or GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62%-66% similarity and 32%-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila (Feng, 2003).

Since the NPFR76F receptor was maximally activated by a short insect NPF-like sequence from Leptinotarsa, genome mining was used to search for Drosophila peptides which might be functionally equivalent to (or better than) the Leptinotarsa I peptide at activating NPFR76F. Initially, one precursor sequence, a gene (npf) encoding Drosophila NPF was identified at chromosome location 89D3. This gene was found by blasting an incomplete version of the Drosophila genome (October 1999) with the amino acid sequence of Aplysia NPY. This revealed a precursor molecule encoding a 36 amino acid peptide with sequence similarity to NPF. The completed Drosophila genome sequence has now been searched and no other potential NPF precursors were identified. Since this peptide (NPF-A1) contained a potential dibasic amino acid cleavage site within its sequence, both the shorter 28 amino acid form (NPF-A2) and the full-length peptide (NPF-A1) were sequenced for testing (Feng, 2003).

A second open reading frame in the Drosophila genome encodes a precursor peptide for two short NPF-like peptides (Drm-sNPF-1, AQRSPSLRLRFamide and Drm-sNPF-2, WFGDVNQKPIRSPSLRLRFamide). The precursor for these peptides is encoded by the short NPF precursor (sNPF) gene (CG13968) and maps to position 38A7 on the left arm of Drosophila chromosome 2. The WFGDVNQKPIRSPSLRLRFamide peptide (Drm-sNPF-2) contains a potential single basic amino acid-processing site. This peptide (Drm-sNPF-2), its shorter form (peptide Drm-sNPF-2 s, PIRSPSLRLRFamide) and the Drm-sNPF-1 peptide (all encoded by the sNPF gene) were synthesized. The same precursor was predicted to include the sequences for two other short peptides, PQRLRWamide and PMRLRWamide, which have been designated Drm-sNPF-3 and Drm-sNPF-4, respectively. These peptides were synthesized and tested (Feng, 2003).

When tested at 1 µm, the shorter NPF-like peptides derived from the sNPF gene (peptides Drm-sNPF-1 and Drm-sNPF-2) were more effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents in Xenopus oocytes expressing NPFR76F and G_alpha16. Shortening of peptide Drm-sNPF-2 to the Drm-sNPF-2s form may slightly increase its effectiveness. The longer Drosophila peptides derived from the precursor gene npf at 89D3 were much less effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents. In addition, the PQRLRWamide (Drm-sNPF-3) and PMRLRWamide (Drm-sNPF4) peptides were much less effective than the original Leptinotarsa I sequence at inducing inward currents (Feng, 2003).

Dose-response curves for the shorter endogenous Drosophila NPF-like peptides encoded by the sNPF gene at 38A7 reveal that AQRSPSLRLRFamide (peptide Drm-sNPF-1) is the most potent peptide tested (pEC50 = -8.84). It showed a threshold for the generation of inward currents between 100 pm and 1 nm and a maximal effect at 100 nm. The second putative endogenous Drosophila NPF-like peptide encoded by the sNPF gene, PIRSPSLRFamide (peptide Drm-sNPF-2 s) (pEC50 = -7.62), and the original Leptinotarsa I NPF-like sequence, ARGPQLRLRFamide (pEC50 = -7.83), were an order of magnitude less potent than AQRSPSLRLRFamide. These results justify the classification of NPFR76F as a NPF-like receptor and suggest that the short peptide AQRSPSLRLRFamide (sNPF-1) may be the endogenous agonist for this receptor. The other two short peptides encoded by the sNPF gene, PQRLRWamide (pEC50 = -6.1) and PMRLRWamide (pEC50 = -7.30), were 2.7 and 1.5 orders of magnitude, respectively, less potent than the AQRSPSLRLRFamide sequence, leading to questioning of their designation as true short NPF-like peptides (Feng, 2003).

Expression of NPFR76F transcripts was assessed by Northern blot analysis of poly(A)⁺ RNA prepared from adult body parts. A single transcript of 6.5 kb was detected in both heads and appendages (legs and antennae), suggesting that NPFR76F is expressed in both the central and peripheral nervous systems. In addition to finding transcript in heads and appendages, trace amounts were also seen in bodies. When compared with the amount of RNA loaded from each body part (as indicated by the ubiquitous rp49 loading control), the relative abundance of transcript in bodies is very low. This distribution of the NPFR76F transcript is consistent with a role for this NPF-like receptor in the Drosophila nervous system (Feng, 2003).

To further refine the NPFR76F receptor transcript expression, in situ hybridization with a digoxigenin-labelled antisense RNA probe to whole mounts of the mature embryos was used. The central nervous system in embryos is composed of two dorsal brain hemispheres and a fused ventral ganglion. The NPFR76F receptor is expressed both in the dorsal brain and in the ventral ganglion. In the brain the receptor is strongly expressed in the specific cells in the dorsal posterior region. It is estimated that there are 22-24 cells in each brain lobe expressing NPFR76F, including the strongly expressing cells. In the ventral ganglion, pairs of cells along the ventral midline, as well as cells found in a bilaterally symmetric pattern in a more lateral position from the midline, also express receptor mRNA. In each full segment of the ventral ganglion, the receptor is strongly expressed in eight to 12 cells, including a pair of cells at the midline in each segment (Feng, 2003).

In the peripheral nervous system the receptor is expressed in a subset of sensilla and in the anterior sensory complex. It is estimated that there are 10-14 cells in the anterior sensory complex, including the antennomaxillary complex, the labral sensory complex and the labial sensory complex, that express NPFR76F. Finally, in the posterior sensilla, there are eight cells that express NPFR76F. The expression pattern of the NPFR76F receptor in many specific cells in the dorsal brain, the ventral ganglion, lateral sensilla, the anterior sensory complex and the posterior sensilla suggests that this receptor is involved in a widespread modulation of neuronal activity (Feng, 2003).

The activation of G-protein gated inwardly rectifying K+ channels by a cloned Drosophila melanogaster neuropeptide F-like receptor

A Drosophila melanogaster G-protein-coupled receptor (NPFR76F) that is activated by neuropeptide F-like peptides has been expressed in Xenopus oocytes to determine its ability to regulate heterologously expressed G-protein-coupled inwardly rectifying potassium channels. The activated receptor produced inwardly rectifying potassium currents by a pertussis toxin-sensitive G-protein-mediated pathway and the effects were reduced in the presence of proteins, such as the betaARK 1 carboxy-tail fragment and alpha-transducin, which bind G-protein betagamma-subunits. Short Drosophila NPF-like peptides are more potent than long NPF-like peptides at coupling the receptor to the activation of inwardly rectifying potassium channels. The putative endogenous short Drosophila NPF-like peptides showed agonist-specific coupling depending on whether their actions were assessed as the activation of the inwardly rectifying potassium channels or as the activation of endogenous inward chloride channels through a co-expressed promiscuous G-protein, Galpha16. As inwardly rectifying potassium channels are known to be encoded in the Drosophila genome and the NPFR76F receptor is widely expressed in the Drosophila nervous system, the receptor could function to control neuronal excitability or slow wave potential generation in the Drosophila nervous system (Reale, 2004).

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

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. This study reports 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 (Lee, 2004).

Various evidence suggests that sNPF and dNPF peptides have different functions. The sNPF peptide is found only in nervous systems, whereas the neuropeptide F (dNPF) is found as the Drosophila brain-gut peptide. The expression patterns of sNPF and dNPF differ in the larval brain. For example, the sNPF expression is found in the anterior dorsal neurons of the brain, whereas the dNPF expression is detected in the four neurons of larval brain. In the feeding behavior analysis, overexpression of sNPF in wandering larvae did not extend the feeding period, contrary to the extension of the feeding period in the wandering larval stage by overexpression of dNPF. At the receptor level, each peptide works in different receptors; for example, the NPFR76F receptor is for sNPF peptides, and the DmNPFR1 receptor is for the dNPF. These differences indicate that the dNPF and sNPF peptides function in different neurons and may regulate different aspects of feeding behaviors in Drosophila (Lee, 2004).

Like other neuropeptides, the sNPF peptide may be involved in regulating various physiological processes other than regulating food intake because the sNPF peptide and transcript were expressed during all developmental stages, and the sNPF is localized in the mushroom body calyx and medulla of the adult brain. The mushroom body is involved in learning and memory. These unknown multi-functions of the sNPF peptide in various biological processes are the subjects of future studies (Lee, 2004).

Structural studies of Drosophila short neuropeptide F: Occurrence and receptor binding activity

Among insects, short neuropeptide Fs (sNPF) have been implicated in regulation of reproduction and feeding behavior. For Drosophila melanogaster, the nucleotide sequence for the sNPF precursor protein encodes four distinctive candidate sNPFs. In the present study, all four peptides were identified by mass spectrometry in body extracts of D. melanogaster; some also were identified in hemolymph, suggesting potential neuroendocrine roles. Actions of sNPFs in D. melanogaster are mediated by the G protein-coupled receptor Drm-NPFR76F. Mammalian CHO-K1 cells were stably transfected with the Drm-NPFR76F receptor for membrane-based radioreceptor studies. Binding assays revealed that longer sNPF peptides comprised of nine or more amino acids are clearly more potent than shorter ones of eight or fewer amino acids. These findings extend understanding of the relationship between structure and function of sNPFs (Garczynski, 2006).

The sNPFs of D. melanogaster differ in their interactions with the sNPF receptor Drm-NPFR76F, as analyzed directly by radioreceptor assay. A wide variety of D. melanogaster sNPFs were assayed for their ability to inhibit the binding of ¹²⁵I-[D-Y¹]-Drm-sNPF1 to membranes prepared from cells stably transfected with Drm-NPFR76F. Two distinctive classes of activity of sNPFs were readily apparent. The sNPF peptides containing nine or more amino acids typically exhibited high affinity, as judged by an IC₅₀ < 1 nM, whereas peptides containing eight for fewer amino acids exhibited IC₅₀ values of >5 nM. Those exhibiting lower affinity included sNPF3 and sNPF4, peptides with the C-terminal RLRWa sequence. The minimum length of the highly active group was represented by sNPF2_11–19. The sequence of this sNPF2_11–19 (RSPSLRLRFa) differs from sNPF1_4–11 (SPSLRLRFa) only by a single arginine residue, which appears to confer a substantial increase in binding affinity. To test this hypothesis, an alanine substituted analog, Drm-sNPF2_11–19R11A, was assayed and found to exhibit a substantial drop in activity, with an IC₅₀ of only 12.5 nM, indicating the crucial role of this arginine residue (Garczynski, 2006).

For further tests of these apparent structure-function relations, putative sNPFs identified in the genomes of A. gambiae and A. aegypti also were examined in the Drm-NPFR76F radioreceptor assay. Each of these mosquito sNPF peptides conformed to the distinctive pattern of length-associated activity established previously for those of D. melanogaster. In contrast, the A. aegypti head peptides which partly resemble sNPFs were either weakly active, Aea-HP-I, or inactive, Aea-HP-III, despite being of sufficient length and having the requisite arginine. Accordingly, additional structural features in the C-terminus common to sNPF peptides appear important for high affinity binding (Garczynski, 2006).

Search PubMed for articles about Drosophila sNPF

Baggerman, G., Liu, F., Wets, G. and Schoofs, L. (2005). Bioinformatic analysis of peptide precursor proteins. Ann. N. Y. Acad. Sci. 1040: 59-65. PubMed Citation: 15891006

Emeson, R. B. and Morabito, M. V. (2005). Food fight: the NPY-serotonin link between aggression and feeding behavior. Sci. STKE pe12. PubMed Citation: 15798100

Feng, G., et al. (2003). Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster. Eur. J. Neurosci. 18: 227-238. PubMed Citation: 12887405

Garczynski, S. F., Brown, M. R. and Crim, J. W. (2006). Structural studies of Drosophila short neuropeptide F: Occurrence and receptor binding activity. Peptides 27(3): 575-82. PubMed Citation: 16330127

Johard, H. A., et al. (2008). Intrinsic neurons of Drosophila mushroom bodies express short neuropeptide F: relations to extrinsic neurons expressing different neurotransmitters. J. Comp. Neurol. 507(4): 1479-96. PubMed Citation: 18205208

Lee, K. S., You, K. H., Choo, J. K., Han, Y. M. and Yu, K. (2004). Drosophila short neuropeptide F regulates food intake and body size. J. Biol. Chem. 279(49): 50781-9. PubMed Citation: 15385546

Lee, K. S., et al. (2008). Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling. Nat. Cell Biol. 10(4): 468-75. PubMed Citation: 18344986

Mertens, I., Meeusen, T., Huybrechts, R., De Loof, A. and Schoofs, L. (2002). Characterization of the short neuropeptide F receptor from Drosophila melanogaster. Biochem Biophys Res Commun. 297(5): 1140-8. PubMed Citation: 12372405

Nässel, D. R., Enell, L. E., Santos, J. G., Wegener, C. and Johard, H. A. (2008). A large population of diverse neurons in the Drosophila central nervous system expresses short neuropeptide F, suggesting multiple distributed peptide functions. BMC Neurosci. 9: 90. PubMed Citation: 18803813

Predel, R., et al (2004). Peptidomics of CNS-associated neurohemal systems of adult Drosophila melanogaster: a mass spectrometric survey of peptides from individual flies. J. Comp. Neurol. 474(3): 379-92. PubMed Citation: 15174081

Reale, V., Chatwin, H. M. and Evans, P. D. (2004). The activation of G-protein gated inwardly rectifying K+ channels by a cloned Drosophila melanogaster neuropeptide F-like receptor. Eur. J. Neurosci. 19(3): 570-6. PubMed Citation: 14984407

Veenstra, J. A. and Lambrou, G. (1995). Isolation of a novel RFamide peptide from the midgut of the American cockroach, Periplaneta americana. Biochem. Biophys. Res. Commun. 213(2): 519-24. PubMed Citation: 7646507

Wahlestedt, C., Pich, E.M., Koob, G.F., Yee, F. and Heilig, M. (1993). Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides. Science 259: 528-531. PubMed Citation: 8380941

Wegener, C., et al. (2006). Direct mass spectrometric peptide profiling and fragmentation of larval peptide hormone release sites in Drosophila melanogaster reveals tagma-specific peptide expression and differential processing. J. Neurochem. 96(5): 1362-74. PubMed Citation: 16441518

date revised: 30 May 2009

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.