fmrfamide and neomyosuppressin

FMRFamide-related peptides (FaRPs) contain a C-terminal RFamide but unique N-terminal extensions. They are expressed throughout the animal kingdom and affect numerous biological activities. Like other animal species, Drosophila melanogaster contains multiple genes that encode different FaRPs. The ease of genetic manipulations, the availability of genomic sequence data, the existence of established bioassays, and its short lifespan make D. melanogaster a versatile experimental organism in which to investigate peptide processing, functions, and signal transduction pathways. Here, the structures, precursor organizations, distributions, and activities of FaRPs encoded by D. melanogaster FMRFamide (dFMRFamide), myosuppressin (Dms), and sulfakinin (Dsk) genes are reviewed, and predictions are made on their signaling pathways and biological functions.

Topics: Amino Acid Sequence; Animals; Drosophila melanogaster; Drosophila Proteins; FMRFamide; Genes, Insect; Insect Hormones; Molecular Sequence Data; Neuropeptides; Signal Transduction

Peptides in the RF-NH2 family are grouped together based on an amidated dipeptide C terminus and signal through G-protein coupled receptors (GPCRs) to influence diverse physiological functions. By determining the mechanisms underlying RF-NH2 signaling targets can be identified to modulate physiological activity; yet, how RF-NH2 peptides interact with GPCRs is relatively unexplored. We predicted conserved residues played a role in Drosophila melanogaster RF-NH2 ligand-receptor interactions. In this study D. melanogaster rhodopsin-like family A peptide GPCRs alignments identified eight conserved residues unique to RF-NH2 receptors. Three of these residues were in extra-cellular loops of modeled RF-NH2 receptors and four in transmembrane helices oriented into a ligand binding pocket to allow contact with a peptide. The eighth residue was unavailable for interaction; yet its conservation suggested it played another role. A novel hydrophobic region representative of RF-NH2 receptors was also discovered. The presence of rhodopsin-like family A GPCR structural motifs including a toggle switch indicated RF-NH2s signal classically; however, some features of the DMS receptors were distinct from other RF-NH2 GPCRs. Additionally, differences in RF-NH2 receptor structures which bind the same peptide explained ligand specificity. Our novel results predicted conserved residues as RF-NH2 ligand-receptor contact sites and identified unique and classic structural features. These discoveries will aid antagonist design to modulate RF-NH2 signaling.

Topics: Animals; FMRFamide; Insect Hormones; Ligands; Neuropeptides; Protein Binding; Protein Structure, Secondary; Receptors, G-Protein-Coupled

Five neuropeptide genes are classified in the FMRF-related (FaRP) group: the Fmrf, dromyosuppressin (Dms), drosulfakinin (Dsk), neuropeptide F (npf) and short neuropeptide F (sNPF) genes coding for 8, 1, 2, 1 and 4 peptides, respectively. In order to compare their effects on the locomotor activity of Drosophila adults, we made RNAi knockdown of the peptides and their specific receptor genes. In addition, we constructed Gal4 drivers with three distinct parts of the Fmrf gene's 5' regulatory sequence (RS8-Gal4, RS11-Gal4, RS17-Gal4), and used them to ablate FMRF-positive neurons inducing apoptosis by expressing the reaper (rpr) gene. We examined the locomotor activity of flies by measuring the mean velocity of movement (MVM) following repeated air-puffs. Locomotor activity was decreased by RNAi knockdown induced in the CNS by the elav-Gal4 driver. According to the MVM curve profiles, RNAi knockdown most effectively decreased the velocity when the DmsR-1 and DmsR-2 genes were silenced together (DmsR-1-RNAi/elav-Gal4; DmsR-2-RNAi/+). Similar effect was observed in Dsk-RNAi/ elav-Gal4; DskR-2-RNAi/+, while moderate effects were found in three other combinations (Fmrf-RNAi/elav-Gal4; FR-RNAi/+, Dms-RNAi/ elav-Gal4;DmsR-2-RNAi/+, CCKLR-17D1-RNAi/elav-Gal4; CCKLR-17D3-RNAi/+), and weak effect in DmsR-2-RNAi/elav-Gal4; DmsR-1-RNAi/+. Male and female flies were not different in this respect. In the cell ablation experiment, the MVM profiles of the female flies were different from the controls when the UAS-rpr transgene was driven by RS8-Gal4 or RS17-Gal4. The RS11-Gal4 and Fmrf-Gal4 drivers were ineffective. In the males only the RS17-Gal4 showed a weak effect. RNAi silencing of the FaRP and FaRP-receptor genes effectively decreased the startle-induced locomotor activity of flies. Ablation of FMRF-positive neurons by the RS8-Gal4 and/or RS17-Gal4 drivers also decreased the flies' activity.

Topics: Animals; Drosophila melanogaster; Female; FMRFamide; Insect Hormones; Male; Microscopy, Confocal; Motor Activity; Neuropeptides; RNA Interference

Neuropeptides are found in both mammals and invertebrates and can modulate neural function through activation of G-protein-coupled receptors (GPCRS). The precise mechanisms by which many of these GPCRs modulate specific signaling cascades to regulate neural function are not well defined. We used Drosophila melanogaster as a model to examine both the cellular and behavioral effects of DPKQDFMRFamide, the most abundant peptide encoded by the dFMRF gene. We show that DPKQDFMRFamide enhanced synaptic transmission through activation of two G-protein-coupled receptors, Fmrf Receptor (FR) and Dromyosupressin Receptor-2 (DmsR-2). The peptide increased both the presynaptic Ca(2+) response and the quantal content of released transmitter. Peptide-induced modulation of synaptic function could be abrogated by depleting intracellular Ca(2+) stores or by interfering with Ca(2+) release from the endoplasmic reticulum through disruption of either the ryanodine receptor or the inositol 1,4,5-trisphosphate receptor. The peptide also altered behavior. Exogenous DPKQDFMRFamide enhanced fictive locomotion; this required both the FR and DmsR-2. Likewise, both receptors were required for an escape response to intense light exposure. Thus, coincident detection of a peptide by two GPCRs modulates synaptic function through effects of Ca(2+)-induced Ca(2+) release, and we hypothesize that these mechanisms are involved in behavioral responses to environmental stress.

Topics: Animals; Behavior, Animal; Calcium Signaling; Drosophila melanogaster; Drosophila Proteins; Escape Reaction; Female; FMRFamide; Insect Hormones; Male; Motor Activity; Neuropeptides; Neurotransmitter Agents; Protein Precursors; Receptors, G-Protein-Coupled; Receptors, Invertebrate Peptide; Receptors, Peptide; Stress, Physiological; Synaptic Transmission

Activation of G protein-coupled receptors (GPCR) leads to the recruitment of beta-arrestins. By tagging the beta-arrestin molecule with a green fluorescent protein, we can visualize the activation of GPCRs in living cells. We have used this approach to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. Here we verify the identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702). We also de-orphan CG6515 and CG7887 by showing these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. We show that CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, we show that dromyosuppressin also stimulated GTPgammaS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the beta-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors.

Topics: Animals; Arrestins; beta-Arrestins; Cell Line; Cloning, Molecular; Cyclic AMP; Dose-Response Relationship, Drug; Drosophila; Drosophila Proteins; FMRFamide; Green Fluorescent Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Insect Hormones; Insect Proteins; Ligands; Luminescent Proteins; Microscopy, Confocal; Neuropeptides; Oligopeptides; Peptides; Protein Transport; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Receptors, Peptide; Receptors, Tachykinin; Transfection

The insect peptides leucomyosuppressin (pEDVDHVFLRFamide) and dromyosuppressin (TDVDHVFLRFamide) have identical chemical sequences with the exception of the N-terminal amino acid; both inhibit spontaneous contraction of insect visceral muscles. Neurons in the hypocerebral ganglion of horn fly, Hematobia irritans (L.), and stable fly, Stomoxys calcitrans (L.), were found to contain material immunoreactive to antiserum produced against the C-terminal of leucomyosuppressin, but not to the N-terminal of dromyossuppressin. Two large lateral clusters containing 8 cells, linked dorsally and ventrally by 2 chains of 6 cells, encircled the anterior surface of the proventriculus and were immunoreactive of leucomyosuppressin and FMRFamide antisera. Axons from these cells were traced to the wall of the aorta and over the surface of the proventriculus. Ultrastructural analysis revealed these cells contained a singular type of elementary secretory granule that contained material of relatively low electron density, both in the cell body and at the axon terminals.

Topics: Animals; Female; FMRFamide; Ganglia, Invertebrate; Immunoenzyme Techniques; Insect Hormones; Male; Muscidae; Neuropeptides