Structure-activity relationship data and ligand-receptor interactions identify novel agonists consistent with sulfakinin tissue-specific signaling in Drosophila melanogaster heart.

BACKGROUND: The structures and activities of invertebrate sulfakinins that influence gut motility and heart rate are like the vertebrate cholecystokinin (CCK) peptides. Typical of sulfakinin precursors Drosophila melanogaster encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2) that bind DSK-R1 and DSK-R2. To explore the role of the nsDSK II N-terminal extension (GGDDQ) in gut we delineated its structure-activity relationship (SAR) and identified novel agonists. We then predicted the nsDSK II extension SAR is tissue specific consistent with cardiac CCK structure activity and signaling being different from gut. METHODS: To evaluate our hypothesis, we tested single-substituted alanine and asparagine analogs in heart. RESULTS: We found alanyl-substituted analogs were less active in heart than nsDSK II; in gut they include a super agonist and a protean agonist. Additionally, we discovered ns[N4]DSK II was more active than nsDSK II in pupal heart, while ns[N3]DSK II was inactive. In contrast, ns[N3]DSK II and ns[N4]DSK II were super agonists in adult heart, yet inactive in larva. Although we reported nsDSK II acts through DSK-R2 in gut, its identity in heart was unknown. CONCLUSIONS: Here we reviewed ligand-receptor interactions in conjunction with SAR data to suggest nsDSK II acts through DSK-R1 in heart consistent with sulfakinin tissue-specific signaling.

In vitro model of ischemic heart failure using human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used extensively to model inherited heart diseases, but hiPSC-CM models of ischemic heart disease are lacking. Here, our objective was to generate an hiPSC-CM model of ischemic heart disease. To this end, hiPSCs were differentiated into functional hiPSC-CMs and then purified using either a simulated ischemia media or by using magnetic antibody-based purification targeting the nonmyocyte population for depletion from the cell population. Flow cytometry analysis confirmed that each purification approach generated hiPSC-CM cultures that had more than 94% cTnT+ cells. After purification, hiPSC-CMs were replated as confluent syncytial monolayers for electrophysiological phenotype analysis and protein expression by Western blotting. The phenotype of metabolic stress-selected hiPSC-CM monolayers recapitulated many of the functional and structural hallmarks of ischemic CMs, including elevated diastolic calcium, diminished calcium transient amplitude, prolonged action potential duration, depolarized resting membrane potential, hypersensitivity to chemotherapy-induced cardiotoxicity, depolarized mitochondrial membrane potential, depressed SERCA2a expression, reduced maximal oxygen consumption rate, and abnormal response to ß1-adrenergic receptor stimulation. These findings indicate that metabolic selection of hiPSC-CMs generated cell populations with phenotype similar to what is well known to occur in the setting of ischemic heart failure and thus provide a opportunity for study of human ischemic heart disease.

Cardiac contractility structure-activity relationship and ligand-receptor interactions; the discovery of unique and novel molecular switches in myosuppressin signaling.

Peptidergic signaling regulates cardiac contractility; thus, identifying molecular switches, ligand-receptor contacts, and antagonists aids in exploring the underlying mechanisms to influence health. Myosuppressin (MS), a decapeptide, diminishes cardiac contractility and gut motility. Myosuppressin binds to G protein-coupled receptor (GPCR) proteins. Two Drosophila melanogaster myosuppressin receptors (DrmMS-Rs) exist; however, no mechanism underlying MS-R activation is reported. We predicted DrmMS-Rs contained molecular switches that resembled those of Rhodopsin. Additionally, we believed DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 interactions would reflect our structure-activity relationship (SAR) data. We hypothesized agonist- and antagonist-receptor contacts would differ from one another depending on activity. Lastly, we expected our study to apply to other species; we tested this hypothesis in Rhodnius prolixus, the Chagas disease vector. Searching DrmMS-Rs for molecular switches led to the discovery of a unique ionic lock and a novel 3-6 lock, as well as transmission and tyrosine toggle switches. The DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 contacts suggested tissue-specific signaling existed, which was in line with our SAR data. We identified R. prolixus (Rhp)MS-R and discovered it, too, contained the unique myosuppressin ionic lock and novel 3-6 lock found in DrmMS-Rs as well as transmission and tyrosine toggle switches. Further, these motifs were present in red flour beetle, common water flea, honey bee, domestic silkworm, and termite MS-Rs. RhpMS and DrmMS decreased R. prolixus cardiac contractility dose dependently with EC50 values of 140 nM and 50 nM. Based on ligand-receptor contacts, we designed RhpMS analogs believed to be an active core and antagonist; testing on heart confirmed these predictions. The active core docking mimicked RhpMS, however, the antagonist did not. Together, these data were consistent with the unique ionic lock, novel 3-6 lock, transmission switch, and tyrosine toggle switch being involved in mechanisms underlying TM movement and MS-R activation, and the ability of MS agonists and antagonists to influence physiology.

Structure-activity relationships of FMRF-NH2 peptides demonstrate A role for the conserved C terminus and unique N-terminal extension in modulating cardiac contractility.

FMRF-NH2 peptides which contain a conserved, identical C-terminal tetrapeptide but unique N terminus modulate cardiac contractility; yet, little is known about the mechanisms involved in signaling. Here, the structure-activity relationships (SARs) of the Drosophila melanogaster FMRF-NH2 peptides, PDNFMRF-NH2, SDNFMRF-NH2, DPKQDFMRF-NH2, SPKQDFMRF-NH2, and TPAEDFMRF-NH2, which bind FMRFa-R, were investigated. The hypothesis tested was the C-terminal tetrapeptide FMRF-NH2, particularly F1, makes extensive, strong ligand-receptor contacts, yet the unique N terminus influences docking and activity. To test this hypothesis, docking, binding, and bioactivity of the C-terminal tetrapeptide and analogs, and the FMRF-NH2 peptides were compared. Results for FMRF-NH2 and analogs were consistent with the hypothesis; F1 made extensive, strong ligand-receptor contacts with FMRFa-R; Y → F (YMRF-NH2) retained binding, yet A → F (AMRF-NH2) did not. These findings reflected amino acid physicochemical properties; the bulky, aromatic residues F and Y formed strong pi-stacking and hydrophobic contacts to anchor the ligand, interactions which could not be maintained in diversity or number by the small, aliphatic A. The FMRF-NH2 peptides modulated heart rate in larva, pupa, and adult distinctly, representative of the contact sites influenced by their unique N-terminal structures. Based on physicochemical properties, the peptides each docked to FMRFa-R with one best pose, except FMRF-NH2 which docked with two equally favorable poses, consistent with the N terminus influencing docking to define specific ligand-receptor contacts. Furthermore, SDNAMRF-NH2 was designed and, despite lacking the aromatic properties of one F, it binds FMRFa-R and demonstrated a unique SAR, consistent with the N terminus influencing docking and conferring binding and activity; thus, supporting our hypothesis.

Structure-activity studies of RFamide-related peptide-1 identify a functional receptor antagonist and novel cardiac myocyte signaling pathway involved in contractile performance.

Human RFamide-related peptide-1 (hRFRP-1, MPHSFANLPLRF-NH(2)) binds to neuropeptide FF receptor 2 (NPFF(2)R) to dramatically diminish cardiovascular performance. hRFRP-1 and its signaling pathway may provide targets to address cardiac dysfunction. Here, structure-activity relationship, transcript, Ca(2+) transient, and phospholabeling data indicate the presence of a hRFRP-1 pathway in cardiomyocytes. Alanyl-substituted and N-terminal truncated analogues identified that R(11) was essential for activity, hRFRP-1((8-12)) mimicked hRFRP-1, and [A(11)]hRFRP-1((8-12)) antagonized the effect of hRFRP-1 in cellular and integrated cardiac performance. RFRP and NPFF(2)R transcripts were amplified from cardiomyocytes and heart. Maintenance of the Ca(2+) transient when hRFRP-1 impaired myocyte shortening indicated the myofilament was its primary downstream target. Enhanced myofilament protein phosphorylation detected after hRFRP-1 treatment but absent in [A(11)]hRFRP-1((8-12))-treated cells was consistent with this result. Protein kinase C (PKC) but not PKA inhibitor diminished the influence of hRFRP-1 on the Ca(2+) transient. Molecules targeting this pathway may help address cardiovascular disease.

Plasticity in the effects of sulfated and nonsulfated sulfakinin on heart contractions.

Neuropeptides regulate the frequency of heart contractions. Drosophila melanogaster sulfakinin (drosulfakinin) encodes FDDYGHMRFamide, DSK I, and GGDDQFDDYGHMRFamide, DSK II. Invertebrate sulfakinins are structurally and functionally related to vertebrate cholecystokinins. Naturally-occurring drosulfakinins contain a sulfated or nonsulfated tyrosine and are designated sDSK I, sDSK II, nsDSK I, and nsDSK II. We developed a novel neural-cardiovascular preparation and investigated mechanisms regulating the effect of sulfakinins on D. melanogaster heart. We established the preparation in larva, pupa, and adult to examine plasticity in neural regulation of cardiovascular parameters. We discovered sDSK I increased the frequency of larval, pupal, and adult heart contractions; nsDSK I only increased the frequency of larval contractions, not pupal or adult. We also discovered sDSK II and nsDSK II increased the frequency of larval and adult contractions, not pupal. This is the first report of nonsulfated sulfakinin activity on heart, and sulfakinin activity examined in 3 developmental stages within the same animal species. Our data demonstrate a role for plasticity in the effects of sulfakinins on heart contractions, and suggest multiple mechanisms are involved.

The different effects of structurally related sulfakinins on Drosophila melanogaster odor preference and locomotion suggest involvement of distinct mechanisms.

Sulfakinins are myoactive peptides and antifeedant factors. Naturally occurring drosulfakinin I (DSK I; FDDYGHMRFNH(2)) and drosulfakinin II (DSK II; GGDDQFDDYGHMRFNH(2)) contain sulfated or nonsulfated tyrosine. We discovered sDSK II and nsDSK II influenced Drosophila melanogaster larval odor preference. However, sDSK I, nsDSK I, MRFNH(2), and saline did not influence odor preference. We discovered sDSK I and nsDSK I influenced larval locomotion. However, sDSK II, nsDSK II, MRFNH(2), and saline did not influence locomotion. Our novel data suggest distinct mechanisms underlie the effects of DSK I and DSK II peptides on odor preference and locomotion, parameters important to many facets of animal survival.

The drosulfakinin 0 (DSK 0) peptide encoded in the conserved Dsk gene affects adult Drosophila melanogaster crop contractions.

We report that the drosulfakinin 0 (DSK 0; NQKTMSFNH2) structure and genomic organization are conserved. The DSK 0 C-terminus, SFNH2, is widely distributed in the animal kingdom suggesting it defines a novel peptide family. We also report the first description of DSK 0 activity. DSK 0, I (DSK I, FDDYGHMRFNH2), and II (DSK II, GGDDQFDDYGHMRFNH2) are encoded in sulfakinin (Dsk). Drosophila erecta, Drosophila sechellia, Drosophila simulans, and Drosophila yakuba shared 62.5-87.5% identity to Drosophila melanogaster DSK 0; Drosophila pseudoobscura shared 37.5% identity; numerous amino acids were one nucleotide different from a corresponding residue in D. melanogaster. DSK I and II were identical among the drosopholids. DSK 0 proteolytic processing sites were RR except D. yakuba contained KR and D. pseudoobscura contained HR, one nucleotide different from RR. DSK I and II processing sites were identical among the drosopholids. We established DSK 0 decreased adult (EC50=237nM and R(2)=0.941), but not larval gut contractions. DSK 0 exists in the central nervous system including the subesophageal ganglion and an abdominal ganglion. Peptide and genomic conservation, activity, and spatial and temporal distribution support the conclusion that DSK 0 plays diverse biological roles in drosopholids including regulating gut muscle contraction.

Localization of leucomyosuppressin in the brain and circadian clock of the cockroach Leucophaea maderae.

The myosuppressins (X1DVX2HX3FLRFamide), which reduce the frequency of insect muscle contractions, constitute a subgroup of the FMRFamide-related peptides. In the cockroach Leucophaea maderae, we have examined whether leucomyosuppressin (pQDVDHVFLRFamide) is present in the accessory medulla, viz., the circadian clock, which governs circadian locomotor activity rhythms. Antisera that specifically recognize leucomyosuppressin stain one to three neurons near the accessory medulla. MALDI-TOF mass spectrometry has confirmed the presence of leucomyosuppressin in the isolated accessory medulla. Injections of 1.15 pmol leucomyosuppressin into the vicinity of the accessory medulla at various circadian times have revealed no statistically significant effects on the phase of circadian locomotor activity rhythms. This is consistent with the morphology of the myosuppressin-immunoreactive neurons, which restrict their arborizations to the circadian clock and other optic lobe neuropils. Thus, leucomyosuppressin might play a role in the circadian system other than in the control of locomotor activity rhythms.

Evidence dromyosuppressin acts at posterior and anterior pacemakers to decrease the fast and the slow cardiac activity in the blowfly Protophormia terraenovae.

The molecular complexity of the simple blowfly heart makes it an attractive preparation to delineate cardiovascular mechanisms. Blowfly cardiac activity consists of a fast, high-frequency signal phase alternating with a slow, low-frequency signal phase triggered by pacemakers located in the posterior abdominal heart and anterior thoracocephalic aorta, respectively. Mechanisms underlying FMRFamide-related peptides (FaRPs) effects on heart contractions are not well understood. Here, we report antisera generated to a FaRP, dromyosuppressin (DMS, TDVDHVFLRFamide), recognized neuronal processes that innervated the blowfly Protophormia terraenovae heart and aorta. Dromyosuppressin caused a reversible cardiac arrest. High- and low-frequency signals were abolished after which they resumed; however, the concentration-dependent resumption of the fast phase differed from the slow phase. Dromyosuppressin decreased the frequency of cardiac activity in a dose-dependent manner with threshold values between 5 fM and 0.5 fM (fast phase), and 0.5 fM and 0.1 fM (slow phase). Dromyosuppressin structure-activity relationship (SAR) for the decrease of the fast-phase frequency was not the same as the SAR for the decrease of the slow-phase frequency. The alanyl-substituted analog TDVDHVFLAFamide ([Ala9] DMS) was inactive on the fast phase, but active on the slow phase, a novel finding. FaRPs including myosuppressins are reported to require the C-terminal RFamide for activity. Our data are consistent with the conclusions DMS acts on posterior and anterior cardiac tissue to play a role in regulating the fast and slow phases of cardiac activity, respectively, and ligand-receptor binding requirements of the abdominal and thoracocephalic pacemakers are different.

FMRFamide-related peptides and serotonin regulate Drosophila melanogaster heart rate: mechanisms and structure requirements.

Drosophila melanogaster FMRFamide-related peptides (FaRPs) include SDNFMRFamide, PDNFMRFamide, and TDVDHVFLRFamide (dromyosuppressin, DMS); each peptide contains a C-terminal FMRFamide but a different N-terminal extension. FaRPs and serotonin (5-HT) each affect the frequency of D. melanogaster heart contractions in vivo. We examined the cellular expression of FaRPs and 5-HT, and the activities of FMRFamide, SDNFMRFamide, PDNFMRFamide, or DMS and 5-HT on heart rate. FaRPs and 5-HT were not co-localized; FaRP-and 5-HT-immunoreactive fibers extended from different brain cells and innervated the anterior D. melanogaster dorsal vessel. However, no neuron expressed both a FaRP and 5-HT. The effect of FMRFamide and 5-HT was not different from the effect of 5-HT alone on heart rate. The effect of PDNFMRFamide and 5-HT showed an additive effect on heart rate. SDNFMRFamide and 5-HT or DMS and 5-HT resulted in non-additive effects on heart rate. Our data provide evidence for the complexity of FaRP and 5-HT interactions to regulate frequency of heart contractions in vivo. Our results also confirm the biological importance of FaRP N-terminal amino acid extensions.

Peptidergic innervation of the crop and the effects of an ingested nonpeptidal agonist on longevity in female Musca domestica (Diptera: Muscidae).

Dromyosuppressin (DMS) immunoreactive neurons were discovered running along the crop duct and on the surface of the crop in the house fly, Musca domestica L. DMS is a myoinhibitory neuropeptide that has been shown to inhibit crop contractions, in vitro, in the blow fly, Phormia regina (Meigen), and in Drosophila melanogaster Meigen. Various concentrations of benzethonium chloride (Bztc), an agonist of DMS with shown inhibitory effects on blow fly crop contractions, were fed to adult female M. domestica. Flies exhibited a dose-dependent mortality; avoidance and subsequent dehydration are probably the cause of the low survivorship at higher Bztc concentrations.

The structure of the FMRFamide receptor and activity of the cardioexcitatory neuropeptide are conserved in mosquito.

Numerous peptides are structurally related to the cardioexcitatory tetrapeptide FMRFamide. One subgroup of FMRFamide-related peptides (FaRPs) contains an FMRFamide C terminus. Searches of the Drosophila melanogaster genome database identified the first invertebrate FMRFamide G-protein coupled receptor (GPCR), DrmFMRFa-R (Cazzamali and Grimmelikhuijzen, Meeusen et al., 2002). In order to explore molecular mechanisms involved in FMRFamide signal transduction we identified a receptor from the malaria mosquito Anopheles gambiae genome (Holt et al., 2002), AngFMRFa-R, and compared its structure to DrmFMRFa-R. The cytoplasmic loops, extracellular loops, and transmembrane regions are highly conserved between these two FMRFamide receptors. Another subgroup of FaRPs is the sulfakinins which are represented by the consensus structure -XDYGHMRFamide, where X is D or E (Nichols, 2003). We compared AngFMRFa-R and DrmFMRFa-R to the A. gambiae sulfakinin receptors, ASK-R1 and ASK-R2 ( Duttlinger et al., 2003), and the D. melanogaster sulfakinin receptors, DSK-R1 and DSK-R2 Brody and Cravchik, 2000; Hewes and Taghert, 2001 ). The cytoplasmic loops, extracellular loops, and the transmembrane regions are not highly conserved between the FMRFamide and sulfakinin receptors. In order to explore the role of FMRFamide in mosquito biology we measured the effect of the tetrapeptide on in vivo heart rate. The tetrapeptide increased the frequency of spontaneous contractions of the larval mosquito heart and, thus, increased heart rate. These data support the conclusion that the structure of the FMRFamide receptor and activity of the cardioexcitatory FMRFamide neuropeptide are conserved in mosquito.

Signaling pathways and physiological functions of Drosophila melanogaster FMRFamide-related peptides.

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.

A nonpeptide provides insight into mechanisms that regulate Drosophila melanogaster heart contractions.

Here we report the effect of a nonpeptide, benzethonium chloride (bztc), on Drosophila melanogaster larval, pupal, and adult heart rates in vivo. Benzethonium chloride reduced the frequency of spontaneous contractions in the D. melanogaster pupal heart, but not in the larval heart or the adult heart as measured in noninvasive whole animal preparations. When applied directly to the D. melanogaster heart, in the absence of hemolymph, bztc reduced the frequency of spontaneous contractions in larval, pupal, and adult hearts. These findings are consistent with the conclusion that bztc acts through or is regulated by different mechanisms in these three developmental stages. An alternative explanation is that larval hemolymph and adult hemolymph contain a material that interferes with the effect of the nonpeptide on heart contractions. Bztc mimicked the effect of the peptide dromyosuppressin (DMS) on the heart at an equivalent concentration; in contrast, 103-fold more nonpeptide is required to mimic the effect of DMS on fly gut. These findings are consistent with the presence of tissue-specific myosuppressin receptors or mechanisms.

The effects of three Drosophila melanogaster myotropins on the frequency of foregut contractions differ.

Myotropic peptides can be grouped into different families based on their structure. Three Drosophila melanogaster myotropin families are represented by TDVDHVFLRFamide, dromyosuppressin (DMS), pEVRYRQCYFNPISCF, an allatostatin C-type peptide named flatline (FLT), and SDNFMRFamide, a FMRFamide-containing peptide. The structures of DMS, FLT, and SDNFMRFamide differ and each peptide is encoded by a different gene. In addition, the spatial and temporal distributions of DMS, FLT, and SDNFMRFamide are dissimilar. DMS, FLT, and SDNFMRFamide each decreases heart rate; however, their effects are profoundly different. Likewise, the effects of these three myotropins on the frequency of the spontaneous contractions of the crop, an anterior portion of the foregut, differ. DMS stops crop movement without recovery for at least a 10-min period after applying the peptide, FLT significantly decreases the frequency of spontaneous contractions, but its effect partially reverses within a few minutes after applying the peptide, and SDNFMRFamide only slightly decreased crop motility, an effect that was not significantly different from the effect of saline. The differences in the structures, distributions, and activities of DMS, FLT, and SDNFMRFamide suggest their synthesis and release are under different sensory inputs and regulatory mechanisms, and that roles in affecting the frequency of crop contractions differ.

Myotropic peptides in Drosophila melanogaster and the genes that encode them.

Myotropic peptides are structurally dissimilar; thus, they comprise different families. The cellular expressions of myotropins suggest they act as hormones, transmitters, and modulators of numerous biological processes. Drosophila melanogaster allatostatin (AST), FMRFamide-containing, dromyosuppressin (DMS), and drosulfakinin (DSK) peptides represent four different myotropin families. A different gene encodes each of these four myotropin families. D. melanogaster AST, FMRFamide-containing, DMS, and DSK peptides are present in neural and gut tissue, but are not all expressed in the same cells. These four families of myotropins affect spontaneous contractions of gut, heart, and/or reproductive tissue, but their effects are dissimilar in magnitude and time course. Based on their structures, genes, distributions, and activities, the synthesis and release of these D. melanogaster myotropins are likely governed by different sensory inputs and regulatory mechanisms. The differences in structures, precursors, cellular expressions, and activities are consistent with the conclusion that they do not play redundant roles in their effects on the frequency of muscle contractions. Orthologs of these D. melanogaster myotropins exist in other animal species; thus, research on the mechanisms involved in their production and processing, functions, and signaling may be widely applicable. Here, we review research on D. melanogaster AST, FMRFamide-containing, myosuppressin, and sulfakinin peptides.

The discovery of novel neuropeptides takes flight.

Structural data are critical for the elucidation of how peptides are synthesized and how they function. Two recent studies have used nanoscale chromatography together with mass spectrometry to determine the structures of novel neuropeptides in rat and Drosophila. The results shed light on neuropeptide synthesis and function(s) in both vertebrates and insects.

Identification in Drosophila melanogaster of the invertebrate G protein-coupled FMRFamide receptor.

We here describe the cloning and characterization of the functionally active Drosophila melanogaster (Drm) FMRFamide receptor, which we designated as DrmFMRFa-R. The full-length ORF of a D. melanogaster orphan receptor, CG 2114 (Berkeley Drosophila Genome Project), was cloned from genomic DNA. This receptor is distantly related to mammalian thyroid-stimulating hormone-releasing hormone receptors and to a set of Caenorhabditis elegans orphan receptors. An extract of 5,000 central nervous systems from the related but bigger flesh fly, Neobellieria bullata (Neb), was used to screen cells expressing the orphan receptor. Successive purification steps, followed by MS, revealed the sequence of two previously uncharacterized endogenous peptides, APPQPSDNFIRFamide (Neb-FIRFamide) and pQPSQDFMRFamide (Neb-FMRFamide). These are reminiscent of other insect FMRFamide peptides, having neurohormonal as well as neurotransmitter functions. Nanomolar concentrations of the Drm FMRFamides (DPKQDFMRFamide, TPAEDFMRFamide, SDNFMRFamide, SPKQDFMRFamide, and PDNFMRFamide) activated the cognate receptor in a dose-dependent manner. To our knowledge, the cloned DrmFMRFa-R is the first functionally active FMRFamide G protein-coupled receptor described in invertebrates to date.

The different effects of three Drosophila melanogaster dFMRFamide-containing peptides on crop contractions suggest these structurally related peptides do not play redundant functions in gut.

A Drosophila melanogaster dFMRFamide gene product, TPAEDFMRFamide, decreased crop contractions. However, DPKQDFMRFamide and SDNFMRFamide, also encoded in dFMRFamide, did not affect crop motility, which suggests these peptides are not functionally redundant in the crop and their unique N-terminal structures are important for activity. TPAEDFMRFamide-specific antisera did not stain the crop, which suggests it acts as a hormone. TDVDHVFLRFamide (DMS), encoded in D. melanogaster myosuppressin, stops crop contractions. TPAEDFMRFamide and DMS each contains a RFamide C-terminus; however, their effects on crop contractions differ, which suggests that unique receptors or different ligand:receptor binding requirements exist for these structurally related peptides.