InteractiveFly: GeneBrief

Myosuppressin and Myosuppressin receptors: Biological Overview | References

Gene name - Myosuppressin, Myosuppressin receptor 1 & Myosuppressin receptor 2

Synonyms -

Cytological map positions - 95F14-95F14 , 62D6-62D6 and 62D5-62D5

Function - neurohormone & G-protein coupled receptors

Keywords -

Myosuppressin is expressed in pars intercerebralis - Ms is a decapeptide that diminishes cardiac contractility and gut motility - enlarges crops in flies that were fed ad libitum, consistent with the relaxant properties of Ms on insect muscles - Myosuppressin receptors are crop muscle receptors through which Ms signals to modulate crop enlargement - MsRs exhibit a unique ionic lock, a novel 3-6 lock, a transmission switch, and a tyrosine toggle switch involved in mechanisms underlying TM movement and MS-R activation
Symbols - Ms, MsR1 & MsR2

FlyBase IDs: FBgn0011581, FBgn0035331, FBgn0264002

Genetic map positions - chr3R:24,341,551-24,343,685, chr3L:2,322,693-2,345,799 & chr3L:2,282,997-2,300,944

Classifications - neurohormone & G-protein coupled receptor

Cellular locations - secreted & surface transmembrane

NCBI links for Ms: EntrezGene, Nucleotide, Protein
NCBI links for MsR1: EntrezGene, Nucleotide, Protein
NCBI links for MsR2: EntrezGene, Nucleotide, Protein

Ms orthologs: Biolitmine

MsR1 and MsR2 orthologs: Biolitmine
Recent literature
Nichols, R., Pittala, K., Leander, M., Maynard, B., Nikolaou, P. and Marciniak, P. (2021). The myosuppressin structure-activity relationship for cardiac contractility and its receptor interactions support the presence of a ligand-directed signaling pathway in heart. Peptides: 170641. PubMed ID: 34453985
The structural conservation and activity of the myosuppressin cardioinhibitory peptide across species suggests it plays an important role in physiology, yet much remains unknown regarding its signaling. Drosophila melanogaster myosuppressin (dromyosuppressin, DMS; TDVDHVFLRF-NH(2)) decreases cardiac contractility through a G protein-coupled receptor, DMS-R2. This study showed the DMS N-terminus amino acids influence its structure-activity relationship (SAR), yet how they act is not established. It was predicted that myosuppressin N-terminal amino acids played a role in activity and signaling. This hypothesis was tested in the beetle, Zophobas atratus, using a semi-isolated heart bioassay to explore SAR in a different Order and focus on cardiac signaling. A series of myosuppressin truncated analogs was generated by removing the N-terminal residue and measuring the activity of each structure on cardiac contractility. While DVDHVFLRF-NH(2) decreased cardiac contractility, it was found VDHVFLRF-NH(2), DHVFLRF-NH(2), and HVFLRF-NH(2) increased activity. In contrast, VFLRF- NH(2) decreased activity and FLRF-NH(2) was inactive. Next, molecular docking data was analyzed, and it was found the active truncated analogs interacted with the 3-6 lock in DMS-R2, the myosuppressin cardiac receptor, disrupting the salt bridge between H114 and E369, and K289 and Q372. Further, the docking results showed the inhibitory effect on contractility may be associated with contact to Y78, while the analogs that increased contractility lacked this interaction. The data from this study demonstrated N-terminal amino acids played a role in myosuppressin activity and signaling suggesting the cardiac receptor can be targeted by biased agonists. These myosuppressin cardiac contractility data and predicted receptor interactions describe the presence of functional selectivity in a ligand-directed signaling pathway in heart.

Reproduction induces increased food intake across females of many animal species, providing a physiologically relevant paradigm for the exploration of appetite regulation. By examining the diversity of enteric neurons in Drosophila melanogaster, this study identified a key role for gut-innervating neurons with sex and reproductive state-specific activity in sustaining the increased food intake of mothers during reproduction. Steroid and enteroendocrine hormones functionally remodel these neurons, which leads to the release of their neuropeptide onto the muscles of the crop-a stomach-like organ-after mating. Neuropeptide release changes the dynamics of crop enlargement, resulting in increased food intake, and preventing the post-mating remodelling of enteric neurons reduces both reproductive hyperphagia and reproductive fitness. The plasticity of enteric neurons is therefore key to reproductive success. These findings provide a mechanism to attain the positive energy balance that sustains gestation, dysregulation of which could contribute to infertility or weight gain (Hadjieconomou, 2020).

Internal state has profound effects on brain function. Despite increasingly recognized roles for the gut-brain axis in maintaining energy balance, links between internal state and gastrointestinal innervation remain poorly characterized. Progress has been hindered by neuroanatomical complexity, which is only beginning to be parsed in mammals. The simpler-yet physiologically complex-intestine of Drosophila provides an alternative entry point into the study of gastrointestinal innervation (Hadjieconomou, 2020).

Innervation of the main digestive portion of the adult fly intestine, which encompasses the anterior midgut and the crop and central neurons of the pars intercerebralis (PI) in the brain. PI neurons directly innervate the anterior midgut and the crop, and include insulin-producing neurons and other peptidergic subtypes. The crop is further populated by processes that emanate from cells of the corpora cardiaca, which produce the glucagon-like adipokinetic hormone and are adjacent to the hypocerebral ganglion (HCG). Also adjacent to both the HCG and the corpora cardiaca are the corpus allatum cells, which produce juvenile hormone and extend short local projections. The thoracico-abdominal ganglion of the central nervous system might not innervate these gut regions (Hadjieconomou, 2020).

The crop-an expandable structure found in the intestines of insects-might be disregarded as a passive food store, but several observations suggest active regulation of its physiology. Refeeding flies after starvation results in enlarged, food-filled crops, pointing to modulation of food ingression into and out of the crop. Live imaging or temporal dissections of flies revealed that food always enters the crop before proceeding to the midgut. Additionally, food transit through the crop is dependent on both its palatability and its nutritional value. Therefore, in adult flies, all food transits through the crop, which is nutrient-sensitive and shows chemically and anatomically diverse innervation (Hadjieconomou, 2020).

The crop and anterior midgut are innervated by myosuppressin (Ms)-positive neurons located in the PI and the HCG. PI Ms neurons are distinct from known neuronal subsets, with the exception of eight Ms neurons that co-express the Taotie-GAL4 marker. Two PI Ms neuron populations can be distinguished by cell size: one comprises 18 large cells and another comprises 12 smaller cells. Single-cell clones of large Ms neurons reveal a single process that bifurcates into a longer, probably axonal projection to the gut-which arborizes in the HCG and extends further to innervate the crop-and a shorter, probably dendritic process that reaches the suboesophageal zone, where the axons of peripheral gustatory sensory neurons terminate. A subset of HCG Ms-expressing neurons also innervates the crop, whereas another subset projects locally. This study confirmed the expression of Ms using an endogenously tagged Ms reporter (Ms-GFP) and in situ hybridization Ms innervation was also observed of the hindgut, the rectal ampulla and the heart, and a subset of peripheral Ms-positive neurons innervating the female reproductive tract (Hadjieconomou, 2020).

This study selectively activated or silenced Ms neurons in adult flies. Activation resulted in greatly enlarged crops in flies that were fed ad libitum, consistent with the relaxant properties of Ms on insect muscles ex vivo. By contrast, silencing of Ms neurons prevented crop enlargement in a starved-refed condition in which the crop normally expands. Genetic downregulation or mutation of Ms (using a new mutant) prevented crop enlargement, albeit to a lesser extent than Ms neuron silencing. This could be due to another Ms-neuron-derived neurotransmitter or neuropeptide contributing to crop enlargement, or to loss of the Ms peptide during development in these experiments, resulting in adaptations that render the crop more active than it would be in response to acute loss of the Ms peptide. A Gal4 insertion into the Ms locus was generated that disrupts Ms production (MsTGEM). In contrast to the crop enlargement resulting from TrpA1-mediated activation from Ms-Gal4, TrpA1 expression from this (Ms mutant) MsTGEM-Gal4 driver failed to induce crop enlargement, further confirming a requirement for Ms. Ms neuron subtype-specific downregulations and activations enabled establishing that the PI Ms neurons (in particular, the Taotie-Gal4-positive subset of large PI Ms neurons) induce, and are indispensable for, crop enlargement through their production of Ms neuropeptide (Hadjieconomou, 2020).

The contributions of myosuppressin receptors 1 and 2 (MsR1 and MsR2) were explored. MsR1 expression was observed in crop muscles, in subsets of neurons including the PI and HCG Ms-positive neurons and neurons innervating the ovary and heart; no MsR1 expression was detected in ovarian or heart muscles. Expression of MsR2 was also detected in crop muscles. To investigate the function of the Ms receptor, MsR1 was downregulated specifically in adult crop muscles using two independent driver lines (vm-Gal4 and MsR1crop-Gal4). Both genetic manipulations led to reduced crop enlargement in a starvation-refeeding assay, comparable to that observed for Ms neuron silencing or Ms mutation. Downregulation of MsR2 did not affect crop enlargement. A role for MsR1 in mediating crop enlargement was confirmed using a MsR1TGEM mutant. MsR1 is therefore identified as the crop muscle receptor through which Ms signals to modulate crop enlargement (Hadjieconomou, 2020).

The physiological regulation of crop enlargement was explored and found that it is dependent on sex and on reproductive status: the crops of mated females fed ad libitum (which were used for all the experiments described above) were consistently more expanded than those of virgin female or mated male flies fed ad libitum. Because post-mating changes were not seen in Ms neuron projections, it was asked whether post-mating crop enlargement might result from the release of Ms preferentially in mated females. Ms peptide levels were lower in the PI neuron cell bodies of females only after mating. In the absence of Ms transcriptional changes this observation is consistent with a post-mating increase in the secretion of Ms peptide in females. This effect of mating on Ms levels was specific to mating: nutrient availability did not affect intracellular Ms levels. It was also observed that the Ms neurons of mated females had higher cumulative calcium levels and a reduced amplitude of calcium oscillations compared to virgin females, as detected both by in vivo GCaMP6 calcium imaging and by the calcium-sensitive reporter CaLexA, in which GFP expression is proportional to cumulative neuronal activity. Physiologically, and in contrast to observations in mated females, a reduction of Ms signalling in males or in virgin female flies failed to impair crop enlargement. Consequently, when Ms signalling to crop muscles was prevented, the size of the crop of mated females no longer differed from that of virgin females. Collectively, these findings support the idea that, in female flies, the activity of PI Ms neurons changes after mating to promote Ms release (Hadjieconomou, 2020).

Levels of the steroid hormone ecdysone, which promotes egg production and intestinal stem-cell proliferation, increase after mating. The ecdysone receptor (EcR) is expressed by all PI Ms neurons, which suggests that they might be sensitive to circulating ecdysone. Expression of a dominant-negative EcR-which targets all EcR isoforms-confined to the Ms neurons of adult flies was found to increase intracellular Ms levels in the Ms PI neuron cell bodies of mated females to the levels observed in virgin females, whereas it had no effect on virgin females. Downregulation of EcR (using RNA interference lines that target all isoforms or the B1 isoform specifically) produced comparable results. In both experiments, the amplitude of in vivo calcium oscillations in Ms neurons was increased to levels seen in virgin females. Compromising EcR signalling in adult Ms neurons significantly reduced crop enlargement preferentially in mated females; this phenotype was also apparent when the PI Ms neurons were targeted using Taotie-Gal4. Ecdysone therefore communicates mating status to Ms neurons through its B1 receptor (Hadjieconomou, 2020).

Previous work showed that the adult intestine is resized and metabolically remodelled after mating (Reiff, 2015), but did not investigate possible effects on its hormone-producing enteroendocrine cells. This study now observe a post-mating increase in the number of enteroendocrine cells, including a subset that expresses the hormone bursicon α (Burs), which is known to signal to adipose tissue through an unidentified neuronal relay. An endogenous protein reporter for the Burs receptor Rickets (Rk, also known as Lgr2) revealed its expression in subsets of neurons including all PI Ms neurons (including the Taotie-Gal4-positive subset) and in projections terminating in the HCG. Expression in a subset of the HCG Ms neurons was observed only sporadically (Hadjieconomou, 2020).

Consistent with the regulation of Ms neurons by the increase in Burs derived from enteroendocrine cells after mating, adult-specific downregulation of the Burs receptor gene rk in Ms neurons reverted intracellular Ms levels in the PI Ms neurons of mated females to levels observed in virgin females; there was no effect in virgin females. Like EcR downregulation, rk downregulation in Ms neurons also increased the amplitude of in vivo calcium oscillations in the Ms neuron cell bodies of mated females to values similar to those observed in virgin females. Functionally, both the downregulation of Burs in intestinal enteroendocrine cells and the adult-specific rk downregulation in Ms neurons-either in all neurons or in the Taotie-Gal4-positive subset in the PI-preferentially reduced crop enlargement in mated females. Conversely, stimulating the intestinal release of enteroendocrine hormones-including Burs-from enteroendocrine cells resulted in reduced Ms levels in the Ms neuron cell bodies of virgin females, similar to those observed in mated females, and greatly enlarged crops (Hadjieconomou, 2020).

Thus, steroid and enteroendocrine hormones communicate mating status to the brain. Acting through their receptors in the PI Ms neurons, these hormones change Ms neuronal activity, promoting the release of Ms after mating (Hadjieconomou, 2020).

To investigate the importance of Ms neuron modulation after mating, post-mating crop enlargement was selectively prevented by downregulating MsR1 in adult crop muscles using two independent strategies. This had no discernible effects in males or virgin females, but specifically prevented the increase in food intake that is normally observed in female flies after mating. Comparable results were obtained by blocking the post-mating ecdysone and Burs inputs into the Ms neurons. Downregulation of MsR2 had no such effect. The post-mating change in crop expandability, mediated by Ms and MsR1 signalling, thus causes the increased food intake observed in females after mating (Hadjieconomou, 2020).

The negative pressures that have been reported in the crops of larger insects suggest that the crop may draw food in by generating suction. The increased crop expandability enabled by Ms release after mating could therefore increase food intake through changes in suction. It was observed that mated females ingest more food per sip than virgin females, which is consistent with mated females needing to generate a higher suction pressure to facilitate bigger sips. Crop enlargement was therefore modeled using the Poiseuille equation for incompressible fluid flow in a pipe and found that the crop would need a suction pressure of the order of -1 kPa to achieve the previously reported intake volume per sip. This is in reasonable agreement with previously reported values measured in cockroach crops of between -0.5 and -1 kPa. The model predicts that mated flies would require a modest increase in suction pressure to -1.3kPa in order to facilitate the increased sip size (Hadjieconomou, 2020).

In the model, the change in crop volume drives food intake through increased suction. A crop that cannot enlarge, or a persistently enlarged crop, should therefore result in a comparable reduction in food intake by preventing the generation of suction. This was tested by persistently preventing crop enlargement (using crop-muscle-specific MsR1 knockdown) or by persistently inducing it (using TrpA1-mediated Ms neuron activation from Ms-Gal4 or Taotie-Gal4), after which the diet of these flies was switched from an undyed to a dye-laced food source to assess food intake. As predicted, both genetic manipulations reduced food intake. Conversely, increasing the rate at which the crop expands should increase food intake. This was tested by activating the Ms neurons as in the previous experiment, but this time the dye-laced food source was provided, and its intake was monitored at the same time as the neurons were activated (that is, as inducing greater crop expansion was being induced) rather than after a persistent activation (when the crop is already maximally expanded). Increased food intake was observed under these conditions in the absence of changes in the number of meals. Although further work will be required to elucidate the full dynamics of crop enlargement, filling and emptying, these experiments support the idea that the Ms-induced enlargement of the crop after mating increases food intake at least partly by increasing the suction power of the crop (Hadjieconomou, 2020).

Finally, given the links between nutrient intake and fecundity, it is proposed that the Ms-driven crop enlargement after mating might be adaptive and support reproduction. Crop enlargement was prevented selectively after mating by downregulating MsR1 from crop muscles, as in previous experiments. This resulted in reduced egg production, and the eggs that were produced had reduced viability. It is therefore conclude that the crop and its Ms innervation sustain the increase in food intake after mating, maximising female fecundity (Hadjieconomou, 2020).

These findings lead to a proposal that the maternal increase in food intake during reproduction is adaptive, that the crop is a key reproductive organ, and that Ms is a major effector of post-mating responses. In support of these ideas, the crop is absent in larvae-the juvenile stage of insects-and other Diptera have co-opted it for reproductive behaviours such as the regurgitation of nuptial gifts or the secretion of male pheromones. Ms receptors are also closely related to the Sex peptide receptor (the 'mating sensor' of female flies), and both diverged after duplication of an ancestral receptor that might have responded to the Myoinhibitory peptide (Mip) in the last common ancestor of protostomes. It will be interesting to explore possible links between Ms and Sex peptide signalling, and whether and how these mating signals affect recently described crop mechanosensing mechanisms that restrain ingestion as the crop expands in order to terminate large meals (Hadjieconomou, 2020).

This study has provided evidence for a gut-to-brain axis in Drosophila by identifying central Ms neurons as targets of the gut-derived hormone Burs. These central neurons innervate the gut, 'closing' a gut-brain-gut loop that connects midgut enteroendocrine signals to the crop, a more anterior gut region. This might allow for the functional coordination of different gut portions, while enabling central modulation by sensory cues (for example, gustatory). This study also identified the Ms neurons as the neural targets of ecdysone, which has been shown to promote food intake. Reproduction has pronounced, and in some cases lasting, effects on the human female brain; Ms neurons provide a tractable and physiologically relevant neural substrate for the investigation of the mechanisms involved (Hadjieconomou, 2020).

The human digestive system might be similarly modulated by reproductive cues to affect food intake. In mammals, enteric neurons express sex and reproductive-hormone receptors, and enteroendocrine hormone levels change during reproduction. It is suggested that pregnancy and lactation represent an attractive and relatively unexplored physiological adaptation for the investigation of nutrient intake regulation, organ remodelling and metabolic plasticity-mechanisms that might eventually be leveraged to curb appetite and/or weight gain (Hadjieconomou, 2020).

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. It was predicted that DrmMS-Rs contained molecular switches that resembled those of Rhodopsin. Additionally, it is believed DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 interactions would reflect structure-activity relationship (SAR) data. It was hypothesized agonist- and antagonist-receptor contacts would differ from one another depending on activity. Lastly, it was expected that this study would apply to other species; this hypothesis was tested 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 SAR data. R. prolixus (Rhp)MS-R was identified and it, too, was found to 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, RhpMS analogs were designed that were 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 (Leander, 2015).

Peptidergic signaling plays numerous critical roles in transmitting and regulating physiological processes. Therefore, delineating the mechanisms that underlie these events is a powerful approach to identifying target molecules to influence health. An important first step in signaling is when a ligand binds to a G protein-coupled receptor protein (GPCR). Ligand-receptor binding disrupts molecular switches which cause TM movement and ultimately results in receptor activation (Leander, 2015).

Myosuppressin dramatically decreases cardiac contractility and gut motility. First isolated as a cockroach brain peptide that affects spontaneous contractions of the gut, myosuppressin was subsequently found to be distributed throughout the invertebrates. The conservation of its structure and activities, and its widespread distribution are consistent with myosuppressin playing an important role in physiology (Leander, 2015).

Myosuppressins are members of a family of peptides with an identical C-terminal RF-NH2, however, the N-terminal extension is unique and differs in length and sequence. The identical C terminus and variant N terminus are both important in binding and activating signaling pathways. FMRF-NH2, the first RF-NH2-containing peptide isolated, was identified from a neural extract applied to a clam heart preparation. This superfamily of peptides is grouped based on XRF-NH2, where myosuppressins often contain X = L. Myosuppressins are typically represented by X1DVX2HX3FLRF-NH2, where X1 = pQ, P, T, A; X2 = D, G, V; X3 = V, S (Leander, 2015).

Drosophila melanogaster myosuppressin (DrmMS; TDVDHVFLRF-NH2) is representative of its peptide family. DrmMS is pleotropic decreasing the frequency of both cardiac contractility and gut motility. The DrmMS structure-activity relationship (SAR) for its effect on cardiac contractility and gut motility is reported; the data are consistent with DrmMS having distinct signaling pathways in heart and gut. In addition, DrmMS binds to two putative GPCR proteins, DrmMS-R1 and DrmMS-R2. Apart from these facts, little is known about MS signaling. No mechanism which underlies MS receptor activation, a crucial step in signal transduction, is described in literature. And, the design and characterization of MS antagonists in a disease vector are molecularly and physiologically limited in scope in the literature (Leander, 2015).

The kissing bug, Rhodnius prolixus, is a vector of the Chagas disease, an important health problem. RhpMS, pQDIDHVFMRF-NH2, contains two substitutions compared to the MS consensus structure; V3 -> I3 and L8 -> M8. The physicochemical characteristics of myosuppressins are conserved with the residue replacements; RhpMS affects R. prolixus heart rate. The unique MS provides an opportunity to further explore a pathway that affects a crucial physiological function in a disease vector. Previously, little was known about RhpMS signaling; its receptor sequence and structure was unidentified and its SAR uncharacterized (Leander, 2015).

This study tested the prediction that MS signaling would mimic mechanisms involved in Rhodopsin activation. Molecular switch motifs were sought across DrmMS-Rs and the unique ionic lock and novel 3-6 lock, in addition to the transmission and tyrosine toggle switches, were discovered. The belief that DrmMS-DrmMS-R1 and DrmMS-DmrMS-R2 interactions would reflect the SAR data was tested, which it did. When DrmMS and its N-terminal truncation and alanyl-substituted analogs were docked to the DrmMS-Rs, the ligand contacts were distinct between receptors consistent with the SAR data. DrmMS interactions with DrmMS-R2 mirrored the cardiac contractility SAR data and DrmMS-DrmMS-R1 interactions reflected the gut motility data. Additionally, it was hypothesized that agonist- and antagonist-receptor contacts would differ from one another reflecting activity and inactivity. The docking data confirmed this prediction; agonists mirrored DrmMS interactions, yet an inactive analog failed to mimic parent peptide contacts (Leander, 2015).

Lastly, it was expected that this study would apply to other species; this hypothesis was tested in R. prolixus. RhpMS-R structure, binding pockets, ligand contacts, and SAR were tested. The R. prolixus receptor was identified, and it was found to share substantial sequence identity to DrmMS-R1 and DrmMS-R2, 56% and 51%, respectively. The predicted protein was modeled to find the receptor contained typical GPCR features. RhpMS-R contained the unique myosuppressin ionic lock and novel 3-6 lock, and the transmission and tyrosine toggle switches, which, upon ligand binding, promoted TM movement and receptor activation. Further support was obtained for the role of the unique and novel locks by identifying and modeling the red flour beetle, common water flea, honey bee, domestic hornworm, and termite MS-Rs; their structures, too, contained the myosuppressin motifs, which were likely involved in TM movement and receptor activation (Leander, 2015).

Due to the conservation of physicochemistry of the amino acids, which differed between the peptides and the high receptor sequence identity, it was predicted that RhpMS and DrmMS would be alike in activity and ligand contacts. RhpMS and DrmMS decreased R. prolixus cardiac contractility dose dependently with EC50 values of 140 nM and 50 nM, respectively. Based on ligand-receptor contacts, analogs were predicted to be an RhpMS agonist or inactive and act as an antagonist. RhpMS mimicked the full-length peptide; RhpMS applied to R. prolixus heart was inactive and blocked the effect of the parent peptide. Together, data from these studies confirmed tissue specificity in MS signaling, and supported the roles of a unique and a novel lock in MS-R activation (Leander, 2015).

This paper has described molecular switches involved in TM movement that underlies MS-R activation; no prior publication reports these motifs and mechanisms. A unique ionic lock and novel 3-6 lock held MS-Rs in an inactive state that, upon ligand binding, may lead to TM6 movement, a crucial step for receptor activation. A conserved TEFP present in MS-Rs mimicked the Rhodopsin transmission switch, CWLP. Lastly, NF(M/I)(I/L)Y in MS-Rs resembled the Rhodopsin tyrosine toggle switch motif, NPVIY (Leander, 2015).

The motifs were present and made contacts consistent with MS-R activation. Even so, the motifs of the MS-Rs were unique compared to Rhodopsin, in line with the MS peptide, receptor structures, and ligand contacts, and consistent with the DrmMS and RhpMS SAR data. The loss of contacts and networks, and weakened interactions observed in MS-Rs compared to Rhodopsin may indicate their transition from the inactive to active state occurs on a different time scale or energy level (Leander, 2015).

Myosuppressins are likely to play crucial roles in physiology; however, ligand-receptor contact data remained unpublished. DrmMS-R1 and DrmMS-R2 share high sequence identity and bind the same ligand, yet, their binding pockets differed physicochemically. The unique DrmMS-DrmMS-R1 and DrmMS-DrmMS-R2 contact data that this study reports and previous SAR data were consistent with tissue-specific signaling in heart and gut. Next, MS ligand binding and receptor activation were explored in R. prolixus. Although RhpMS-R shared high sequence identity with the DrmMS-Rs, its binding pocket was different in shape and size, and the residues which projected into it. These data were in line with the unique RhpMS SAR compared to DrmMS data, in particular, the differences in the RhpMS active core and antagonist structures for R. prolixus cardiac contractility (Leander, 2015).

Together, the data from this study describe molecular switches involved in receptor activation and ligand contacts which provide insight into how the motifs are involved in MS signaling. Additionally, a bioassay, and binding pockets, size and physicochemistry of the residues available to make contacts supported published data demonstrating tissue-specificity of myosuppressin signaling and yielded information on MS SAR and its receptor in a disease vector (Leander, 2015).

Conserved molecular switch interactions in modeled cardioactive RF-NH2 peptide receptors: Ligand binding and activation

Peptides may act through G protein-coupled receptors to influence cardiovascular performance; thus, delineating mechanisms involved in signaling is a molecular-based strategy to influence health. Molecular switches, often represented by conserved motifs, maintain a receptor in an inactive state. However, once the switch is broken, the transmembrane regions move and activation occurs. The molecular switches of Drosophila melanogaster myosuppressin (MS) receptors were previously identified to include a unique ionic lock and novel 3-6 lock, as well as transmission and tyrosine toggle switches. In addition to MS, cardioactive ligands structurally related by a C-terminal RF-NH2 include sulfakinin, neuropeptide F (NPF), short NPF, and FMRF-NH2-containing peptide subfamilies. It was hypothesized receptor molecular switch motifs were conserved within a RF-NH2 subfamily and across species. Thus, RF-NH2 receptor (RFa-R) molecular switches were investigated in D. melanogaster, Tribolium castaneum, Anopheles gambiae, Rhodnius prolixus, and Bombyx mori. Adipokinetic hormone (AKH), which does not contain a RF-NH2, was also examined. The tyrosine toggle switch and ionic lock showed a higher degree of conservation within a RF-NH2 subfamily than the transmission switch and 3-7 lock. AKH receptor motifs were not representative of a RF-NH2 subfamily. The motifs and interactions of switches in the RFa-Rs were consistent with receptor activation and ligand-specific binding (Rasmussen, 2015).

Structure-activity and immunochemical data provide evidence of developmental- and tissue-specific myosuppressin signaling

Myosuppressin peptides dramatically diminish contractions of the gut and heart. Thus, delineating mechanisms involved in myosuppressin signaling may provide insight into peptidergic control of muscle contractility. Drosophila myosuppressin (DMS, TDVDHVFLRFamide) structure-activity relationship (SAR) was investigated to identify an antagonist and explore signaling. Alanyl-substituted, N-terminal truncated, and modified amino acid analogs identified residues and peptide length required for activity. Immunochemistry independently provided insight into myosuppressin mechanisms. DMS decreased gut motility and cardiac contractility dose dependently; the different effective concentrations at half maximal-response were indicative of tissue-specific mechanisms. Replacement of aspartic acid 2 (D2) generated an analog with different developmental- and tissue-specific effects; [A2] DMS mimicked DMS in adult gut (100% inhibition), yet decreased larval gut contractions by only 32% with increased potency in pupal heart (126% inhibition). The DMS active core differed across development and in tissues; adult (DHVFLRFamide) and larval gut (TDVDHVFLRFamide), and adult (VFLRFamide) and pupal heart (VFLRFamide). Substitution of D2 and D4 with a modified amino acid, p-benzoyl-phenylalanine, produced developmental- and tissue-specific antagonists. In the presence of protease inhibitors, DMS and VFLRFamide were more effective in adult gut, but lower or unchanged in pupal heart compared to peptide or analog alone, respectively. DMS-specific antisera stained neurons that innervated the gut or heart. This study describes novel antagonists and data to identify developmental- and tissue-specific mechanisms underlying the pleotropic effects of myosuppressin in muscle physiology (Dickerson, 2012).

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

This study reports 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 (Mispelon, 2003).

Molecular cloning and functional expression of the first two specific insect myosuppressin receptors

The Drosophila Genome Project database contains the sequences of two genes, CG8985 and CG13803, which are predicted to code for G protein-coupled receptors. The cDNAs corresponding to these genes were purified, and it was found that their gene structures had not been correctly annotated. Subsequently, the coding regions of the two corrected receptor genes were expressed in Chinese hamster ovary cells; each of them coded for a receptor that could be activated by low concentrations of Drosophila myosuppressin (EC50,4 x 10(-8) M). The insect myosuppressins are decapeptides that generally inhibit insect visceral muscles. Other tested Drosophila neuropeptides did not activate the two receptors. In addition to the two Drosophila myosuppressin receptors, a sequence in the genomic database from the malaria mosquito Anopheles gambiae was identified that also very likely codes for a myosuppressin receptor. This paper is the first report on the molecular identification of specific insect myosuppressin receptors (Egerod, 2003).

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 (Duttlinger, 2002).


Search PubMed for articles about Drosophila Myosuppressin

Dickerson, M., McCormick, J., Mispelon, M., Paisley, K. and Nichols, R. (2012). Structure-activity and immunochemical data provide evidence of developmental- and tissue-specific myosuppressin signaling. Peptides 36(2): 272-279. PubMed ID: 22613084

Duttlinger, A., Berry, K. and Nichols, R. (2002). 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. Peptides 23(11): 1953-1957. PubMed ID: 12431733

Egerod, K., Reynisson, E., Hauser, F., Cazzamali, G., Williamson, M. and Grimmelikhuijzen, C. J. (2003). Molecular cloning and functional expression of the first two specific insect myosuppressin receptors. Proc Natl Acad Sci U S A 100(17): 9808-9813. PubMed ID: 12907701

Hadjieconomou, D., King, G., Gaspar, P., Mineo, A., Blackie, L., Ameku, T., Studd, C., de Mendoza, A., Diao, F., White, B. H., Brown, A. E. X., Placais, P. Y., Preat, T. and Miguel-Aliaga, I. (2020). Enteric neurons increase maternal food intake during reproduction. Nature 587(7834): 455-459. PubMed ID: 33116314

Leander, M., Bass, C., Marchetti, K., Maynard, B. F., Wulff, J. P., Ons, S. and Nichols, R. (2015). Cardiac contractility structure-activity relationship and ligand-receptor interactions; the discovery of unique and novel molecular switches in myosuppressin signaling. PLoS One 10(3): e0120492. PubMed ID: 25793503

Mispelon, M., Thakur, K., Chinn, L., Owen, R. and Nichols, R. (2003). A nonpeptide provides insight into mechanisms that regulate Drosophila melanogaster heart contractions. Peptides 24(10): 1599-1605. PubMed ID: 14706539

Rasmussen, M., Leander, M., Ons, S. and Nichols, R. (2015). Conserved molecular switch interactions in modeled cardioactive RF-NH2 peptide receptors: Ligand binding and activation. Peptides 71: 259-267. PubMed ID: 26211890

Reiff, T., Jacobson, J., Cognigni, P., Antonello, Z., Ballesta, E., Tan, K. J., Yew, J. Y., Dominguez, M. and Miguel-Aliaga, I. (2015). Endocrine remodelling of the adult intestine sustains reproduction in Drosophila. Elife 4: e06930. PubMed ID: 26216039

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date revised: 25 May 2021

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