Signaling properties and pharmacological analysis of two sulfakinin receptors from the red flour beetle, Tribolium castaneum (original) (raw)
Related papers
Peptides, 2014
Insects acquire essential nutrients from their food to support a diverse range of biological processes such as development and reproduction. An important role in the food intake regulation is attributed to the neurohormone sulfakinin signaling pathway. Sulfakinins (SKs) elicit satiety together with sulfakinin receptors (SKRs). In this project, the gene coding a second putative SKR, namely skr2, was cloned, characterized and functionally studied in the red flour beetle Tribolium castaneum, with the purpose to understand its role in food intake regulation. The gene skr2 encoded a seven-transmembrane SKR2 protein with 420 amino acids and is evolutionarily close to the two SKRs in Drosophila. A distribution analysis by means of quantitative real-time PCR revealed a tissue-and stage-specific expression pattern with skr2 being dominantly expressed in head, and in the pupal and adult stages. Besides, its expression in gut was notable. With use of dsRNA of skr2, larval food intake was stimulated in an injection RNAi assay. Based on the data obtained, skr2 is considered as an indispensable component in the regulation of food intake in T. castaneum.
Peptides, 2013
The insect sulfakinins (SKs) constitute a family of neuropeptides that display both structural and functional similarities to the mammalian hormones gastrin and cholecystokinin (CCK). As a multifunctional neuropeptide, SKs are involved in muscle contractions as well as food intake regulation in many insects. In the red flour beetle Tribolium castaneum, the action on food intake by a series of synthetic SK analogs and one putative antagonist was investigated by injection in beetle adults. The most remarkable result was that both sulfated and non-sulfated SKs [FDDY(SO 3 H)GHMRFamide] inhibited food intake by about 70%. Strong activity observed for SK analogs featuring a residue that mimics the acidic nature of Tyr(SO 3 H) but lack the phenyl ring of Tyr, indicate that aromaticity is not a critical characteristic for this position of the peptide. SK demonstrated considerable tolerance to Ser and Ala substitution in position 8 (basic Arg), as analogs featuring these uncharged substitutions retained almost all of the food intake inhibitory activity. Also, the Phe in position 1 could be replaced by Ser without complete loss of activity. Conversely, substitution of Met by Nle in position 3 led to inactive compounds. Finally, the Caenorhabditis elegans sulfated neuropeptide-like protein-12 (NLP-12), that shares some sequence similarities with the SKs but features a Gln-Phe-amide rather than an Arg-Phe-amide at the C-terminus, elicited increased food intake in T. castaneum, which may indicate an antagonist activity. Co-injection NLP-12 with nsSK blocked the food intake inhibitory effects of nsSK. (G. Smagghe). As hormones, SKs require certain receptors. Sulfakinin recep-38 tors (SK-Rs) were predicted and cloned in Drosophila melanogaster, 39 based on their sequence similarity to the CCK receptors, and 40 designated as DSK-R1 and DSK-R2 [15,18]. In the red flour bee-41 tle Tribolium castaneum, two SK-Rs were predicted based on the 42 genome sequence [14]. These receptors were identified as G 43 protein-coupled receptors. 44 Studies on function reported that SKs influence the frequency 45 and amplitude of foregut and hindgut visceral muscle contractions 46 [26,28,33,36]. Similar to the satiety effect of gastrin/CCK in mam-47 mals, insect SKs significantly inhibited food intake in the locust 48 Schistocerca gregaria [46], the cockroach Blattella germanica [19] 49 and in the cricket Gryllus bimaculatus [22]. In the blow fly Phormia 50 regina, carbohydrate ingestion was decreased by feeding SK [6]. In 51 addition, SKs stimulate release of the digestive enzyme ␣-amylase 52 in the red palm weevil Rhynchophorus ferrugineus [24] and the 53 coconut pest Opisina arenosella [13]. 54 The insect SKs share a conserved C-terminus (DYGHMRFamide) 55 among various species [5,8,22]. Two SK peptides exist in many 56 0196-9781/$ -see front matter PEP 68869 1-6 2 N. Yu et al. / Peptides xxx (2012) xxx-xxx insects, which were designated as SK I and SK II, differing in the N-57 terminal sequences. Previous studies showed that the sulfated Tyr 58 is required for the activities of SKs [18,19,46]. In Drosophila, a 3000-59 fold higher concentration of the nonsulfated sulfakinin (nsDSK) 60 than the sulfated sulfakinin (sDSK) was required to stimulate the SK 61 receptor (DSK-R1) as expressed in mammalian cells [18]. In addi-62 tion, sDSK and nsDSK were reported to display different activities 63 in odor preference and locomotion or in different tissues or stages, 64 phenomena which indicate that distinct mechanisms may underlie 65 the effect of SKs [33,34]. Thus, studies on the SK activity related to 235 Tyr is not necessary for this activity. In the American cockroach 236 Periplaneta americana, both sulfated and nonsulfated SKs (Lem-237 SKs) were detected in the corpora cardiac/corpora allata complexes, 238 providing evidence that the two forms of SK naturally co-exist in 239 insects [39]. Previous studies on food intake suggested that the 240 sulfate moiety is critical for this modulatory activity, which is oppo-241 site to the case in T. castaneum. In the desert locust, S. gregaria, 242 nsLom-SK had no effect on food intake [46]. Maestro et al. reported 243 that in B. germanica, the sLem-SK-II and sPea-SK could inhibit food 244 intake at low concentrations, while the nonsulfated peptides were 245 inactive even at higher concentrations [19]. In addition, the nsDSK 246 was 3000-fold less potent than the sDSK to activate DSK-R1 in D. 247 melanogaster [18]. Nevertheless, nsSK activities were also reported 248 in Drosophila, mainly focusing on myoactivity, odor preference and 249 Please cite this article in press as: Yu N, et al. Analogs of sulfakinin-related peptides demonstrate reduction in food intake in the red flour beetle, Tribolium castaneum, while putative antagonists increase consumption. Peptides (2012), http://dx.
Scientific Reports, 2015
Despite their fundamental importance for growth, the mechanisms that regulate food intake are poorly understood. Our previous work demonstrated that insect sulfakinin (SK) signaling is involved in inhibiting feeding in an important model and pest insect, the red flour beetle Tribolium castaneum. Because the interaction of SK peptide and SK receptors (SKR) initiates the SK signaling, we have special interest on the structural factors that influence the SK-SKR interaction. First, the threedimensional structures of the two T. castaneum SKRs (TcSKR1 and TcSKR2) were generated from molecular modeling and they displayed significance in terms of the outer opening of the cavity and protein flexibility. TcSKR1 contained a larger outer opening of the cavity than that in TcSKR2, which allows ligands a deep access into the cavity through cell membrane. Second, normal mode analysis revealed that TcSKR1 was more flexible than TcSKR2 during receptor-ligand interaction. Third, the sulfated SK (sSK) and sSK-related peptides were more potent than the nonsulfated SK, suggesting the importance of the sulfate moiety. Insect sulfakinin (SK) signaling is active in a variety of biological processes in insects, such as the regulation of feeding 1-5 and muscle contraction 6-8. SK signaling consists of SK peptides, SK receptors (SKRs) and other molecules. The interaction of SK peptides and SKRs initiates the SK signaling transduction. SK peptides share a conserved carboxyl-terminal amino acid sequence YGHMRF-NH 2 with different amino-terminal extensions 9. Two forms of SK peptides are often found in insects as sulfated SK (sSK) and nonsulfated SK (nsSK), depending on the presence of a sulfate group on the tyrosyl residue 8. SKRs are G-protein coupled receptors (GPCRs) that can convert the extracellular signals into intracellular signals 10. Insect SK signaling is found to be homologous to the cholecystokinin (CCK) signaling in humans since they show homology over their components, signaling transduction and functions 2,6,7,9,11,12. Human CCK signaling has been investigated extensively. It involves the CCK peptides and CCK receptors 13. CCK peptides exist in two forms: sulfated CCK (sCCK) and nonsulfated CCK (nsCCK). In human CCK signaling, two CCK receptors (CCKRs), namely CCK1R and CCK2R, show different affinities to sCCK and nsCCK. CCK1R is activated by sCCK 500-to 1000-fold more than by nsCCK, while CCK2R responds to both sCCK and nsCCK similarly 13. The ligand binding sites in CCKRs have been examined via site-directed mutagenesis 14-18 , photoaffinity labeling 19,20 and molecular modeling 16-19. Several amino
ACS Omega, 2020
G protein-coupled receptors (GPCRs) play a pivotal role in regulating key physiological events in all animal species. Recent advances in collective analysis of genes and proteins revealed numerous potential neuropeptides and GPCRs from insect species, allowing for the characterization of peptide−receptor pairs. In this work, we used fluorescence resonance energy transfer (FRET)-based genetically encoded biosensors in intact mammalian cells to study the pharmacological features of the cognate GPCR of the type-C allatostatin (AST-C) peptide from the stick insect, Carausius morosus. Analysis of multiple downstream pathways revealed that AST-C can activate the human Gi 2 protein, and not Gs or Gq, through AST-C receptor (AlstRC). Activated AlstRC recruits β-arrestin2 independent of the Gi protein but stimulates ERK phosphorylation in a Gi protein-dependent manner. Identification of Gαi-, arrestin-, and GRK-like transcripts from C. morosus revealed high evolutionary conservation at the G protein level, while β-arrestins and GRKs displayed less conservation. In conclusion, our study provides experimental and homology-based evidence on the functionality of vertebrate G proteins and downstream signaling biosensors to characterize early signaling steps of an insect GPCR. These results may serve as a scaffold for developing assays to characterize pharmacological and structural aspects of other insect GPCRs and can be used in deorphanization and pesticide studies.
Insect Biochemistry and Molecular Biology, 2011
Following a reverse pharmacology approach, we identified an allatotropin-like peptide receptor in Tribolium castaneum. Allatotropins are multifunctional neuropeptides initially isolated from the tabacco hornworm, Manduca sexta. They have been shown to be myoactive, to be cardio-acceleratory, to inhibit active ion transport, to stimulate juvenile hormone production and release and to be involved in the photic entrainment of the circadian clock. A tissue distribution analysis of the T. castaneum allatotropinlike peptide receptor by means of qRT-PCR revealed a prominent sexual dimorphism, the transcript levels being significantly higher in the male fat body and reproductive system. The endogenous ligand of the receptor, Trica-ATL, is able to increase the frequency and tonus of contractions in the gut and in the reproductive tract of mature red flour beetles.
Insect Biochemistry and Molecular Biology, 2013
The insect PISCF/allatostatins (ASTs) are pleiotropic peptides that are involved in the regulation of juvenile hormone biosynthesis, are myoinhibitory on the gut and the heart, and suppress feeding in various insects, but their roles in beetles are poorly understood. To provide further insight into the significance of PISCF/ASTs in beetles, the PISCF/AST receptor from Tribolium castaneum has been characterised and its tissue distribution determined. The biological activity of the T. castaneum PISCF/AST (Trica-AS) was also investigated. The Trica-AS receptor shows high sequence homology to other insect PISCF/AST receptors, which are related to the mammalian somatostatin/opioid receptors, a family of G protein-coupled receptors. The Trica-AS receptor was activated in a dose-dependent manner by both Trica-AS and T. castaneum allatostatin double C (Trica-ASTCC) as well as Manduca sexta-allatostatin (Manse-AS). Other allatoregulatory peptides (a FLG/AST, a MIP/AST and an allatotropin) and somatostatin 14 were inactive on this receptor. Receptor transcript levels in tissues, determined by qRT-PCR, were highest in the head and the gut, with variable amounts in the fat body and reproductive organs. There were measurable differences in receptor levels of the head, fat body and reproductive organs between males and females. There was also a widespread distribution of Trica-AS in various tissues of T. castaneum. The Trica-AS peptide precursor was most abundant in the head and there was a significant difference between levels in the heads and reproductive organs of males and females. Whole mount immunocytochemistry localised Trica-AS in the median and lateral neurosecretory cells of the brain, in the corpus cardiacum and throughout the ventral nerve cord. The peptide was also present in midgut neurosecretory cells, but no immunostaining was detected in the reproductive organs or Malpighian tubules. The widespread distribution of both Trica-AS and its receptor suggest this peptide may have multiple roles in beetles. However, Trica-AS had no effect on the spontaneous contractions of the gut or ovaries of T. castaneum but this peptide did stimulate the release of proteases from the anterior midgut of another beetle, Tenebrio molitor. The activation of the Trica-AS receptor by Trica-ASTCC implies a physiological role for this peptide in beetles, which remains to be identified.
Advances in Insect Physiology, 2014
In metazoans, neuronal and endocrine communication is based on the release of extracellular signaling molecules that are recognized in a physiological concentration range by specific receptor proteins present in the target cells. These receptors will elicit a cellular response upon activation by their physiological agonist. A highly diverse repertoire of naturally occurring receptor agonists has already been discovered. Peptides, proteins and biogenic amines constitute the most diverse agonist classes. Most of these interact with G protein-coupled receptors (GPCRs), the largest category of signal transducing receptors that controls virtually every physiological process in metazoans. For more than two decades, insect GPCRs have been hailed for their potentially excellent aptitude to serve as pharmacological targets for the development of novel products for insect pest control. In this review, we will address this issue and enumerate reasons why it would be worth investing more in these targets. -HT 5-hydroxytryptamine (serotonin) AKH adipokinetic hormone AR adrenergic receptor ARF ADP-ribosylation factor ARNO ARF nucleotide binding site opener AST allatostatin AT allatotropin ATL AT-like peptide ATR AT receptor BmNPV Bombyx mori nuclear polyhedrosis virus CA corpora allata CC corpora cardiaca CCAP crustacean cardioactive peptide CCK cholecystokinin CRF corticotrophin releasing factor CRF/DH CRF-like diuretic hormone CT/DH calcitonin-like diuretic hormone DLGR Drosophila melanogaster leucine-rich repeats containing GPCR E ecdysone ECL extracellular loop EH eclosion hormone ERK extracellular signal-regulated kinase ETH ecdysis-triggering hormone FOXO forkhead transcription factor GTPase activating protein GIT GRK-interacting protein GnRH gonadotropin-releasing hormone Grb2 growth factor receptor bound protein-2 GRK GPCR kinase ICL intracellular loop ILP insulin-like peptide InR insulin receptor IRP insulin-related peptide JH juvenile hormone MAPK mitogen-activated protein kinase MIP myoinhibiting peptide NPF neuropeptide F NPY neuropeptide Y NSF N-ethylmaleimide-sensitive fusion protein OMP ovary maturating parsin PBAN pheromone biosynthesis activating neuropeptide PDF pigment dispersing factor PDK1 3-phospoinositide-dependent protein kinase PI3K phosphoinositide 3-kinase PK pyrokinin PKA cAMP-dependent protein kinase PKB protein kinase B PKC Ca 2+ -dependent protein kinase PTEN phosphatase and tensin homolog PTTH prothoracicotropic hormone RPCH red pigment concentrating hormone
European Journal of Biochemistry, 2001
The feeding cycle of the adult female cockroach Blattella germanica parallels vitellogenesis. The study of the mechanisms that regulate this cycle led us to look for food-intake inhibitors in brain extracts. The antifeedant activity of brain extracts was tested in vivo by injecting the extract and measuring the carotenoids contained in the gut from carrot ingested after the treatment. By HPLC fractionation and tracking the biological activity with the carrot test, we isolated the sulfakinin EQFDDY(SO 3 H) GHMRFamide (Pea-SK). A synthetic version of the peptide inhibited food intake when injected at doses of 1 mg (50% inhibition) and 10 mg (60% inhibition). The sulfate group was required for food-intake inhibition. These biological and structural features are similar to those of the gastrincholecystokinin (gastrin-CCK) family of vertebrate peptides. However, heterologous feeding assays (human CCK-8 tested on B. germanica, and Pea-SK tested on the goldfish Carassius auratus ) were negative. In spite of this, alignment and cluster analysis of these and other structurally similar peptide families suggest that sulfakinins and gastrin -CCKs are homologous, and that mechanisms of feeding regulation involving these regulatory peptides may have been conserved during evolution between insects and vertebrates.
Sulfakinins reduce food intake in the desert locust, Schistocerca gregaria
Journal of Insect Physiology, 2000
In vertebrates, the peptides cholecystokinin (CCK), neuropeptide Y, galanin, and bombesin are known to be involved in the control of food intake. We report here that insect sulfakinins, peptides which display substantial sequence similarities with the vertebrate gastrin/CCK peptide family, significantly inhibit food uptake in fifth instar nymphs of the locust, Schistocerca gregaria. Upon injection of Lom-sulfakinin, a neuropeptide present in the corpus cardiacum of locusts, food intake was significantly reduced in a dose-dependent manner within a fixed 20 min time period. The induced effect ranged from 13% inhibition (10 pmol of injected peptide) to over 50% inhibition at 1 nmol. Other naturally occurring sulfakinins from different insect species also elicited this satiety effect. Analogous to the satiety effect of CCK in vertebrates, the sulfate group is required for activity. No effect on the palptip resistance was found after injection with sulfakinin. Therefore it seems unlikly that sulfakinins reduce food intake by decreasing the sensitivity of the taste receptors.
Tissue and Cell, 1997
We describe the distribution of sulfakinin-like neuropeptides in the central and sympathetic nervous system of the American cockroach Periplaneta americana (L.) (Blattodea) and the field cricket Teleogryllus commodus (Walker) (Orthoptera), using an antisulfakinin primary antibody and confocal laser scanning microscopy. We conclude that, in the cockroach, sulfakinin-like material is produced in ten pairs of anterior cells in the pars intercerebralis, as well as two pairs of medial and one major pair of lateral posterior brain cells. This contrasts with findings in other insects, including the cricket, where only the posterior cell groups express sulfakinin-immunoreactive material. Extensive arborization of dendrites containing sulfakinin-like peptides occurs within the neuropile of both species, suggesting a neurotransmitter/ neuromodulator function. In the cockroach, there is clear evidence of direct distribution of sulfakinin-like peptides along axons to the foregut tissue, and a plexus of retrocerebral nerves is likely to serve as a neurohaemal release site. Neurohaemal release into the dorsal aorta is also postulated. Sulfakininimmunoreactive axons do not innervate the hindgut in either cockroaches or crickets. Sulfakinin may function as a gut myotropin in the Blattodea, in addition to functioning as a neurotransmitter within the central nervous system. This latter function appears to be general across insect orders, while the neurohaemal distribution and myotropic activity are restricted to the Blattodea.