Potent activity of a PK/PBAN analog with an (E)-alkene,trans-Pro mimic identifies the Pro orientation and core conformation during interaction with HevPBANR-C receptor

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a multifunctional role in an array of important physiological processes in insects, including regulation of sex pheromone biosynthesis in moths. A cyclic PK/PBAN analog (cyclo[NTSFTPRL]) retains significant activity on the pheromonotropic HevPBANR receptor from the tobacco budworm Heliothis virescens expressed in CHO-K1 cells. Previous studies indicate that this rigid, cyclic analog adopts a type I β-turn with a transPro over residues TPRL within the core PK/PBAN region. An analog containing an (E)-alkene, trans-Pro mimetic motif was synthesized, and upon evaluation on the HevPBANR receptor found to have an EC₅₀ value that is not statistically different from a parent C-terminal PK/PBAN hexapeptide sequence. The results, in aggregate, provide strong evidence for the orientation of Pro and the core conformation of PK/PBAN neuropeptides during interaction with the expressed PBAN receptor. The work further identifies a novel scaffold with which to design mimetic PBAN analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting PK/PBAN-regulated pheromone signaling systems.     

Structural and functional differences between pheromonotropic and melanotropic PK/PBAN receptors

Background

The pyrokinin/pheromone biosynthesis-activating neuropeptide (PK/PBAN) plays a major role in regulating a wide range of physiological processes in insects. The ubiquitous and multifunctional nature of the PK/PBAN peptide family raises many questions regarding the mechanisms by which these neuropeptides elicit their effects and the nature of the receptors that mediate their functions.

Methods

A sex pheromone gland receptor of the PK/PBAN family from Heliothis peltigera female moth and a Spodoptera littoralis larval receptor were cloned and stably expressed, and their structural models, electrostatic potentials and cellular functional properties were evaluated.

Results

Homology modeling indicated highly conserved amino-acid residues in appropriate structural positions as experimentally shown for class A G-protein coupled receptors. Structural differences could be proposed and electrostatic potentials of the two receptor models revealed net charge differences. Calcium mobilization assays demonstrated that both receptors were fully functional and could initiate extracellular calcium influx to start PK/PBAN signal transduction. Evaluation of the signaling response of both receptors to PBAN and diapause hormone (DH) revealed a highly sensitive, though differential response. Both receptors responded to PBAN whereas only Spl-PK/PBAN-R exhibited a high response toward DH.

Conclusions

The structural, electrostatic and cellular functional differences indicate that different PK/PBAN in vivo functions may be mediated by different PK/PBAN receptors and elicited by different peptide(s).

General significance

The results advance our understanding of the mode of action of the PK/PBAN family, and might help in exploring novel high-affinity receptor-specific antagonists that can serve as a basis for the development of new families of insect-control agents.     

Insect neuropeptide antagonists

The development of a new integrated approach to the generation of a novel type of insect neuropeptide (Np) antagonists and putative insect control agents based on backbone cyclic compounds is described. The approach, termed the backbone cyclic neuropeptide-based antagonist (BBC-NBA), was applied to the insect pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) family as a model, and led to the discovery of a potent linear lead antagonist and several highly potent, metabolically stable BBC antagonists, devoid of agonistic activity, which inhibited PBAN-mediated activities in moths in vivo. This review briefly summarizes our knowledge of insect Nps, describes the PK/PBAN Np family, presents the basic concepts behind the BBC-NBA approach, and introduces the advantages of this method for generation of Np agonists, antagonists and insecticide prototype molecules.     

Role of neuropeptides in sex pheromone production in moths

Sex pheromone biosynthesis in many moth species is controlled by a cerebral neuropeptide, termed pheromone biosynthesis activating neuropeptide (PBAN). PBAN is a 33 amino acid C-terminally amidated neuropeptide that is produced by neuroendocrine cells of the subesophageal ganglion (SEG). Studies of the regulation of sex pheromone biosynthesis in moths have revealed that this function can be elicited by additional neuropeptides all of which share the common C-terminal pentapeptide FXPRL-amide (X = S, T, G, V). In the past two decades extensive studies were carried out on the chemical, cellular and molecular aspects of PBAN and the other peptides (termed the pyrokinin (PK)/PBAN family) aiming to understand the mode of their action on sex pheromone biosynthesis. In the present review we focus on a few of these aspects, specifically on the: (i) structure-activity relationship (SAR) of the PK/PBAN family, (ii) characterization of the PK/PBAN receptor and (iii) development of a novel strategy for the generation of PK/PBAN antagonists and their employment in studying the mode of action of the PK/PBAN peptides.     

Site-directed mutagenesis and PBAN activation of the Helicoverpa zea PBAN-receptor

Pheromone biosynthesis-activating neuropeptide (PBAN) and pyrokinins belong to a family of insect peptide hormones that have a common FXPRLamide C-terminal ending. The G-protein-coupled receptors (GPCRs) for this peptide family were first identified from a moth and Drosophila with sequence similarity to neuromedin U receptors from vertebrates. We have characterized the PBAN-receptor (PBAN-R or PR) active binding domains using chimeric GPCRs and proposed that extracellular loop 3 is critical for ligand selection. Here, we characterized the 3rd extracellular domain of PBAN-R through site-directed point mutations. Results are discussed in context of the structural features required for receptor activation using receptor activation experiments and in silico computational modeling. This research will help in characterizing these receptors towards a goal of finding agonists and/or antagonists for PBAN/pyrokinin receptors.     

An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect

A linear pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) antagonist lead (RYF[dF]PRLa) was structurally modified to impart amphiphilic properties to enhance its ability to transmigrate the hydrophobic cuticle of noctuid moth species and yet retain aqueous solubility in the hemolymph to reach target PK/PBAN receptors within the internal insect environment. The resulting novel PK/PBAN analog, Hex-Suc-A[dF]PRLa (PPK-AA), was synthesized and evaluated as an antagonist in a pheromonotropic assay in Heliothis peltigera against 4 natural PK/PBAN peptide elicitors (PBAN; pheromonotropin, PT; myotropin, MT; leucopyrokinin, LPK) and in a melanotropic assay in Spodoptera littoralis against 3 natural PK/PBAN peptide elicitors (PBAN, PT, LPK). The analog proved to be a potent and efficacious inhibitor of sex pheromone biosynthesis elicited by PBAN (84% at 100 pmol) and PT (54% at 100 pmol), but not by MT and LPK. PPK-AA is a selective pure antagonist (i.e., does not exhibit any agonistic activity) as it failed to inhibit melanization elicited by any of the natural PK/PBAN peptides. The analog was shown to transmigrate isolated cuticle dissected from adult female Heliothis virescens moths to a high extent of 25-30% (130-150 pmol), representing physiologically significant quantities. PPK-AA represents a significant addition to the arsenal of tools available to arthropod endocrinologists studying the endogenous mechanisms of PK/PBAN regulated processes, and a prototype for the development of environmentally friendly pest management agents capable of disrupting the critical process of reproduction.     

Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor gene in Spodoptera littoralis larvae

In noctuid moths cuticular pigmentation is regulated by the pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family, which also mediates a variety of other functions in moths and other insects. Numerous studies have shown that these neuropeptides exert their functions through activation of the PBAN receptor (PBAN-R), with subsequent Ca(2+) influx, followed by either activation of cAMP or direct activation of downstream kinases. Recently, several PBAN-Rs have been identified, all of which are from the pheromone gland of adult female moths, but evidence shows that functional PK/PBAN-Rs can also be expressed in insect larvae, where they mediate melanization and possibly other functions (e.g., diapause). Here, we identified a gene encoding a G-protein-coupled receptor from the 5th instar larval tissue of the moth Spodoptera littoralis. The cDNA of this gene contains an open reading frame with a length of 1050 nucleotides, which translates to a 350-amino acid, 42-kDa protein that shares 92% amino acid identity with Helicoverpa zea and Helicoverpa armigera PBAN-R, 81% with Bombyx mori PBAN-R and 72% with Plutella xylostella PBAN-R. The S. littoralis PBAN-R gene was stably expressed in NIH3T3 cells and transiently in HEK293 cells. We show that it mediates the dose-dependent PBAN-induced intracellular Ca(2+) response and activation of the MAP kinase via a PKC-dependent but Galphai-independent signaling mechanism. Other PK/PBAN family peptides (pheromonotropin and a C-terminally PBAN-derived peptide PBAN(28-33)NH(2)) also triggered MAP kinase activation. This receptor, together with the previously cloned PBAN-R, may facilitate our understanding of the cell-specific responses and functional diversities of this diverse neuropeptide family.     

The Drosophila genes CG14593 and CG30106 code for G-protein-coupled receptors specifically activated by the neuropeptides CCHamide-1 and CCHamide-2

Recently, a novel neuropeptide, CCHamide, was discovered in the silkworm Bombyx mori (L. Roller et al., Insect Biochem. Mol. Biol. 38 (2008) 1147–1157). We have now found that all insects with a sequenced genome have two genes, each coding for a different CCHamide, CCHamide-1 and -2. We have also cloned and deorphanized two Drosophila G-protein-coupled receptors (GPCRs) coded for by genes CG14593 and CG30106 that are selectively activated by Drosophila CCH-amide-1 (EC₅₀, 2 × 10⁻⁹ M) and CCH-amide-2 (EC₅₀, 5 × 10⁻⁹ M), respectively. Gene CG30106 (symbol synonym CG14484) has in a previous publication (E.C. Johnson et al., J. Biol. Chem. 278 (2003) 52172–52178) been wrongly assigned to code for an allatostatin-B receptor. This conclusion is based on our findings that the allatostatins-B do not activate the CG30106 receptor and on the recent findings from other research groups that the allatostatins-B activate an unrelated GPCR coded for by gene CG16752. Comparative genomics suggests that a duplication of the CCHamide neuropeptide signalling system occurred after the split of crustaceans and insects, about 410 million years ago, because only one CCHamide neuropeptide gene is found in the water flea Daphnia pulex (Crustacea) and the tick Ixodes scapularis (Chelicerata).     

A neuromedin-pyrokinin-like neuropeptide signaling system in Caenorhabditis elegans

Neuromedin U (NMU) in vertebrates is a structurally highly conserved neuropeptide of which highest levels are found in the pituitary and gastrointestinal tract. In Drosophila, two neuropeptide genes encoding pyrokinins (PKs), capability (capa) and hugin, are possible insect homologs of vertebrate NMU. Here, the ligand for an orphan G protein-coupled receptor in the nematode Caenorhabditis elegans (Ce-PK-R) was found using a bioinformatics approach. After cloning and expressing Ce-PK-R in HEK293T cells, we found that it was activated by a neuropeptide from the C. elegans NLP-44 precursor (EC₅₀ = 18 nM). This neuropeptide precursor is reminiscent of insect CAPA precursors since it encodes a PK-like peptide and two periviscerokinin-like peptides (PVKs). Analogous to CAPA peptides in insects and NMUs in vertebrates, whole mount immunostaining in C. elegans revealed that the CAPA precursor is expressed in the nervous system. The present data also suggest that the ancestral CAPA precursor was already present in the common ancestor of Protostomians and Deuterostomians and that it might have been duplicated into CAPA and HUGIN in insects. In vertebrates, NMU is the putative homolog of a protostomian CAPA-PK.     

Pyrokinin/PBAN radio-receptor assay: development and application for the characterization of a putative receptor from the pheromone gland of Heliothis peltigera

A radio-receptor assay (RRA) for the insect pyrokinin/PBAN family has been developed. The development involved examination of the ligand (3H-tyrosyl-PBAN28-33NH2)-receptor interaction under various incubation conditions and variations on sex pheromone gland membrane preparation. Application of the RRA for a partial characterization of the putative pyrokinin/PBAN receptor in the pheromone gland of H. peltigera revealed age-dependence of its expression. Pharmacological characterization revealed a high correlation between the binding-affinity to the receptor of various PBAN-derived peptides and their in vivo pheromonotropic bioactivity, and shed light on the interaction of backbone cyclic and linear ([Arg27,D-Phe30]PBAN28-33NH2) PBAN antagonists with the receptor.     

Molecular diversity of PBAN family peptides from fire ants

The PBAN/Pyrokinin peptide family is a major neuropeptide family characterized with a common FXPRLamide in the C‐termini. These peptides are ubiquitously distributed in the Insecta and are involved in many essential endocrinal functions, e.g., pheromone production. Previous work demonstrated the localization of PBAN in the fire ant central nervous system, and identified a new family of PBAN from the red imported fire ant, Solenopsis invicta. In this study, we identified five more PBAN/Pyrokinin genes from S. geminata, S. richteri, S. pergandii, S. carolinensis, and a hybrid of S. invicta and S. richteri. The gene sequences were used to determine the phylogenetic relationships of these species and hybrid, which compared well to the morphologically defined fire ant subgroup complexes. The putative PBAN and other peptides were determined from the amino acid sequences of the PBAN/pyrokinin genes. We summarized all known insect PBAN family neuropeptides, and for the first time constructed a phylogenetic tree based on the full amino acid sequences translated from representative PBAN cDNAs. The PBAN/pyrokinin gene is well conserved in Insecta and probably extends into the Arthropod phylum; however, translated pre‐propeptides may vary and functional diversity may be retained, lost, or modified during the evolutionary process. Published 2010 Wiley Periodicals, Inc.     

Histochemical localization of the PBAN receptor in the pheromone gland of Heliothis peltigera

The presence of the pyrokinin (PK)/ Pheromone biosynthesis activating neuropeptide (PBAN) receptor in pheromone gland cells of Heliothis peltigera females was demonstrated, and its spatial distribution in the ovipositor was visualized with two photo-affinity biotinilated ligands: BpaPBAN1-33NH(2) and BpaArg(27)-PBAN28-33NH(2). Light microscopy histological studies revealed that the gland is contained within the inter-segmental membrane (ISM) between the 8th and 9th abdominal segments. The gland was found to be composed of a single layer of columnar epithelial cells positioned under the inter-segmental cuticle. Similar epithelial cells were also found in the dorsal and ventral regions of the 9th abdominal segment. All regions containing the glandular cells bound both ligands, indicating presence of the PK/PBAN receptor. The patterns obtained with both ligands were similar, hinting at the possibility that either both ligands bind to the same receptor, or, that if there are two distinct receptors, their spatial distribution throughout the gland is very similar.     

Identification of the Drosophila and Tribolium receptors for the recently discovered insect RYamide neuropeptides

One year ago, we discovered a new family of insect RYamide neuropeptides, which has the C-terminal consensus sequence FFXXXRYamide, and which is widely occurring in most insects, including the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum (F. Hauser et al., J. Proteome Res. 9 (2010) 5296–5310). Here, we identify a Drosophila G-protein-coupled receptor (GPCR) coded for by gene CG5811 and its Tribolium GPCR ortholog as insect RYamide receptors. The Drosophila RYamide receptor is equally well activated (EC₅₀, 1 × 10⁻⁹ M) by the two Drosophila RYamide neuropeptides: RYamide-1 (PVFFVASRYamide) and RYamide-2 (NEHFFLGSRYamide), both contained in a preprohormone coded for by gene CG40733. The Tribolium receptor shows a somewhat higher affinity to Tribolium RYamide-2 (ADAFFLGPRYamide; EC₅₀, 5 × 10⁻⁹ M) than to Tribolium RYamide-1 (VQNLATFKTMMRYamide; EC₅₀, 7 × 10⁻⁸ M), which might be due to the fact that the last peptide does not completely follow the RYamide consensus sequence rule. There are other neuropeptides in insects that have similar C-terminal sequences (RWamide or RFamide), such as the FMRFamides, sulfakinins, myosuppressins, neuropeptides F, and the various short neuropeptides F. Amazingly, these neuropeptides show no cross-reactivity to the Tribolium RYamide receptor, while the Drosophila RYamide receptor is only very slightly activated by high concentrations (>10⁻⁶ M) of neuropeptide F and short neuropeptide F-1, showing that the two RYamide receptors are quite specific for activation by insect RYamides, and that the sequence FFXXXRYamide is needed for effective insect RYamide receptor activation. Phylogenetic tree analyses and other amino acid sequence comparisons show that the insect RYamide receptors are not closely related to any other known insect or invertebrate/vertebrate receptors, including mammalian neuropeptide Y and insect neuropeptide F and short neuropeptide F receptors. Gene expression data published in Flybase (www.flybase.org) show that the Drosophila CG5811 gene is significantly expressed in the hindgut of adult flies, suggesting a role of insect RYamides in digestion or water reabsorption.     

PBAN receptor: Employment of anti-receptor antibodies for its characterization and for development of a microplate binding assay

This study describes generation of an anti-PBAN receptor (PBAN-R) antiserum and its employment for the characterization of the PK/PBAN-R(s). The antiserum recognized, in a specific and dose-dependent manner, the presence of PBAN-R in pheromone gland membrane preparations of three female moths: Heliothis peltigera, Helicoverpa armigera and Spodoptera littoralis. It also reacted specifically with the S. littoralis larval receptor in vivo, most likely by competing with the ligand on the binding site and consequently inhibiting cuticular melanization. Despite its ability to react with the receptor of H. peltigera in dot blot experiments, the antiserum did not react with the receptor in vivo and failed to inhibit sex pheromone biosynthesis. The antiserum was also used to develop two microplate binding assays. The Ab described in this study is the first raised against an insect neuropeptide (Np) receptor to be used in vivo, and its employment for characterization of the PK/PBAN-R(s) may thus provide important information on the mode of action of this Np family. The present study adds important information on the difference between the receptors in the two moth species, hints at the possible existence of receptor subtypes, and provides a platform for the development of a high-throughput assay (HTA) for screening of PK/PBAN agonists and antagonists.     

PBAN/pyrokinin神经肽类及其基因的研究进展

昆虫在其生长发育过程中,如胚胎发育、蜕皮变态、滞育、迁飞、代谢、生殖等都离不开神经肽的调控。信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)和Pyrokinin神经肽是C端具有五肽FXPRL(X=S,V,T,G等)(苯丙-X-脯-精-亮氨酸)序列的一类神经肽,在昆虫的生长发育中起重要的生理功能,如性信息素的合成、控制表皮色素、促进胚胎滞育和刺激内脏肌肉收缩等重要的生理功能。因此近几年对PBAN/pyrokinin神经肽的鉴定、加工、作用和降解方式的研究成为研究的热点,为研制高效、低毒、专一性强、无公害的杀虫剂提供了思路。介绍了PBAN/pyrokinin神经肽类及其基因的研究进展,并对PBAN/pyrokinin神经肽在害虫防治中的应用进行了展望。     

The Red Imported Fire Ant (Solenopsis invicta Buren) Kept Y not F: Predicted sNPY Endogenous Ligands Deorphanize the Short NPF (sNPF) Receptor

Neuropeptides and their receptors play vital roles in controlling the physiology and behavior of animals. Short neuropeptide F (sNPF) signaling regulates several physiological processes in insects such as feeding, locomotion, circadian rhythm and reproduction, among others. Previously, the red imported fire ant (Solenopsis invicta) sNPF receptor (S. invicta sNPFR), a G protein-coupled receptor, was immunolocalized in queen and worker brain and queen ovaries. Differential distribution patterns of S. invicta sNPFR protein in fire ant worker brain were associated both with worker subcastes and with presence or absence of brood in the colony. However, the cognate ligand for this sNPFR has not been characterized and attempts to deorphanize the receptor with sNPF peptides from other insect species which ended in the canonical sequence LRLRFamide, failed. Receptor deorphanization is an important step to understand the neuropeptide receptor downstream signaling cascade. We cloned the full length cDNA of the putative S. invicta sNPF prepropeptide and identified the putative “sNPF” ligand within its sequence. The peptide ends with an amidated Tyr residue whereas in other insect species sNPFs have an amidated Phe or Trp residue at the C-terminus. We stably expressed the HA-tagged S. invicta sNPFR in CHO-K1 cells. Two S. invicta sNPFs differing at their N-terminus were synthesized that equally activated the sNPFR, SLRSALAAGHLRYa (EC₅₀ = 3.2 nM) and SALAAGHLRYa (EC₅₀ = 8.6 nM). Both peptides decreased the intracellular cAMP concentration, indicating signaling through the G_αi-subunit. The receptor was not activated by sNPF peptides from other insect species, honey bee long NPF (NPY) or mammalian PYY. Further, a synthesized peptide otherwise identical to the fire ant sequence but in which the C-terminal amidated amino acid residue ‘Y’ was switched to ‘F’, failed to activate the sNPFR. This discovery will now allow us to investigate the function of sNPY and its cognate receptor in fire ant biology.