Pheromonotropic stimulation of moth pheromone gland cultures in vitro

The direct neurohormonal control of pheromone biosynthesis by pheromone biosynthesis activating neuropeptide (PBAN) was demonstrated in Helicoverpa (Heliothis) spp. using pheromone gland cultures in vitro. Pheromone gland activation involved the de novo production of the main pheromone component (Z)-11-hexadecenal as revealed by radio-TLC, radio-HPLC, and radio-GC. Activation was found to be a specific response attributed to pheromone gland cultures alone. Specificity of pheromonotropic activation was demonstrated to be limited to nervous tissue extracts. A sensitive and specific radioimmunoassay was developed using [³H]-PBAN, and the spatial and temporal distribution of PBAN-immunore-activity was studied. PBAN-immunoreactivity in brain complexes was found throughout the photoperiod and in all ages. From the distribution of PBAN-immunoreactivity it appears that PBAN release is affected by photoperiod. Pheromone gland cultures were found to be competent to pheromone production irrespective of age and photoperiod. Therefore, the neuroendocrine control of pheromone production operates at the level of neuropeptide synthesis and/or release and not at the level of the target tissue itself. The involvement of cyclic-AMP as a second messenger system was demonstrated. Brain extracts and PBAN were shown to stimulate dose- and time-dependent changes in intracellular cyclic-AMP levels. The role of cyclic-AMP in this mechanism was further verified by the ability of cyclic-AMP mimetics to mimic the pheromonotropic effect of brain extracts and PBAN. However, dose-response studies using PBAN and a hexapeptide C-terminal fragment of PBAN suggested that PBAN induces a two mechanism response, one occurring at low PBAN concentrations (high affinity receptor) and another at higher PBAN concentrations (low affinity receptor). Further evidence indicating a dual receptor system was obtained with the observation that the active phorbol ester (phorbol-12-myristate 13-acetate), the diacyl-glycerol analog (1,2-dioleolyl-sn-glycerol), and the intracellular calcium ionophore (ionomycin) mimicked the physiological action of PBAN and that lithium chloride had a pheromonostatic effect. The results indicate that pheromone glands also possess receptors that are linked to inositol phosphate hydolysis. © 1994 Wiley-Liss, Inc.     

Neuroendocrine control of pheromone production in moths

In many moth species regulation of pheromone production has been attributed to the timely release of a pheromone biosynthesis activating neuropeptide (PBAN). The gene encoding PBAN has been sequenced in two moth species. Immunochemical studies as well asin situ hybridization and Northern analysis of PBAN encoding mRNA have localized the neuroendocrine cells responsible for the production of PBAN and have traced the neuronal network of PBAN immunoreactivity. Release into the bloodstream has been demonstrated, the target tissue delineated, and the signal transduction pathway and its modulation analyzed. This paper reviews the current status of research concerning the neuroendocrine control of pheromone production in Lepidopterans and presents some recent developments concerning the receptors involved in the pheromonotropic activity. In this study, we report on the use of a biologically active photoaffinity-biotin-labeled derivative of PBAN N-[N-(4-azido-tetrafluorobenzoyl-biocytinyloxyl-succinimide) and show the presence of a protein (estimated molecular weight of 50 kDa) which specifically binds to PBAN in membrane preparations of pheromone glands. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No.2279-E, 1997 series     

PBAN stimulation of pheromone biosynthesis by inducing calcium influx in pheromone glands of Helicoverpa zea

Isolated pheromone glands of Helicoverpa zea were utilized to investigate the physiological action of pheromone biosynthesis activating neuropeptide (PBAN) with regard to the role of calcium ions in stimulating pheromone biosynthesis under various incubation conditions. Incubation of glands with 1 microM or 1 nM PBAN produced a significant amount of pheromone after a 5 min incubation period and reached maximum pheromone production after 30 min. Glands incubated with PBAN for 1 min, and then without PBAN for 30 min, produced pheromone whether or not extracellular calcium was present during the first 1 min. The presence of lanthanum as a calcium channel blocker did not affect pheromone production if present during the first 1 min of incubation with PBAN. However, if calcium was absent or lanthanum ion was present during the 30 min of incubation, no pheromone was produced. A maximum amount of pheromone was reached when glands were incubated for 1 min with PBAN and for 10 min without PBAN, and repeated three times. The present results indicate that a time interval exists between PBAN binding to a receptor and opening of extracellular calcium channels. Calcium influx into the cytosol from extracellular stores is required for PBAN to stimulate pheromone production. This could be achieved by PBAN either binding periodically to the receptor or the plasma membrane calcium channel could remain activated for a period of time after the initial activation.     

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.     

Involvement of calcineurin in the signal transduction of PBAN in the silkworm, Bombyx mori (Lepidoptera)

In several moth species sex pheromone production in the pheromone gland is regulated by a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori it is suggested that PBAN, after binding to the cell-surface receptor, primarily activates a plasma membrane receptor-activated Ca2+ channel to increase cytosolic levels of Ca2+, and Ca2+/calmodulin complex directly or indirectly activates a phosphoprotein phosphatase, which in turn elicits activation of acyl CoA reductase (the key enzyme under PBAN control) through dephosphorylation, resulting in pheromone (bombykol) production. The effect of cyclosporin A (CsA) and FK 506, specific inhibitors of calcineurin (phosphoprotein phosphatase 2B) was studied on the sex pheromone production, in B. mori. The in vitro experiments showed that both chemicals exerted a dose-dependent inhibitory action when they were co-incubated with TKYFSPRL amide (Hez-PBAN fragment peptide). Practically, no difference was detected between the two chemicals in the tested doses (0.025-1250 microM). When effects of CsA or FK 506 were studied on cell-free production of bombykol by using microsomal fraction no inhibition was detected. Since microsomal fraction contains the acyl CoA synthetase, the rate-limiting acyl CoA reductase and the precursor, bombykol is produced if supplied with CoA, ATP and NADPH. Thus, the inhibitory action of CsA and FK506 under in vitro conditions should occur before the step of acyl group reduction and the effect is likely to be attributable to the inhibition of calcineurin in the signal transduction cascade mechanism of PBAN, in B. mori. The existence of calcineurin in the pheromone gland by using Western blot analysis is also demonstrated.     

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.     

The identification of an age- and female-specific putative PBAN membrane-receptor protein in pheromone glands of Helicoverpa armigera: possible up-regulation by Juvenile Hormone

The present study was designed to determine the age and female specificity of a membrane protein that binds to a pheromone biosynthesis activating neuropeptide (PBAN) ligand and to elucidate the effect of Juvenile Hormone (JH) on binding as well as pheromone activation. The precise age at which developing adult females of Helicoverpa armigera begin to respond to PBAN was determined. PBAN activates in vitro pheromone biosynthesis as well as its intracellular second messenger, cAMP, only in intersegments of newly emerged adult female pheromone glands (i.e. 1-day-old females). An increase in response was observed in 2-day-old females. Intersegments of female pupae and the homologous tissues of adult males do not respond to PBAN. However, in the presence of Juvenile Hormone II (JH II) PBAN induced a response in females, 1 day before emergence (pharate females), but not in younger female pupae. This phenomenon was also observed after topical applications of the JH analog fenoxycarb (FX). In addition the response to PBAN by intersegments of FX-treated emerged adults increased significantly to the level of 2-day-old females. JH II also stimulated the level of incorporation of (35)S-labelled amino acids in female pupae into membrane proteins that are typical in adult intersegments. Using a photoaffinity-biotin labelled PBAN analog we demonstrate specific binding of a membrane protein (estimated MW: 50 kD) in adult females. This binding was not detected in female pupae 3 days before emergence. However, in such female pupae specific binding of the 50 kD protein by the photoaffinity-biotin labelled PBAN analog was induced after JH II or FX treatments thereby providing evidence that JH may up-regulate this putative receptor protein.     

Molecular modeling of the binding of pheromone biosynthesis activating neuropeptide to its receptor

Moth sex-pheromone biosynthesis follows a circadian cycle, which is cued by the release of the neurohormone pheromone biosynthesis activating neuropeptide (PBAN) to the hemolymph. PBAN binds to a G protein-coupled receptor (GPCR), in pheromone glands, (PG) initially identified by us in Helicoverpa zea moths (HezPBAN-R). In this study, the sequences of the seven transmembrane helices of HezPBAN-R were identified, built, packed and oriented correctly after multiple sequence alignment of the HezPBAN-R and several other GPCRs using the X-ray structure of rhodopsin as a template. Molecular dynamics simulations were run on three different beta-turn types of the C-terminal hexapeptide of PBAN and the results clustered into 12 structurally distinct groups. The lowest energy conformation from each group was used for computer-simulated docking with the model of the HezPBAN-R. Highest scoring complexes were examined and putative binding sites were identified. Experimental studies, using in vitro PG, revealed lower levels of pheromonotropic activity when challenged with pyrokinin-like peptides than with HezPBAN as ligand. Thus, the Drosophila melanogaster pyrokinin-1 receptor (CG9918) was chosen to create chimera receptors by exchanging between the three extracellular loops of the HezPBAN-R and the CG9918 for in silico mutagenesis experiments. The predicted docking model was validated with experimental data obtained from expressed chimera receptors in Sf9 cells.     

Mechanisms involved in the control of pheromone production in female moths: recent developments

Species‐specific pheromone blends of nocturnal female moths, derived from fatty acid precursors, are produced and released for mate‐finding, and are initiated by the circadian, trophic hormone, Pheromone Biosynthesis Activating Neuropeptide (PBAN). PBAN, produced in the sub‐oesophageal ganglion, is a 33 amino acid neuropeptide with a minimum active core in its FXPRLamide C‐terminal. PBAN acts directly on pheromone gland cells of mature females by binding to a specific G‐protein‐coupled membrane receptor (GPCR), and thereby initiating a signal transduction cascade involving calcium and cAMP. This discussion will review recent developments concerning the identification of the PBAN GPCR, its regulation by juvenile hormone (JH), and its mode of action at the level of the pheromone biosynthetic pathway. The discussion will also include recent developments concerning events occurring as a result of the transfer of pheromonostatic compounds of male origin after mating.     

PBAN-induced sex pheromone biosynthesis in Heliothis peltigera: Structure,dose, and time dependent analysis

A structure-activity relationship study of Hez-PBAN was performed with respect to its pheromonotropic activity, using Heliothis peltigera as the test animal. The activity of N- and C-terminally derived sequences was examined in a time- and dose-dependent mode. Using a variety of Hez-PBAN-derived fragments at two doses (1 and 10 pmol) and at different times post-injection (5–120 min), we were able to demonstrate that peptides lacking 12 and 16 amino acids from their N-terminus are as potent as the full length PBAN, and that the C-terminally derived hexapeptide was capable of stimulating sex pheromone production to a similar extent as PBAN 1–33NH₂, when its activity was analyzed at shorter post-injection times. Within the C-terminal sequence, the amide was found to play a crucial role. In addition, it was observed that the region between amino acids 9 and 13 is important for the biological activity of the full length PBAN. The fact that the pheromonotropic activity of the hexapeptide was similar to that of the full length PBAN, under specific conditions, suggests that this sequence constitutes the biologically active site of the neuropeptide. The discovery that PBAN-derived peptides reacted in a time- and dose-dependent mode, strengthens the assumption that proteolytic enzymes interfere with the pheromonotropic activity of the PBAN-derived fragments. The ability of a variety of peptides to stimulate sex pheromone biosynthesis suggests two possible mechanisms: (1) Existence of multiple pheromonotropic mechanisms which may be mediated by multiple PBAN receptors that are activated at different kinetics; (2) Existence of only one mechanism mediated by short C-terminally derived peptides. In the first case, the C-terminally derived sequences fulfill the conformational requirement of only one class of receptors, and other regions in the PBAN molecule (e.g., 9–13) fulfill the conformational requirements of a second (or other) class of receptors. In the second case, the C-terminally derived sequence is the only conformationally important sequence, and other sequences, which were found to be essential for the biological activity, serve other non-conformational purposes (e.g., protection against proteolytic degradation). © 1995 Wiley-Liss, Inc.     

Ant Trail Pheromone Biosynthesis Is Triggered by a Neuropeptide Hormone

Our understanding of insect chemical communication including pheromone identification, synthesis, and their role in behavior has advanced tremendously over the last half-century. However, endocrine regulation of pheromone biosynthesis has progressed slowly due to the complexity of direct and/or indirect hormonal activation of the biosynthetic cascades resulting in insect pheromones. Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN) was identified that stimulated sex pheromone biosynthesis in a lepidopteran moth. Since then, the physiological role, target site, and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in moths. Despite that PBAN-like peptides (∼200) have been identified from various insect Orders, their role in pheromone regulation had not expanded to the other insect groups except for Lepidoptera. Here, we report that trail pheromone biosynthesis in the Dufour''s gland (DG) of the fire ant, Solenopsis invicta, is regulated by PBAN. RNAi knock down of PBAN gene (in subesophageal ganglia) or PBAN receptor gene (in DG) expression inhibited trail pheromone biosynthesis. Reduced trail pheromone was documented analytically and through a behavioral bioassay. Extension of PBAN''s role in pheromone biosynthesis to a new target insect, mode of action, and behavioral function will renew research efforts on the involvement of PBAN in pheromone biosynthesis in Insecta.     

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.     

Hormonal control of female sex pheromone biosynthesis in the redbanded leafroller moth,Argyrotaenia velutinana

Pheromone biosynthesis in female redbanded leafroller moths (RBLR) is under control of a neuropeptide produced in the brain. A bioassay consisting of isolated abdomens was developed to test the mode of action of the pheromone biosynthesis activating neuropetide (PBAN). Pheromone titer and incorporation of radiolabeled acetate into pheromone could be monitored with this bioassay. Synthetic PBAN with sequences identical to PBAN isolated from Heliothis zea and Bombyx mori were active in inducing synthesis of pheromone in RBLR. Removal of the ventral nerve cord in isolated abdomens did not inhibit the action of PBAN. Small amounts of PBAN-like activity was found in hemolymph collected from normal females but not from decapitated females. Severing the VNC in vivo in normal females did not lower the pheromone titer. These data indicate that PBAN is released into the hemolymph and then travels to its site of action. A two-fold increase in both pheromone titer and radiolabeled acetate incorporation upon incubation with PBAN was shown with isolated pheromone glands. However, the differences between control and PBAN-induced values were smaller than those obtained with the isolated abdomen culture bioassay where a seven-fold increase was observed. A decrease in pheromone titer was seen upon the in vivo removal of the corpus bursae from normal females. Removal of the corpus bursae in the isolated abdomen cultures also abolished the activity of PBAN. However, cutting the cervix bursae and leaving the corpus bursae in the abdomen culture increased both titer and radiolabeled acetate incorporation into pheromone without the presence of PBAN. An aqueous extract made from the corpus bursae of 5-day-old females was also active by itself in inducing pheromone biosynthesis in the isolated abdomen cultures. Experiments performed using newly emerged females confirmed that the corpus bursae extracts will induce pheromone biosynthesis. These results indicate that both PBAN and the corpus bursae are involved in controlling pheromone biosynthesis in RBLR.     

水稻螟虫神经肽PBAN及其受体序列的生物信息学分析

【目的】性信息素合成激活肽(PBAN)是控制昆虫产生性信息素的激素,本文旨在分析水稻螟虫神经肽PBAN及其受体的序列。【方法】通过t Blastn同源检索从水稻螟虫基因组和转录组数据库中鉴定水稻螟虫PBAN神经肽及其受体序列,在此基础上进行序列比对及系统发生分析。【结果】发现二化螟Chilo suppressalis、三化螟Tryporyza incertulas和大螟Sesamia inferens的PBAN成熟肽序列均含有33个氨基酸残基,其C端五肽序列完全相同,3种水稻螟虫PBAN多肽相似度为54.55%~63.64%;发现二化螟PBAN受体3个异构体全长氨基酸序列(PBANR-A、PBANR-B和PBANR-C),均含有7个跨膜区域。【结论】进化树分析发现不同昆虫PBAN神经肽及其受体存在一定的保守性和多样性,并且在进化树上的位置几乎与昆虫系统发育分类一致,推测PBAN神经肽和PBAN受体在昆虫系统进化过程中可能存在协同进化现象。本研究为水稻螟虫PBAN神经肽及其受体的结构和功能分析提供基础。     

RNA interference of pheromone biosynthesis-activating neuropeptide receptor suppresses mating behavior by inhibiting sex pheromone production in Plutella xylostella (L.)

Sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN) in many lepidopteran species. We cloned a PBAN receptor (Plx-PBANr) gene from the female pheromone gland of the diamondback moth, Plutella xylostella (L.). Plx-PBANr encodes 338 amino acids and has conserved structural motifs implicating in promoting G protein coupling and tyrosine-based sorting signaling along with seven transmembrane domains, indicating a typical G protein-coupled receptor. The expression of Plx-PBANr was found only in the pheromone gland of female adults among examined tissues and developmental stages. Heterologous expression in human uterus cervical cancer cells revealed that Plx-PBANr induced significant calcium elevation when challenged with Plx-PBAN. Female P. xylostella injected with double-stranded RNA specific to Plx-PBANr showed suppression of the receptor gene expression and exhibited significant reduction in pheromone biosynthesis, which resulted in loss of male attractiveness. Taken together, the identified PBAN receptor is functional in PBAN signaling via calcium secondary messenger, which leads to activation of pheromone biosynthesis and male attraction.     

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.     

The pheromone biosynthesis activating neuropeptide (PBAN) receptor of Heliothis virescens: identification, functional expression, and structure-activity relationships of ligand analogs

Pheromone biosynthesis activating neuropeptide (PBAN) promotes synthesis and release of sex pheromones in moths. We have identified and functionally expressed a PBAN receptor from Heliothis virescens (HevPBANR) and elucidated structure-activity relationships of PBAN analogs. Screening of a larval CNS cDNA library revealed three putative receptor subtypes and nucleotide sequence comparisons suggest that they are produced through alternative splicing at the 3'-end. RT-PCR amplified preferentially HevPBANR-C from female pheromone glands. CHO cells expressing HevPBANR-C are highly sensitive to PBAN and related analogs, especially those sharing the C-terminal pentapeptide core, FXPRLamide (X=T, S or V). Alanine replacements in the C-terminal hexapeptide (YFTPRLamide) revealed the relative importance of each residue in the active core as follows: R5>L6>F2>P4>T3>Y1. This study provides a framework for the rational design of PBANR-specific agonists and/or antagonists that could be exploited for disruption of reproductive function in agriculturally important insect pests.     

Sex pheromone production in the silkworm, Bombyx mori, is mediated by store-operated Ca2+ channels

In most female moths, pheromone biosynthesis activating neuropeptide (PBAN) regulates sex pheromone production by stimulating an influx of extracellular Ca(2+). Little is known about the plasma membrane channel or how the PBAN stimulus is communicated to the channel. Fluorescent Ca(2+) imaging techniques confirmed PBAN-induced Ca(2+) influx in the silkworm, Bombyx mori, and showed that the PBAN response is reduced with repeated stimulation. Compounds known to impact Ca(2+) signaling were examined for their effects on sex pheromone production. These experiments demonstrated that the PBAN signal is likely mediated by a store-operated channel (SOC). SOC blockers, SKF-96365 and 2-aminoethoxydiphenyl borate, abolished sex pheromone production, as did flufenamic acid, a blocker of transient receptor potential (TRP) channels. Thapsigargin mimicked the pheromonotropic effects of PBAN. Similar results were seen when PBAN-induced lipase activity was assayed. Conversely, 1-oleoyl-2-acetyl-sn-glycerol and arachidonic acid, activators of diacylglycerol-dependent Ca(2+) channels, had no effect on bombykol production.     

Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1 (BmLsd1)

Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via the fatty acid biosynthetic pathway. This pathway is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-amino acid peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets, which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells. PBAN stimulates lipolysis of the stored lipid droplet triacylglycerols (TAGs) and releases the precursor for final modification. PBAN exerts its physiological function via the PG cell-surface PBAN receptor, a G protein-coupled receptor that belongs to the neuromedin U receptor family. The PBAN receptor-mediated signal is transmitted via a canonical store-operated channel activation pathway utilizing Gq-mediated phospholipase C activation (Hull, J. J., Kajigaya, R., Imai, K., and Matsumoto, S. (2007) Biosci. Biotechnol. Biochem. 71, 1993-2001; Hull, J. J., Lee, J. M., Kajigaya, R., and Matsumoto, S. (2009) J. Biol. Chem. 284, 31200-31213; Hull, J. J., Lee, J. M., and Matsumoto, S. (2010) Insect Mol. Biol. 19, 553-566). Little, however, is known about the molecular components regulating TAG lipolysis in PG cells. In the current study we found that PBAN signaling involves phosphorylation of an insect PAT family protein named B. mori lipid storage droplet protein-1 (BmLsd1) and that BmLsd1 plays an essential role in the TAG lipolysis associated with bombykol production. Unlike mammalian PAT family perilipins, however, BmLsd1 activation is dependent on phosphorylation by B. mori Ca(2+)/calmodulin-dependent protein kinase II rather than protein kinase A.