Pigment Dispersing Factor Regulates Ecdysone Biosynthesis via Bombyx Neuropeptide G Protein Coupled Receptor-B2 in the Prothoracic Glands of Bombyx mori

Abstract

Ecdysone is the key hormone regulating insect growth and development. Ecdysone synthesis occurs in the prothoracic glands (PGs) and is regulated by several neuropeptides. Four prothoracicotropic and three prothoracicostatic factors have been identified to date, suggesting that ecdysone biosynthesis is intricately regulated. Here, we demonstrate that the neuropeptide pigment dispersing factor (PDF) stimulates ecdysone biosynthesis and that this novel signaling pathway partially overlaps with the prothoracicotropic hormone (PTTH) signaling pathway. We performed transcriptome analysis and focused on receptors predominantly expressed in the PGs. From this screen, we identified a candidate orphan G protein coupled receptor (GPCR), Bombyx neuropeptide GPCR-B2 (BNGR-B2). BNGR-B2 was predominantly expressed in ecdysteroidogenic tissues, and the expression pattern in the PGs corresponded to the ecdysteroid titer in the hemolymph. Furthermore, we identified PDF as a ligand for BNGR-B2. PDF stimulated ecdysone biosynthesis in the PGs, but the stimulation was only observed in the PGs during a specific larval stage. PDF did not affect the transcript level of known ecdysone biosynthetic enzymes, and inhibiting transcription did not suppress ecdysone biosynthesis, suggesting that the effects of PDF might be mediated by translational regulation and/or post-translational modification. In addition, the participation of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), target of rapamycin (TOR) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) in the PDF signaling pathway was discovered.

Peptidomics of Neuropeptidergic Tissues of the Tsetse FlyGlossina morsitans morsitans

“…It is well known as an important regulator of the insect circadian clock and has been associated with several other processes like activity, reproduction, and geotactic behavior (reviewed in [99]). More recently, PDF has also been associated with ecdysteroid biosynthesis, pheromone production and mating behavior [100,101]. The PDF neuropeptide occurs in tsetse brain and PM samples.…”

Calcium influx enhances neuropeptide activation of ecdysteroid hormone production by mosquito ovaries

“…The most likely candidates, however, would be neuropeptides that activate G protein coupled receptors, which often function through Ca 2+ /cAMP signaling pathways (Vogel et al, 2013). Many such neuropeptides exist in mosquitoes (Predel et al, 2010; Vogel et al, 2013) including orthologs of pigment dispersing factor, which was recently reported to stimulate PTGs from B. mori to produce ECD (Iga et al, 2014). Several other peptide hormones in B. mori have also been suggested to play roles in PTG activation (Marchal et al, 2010).…”

Identification and characterization of the diuretic hormone 31 receptor in the silkwormBombyx mori

“…Email: kataoka@k.u-tokyo.ac.jp Bioscience, Biotechnology, and Biochemistry, 2015 measured using the cAMP-Screen Chemiluminescent Immunoassay System (Applied Biosystems Foster City, CA, USA) with a Wallac ARVO SX 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA) as previously described. 7) The BNGR-B1-expressed cells only responded to DH31, and a significant increase in the intracellular cAMP was observed ( Fig. 1(B)).…”

“…In Drosophila, PDFR showed a weak affinity to DH31, 6) whereas Bombyx DH31 did not show any affinity to Bombyx PDFR, BNGR-B2. 7) In addition, the receptor of DH41 (DHR) has been reported in Manduca sexta, 8) and the homologous receptor was identified in B. mori. 9) Thus, BNGR-B2 (PDFR) and DHR were excluded from subsequent analysis, which focused on the three remaining orphan G-protein coupled receptors (GPCRs): BNGR-B1, B3, and B4.…”

“…An empty pME18S vector or the full-length ORF of the candidate receptor (BNGR-B1, B3 or B4) inserted into the pME18S vector was transfected into HEK293 cells using Lipofectamine LTX with PLUS reagent (Invitrogen, Carlsbad, CA, USA) as previously described. 7) The transfected cells were treated with 1 μM DH31 in the presence of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) at 37°C for 30 min. The amount of intracellular cAMP was *Corresponding author.…”