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Pigment dispersing factor regulates ecdysone biosynthesis via bombyx neuropeptide G protein coupled receptor-B2 in the prothoracic glands of Bombyx mori.

Iga M, Nakaoka T, Suzuki Y, Kataoka H - PLoS ONE (2014)

Bottom Line: Furthermore, we identified PDF as a ligand for BNGR-B2.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.

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.

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Characterization of BNGR-B2.(A) Phylogenetic relationship of BNGR-B2 and highly homologous receptors. The tree was generated based on the amino acid sequences of selected regions with the neighbor-joining method using the ClustalX multiple alignment program and a bootstrap value of 1000 trials for each branch position. The indicated numbers are the bootstrap values as a percentage of 1000 replicates, and the scale bar indicates 0.05 changes per residue. Bootstrap values greater than 50% are indicated. The Mus musculus calcitonin receptor (CR) was used as an outgroup. (B) Ligand-binding analysis of BNGR-B2 by examining the change in intracellular cAMP levels. BNGR-B2-expressing HEK293 cells were treated with 1 µM of the candidate BNGR-B2 ligands (PDF and DH31). Each datum point represents the mean ±SEM (n = 5). Statistically significant differences were evaluated by Student's t-test (***P<0.001).
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pone-0103239-g002: Characterization of BNGR-B2.(A) Phylogenetic relationship of BNGR-B2 and highly homologous receptors. The tree was generated based on the amino acid sequences of selected regions with the neighbor-joining method using the ClustalX multiple alignment program and a bootstrap value of 1000 trials for each branch position. The indicated numbers are the bootstrap values as a percentage of 1000 replicates, and the scale bar indicates 0.05 changes per residue. Bootstrap values greater than 50% are indicated. The Mus musculus calcitonin receptor (CR) was used as an outgroup. (B) Ligand-binding analysis of BNGR-B2 by examining the change in intracellular cAMP levels. BNGR-B2-expressing HEK293 cells were treated with 1 µM of the candidate BNGR-B2 ligands (PDF and DH31). Each datum point represents the mean ±SEM (n = 5). Statistically significant differences were evaluated by Student's t-test (***P<0.001).

Mentions: The deduced amino acid sequence of BNGR-B2 was highly similar to the PDFR in other insect species and nematodes and to the pigment-dispersing hormone receptor (PDHR) in Crustacea (Figure 2A). In Drosophila, PDF and diuretic hormone 31 (DH31) have been reported as PDFR ligands [25]; therefore, Bombyx PDF and DH31 were selected as candidate ligands for BNGR-B2. A heterologous expression system was employed for ligand screening [26]. HEK293 cells were transfected with the BNGR-B2/pME18S vector, and binding of the candidate ligands was evaluated by changes to the intracellular cAMP level. PDF only stimulated an increase in the intracellular cAMP level in BNGR-B2-transfected cells (Figure 2B), whereas DH31 had no effect (Figure 2C).


Pigment dispersing factor regulates ecdysone biosynthesis via bombyx neuropeptide G protein coupled receptor-B2 in the prothoracic glands of Bombyx mori.

Iga M, Nakaoka T, Suzuki Y, Kataoka H - PLoS ONE (2014)

Characterization of BNGR-B2.(A) Phylogenetic relationship of BNGR-B2 and highly homologous receptors. The tree was generated based on the amino acid sequences of selected regions with the neighbor-joining method using the ClustalX multiple alignment program and a bootstrap value of 1000 trials for each branch position. The indicated numbers are the bootstrap values as a percentage of 1000 replicates, and the scale bar indicates 0.05 changes per residue. Bootstrap values greater than 50% are indicated. The Mus musculus calcitonin receptor (CR) was used as an outgroup. (B) Ligand-binding analysis of BNGR-B2 by examining the change in intracellular cAMP levels. BNGR-B2-expressing HEK293 cells were treated with 1 µM of the candidate BNGR-B2 ligands (PDF and DH31). Each datum point represents the mean ±SEM (n = 5). Statistically significant differences were evaluated by Student's t-test (***P<0.001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114559&req=5

pone-0103239-g002: Characterization of BNGR-B2.(A) Phylogenetic relationship of BNGR-B2 and highly homologous receptors. The tree was generated based on the amino acid sequences of selected regions with the neighbor-joining method using the ClustalX multiple alignment program and a bootstrap value of 1000 trials for each branch position. The indicated numbers are the bootstrap values as a percentage of 1000 replicates, and the scale bar indicates 0.05 changes per residue. Bootstrap values greater than 50% are indicated. The Mus musculus calcitonin receptor (CR) was used as an outgroup. (B) Ligand-binding analysis of BNGR-B2 by examining the change in intracellular cAMP levels. BNGR-B2-expressing HEK293 cells were treated with 1 µM of the candidate BNGR-B2 ligands (PDF and DH31). Each datum point represents the mean ±SEM (n = 5). Statistically significant differences were evaluated by Student's t-test (***P<0.001).
Mentions: The deduced amino acid sequence of BNGR-B2 was highly similar to the PDFR in other insect species and nematodes and to the pigment-dispersing hormone receptor (PDHR) in Crustacea (Figure 2A). In Drosophila, PDF and diuretic hormone 31 (DH31) have been reported as PDFR ligands [25]; therefore, Bombyx PDF and DH31 were selected as candidate ligands for BNGR-B2. A heterologous expression system was employed for ligand screening [26]. HEK293 cells were transfected with the BNGR-B2/pME18S vector, and binding of the candidate ligands was evaluated by changes to the intracellular cAMP level. PDF only stimulated an increase in the intracellular cAMP level in BNGR-B2-transfected cells (Figure 2B), whereas DH31 had no effect (Figure 2C).

Bottom Line: Furthermore, we identified PDF as a ligand for BNGR-B2.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.

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.

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Related in: MedlinePlus