Figure 1.
Screening of candidate receptors.
(A) Tissue distribution of BNGR-B2. The expression of BNGR-B2 was measured by RT-PCR in the selected tissues from gut-purged fifth instar larvae (p50T strain). PG: prothoracic gland, BR: brain, FB: fat body, MT: Malpighian tubule, ASG: anterior silk gland, MG: midgut, TE: testis and OV: ovary. (B) Developmental profile of BNGR-B2 in the PGs. The expression of BNGR-B2 was measured by Q-PCR. The timing of molting, gut purge and pupation in our rearing conditions is indicated with arrows. Each datum point represents the mean ±SEM (n = 3). The dashed line indicates the outline of the hemolymph ecdysteroid titer described by Koyama et al., 2004 (4th instar), Sakurai et al., 1998 (5th instar) and Kaneko et al., 2006 (5th instar). (A, B) RpL3 was used as an internal standard.
Figure 2.
(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).
Figure 3.
Prothoracicotropic activities of PDF in vitro.
(A) Effect of PDF (1 µM) and DH31 (1 µM) on ecdysone biosynthesis in the PGs of V7 larvae (n = 14 and 12). (B) Dose-response curves for PDF and PTTH on ecdysone biosynthesis in the PGs of V7 larvae. Closed circles indicate PDF (n = 4–24) and open squares indicate PTTH (n = 10–38). (C, D) Developmental changes in PDF responsiveness on (C) ecdysone biosynthesis (n = 8–14) and (D) intracellular cAMP level (n = 5–6). (E) Effect of extracellular Ca2+ on ecdysone biosynthesis (n = 6). (F) Effect of extracellular Ca2+ on the change in intracellular cAMP levels (n = 4–8). Statistically significant differences were evaluated by Student's t-test (***P<0.001, **P<0.01, *P<0.05).
Figure 4.
Signaling pathways involved in PDF-induced ecdysone biosynthesis.
(A) Effect of PDF on the transcript level of ecdysone biosynthesis-related enzymes in the PGs. PGs of V7 larvae were treated with or without PDF. The transcript levels of nvd, nm-g, spo, phm, dib and sad were quantified with Q-PCR. Each datum point represents the mean ±SEM (n = 3). (B-D) Effect of (B) transcript inhibitor (actinomycin D: ActD, 10 µM), (C) PKA inhibitor (H-89, 0.1 mM) and (D) translation inhibitor (cycloheximide: CHX, 0.2 mM) on PDF-induced ecdysone biosynthesis. Each datum point represents the mean ±SEM (n = 10). (E, F) Effect of (E) PI3K inhibitor (LY294002: LY, 50 µM) and (F) TOR inhibitor (rapamycin: Rap, 10 µM) on PDF-induced ecdysone biosynthesis. Each datum point represents the mean ±SEM (n = 3 and 4). Statistically significant differences were evaluated by Student's t-test (***P<0.001, **P<0.01). (G) Effect of PDF on the levels of p-ERK and p-4E-BP in cultured PGs. The phosphorylated proteins were examined by immunoblotting, and α-tubulin was used as a loading control. (H, I) Effect of (H) PI3K inhibitor (LY294002: LY, 50 µM) and (I) PKA inhibitor (H-89, 0.1 mM) on p-4E-BP levels in cultured PGs. The phosphorylated proteins were examined by immunoblotting, and α-tubulin was used as a loading control.
Figure 5.
Integration of the PDF signaling model with the known PTTH signaling pathway.
Solid lines indicate demonstrated or highly likely pathways, and dashed lines indicate hypothetical pathways. Gαs: G protein αs subunit, AC: adenylate cyclase, AMP: adenosine monophosphate, cAMP: cyclic AMP, EPAC: exchange protein directly activated by cAMP, eIF4e: eukaryotic translation initiation factor 4E, 4E-BP: eIF4E binding protein, TOR: target of rapamycin, PKA: protein kinase A, PKC: protein kinase C, PI3K: phosphatidylinositol 3-kinase, AKT: protein kinase B, CREB: cAMP response element-binding protein, MAPK: mitogen-activated protein kinase, ERK: extracellular signal-regulated kinase, MEK: MAP kinase kinase, Raf: MAP kinase kinase kinase, S6: ribosomal protein S6, p70S6K: 70 kDa S6 kinase, PLC: phospholipase C, DAG: diacylglycerol, IP3: inositol 1,4,5-trisphosphate, IP3R: IP3 receptor, CaM: calmodulin.