Fig 1.
(A) Tissue distribution and expression profile of the tachykinin gene (tk) in B. mori larvae. Tissues from six individual fifth-instar day 2 larvae fed ad libitum or starved for 24 h were used for this semi-quantitative RT-PCR analysis. Ribosomal protein L32 gene (rpl32) was used as an experimental and expression control. The left panel is a representative image of the analyses. The right panel shows the densitometric analysis results from three independent experiments. The starved larvae data are depicted as the fold change over that in the same tissue of fed larvae (mean + S.D.). Asterisks indicate significant differences between the two groups for the same tissue (Fed vs. Starved; P < 0.05, unpaired t-test). Br, brain; CNS, central nervous system; Pg, prothoracic glands; Sg, silk gland; Fg, foregut; Mg, midgut; Hg, hindgut; Mt, Malpighian tubules; Fb, fat body; Ms, smooth muscle from the 8th body segment; Hm, hemocytes; Ts, testis; Ov, ovary. (B) Amino acid sequence of the polypeptide encoded by the tachykinin gene; this precursor is processed to produce five premature peptides (boxed), which are subsequently amidated with the C-terminal Gly residue serving as a donor (underlined) that requires maturation. (C) Sequence comparison of the five mature TRPs and ITPL present in B. mori. ITPL contains three intramolecular disulfide bonds (solid lines). The amino acid sequences of the five TRPs were aligned using ClustalW (http://clustalw.ddbj.nig.ac.jp); these peptides contain the consensus sequence (-Phe-X1-Gly-X2-Arg-NH2) of TRPs found in invertebrates. Numbers in parentheses indicate the number of amino acid residues.
Fig 2.
Responses of BNGR-A24 and BNGR-A32 to B. mori TRPs.
The response of HEK293T cells co-expressing BNGR-A24 (A) or BNGR-A32 (B) and promiscuous mouse Gα15 to the TRPs was monitored using the Ca2+ imaging technique. Dose-response curves are depicted as relative fluorescent intensity (mean ± S.D.; n = 3).
Table 1.
EC50 of BNGR responses to B. mori TRPs.
Fig 3.
Effect of spantide I on the response of BNGR-A24 to rITPL and TK-4.
Spantide I (SP), a potential competitor, was added together with rITPL or TK-4 and the response of BNGR-A24 was monitored using the Ca2+ imaging assay. Dose-response curves of the responses are presented as relative fluorescent intensity (mean ± S.D.; n = 3). The response curve of TK-4 with no competitor is the same as the one shown in Fig 2A. Asterisks indicate significant differences for treatment with the same ligand without spantide I (P < 0.05; Dunnett’s test).
Fig 4.
In vitro competitive binding assay of rITPL and TK-4 to BNGR-A24.
(A–D) Microscopic imaging. The binding of RR-labeled ligands (A and C, rITPL; B and D, TK-4) and CHO cells expressing EGFP-fused BNGR-A24 was examined in the presence of potential competitors (A, TK-4; B, rITPL; C and D, spantide I [SP]). EGFP and RR fluorescences were observed by confocal microscopy. Co-localization is presented as yellow in the merged images (Merge). Representative images of cells from at least two independent experiments are shown. (E–H) Relative fluorescent intensity obtained from experiment images (A)–(D), respectively. RR fluorescent intensity was normalized by EGFP intensity per image and is indicated as fold change over that without competitor (mean + S.D.; n = 6). Asterisks indicate significant differences compared to ‘no competitor’ data (P < 0.05, Dunnett’s test).
Fig 5.
Effects of TK-4, rITPL, and spantide I on cyclic nucleotide levels in BmN cells.
Intracellular cGMP (A) and cAMP (B) levels in BmN cells following 30-min incubation with the peptide and reagent of interest at the indicated concentrations were measured. (A) Effects of TK-4 and spantide I on the response of BmN cells to rITPL. The cGMP levels of the cells exposed to 100 nM rITPL with TK-4 or spantide I (SP) were determined. Responses to TK-4 alone, spantide I alone, rITPL alone, and vehicle treatment (-) were also examined. (B) The cAMP levels of the cells after incubation with 1 μM of five TRPs or rITPL. Responses to vehicle treatment (-) and 10 μM forskolin (FSK; positive control) were also examined. Data are presented as the mean + S.D. (n = 3 or 4). Different letters indicate significant differences (P < 0.05; Tukey’s HSD).