Abstract
The short neuropeptide F (sNPF) neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects. In the silkworm (Bombyx mori) two orphan G protein-coupled receptors, Bombyx neuropeptide G protein-coupled receptor (BNGR) A10 and A11, have been identified as cognate receptors for sNPFs, but other sNPF receptors and their signaling mechanisms in B. mori remain unknown. Here, we cloned the full-length cDNA of the orphan receptor BNGR-A7 from the brain of B. mori larvae and identified it as a receptor for Bombyx sNPFs. Further characterization of signaling and internalization indicated that BNGR-A7, -A10, and -A11 are activated by direct interaction with synthetic Bombyx sNPF-1 and -3 peptides. This activation inhibited forskolin or adipokinetic hormone-induced adenylyl cyclase activity and intracellular Ca2+ mobilization via a Gi/o-dependent pathway. Upon activation by sNPFs, BNGR-A7, -A10, and -A11 evoked ERK1/2 phosphorylation and underwent internalization. On the basis of these findings, we designated the receptors BNGR-A7, -A10, and -A11 as Bommo-sNPFR-1, -2, and -3, respectively. Moreover, the results obtained with quantitative RT-PCR analysis revealed that the three Bombyx sNPF receptor subtypes exhibit differential spatial and temporal expression patterns, suggesting possible roles of sNPF signaling in the regulation of a wide range of biological processes. Our findings provide the first in-depth information on sNPF signaling for further elucidation of the roles of the Bombyx sNPF/sNPFR system in the regulation of physiological activities.
Highlights
The short neuropeptide F neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects
We have reported on the cloning of the cDNA encoding a Bombyx neuropeptide G protein– coupled receptor (GPCR) A7 (BNGR-A7) sequence, which, as based on genomic data mining and phylogenetic analysis, is closely related to Drosophila receptor termed NPFR76F and the mammalian Y2 NPY receptor [23, 25]
Bombyx neuropeptide G protein– coupled receptor (BNGR)-A7, -A10, and -A11 have been identified in silico as short neuropeptide F (sNPF)-like receptors by using genomic data mining and phylogenetic analysis [23, 25], and BNGR-A10 and -A11 have been confirmed as sNPF receptors by molecular and biochemical methods [23]
Summary
BNGR-A7, -A10, and -A11 have been identified in silico as sNPF-like receptors by using genomic data mining and phylogenetic analysis [23, 25], and BNGR-A10 and -A11 have been confirmed as sNPF receptors by molecular and biochemical methods [23]. Upon stimulation by sNPF peptides, FLAG–BNGR-A11 and BNGR-A11–EGFP exhibited significant inhibition of forskolin-evoked CRE–luciferase activity with EC50 values of 33.41 and 36.06 nM, respectively, comparable with the wild-type receptor (EC50 57.09 nM) (Fig. 1D) These data suggest that N-terminally FLAGtagged or C-terminally EGFP-fused BNGR-A7, -A10 and -A11 exhibited normal expression and membrane translocation in HEK293 and sf cells. Upon stimulation by sNPF-1 and sNPF-3, cells expressing BNGR-A7, -A10, and -A11, respectively, exhibited inhibitory effects on forskolin-induced CRE–luciferase activity in a dose-dependent manner with EC50 values of 8.71 (sNPF-1) and 2.08 nM (sNPF-3) for BNGR-A7, 0.15 (sNPF-1) and 0.98 nM (sNPF-3) for BNGR-A10, and 45.16 pM (sNPF-1) and 4.91 nM (sNPF-3) for BNGR-A11 in transfected HEK293 cells. Functional analysis using a CREdriven luciferase assay indicated that FITC–sNPF-3 could lead to inhibition of forskolin-stimulated luciferase activity with an EC50 value of 27.34 nM in BNGR-A7– expressing HEK293 cells, comparable with the wild-type sNPF-3 (Fig. 4A). Our data suggest that Bombyx sNPF signaling is likely to function as a pleiotropic regulator
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