Functional Characterization Of Signaling And Internalization Of Bombyx.mori Neuropeptide F Receptor

Neuropeptides are ubiquitous in the nervous system of all levels of organization from hydrozoans to man and they play a very important role in the control of development, reproduction, behavior, feeding and many other physiological processes in animals. Most of the neuropeptides activate GPCRs. Silkworm, a4000-year-old domesticated animal in China, have economic and medicinal value, researches on physiological function of neuropeptides and the underlying mechanism in silkworm are thus of great significance. Silkworm is a large Lepidoptera insect and could be an ideal model to study neuropeptide signaling system. Neuropeptide F, as a member of neuropeptide family, has regulatory roles in foraging, feeding and motivation, ethanol sensitivity, stress responses including nociception, aggression, reproduction, clock function and learning in insects. However, the NPF/NPFR signaling pathways in Bombyx mori and its physiological function have not been clarified so far.Based on the database of domesticated silkworm expressed sequence tag, we made sequence alignment of NPFs and NPFRs in insects and found the conservatively evolved NPF/NPFR system in silkworm, BNGR-A4receptor and three predicted BmNPFs, which is highly homologous to that of Drosophila melanogaster’s, and then a series of experiments (cAMP measurement, internalization, ERK phosphorylation) were performed to clarify the signaling mechanisms and physiological roles of this BmNPF/NPFR system.Our research showed that BNGR-A4in response to BmNPFla/2could decrease the level of3’,5’-cyclic adenosine monophosphate (cAMP). This effect can be inhibited by PTX, indicating that the BNGR-A4is coupled to Gi protein and BNGR-A4can significantly evoke calcium flow as well. Furthermore, we also found that BmNPF1a with RPRFamide-C terminal is more active than BmNPF2with RGRYamide-C terminal, which suggested that BmNPF1a is the endogenous ligand for BNGR-A4, so we named the receptor as BmNPFR. Internalization of GPCR is an essential mechanism for receptor desensitization and (3-arrestins play an important role in the GPCR internalization. We thus cloned the homolog of the only β-arrestin in Drosophila melanogaster from Bombyx mori, Bm-Kurtz. Our results revealed that the internalization of BmNPFR stimulated by BmNPFs was time-and dose-dependent and can be inhibited by PTX, clathrin is essential for BmNPFR internalization. However, the internalization didn’t decrease notablely when the β-arrestins silenced by siRNA, suggesting that arrestins are not the primary physiologic regulators of BmNPFR. MAPK (mitogen-activated protein kinase pathways) play an important role in regulation of physiological process as signal transduction molecules. Our research demonstrated that BmNPFR can evoke ERK1/2phosphorylation in time-and dose-dependent ways, and the phosphorylation was regulated by MEK1/2phosphorylation, PI3K and PKC signaling pathway and this activation can also be inhibited by PTX. In order to access the physiological processes of BmNPFR in feeding and development, we used in vitro siRNA injection to inhibit the BmNPFR expression in domesticated silkworm. Under per10μg/48h injection for4times, the weight of siRNA injection group was10%lighter than control group,while the BmNPFR expression of siRNA injected group in fat body and ovary decreased30%and10%respectively compared to control group. These results indicated that BmNPFR may involve in the energy expenditure and storage.In summary, our research expanded the understanding of NPF family in Bombyx mori, demonstrated the signaling mechanisms such as internalization of the G protein-coupled receptor BmNPFR and its elevation of intracellular calcium and ERK1/2phosphorylation, which also pave for the studies on the BmNPFR signaling and physiological role.