Indentification And Functional Characterization Of The Bombyx Neuropeptide Receptor GPCR A9 For Sulfakinin From Silkworm,Bombyx Mori

Sulfakinins,structural and functional homolog of the mammalian peptides gastrin and cholecystokinin?CCK?,are multifunctional neuropeptides widely found in insects.They are involved in diverse biological processes via G protein-coupled receptors?GPCRs?,including food intake,locomotion,metabolism,growth and development,as well as learning and memory.In this paper,we studied the related regulation mechanism of sulfakinin and its receptor in silkworm Bombyx mori.1.Cloning and functional identification of the neuropeptide receptor BNGR-A9from silkworm,Bombyx moriThrough bioinformatics analysis,it was found that the silkworm orphan neuropeptide receptor BNGR-A9 has high homology and sequence similarity with known insect sulfakinin receptors.Using both mammalian cell line HEK293 and insect cell line BmN,the expressed BNGR-A9 was specifically activated by Bombyx sulfakinin?BmSK?,resulting in intracellular Ca²⁺mobilization and the reaction can be significantly inhibited by the specific inhibitor of G_q protein.Moreover,our research found that the sulfated BmSK was more active than the non-sulfated one.So,Bombyx sulfakinin was identified as a specific endogenous ligand for the Bombyx neuropeptide GPCR A9?BNGR-A9?and thus designated BNGR-A9 as BmSKR.Through pretreatment with a specific inhibitor,it was also found that BmSKR mainly binds to G_q protein after being activated by the ligand.2.The MAPK signaling pathway mediated by the receptor BmSKRAfter stimulated by BmSK,BmSKR activated the MAPK signaling pathway that regulates cell proliferation and differentiation.In BmSKR-HEK293 cells,BmSKR was found to mediate ERK1/2 phosphorylation in time gradient and concentration gradient-dependent ways.The results of specific inhibitor treatment showed that PLC?/Ca²⁺/PKC were involved in the activation of MAPK/ERK signaling pathway mediated by BmSK/BmSKR system,and that PKA was not involved in the regulation of BmSKR-mediated ERK1/2 phosphorylation.3.Physiological functions mediated by BmSK/BmSKR system in the silkworm neuronsWe studied the expression pattern of BmSKR in different tissues of silkworm.To know the role of BmSKR playing in the physiological processes,we used in vitro dsRNA injection to inhibit the expression of BmSKR in silkworm.The results demonstrated the feeding behavior of the silkworm was affected and sulfakinin could not play its role as satiety factor.Moreover,BmSK injection made the concentration of trehalose in the hemolymph significantly increase.Therefore,it is speculated that BmSKR may be involved in regulation of physiological functions such as feeding and energy metabolism.In summary,the paper carried out a detailed study on the molecular mechanism of the signal transduction of the neuropeptide sulfakinin and its receptor and the physiological function of BmSK/BmSKR system.These results of the study may provide new insights into the nature of molecular mechanisms underlying sulfakinin-involved functions and clarify the physiological regulatory of Bombyx mori.