| Insect neuropeptides represent the largest and most diverse class of extracellular signaling molecules,which are involved in almost all physiological processes of insect life.RYamide constitute a novel family of neuropeptides newly identified in most insects,which has the C-terminal FFXXXRYamide and play important roles in regulating a variety of physiological processes.However,the signaling characteristics and physiological actions of RYamide signaling system remain largely unknown.In the present study,we cloned the full-length cDNA of the putative RYamide receptor BNGR-A19 from Bombyx mori larvae.After expression in mammalian HEK293T and insect Sf9cells,functional assays demonstrated that BNGR-A19 was activated by synthetic RYamide peptides,triggering a significant increase in cAMP-response element-controlled luciferase activity and Ca2+mobilization in a Gq inhibitor–sensitive manner.Upon activation by RYamides,BNGR-A19 elicited ERK1/2 phosphorylation via a Gq-PLC-PKC pathway,and also underwent a rapid internalization from the cell surface to the cytoplasm.The synthetic RYamide-2 exhibited more potent in activating BNGR-A19 than that of RYamide-1.Further cross-activity analysis revealed that BNGR-A19 exhibited very weak response upon stimulation by high concentrations?1?M?of Bombyx sulfakinin-1,neuropeptide F-1 and short neuropeptide F-1,and vice versa,Bombyx RYamides also showed slight potency for activating Bombyx NPF receptor?BNGR-A4?and sNPF receptor?BNGR-A11?,suggesting that BNGR-A19 is quite specifically activated by RYamides.In addition,the qRT-PCR results showed that the high level expression of BNGR-A19 was detected in the hindgut and testis,suggesting that the RYamide signaling is likely involved in the regulation of feeding,water homeostasis and testis development.This study provides the first indepth information on the RYamide signaling system,facilitating the further clarification of its endocrinological roles in insect physiology. |
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