Activation of Bombyx mori neuropeptide G protein-coupled receptor A19 by neuropeptide RYamides couples to Gq protein-dependent signaling pathways

RYamides constitute a novel family of neuropeptides newly identified in insects, 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 complementary DNA of the RYamide receptor BNGR-A19 from Bombyx mori larvae. After expression in mammalian HEK293T and insect Sf9 cells, functional assays revealed 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 G(q) inhibitor-sensitive manner. Upon activation by RYamide peptides, BNGR-A19 elicited ERK1/2 phosphorylation via a G(q)-PLC-PKC pathway, and also underwent a rapid internalization from the cell surface to the cytoplasm. Further cross-activity analysis indicated that BNGR-A19 exhibited very weak response upon stimulation by high concentration (1 mu 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). In addition, the quantitative reverse-transcription polymerase chain reaction 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 in-depth information on the insect RYamide signaling system, facilitating the further clarification of its endocrinological roles in insect physiology.

Automated summary

RYamides are a newly identified family of neuropeptides in insects playing important roles in physiological regulation. This study cloned the RYamide receptor BNGR-A19 from Bombyx mori larvae and uncovered its signaling mechanism and physiological functions, shedding light on its potential roles in feeding, water homeostasis, and testis development in insects.