Molecular Mechanism Of Diapause Signaling Transduction In The Silkworm,Bombyx Mori

Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter.Silkworm diapause is regulated by Bombyx mori diapause hormone(BmDH).BmDH is a 24-aa amidated neuropeptide that elicits the embryonic diapause of the silkworm,via specific and selective interaction with its receptor,B.mori DH receptor(BmDHR).Here,we study the expression and characteristics of BmDHR;BmDHR-induced MAPK/ERK signaling pathways;structural basis for the interaction of BmDHR with BmDH;silkworm neuropeptide capacity(CAPA)receptor-mediated signaling pathways crosstalk with BmDH/BmDHR signaling pathways.The main results are as follows:1.We combined a series of cellular functional experiments to investigate specific signal transduction pathways mediated by BmDHR.The results showed that BmDHR is mainly localized on the cell membrane.Upon stimulation of BmDH,BmDHR is coupled to the Gaq protein,resulting in a significant increase of intracellular Ca2+flow and cAMP response element(CRE)-driven luciferase activity,but no cAMP accumulation.2.BmDHR-mediated ERK1/2 phosphorylation in a dose-and time-dependent manner in response to BmDH.This effect could be significant inhibited by Gaq inhibitor,PLC inhibitor,PKC inhibitor,Ca2+chelator,G?? specific inhibitor and the PI3K specific inhibitor.Transactivation pathways of receptor tyrosine kinase(RTK)are not involved in the BmDHR-mediated ERK1/2 phosphorylation.3.We performed Cy5.5 labeled BmDH competitive binding assay,a series of cellular functional assays based on the activation of BmDHR and protein structure simulation to study the structural basis for the specific interaction of BmDH with BmDHR.The results showed that the C-terminal hexapeptide of BmDH is essentially required for the activation of BmDHR.C-terminal residues of Arg23 and Leu24 of BmDH are essential for the binding and activation of BmDHR.Trp19 and Phe20 contribute to the functional activity of BmDH.Residues of Glu89,Phe172,Phe194,and Tyr299 in BmDHR are critically involved in the interaction with BmDH and downstream signaling transduction pathways.The C-terminal hexapeptide of BmDH bound to a pocket that was enclosed by the N terminus,ECL1,ECL2,ECL3,TM2,TM3,and TM5-TM7 of BmDHR.A disulfide bridge between Cys106 at TM3 and Cys183 at ECL2 of BmDHR anchored ECL2 for its interaction with BmDH4.Upon stimulation of Bm-CAPA-PVK-1 or-PVK-2,Bm-CAPA-PVK receptor 2 significantly increases intracellular cAMP-response element-drived luciferase activity and Ca2+mobilization in a Gaq inhibitor-sensitive manner.Bm-CAPA-PVK receptor 2 stimulated ERK1/2 phosphorylation also in a dose-and time-dependent manner in response to Bm-CAPA-PVK-1 or-PVK-2 with similar potencies.ERK1/2 phosphorylation can be inhibited by Gaq inhibitor,PLC inhibitor,protein kinase C(PKC)inhibitor,phospholipase D(PLD)inhibitor,Ca2+chelator,G??-specific inhibitors,phosphatidylinositol 3-kinase(PI3K)-specific inhibitor and Src-specific inhibitor.Receptor tyrosine kinase(RTK)transactivation pathways are involved in the mechanisms of Bm-CAPA-PVK receptor to ERK 1/2 phosphorylation?-arrestin1/2 is not involved in Bm-CAPA-PVK-R2-mediated ERK1/2 activation but required for the agonist-independent,ERK1/2 activation-dependent receptor internalization.In addition,Bm-CAPA-PK could activate BmDHR-indueced downstream signaling pathways and BmDH also could activate Bm-CAPA-P VK-R1?341(BNGR-A25S)-mediated downstream signaling transduction pathways.Above all,we elucidated BmDHR-induced downstream signaling pathways and structural basis for the interaction of BmDHR with DH.Bm-CAPA receptor-mediated signaling pathways have crosstalk activation with BmDH/BmDHR signaling pathways.The results lay the foundation for comprehensively understanding the molecular mechanism and providing new ideas for the further study of other different insect diapause mechanisms.