Identification And Functional Charaderization Of Bombvx Mori Tachykinn-related Peptide Receptor

Neuropeptides are one of the most important signaling molecules in multicellular organisms, which are involved in regulating a variety of physiological functions. The tachykinins and tachykinin-related peptides is one of the largest peptide families in the animal kingdom and exert their diverse actions via a subfamily of structurally related G protein-coupled receptors. In this present study, we paired the Bombyx tachykinin-related peptides asspecificendogenousligandsfor the Bombyx neuropeptide G protein-coupled receptor (BNGR)-A24, and the Bombyx natalisin10carrying the YxxxRa consensus sequence for BNGR-A33. We also identified the Bombyx natalisin1with the C-terminal FxxxRa motif as a specific endogenous ligand for BNGR-A32, which was mutant to a16-base pair deletion, loss-of-function receptor in23out of27domesticated silkworm strains that we tested. We designated BNGR-A24, BNGR-A33and BNGR-A32as BmTRPR, BmNTLRl and BmNTLR2, respectively. Further characterization demonstrated that BmTRPR and BmNTLRs were activated in a Gs and Gq inhibitors-sensitive manner. Moreover, our data derived from quantitative RT-PCR analysis and dsRNA-mediated knockdown experiments suggested the possible role of BomTRPR in the regulation of feeding and growth.Endocytosis is an important mechanism for GPCR desensitization. Our research revealed that upon TRP1treatment, the BmTRPR receptor was rapidly and dramaticlly translocated to the early endosome via clathrin-dependent pathway in a dose-dependent and time-dependent manner. Further investigation demonstrated that internalized BmTRPR was recycled to the cell surface after the removal of agonist. Through siRNA interference, we can deduce GRK2, GRK5, and β-arrestin2are involved in the internalization process of BmTRPR. Moreover, TRP1-mediated endocytosis of BmTRPR is in a PKC, PKA and Src-dependent way.Most GPCR signal via mitogen-activated protein kinase (MAPK) cascades, which is involved in regulation of diverse processes ranging from proliferation and differentiation to apoptosis. Using HEK293cells stably expressing BmTRPR, we found that activation of extracellular signal-regulated kinases1and2(ERK1/2) by TRP1was rapid, peaking within5min. furthermore, by specific inhibitor treatment, we found the BmTRPR mediated phosphrylation of ERK1/2via Gs/cAMP/PKA and Gq/PLCβ/PKC pathway. In addition, EGFR tranactivation was involved in the BmTRPR-mediated ERK1/2phosphorylation later time points (>5min). Using β-arrestinl/2specific siRNA, we found that P-arrestins were not involved in BmTRPR-mediated ERK1/2activation.In the present study, we clarified the detailed mechanism of internalization of BmTRPR and activation of ERK1/2pathway. On this basis, our findings will facilitate the understanding of Bombyx TRP/BmTRPR system in the regulation of fundamental physiological processes.