Characterization Of Signaling Of Tachykinin Receptor A32of Silkworm,Bombyx Mori

The receptor of tachykinin （TKR） is a typical G-protein coupled receptors （theG-protein coupled receptor）, mediated by a series of intracellular signal transductionpathways, lack of TKR cause disruption of normal metabolism, so tachykinin receptorsystem is a good model for the study of human metabolism. Studies have shown thattachykinin immunomodulatory functions in the human body, Bombyx mori classic modelorganism as a human tachykinin receptor A32study is of great significance. Therefore, westudied the the silkworm tachykinin receptor A32signal transduction mechanism. Theresults showed that the16bp deletion, we cloned the A32receptor in995¹010bp in orderto determine the new discoveries, from the27silkworm varieties of the fifth instar larvae fatbody RNA was extracted, RT-PCR method found only four species of wild-type, and theremaining23species are mutant. In order to analyze the difference of the wild-type andmutant physiological function, we constructed a GFP expression vector into the wild-typeand mutant A32fragment of both plasmids were transfected into HEK293cells, andobserved under a fluorescence microscope wild-type receptor A32located on the cellmembrane, only a small amount of the distribution of intracellular mutant receptor A32isnot positioned in the cell membrane, but distributed in the cytoplasm. In the silkwormtachykinin wild-type receptor A32distribution location is changed, shifted to a perinuclear,punctate clumped TK4, TK5, TK6role is particularly evident, while the mutant receptorA32did not change significantly.MAPK is an important cell regulatory factors, the regulation of many importantphysiological processes such as cell growth, development, reproduction, death, metabolism.Our results show that the receptor A32by the ligand TK4, TK5, in TK6stimulate wouldlead to intracellular Ca²⁺transient increase and increase in cAMP concentration, followedby MAPK activation. In order to clarify the activation pathway, we constructed HEK293cell lines stably expressing the A32receptor, results showed that pretreatment of cells withpertussis toxin had no significant effect on cAMP and cholera toxin processing overnightbefore the the silkworm speed bradykinin stimulate cAMP. The level is still significantlyimproved. These results indicate that in HEK293cells, the receptor A32-mediated signaltransduction pathway is coupled Gs protein to activate downstream signaling. Theextracellular signal-regulated kinase （ERK1/2） is a core member of the MAPK, we foundthat the TK-mediated signaling pathways to activate ERK1/2, but its mechanism is unclear. The third chapter we study the ERK1/2mediated phosphorylation, with the A32receptorstably transfected HEK293cells, we found that TK4, TK5, in TK6stimulate activation ofERK1/2phosphorylation can cause BNGR-A32transient increase in the silkworm fatmoving hormone AKH1, are not caused by the ERK1/2phosphorylation the Corazonin andsuppression of thymosin1. Taking into account the tachykinin receptor is highly conserved,our studies will provide an important reference for the tachykinin receptor family andexperimental evidence.