Juvenile Hormone Triggers Receptor Mediated Vitellogenin Uptake Through Enhancing Protein Kinase C-catalyzed Phosphorylation Of Vitellogenin Receptor

The migratory locust,Locusta migratoria(Lm)is one of the most destructive insect pests in the agricultural production in China.Due to its high fecundity,the migratiory lcoust can cause tremendous damage to agricultural production.Vitellogenesis is one of the most important parts in insect egg production.In vitellogenesis,vitellogenin(Vg)is synthesized in the fat body,released into the hemolymph,and taken up by the ovary.The uptake of Vg into maturing oocytes is mediated by a specific receptor,vitellogenin receptor(VgR)that is located on the membrane of occytes.In the maturing oocytes,Vg is processed into vitellin(Vn),the major nutrients for later embryonic development.Though VgR plays an important role in the uptake of Vg,the molecular mechanisms of JH regulation in VgR expression and function is not fully understood.Insect VgRs belong to the low density lipoprotein receptor(LDLR)superfamily.Recently,more than 30 insect VgRs have been reported.Vitellogenesis in many insects including the migratory locust is controlled by juvenile hormone(JH).However,the underlying mechanisms of VgR-mediated Vg uptake into oocytes remain unclear.In this dissertation study,we explored LmVgR function and regulation by the JH pathway in locusts using the approaches of bioinformatics,molecular biology,biochemistry and cell biology.We aimed to reveal the molecular mechanisms of LmVgR-mediated LmVg uptake in the maturing oocytes under JH induction.Initially,the full-length of LmVgR gene sequence of locust was cloned by RACE PCR using the known VgR gene sequence fragments.The polyclonal LmVgR antibody was rasied by cDNA cloning,protein expression and purification using the prokaryotic expression system,and immunization with New Zealand rabbits.We found that the expression of LmVgR and the uptake of Vg in the ovary are positively correlated,considering the spatio-temporal expression of LmVgR and Vg in the levels of both mRNA and protein.Knockdown of LmVgR resulted in significant reduction of LmVg uptake by the ovary.We demonstrated that JH significantly enhanced the uptake of LmVg by the ovary.The results indicate that JH promotes the uptake of LmVg in the locust ovary through LmVgR.Interestingly,Western blot analysis showed that the posttranslational modification of LmVgR correlated with ovarian Vg uptake during vitellogenic stage.LmVgR phosphorylation was further confirmed by alkaline phosphatase treatment and immunoprecipitation(IP)with antibodies against LmVgR and protein kinase C(PKC)substrates.Pharmacological experiments demonstrated that JH stimulated LmVgR phosphorylation,and PKC inhibitor(Staurosporine)but not calcium/calmodulin-dependent protein kinase II(CaMK II)inhibitor blocked JH-induced VgR phosphorylation.Interestingly,G-protein coupled receptor(GPCR)inhibitor Suramin and phospholipase C(PLC)inhibitor U73122 rather than the receptor tyrosine kinase(RTK)inhibitor SU6668 inhibited LmVgR phosphorylation triggered by JH.These observations indicate that JH promotes ovarian uptake of LmVg via regulating the interaction of LmVg to LmVgR,and PKC phosphoryation of LmVgR is involved in their binding affinity.It is likely that JH induces LmVgR phosphorylation through GPCR,PLC,and PKC pathways.Taken together,data of this dissertation research demonstrate that JH stimulates the binding of LmVgR to LmVg via PKC-mediated phosphorylation of LmVgR.The results provide new insights into the understanding of JH regulation in insect vitellogenesis,oocyte maturation and egg production.