Function Studies On Methoprene-tolerant 1 And G-protein-coupled Receptor Kinase 2 During Development In Helicoverpa Armigera

BackgroundsWith the development of molecular biology techniques, we have a better platform to investigate insect growth and development. By clarifying the molecular mechanism of insect development, we can provide reference and theoretical basis for the study of human physiological processes associated with disease. The life cycle of holometabolous insects undergo several times molting.20-hydroxyecdysone (20E) and juvenile hormone (JH) regulate the development, molting and metamorphosis. It is meaningful to elucidate the mechanism of 20E signal termination and JH controls Metl to maintain larval status.Progress and scientific questionsCurrent researches on JH focuse on its receptor; Methoprene tolerant is the most likely JH receptor. JH-Met combines with other bHLH-PAS family members together as a complex to binds to JH response element (JHRE), and then initiate the transcription of Kruppel homolog 1. Knockdown Met in Tribolium castaneum causes precocious metamorphosis in larvae. USP is a key fator in both 20E pathway and JH pathway. The mechanism for Met conducts JH signal and USP1 function in JH pathway are the research hotspots. The best-known function of G protein coupled receptor kinase (GRK2) is regulating the termination of a G protein coupled receptor (GPCR) signal.20E membrane receptor is the research hotspot; GPCR is the most likely 20E membrane receptor. It is not clear about the mechanism of GRK2 translocating to the cell membrane to terminate 20E signal. The researches on Metl and GRK2 can provide further explain for the growth and hormonal regulation of insect metamorphosis and a theoretical basis for the pest control target gene.Acquired ResultsBy using an epidermal cell line (HaEpi) and Helicoverpa armigera larvae, in conjunction with molecular techniques, we have investigated the function of Metl and GRK2 in the development and metamorphosis of H.armigera. Results are as follow: 1.Phosphorylated-Methoprene-tolerant1 and non-phosphorylated-ultraspiracle proteinl form transcription complex to promote gene transcription in juvenile hormone pathwayMetl protein is increased during feeding stage, decreased during molting and metamorphic stages. Metl is upregulated by JH Ⅲ and low concentration of 20E independently, but is downregulated by high concentration of 20E. Knockdown of Met1 in larvae causes precocious pupation. Met1 upregulates JH pathway gene expression and downregulates 20E pathway gene expression to maintain larval status. Metl interacts with Met1 and USP1 as a complex without hormone. In the induction of JH Ⅲ, the PAC domain of Met1 is phosphorylated, Met1-Met1 is separated, Met1 interacts with phosphorylated-Hsp90 and USP1 as complex to binds with JHRE to regulate Krh1 transcription. The 21st serine of USP1 is phosphorylated in the induction of 20E; the phosphorylation of USP1 break the combination between USP1 and Met1, and then break the Met1-Met1 homodimer, phosphorylated-USP1 combines with EcR-B1 as complex to participate the 20E pathway.2. G-protein-coupled receptor kinase 2 terminates G-protein-coupled receptor function in steroid hormone 20-hydroxyecdysone signaling in the cell membraneGRK2 protein expression is increased during the metamorphic stage as a result of induction by 20E. Knockdown of GRK2 in larvae causes accelerated pupation, an increase in 20E-pathway gene expression, and advanced apoptosis and metamorphosis. 20E induces translocation of GRK2 to the cell membrane from the cytosol via ecdysone-responsive GPCR (ErGPCR-2). GRK2 is phosphorylated by PKC on serine 680 after induction by 20E, which leads to the translocation of GRK2 to the cell membrane. GRK2 interacts with ErGPCR-2 and phosphorylates ErGPCR-2 to induce the internalization of ErGPCR-2. These data indicate that GRK2 terminates the ErGPCR-2 function in 20E signaling in the cell membrane by negative feedback way.Conclusions and scientific significancesMet1 promotes JH pathway and represses 20E pathway to maintain larval status, and provide new experimental evidence and theoretical support for JH regulating insect growth and inhibit metamorphosis. Phosphorylated-Met1 and nonphosphorylated-USP1 together transmit JH signal. We clarified the mechanism for steroid hormone 20E termination by the study on GRK2.We provided new target for pest control.