Ecdysone Regulates Insect Metamorphosis And Facilitates Diffusion Into Cells Via G Protein-coupled Receptors

Background,scientific questions,and significanceInsect growth and development are jointly regulated by a variety of hormones,including 20-hydroxyecdysone(20E),juvenile hormone(JH)and insulin.20E interacts with JH and insulin to regulate insect growth,molting,and metamorphosis.During the metamorphosis of insects,violent changes occur in the body,such as the apoptosis of the larval midgut,the formation of the imaginal midgut,the disintegration and reconstruction of the fat body,the growth of the adult discs,and the remodeling of the brain structure.In general,ecdysone acts by binding to the ecdysone nuclear receptor(EcR)to initiate the expression of early and late 20E genes and initiate the metamorphosis process,which is the 20E genome signaling pathway.In recent years,the 20E non-genomic pathway has been proved in addition to the classic 20E genomic signaling pathway.20E triggers rapid changes in intracellular calcium and cAMP levels by activating cell membrane receptors,which causes post-translational modification of a series of proteins,regulating gene transcription and metamorphosis.G protein-coupled receptors(GPCRs)are a family of seven transmembrane proteins that play an important role in transmitting various signals and are the targets of various drugs.Studies have found that GPCRs,as receptors for steroids,play a role in the regulation of insect metamorphosis by 20E.The dopamine ecdysone receptor(DmDopEcR)in Drosophila can bind to the 20E analog,which is considered to be the membrane receptor of 20E,but the function of DopEcR in 20E signaling pathway is unclear.Two other GPCRs transmitting 20E signal were reported in Helicoverpa armigera,but they were not detected to bind 20E.Previous studies also found that the amount of 20E entering the cell was controlled,but the mechanism and biological effects were not clear.These studies have broken the classic theory that 20E initiates the transcription of related genes by free diffusion into cells and binding to nuclear receptors.The scientific issues yet to be clarified are of great significance for establishing new theories of steroid cell membrane receptors and their signaling pathways.In this paper,the major agricultural pest H.armigera is used as a model to study the function of DopEcR in the 20E signaling pathway and prove that GPCR can bind 20E.To study the mechanism and biological effects of GPCR controlling the entry of 20E into cells.These studies can enrich and improve the non-genomic signaling pathways of 20E and the molecular mechanisms regulating insect metamorphosis.ResultsHolometabolous insects stop feeding at the final larval instar stage,and then metamorphosis occurs.However,the mechanism by which the larvae stop feeding is unclear.Using the agricultural pest H.armigera as a model,we revealed that 20E binds to dopamine receptor(DopEcR),a G protein-coupled receptor,to stop larval feeding and promote pupation.DopEcR was expressed in various tissues,and its level increased during metamorphosis under 20E regulation.The 20E titer was low during larval feeding stages and high during wandering stages.In contrast,dopamine(DA)titers were higher during the larval feeding stage and lower during the wandering stages.Injection of 20E or blocking dopamine receptors using the inhibitor flupentixol decreased larval food consumption and body weight.Knockdown of DopEcR repressed larval feeding,growth,and pupation.20E,via DopEcR,promoted apoptosis;and DA,via DopEcR,induced cell proliferation.20E opposed DA function by repressing DA-induced cell proliferation and AKT phosphorylation.20E,via DopEcR,induced gene expression and a rapid increase in intracellular calcium ions and cAMP.20E induced the interaction of DopEcR with G proteins as and aq.20E,via DopEcR,induced protein phosphorylation and binding of the EcRB1-USP1 transcription complex to the ecdysone response element.DopEcR could bind 20E in the cell membrane or after being isolated from the cell membrane.Mutation of DopEcR decreased 20E binding levels and related cellular responses.The results suggested that 20E competes with DA to bind DopEcR,arresting larval feeding and promotes pupation.We revealed that a GPCR,named GPCR-3,transmits the steroid hormone 20-hydroxyecdysone(20E)signal by triggering a G protein-mediated signal cascade and forming a tetramer to promote 20E into the cell in H.armigera.GPCR-3 was screened as being involved in the 20E pathway by RNA interference.Knockdown of GPCR-3 via RNA interference caused delayed and abnormal pupation,inhibited remodeling of the larval midgut and fat body,and repressed 20E-induced gene expression.20E induced GPCR-3 interacting with G proteins aq and as and a rapid increase in intracellular calcium,cAMP,and protein phosphorylation.GPCR-3 was located in the plasma membrane and was internalized after being phosphorylated by GPCR kinase 2 and was degraded under 20E induction to desensitize 20E signaling.?-arrestin-1 and clathrin mediated the internalization of GPCR-3.GPCR-3 can bind 20E in the cell membrane and in vitro after being isolated.The binding of 20E to GPCR-3 induced the homo-dimer of GPCR-3 to form a homo-tetramer.The homo-tetramer of GPCR-3 facilitated 20E entry and transmitted 20E signaling.The homo-tetrameric GPCR-3 acts as a cell membrane receptor and transporter of 20E,controls the amount of 20E in different tissues,and regulates the developmental fate of different tissues during metamorphosis-apoptosis or proliferation.Conclusions and scientific significances1.This paper studies the dual function of DopEcR in insect feeding and pupation.DA promotes the feeding and growth of larvae through DopEcR.20E competes with DA to bind DopEcR,transmits 20E signal through DopEcR,and promotes the metamorphosis of insects,revealing the interaction of steroid.hormones and the dopamine system.It elucidated the mechanism of 20E inhibiting lepidopteran insects feeding and promoting metamorphosis,providing a new theory of steroid hormone signaling pathway and its interaction with the nervous system.It provides a new target of the growth regulators for pest control.2.It is proved that ErGPCR-2 can bind 20E,which supports the previous research conclusion-ErGPCR-2 is a cell membrane receptor of 20E.3.It is proved that GPCR-3 is a cell membrane receptor of 20E.20E binding induced homo-tetramerization of GPCR-3.GPCR-3 homo-tetramer transmits 20E signaling,and functions as a transporter to facilitate diffusion of 20E.This is the first example of GPCR functions as 20E transporter by forming homo-tetramer.