The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation

Abstract

Holometabolous insects stop feeding at the final larval instar stage and then undergo metamorphosis; however, the mechanism is unclear. In the present study, using the serious lepidopteran agricultural pest Helicoverpa armigera as a model, we revealed that 20-hydroxyecdysone (20E) binds to the 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 metamorphic molting under 20E regulation. The 20E titer was low during larval feeding stages and high during wandering stages. By contrast, the dopamine (DA) titer was high during larval feeding stages and low 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 αs and αq. 20E, via DopEcR, induced protein phosphorylation and binding of the EcRB1-USP1 transcription complex to the ecdysone response element. DopEcR could bind 20E inside the cell membrane or after being isolated from the cell membrane. Mutation of DopEcR decreased 20E binding levels and related cellular responses. 20E competed with DA to bind to DopEcR. The results of the present study suggested that 20E, via binding to DopEcR, arrests larval feeding and promotes pupation.

G protein-coupled receptors function as cell membrane receptors for the steroid hormone 20-hydroxyecdysone

“…A recent study shows that ErGPCR-2 and DopEcR in H. armigera could bind 20E in the cell membrane or as isolated proteins, using a 20E enzyme immunoassay (20E-EIA). That study also demonstrates one of the mechanisms by which 20E represses larval feeding and promotes metamorphosis: 20E competes with dopamine to bind to DopEcR to block the dopamine-mediated motor function and reward-motivated behavior, and initiates the 20E pathway [49].…”

“…A suitable method to detect the dynamic binding of small lipid ligands to GPCRs needs to be established. 20E-EIA detects the binding of GPCRs to 20E directly, presenting an alternative method to that using the isotope-labeled analog Pon A [49].…”

“…DmDopEcR in Drosophila is reported to bind the 20E analog [ 3 H]-labeled Pon A [ 30 ]. DopEcR in H. armigera can bind 20E in the cell membrane or as an isolated protein [ 49 ]. To prove the GPCRs binding steroid hormone is very difficult.…”

“…Phylogenetic analysis using amino acid sequences show that ErGPCR-1 and ErGPCR-2 differ from GPR30, beta-2 AR, or Drosophila DmDopEcR [ 48 ]. Studies on ErGPCR-1, ErGPCR-2, and DopEcR suggest the possibility that several GPCRs are involved in 20E signaling via the participation of several downstream cascades or the differential expression and distribution of GPCRs in tissues [ 49 ].…”

Label-free quantitative proteomic analysis of insect larval and metamorphic molts

“…Fifth instar larvae eat for more than 30 h, and the head capsule slippage (HCS) occurred at about 5th 36 h. Then larvae shed the old cuticle and enter 6th instar, the final larval instar. After eating for more than 48 h, 6th larvae begin wandering at 72 h, then the legs contract and pupation at about 140 h. Considering 20E titer pulse [45] and according the phenotype, we prepared the samples at 5th instar feeding (5F, 12-24 h after ecdysis into 5th instar), 5th molting (5 M, 36 h after ecdysis into 5th instar), 6th instar feeding (6F, 12-60 h after ecdysis into 6 h instar), and 6th instar metamorphosis (72-120 h after ecdysis into 6 h instar) stages.…”