Characterization of the Drosophila Methoprene ‐tolerant gene product

Abstract

Insect development and reproduction are regulated by two classes of lipid-soluble hormones, the ecdysteroids and juvenile hormones (JHs). The ecdysteroids activate target genes through a heterodimeric receptor complex composing the ecdysone receptor and ultraspiracle (USP) proteins, both of which are members of the nuclear steroid ⁄ thyroid ⁄ retinoid receptor superfamily [1]. During insect development, ecdysteroids induce molting while JH determines the nature of each molt by modulating the ecdysteroid-induced gene expression cascade [2][3][4]. In addition, in adult insects, JH has a wide variety of actions related to reproduction, including oogenesis, migratory behaviour and diapause [2,5,6]. The mode of molecular action of JH, however, is still obscure [7]. JHs are a family of esterified sesquiterpenoids, whose lipid-soluble nature has suggested action directly on the genome through nuclear receptors such as ecdysteroids and the vertebrate steroid ⁄ thyroid ⁄ retinoid hormones [5,8] although actions of JH through the cell membrane are also documented [9,10].Many attempts have been made to identify nuclear JH receptors. Jones and Sharp [11] showed that JH III binds to the Drosophila USP protein, which is a homologue of the vertebrate retinoid X receptor, promoting Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a great diversity of processes in development and reproduction. As yet the molecular modes of action of JH are poorly understood. The Methoprenetolerant (Met) gene of Drosophila melanogaster has been found to be responsible for resistance to a JH analogue (JHA) insecticide, methoprene. Previous studies on Met have implicated its involvement in JH signaling, although direct evidence is lacking. We have now examined the product of Met (MET) in terms of its binding to JH and ligand-dependent gene regulation. In vitro synthesized MET directly bound to JH III with high affinity (K d ¼ 5.3 ± 1.5 nm, mean ± SD), consistent with the physiological JH concentration. In transient transfection assays using Drosophila S2 cells the yeast GAL4-DNA binding domain fused to MET exerted JH-or JHAdependent activation of a reporter gene. Activation of the reporter gene was highly JH-or JHA-specific with the order of effectiveness: JH III JH II > JH I > methoprene; compounds which are only structurally related to JH or JHA did not induce any activation. Localization of MET in the S2 cells was nuclear irrespective of the presence or absence of JH. These results suggest that MET may function as a JH-dependent transcription factor.

Juvenile hormone resistance gene Methoprene-tolerant controls entry into metamorphosis in the beetle Tribolium castaneum

“…Met mutants tolerate harmful effects of JH (21,28), and their tissues display reduced affinity to JH (23). The recombinant Met protein has been shown to bind JH and to stimulate transcription in response to JH-III (24). Finally, Met can form homodimers and heterodimers with Gce in the absence of JH (26).…”

Insect juvenile hormone action as a potential target of pest management

“…55,56) Recently Met protein was reported to bind to JH III with high affinity (K d ϭ5.3 nM), suggesting that Met may function as a JH-dependent transcription factor. 57) Importantly, protein kinase C-mediated phosphorylation of nuclear proteins has been found to decrease their binding to the putative JH-responsive element in the promoters of two JH-responsive genes, jhp21 in the locust 58) and JHE in the spruce budworm Choristoneura fumiferana. 59) The signaling pathway of JH is unknown yet, but the expression of some ecdysone-regulated early genes such as broad and E75 is modulated by JH in both D. melanogaster and M. sexta.…”

Drosophila melanogaster Methoprene-tolerant (Met) gene homologs from three mosquito species: Members of PAS transcriptional factor family

“…Isolation and sequence determination of Met showed this gene to be a member of the bHLH-PAS family of transcriptional regulators (Ashok et al, 1998). Met is proposed to be a component of the elusive JH receptor, based on the mutant phenotype (Wilson and Fabian, 1986), the binding of JH III by MET, its ability for transcriptional activation (Miura et al, 2005), and its genetic interaction with the 20-hydroxyecdysone primary response gene, Broad-Complex . Another D. melanogaster bHLH-PAS gene, germ cell-expressed (gce), shows more than 50% homology to Met (Moore et al, 2000); however, a function for gce has not been reported.…”