The Methoprene-tolerant (Met) bHLH-PAS gene also termed Resistance to Juvenile Hormone (Rst(1)JH) -- is involved in juvenile hormone (JH) action in Drosophila as a likely component of a JH receptor. Met was expressed in Drosophila S2 cells, and MET partners were sought using pull-down assays. MET-MET interaction was found to occur. The germ-cell expressed (gce) gene is another Drosophila bHLH-PAS gene with high homology to Met; GCE forms heterodimers with MET. In the presence of JH or either of two JH agonists, MET-MET and MET-GCE formation is drastically reduced. Interaction between GCE and MET having N- or C-terminus truncations, bHLH or PAS-A domain deletions, or a point mutation in the PAS-B domain fail to occur. However, JH-dependent interaction occurs between GCE and MET having point mutations in bHLH or PAS-A. During development, changes in JH titer may alter partner binding by MET and result in different gene expression patterns (Godlewski, 2006).

Hormonal regulation of insect development involves the steroid 20-hydroxyecdysone (20-HE) and the sesquiterpenoid juvenile hormone (JH). The molecular biology and action of 20-HE are relatively well described. In contrast, neither the JH receptor nor the mechanism of action is well understood. JH is present during larval development of probably all insects; its role during this time period is best understood in lepidopteran and hemimetabolous insects, when it acts to maintain the developmental 'status quo' prior to metamorphosis, probably to allow proper larval molting and prevent premature metamorphosis. Its absence at the end of larval development is thought to permit 20-HE control of metamorphosis. JH can act at the molecular level to regulate gene expression (Godlewski, 2006).

One gene, Methoprene-tolerant (Met), is known to be directly involved in the manifestation of the JH effects in D. melanogaster. Met mutants show resistance to both the toxic and morphogenetic effects of JH and several JH analogs (Wilson, 1986; Riddiford, 1991; Restifo, 1998; Wilson, 1996). Ligand-binding studies have shown MET to bind JH III with specificity and nanomolar affinity (Shemshedini, 1990a; Miura, 2005), suggesting that MET is a component of a JH receptor. Met encodes a bHLH-PAS transcriptional regulator family member (Ashok, 1998), and MET can activate a reporter gene in transfected Drosophila S2 cells (Miura, 2005). Although Met is expressed throughout development (Ashok, 1998), roles for MET during embryonic and larval development are not readily apparent. The gene germ-cell expressed (gce) is another bHLH-PAS gene in D. melanogaster with high Met homology (70%-86% amino acid identity in the conserved domains) but unknown function (Godlewski, 2006).

bHLH-PAS proteins typically function as heterodimers with other bHLH-PAS proteins (Gu, 2000). Binding partners for MET were explored by co-expressing Met with either gce (heterodimer) or another Met construct (homodimer) and examining binding interaction using pull-down assays. Except in certain Met mutant constructs, MET-GCE interaction was apparent, and MET-MET interaction was also detected. However, in the presence of JH, both interactions were greatly diminished. These results suggest how JH might act during preadult development in insects (Godlewski, 2006).

The Met gene is clearly involved in the response of D. melanogaster to JH or JH agonist exposure during the sensitive period at the onset of metamorphosis. At this time of development, endogenous JH is absent, and secretion of 20-HE is initiating metamorphosis by activation of the primary-response genes. The presence of JH or JH analog (JHA) during this time disrupts metamorphosis probably by disrupting expression (Zhou, 2002) or function (Wilson, 2006) of one or more genes necessary for the completion of the adult form, resulting in the JH pathology seen in the pharate adult. Met mutants are resistant to the effects of JH (Wilson, 1986), and overexpression of Met⁺ increases sensitivity to exogenous hormone (TGW, unpublished), clearly implicating MET in the molecular action of JH/JHA. Since MET binds JH III at physiological concentration and can act as a transcriptional regulator (Miura, 2005), this protein possesses characteristics expected of a JH receptor component (Godlewski, 2006).

This work has demonstrated interaction between MET and GCE proteins co-expressed in Drosophila S2 cells. Heterodimer formation between very similar bHLH-PAS proteins is not unique: the vertebrate aryl hydrocarbon receptor functions as a heterodimer of two homologous proteins, AHR and ARNT (Gu, 2000). Since PAS proteins normally function as heterodimers, it was surprising to find MET homodimer formation. Again, MET is not unique in this property: ARNT homodimer formation has been shown (Godlewski, 2006).

JH or JHAs can block MET homodimer and heterodimer formation or stability. The effect is both time- and dose-dependent. Since MET has been shown to bind JH III, the expressed MET seems likely to be the JH binder, presumably resulting in disrupted partner protein binding. However, an effect of the added hormone on S2 cell physiology that results in disrupted binding cannot be ruled out (Godlewski, 2006).

The MET mutants that were evaluated in this study served two purposes. First, the failure of many of them to heterodimerize with GCE provided further evidence that the MET-GCE interaction is specific. Second, they provided information about the conserved domains in the MET protein. As expected, the severe N- and C-terminal deletions failed to heterodimerize. The mutations having small deletions in the bHLH and PAS-A domains also failed to heterodimerize. Other studies have demonstrated involvement of these domains in PAS protein heterodimer formation, corroborated here for MET and GCE. Met¹²⁸ (with a point mutation in PAS-B) showed poor dimer formation, but Met³ (having a point mutation in bHLH) and Met¹ (having a point mutation in PAS-A) heterodimerized. Both Met³ and Met¹ flies show strong methoprene resistance (Wilson, 1986), suggesting that the mutant phenotype results from a separate problem, such as faulty interaction with DNA, but not from faulty JH binding, since MET-GCE heterodimerization was blocked by methoprene in both of these mutants. Future studies examining methoprene-sensitive heterodimerization following site-directed mutagenesis may be valuable in identifying the JH-binding domain of MET (Godlewski, 2006).

These results have suggested how MET and GCE might function during insect development as JH receptor components. During larval development when the JH titer is high, MET either acts as a monomer or forms a dimer with an unknown protein (Godlewski, 2006).

Obviously, this dimer would be hormone insensitive, unlike MET-GCE. MET could then act as a transcriptional regulator for one or more genes involved in larval development, allowing JH to fulfill its 'status quo' role. At the end of larval development when the JH titer is depleted, MET could then homodimerize or dimerize with GCE (or both) to regulate either a set of pupal genes involved in metamorphosis or express a different pattern, having a different function, from the larval genes. If JH/JHA were artificially present during early metamorphosis, then a larval-state MET is produced, leading to either larval gene expression or failure of correct pupal gene expression, resulting in the pathology seen. Thus, in this scenario, JH presence or absence provides the signal for a change in gene expression through MET and probably other transcription factors and the resultant physiology in larvae or pupae (Godlewski, 2006).

GENE STRUCTURE

Exons - 2

PROTEIN STRUCTURE

Amino Acids - 716

Structural Domains

MET shows three regions of homology to members of a family of transcriptional activators known as bHLH-PAS proteins. The bHLH-PAS gene family was named for the three founding members: period (per) gene, Aryl hydrocarbon receptor (Ahr), and single-minded (sim) gene. per is a biological clock gene, and sim is a transcription factor gene, both originally isolated from Drosophila. Ahr has been isolated from several vertebrates, and its product functions to bind xenobiotic compounds and a partner protein, the product of the Ah receptor nuclear translocator (arnt) gene. Most of the bHLH-PAS proteins function either as transcription factors or as interactive partners with transcription factor proteins. MET generally has higher homology to the vertebrate bHLH-PAS proteins than to those identified in D. melanogaster. A Drosophila ARNT-like gene has been cloned, and DARNT has higher homology to vertebrate ARNT than does MET, suggesting that DARNT, not MET, may function like ARNT in flies. MET homology to these proteins includes the bHLH region that is involved in DNA binding (30%-38% identity), the PAS-A region (28%-40%), and the PAS-B region (22%-35%). The arrangement of these domains in the Met gene is the same as for other bHLH-PAS genes (Ashok, 1998).

date revised: 28 September 2006

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