Báo cáo khoa học: Characterization of the Drosophila Methoprene -tolerant gene product Juvenile hormone binding and ligand-dependent gene regulation potx

Characterization of the Drosophila Methoprene -tolerant gene product Juvenile hormone binding and ligand-dependent gene regulation Ken Miura, Masahito Oda, Sumiko Makita and Yasuo Chinzei Department of Medical Zoology, School of Medicine, Mie University, Tsu City, Japan 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 nuc- lear steroid ⁄ thyroid ⁄ retinoid receptor superfamily [1]. During insect development, ecdysteroids induce molting while JH determines the nature of each molt by modu- lating the ecdysteroid-induced gene expression cascade [2–4]. 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 sesquiterpe- noids, whose lipid-soluble nature has suggested action directly on the genome through nuclear receptors such as ecdysteroids and the vertebrate steroid ⁄ thyroid ⁄ reti- noid 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 homo- logue of the vertebrate retinoid X receptor, promoting Keywords juvenile hormone; juvenile hormone receptor; Methoprene-tolerant; Drosophila; transcription factor Correspondence K. Miura, Department of Medical Zoology, School of Medicine, Mie University, Edobashi 2-174, Tsu514-8507, Japan Fax: +81 59 231 5215 Tel: +81 59 231 5013 E-mail: k-miura@doc.medic.mie-u.ac.jp (Received 27 October 2004, revised 20 December 2004, accepted 4 January 2005) doi:10.1111/j.1742-4658.2005.04552.x 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 Methoprene- tolerant (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 regu- lation. 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 JHA- dependent 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 structur- ally 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 tran- scription factor. Abbreviations Ahr, aryl hydrocarbon receptor; Arnt, Ahr nuclear translocator; bHLH, basic helix-loop-helix; DBD, DNA binding domain; DCC, dextran-coated charcoal; EGFP, enhanced green fluorescent protein; JH, juvenile hormone; JHA, synthetic analogue of JH; Met, Methoprene-tolerant gene; MET, Met protein; PAS, period-aryl hydrocarbon receptor ⁄ aryl hydrocarbon receptor nuclear translocator-single-minded; Per, Drosophila period clock protein; Sim, Drosophila single-minded protein; SFM, serum-free medium; TNT, coupled in vitro transcription ⁄ translation; UAS, upstream activating sequence; USP, ultraspiracle protein. FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS 1169 its homodimerization, but the concentrations of JH required are several orders of magnitude higher than its physiological titre [12]. In transiently transfected cultured cells, a high dose of JH led to transcriptional activation through the binding of USP to a DNA response element upstream of a core promoter [13]. In other insect species, gene regulation by JH through DNA sequences resembling nuclear hormone response elements has been reported [14–16], suggesting the involvement of nuclear receptor family members in JH signaling. Because the strict regulation of JH titre in the insect body is crucial [17], the application of exogenous JH or analogues (JHAs) can disrupt normal development, and a number of JHAs have been synthesized and used as insecticides as well as research tools. The genetic and biochemical studies on resistance to JHA insecti- cides have led to the implication of another class of transcriptional regulator in JH signaling. Wilson and coworkers examined the resistance mechanism of Dro- sophila to the JHA, methoprene, and isolated mutant lines of flies that are resistant to morphogenetic and lethal effects of natural JH or methoprene [18]. The allele responsible for this resistance, named Metho- prene-tolerant (Met), encodes a basic helix-loop-helix (bHLH)-PAS protein, MET [19]. The bHLH-PAS fam- ily comprises transcriptional regulator proteins that are key players in a wide array of developmental and physiological pathways such as neurogenesis, circadian rhythms, hypoxia response, and toxin metabolism [20,21]. PAS is an acronym from the initial members of the family: Drosophila period clock protein (Per), vertebrate aryl hydrocarbon receptor (Ahr, also known as dioxin receptor) ⁄ Ahr nuclear translocator (Arnt), and Drosophila single-minded protein (Sim) [22,23]. The bHLH-PAS transcription factors share a com- mon overall structure. The bHLH domain is located near the N terminus. The basic region binds to a con- sensus palindromic hexanucleotide E-box (CANNTG) [24] or its derivatives [25,26]. The HLH domain allows these proteins to form a hetero- or homodimer. The bHLH domain is followed by PAS-1 and PAS-2 domains, which are used for dimerization between PAS proteins, small molecule binding, and also for binding to non-PAS proteins. The C-terminal half residues, which are not well conserved, harbour transcription activation ⁄ repression domains [20]. These structural features are found in MET [19]. Met mutant flies exhi- bit low JH binding affinity in fat body cytosolic extracts while an 85-kDa protein seems to be respon- sible for this binding [27,28]. Localization of MET in Drosophila tissues is exclusively nuclear [29]. Met females show reduced oogenesis [30], and the males have some defects in reproduction [28,31]. The Met null mutant flies are viable, showing that Met is not a vital gene, but this might be explained by redundancy provi- ded by cognate genes [30]. These observations suggest the involvement of MET in at least one pathway of JH signaling. The direct evidence, however, is still lacking: does JH bind directly to MET?; does MET function as a JH-dependent transcriptional regulator? We have now examined the binding of radiolabeled JH III to MET protein. Using a heterologous system in Drosophila S2 cells, we have characterized ligand- dependent gene regulation by MET. Our results suggest that MET may function as a JH-dependent transcription factor. Results MET binds to JH III with high affinity The MET protein was obtained by using coupled in vitro transcription ⁄ translation (TNT) reaction. The production of the full-length polypeptide was confirmed by analysing the product of reaction in the presence of 35 S-methionine by SDS ⁄ PAGE and autoradiography (Fig. 1). As a negative control, mock-programmed lysate was processed in parallel. As evident in the figure, the principal product had the expected full-length molecular mass of 79 kDa. Faster migrating minor pro- tein bands are also visible, which were not eliminated by the addition of protease inhibitor mixture in the reac- tion (data not shown). Then, the programmed lysate was used as a protein source for binding assay by the dextran-coated charcoal (DCC) method. As seen in Fig. 2, specific binding of 3 H-labeled JH III to the TNT protein showed saturable profile. The experiment was repeated three times, and the K d value by Scatchard analysis was calculated to be 5.3 ± 1.5 nm (mean ± SD). By these experiments, it was demonstrated that MET binds to JH III directly with a nanomolar K d value although we do not rule out the possibility that factors in the rabbit reticulocyte lysate may influence binding. The specific binding was competed away by 100-fold molar excess of cold JH III (data not shown). MET regulates transcription in a JH-dependent manner The Met gene product was examined for its transacti- vation ability. The binding sequence motif of MET is presently uncertain. In addition, it is unknown whether MET functions as a homo- or heterodimer. So, we utilized a heterologous approach with the yeast GAL4–DBD (DNA binding domain) fusion ⁄ UAS Characterization of Drosophila Met gene product K. Miura et al. 1170 FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS (upstream activating sequence) system. The GAL4– DBD possesses a zinc finger that directs homodimeri- zation and binding to UAS elements, and a potent nuclear localization sequence [32,33]. By using this system, we were able to assess the transactivation potential of MET independently of its dimerization properties or nuclear localization signals. The MET protein was expressed in S2 cells as a fusion with GAL4–DBD, and a luciferase reporter construct pos- sessing five tandem copies of the UAS in its regulatory region was used. In this system, the effect of JH on transcription from the reporter gene was tested. As shown in Fig. 3, when an empty expression vec- tor was transfected, the addition of 5 lm JH III to the culture medium caused no elevation of reporter activ- ity over that of controls given the vehicle ethanol. Expression of native MET lacking GAL4–DBD slightly elevated the reporter activity, but did not show any JH dependency. Next, only the GAL4–DBD was expressed. In this case, the reporter activity was eleva- ted about twofold above the empty vector control in either the presence or absence of JH III, indicating that the GAL4–DBD translocates into the nucleus and functions as a moderate, constitutive activator of transcription in a JH-independent manner. The GAL4–DBD–MET fusion in the presence of ethanol did not bring about any enhanced reporter activity relative to the empty vector control, but when JH III was added, the reporter activity was elevated about fivefold over the case of the empty vector control or the case of the GAL4–DBD–MET with ethanol. This activation by JH III can also be described as about twofold when compared to the case of GAL4–DBD with JH III. This indicates that MET has transactiva- tion domain(s), and its transactivation function is JH dependent. It is noteworthy that in the absence of JH III the MET moiety of the fusion protein repressed the moderate transactivation produced by GAL4– DBD. This suggests that unliganded MET may func- tion as a transcriptional repressor. Fig. 1. Autoradiogram of TNT lysate programmed with Met cDNA. The TNT reaction was performed with 400 ng PCR fragment con- taining T7 promoter and Met full ORF in the presence of 35 S-methio- nine. A portion of the lysate was separated by 10% SDS ⁄ PAGE and autoradiographed. A mock-programmed lysate was run in paral- lel. Molecular mass markers are shown at the left. An arrowhead indicates the position of full-length MET (79 kDa). Fig. 2. MET binds to JH III with high affinity. (A) Binding of 3 H-labe- led JH III to MET. MET was obtained by TNT reaction and subjec- ted to DCC assay with 3 H-labeled JH III as described in Experimental procedures. Specific binding was calculated by sub- tracting the counts of mock-programmed lysates from those of cor- responding programmed lysates. The specific binding is shown in the figure. (B) Scatchard analysis of JH III binding to MET. The K d value was calculated from the slope of the regression line. These analyses were performed three times and representative data are shown. K. Miura et al. Characterization of Drosophila Met gene product FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS 1171 Ligand specificity of transactivation by MET If MET represents a JH-dependent transcription fac- tor, it should show stringent ligand specificity. To test this, several compounds that are structurally related to JH or JHA but show no JH activity were examined in the GAL4-MET fusion ⁄ UAS system. The effects of these potential ligands on the reporter activity are shown as fold induction by dividing the activity obtained with the pAcGAL4–DBD–Met by that in negative controls using empty pAc vectors (Fig. 4). As is evident here, addition of squalene, farnesol, farnesyl acetate and geraniol at a final concentration of 5 lm did not result in any activation of the reporter. JH III, however, again brought about enhanced reporter activity. Interestingly, a JHA ) methoprene ) showed weaker ligand activity than JH III. Thus, the trans- activation exerted by MET shows stringent ligand specificity apparently related to JH activity, ruling out nonspecific transactivation by lipid-soluble compounds. Dose–responses of natural JHs and JHA on MET transactivation The binding assay showed that MET has a nanomolar level K d for JH III and we used several potential lig- ands at 5 lm in the experiments described above. If MET functions as a JH-dependent transcription factor, it should respond to nanomolar levels of ligand, con- sistent with its high affinity for JH III. Here, we tested three natural JHs, JH I, JH II, and JH III, and the JHA methoprene in varying concentrations using the GAL4-MET fusion ⁄ UAS transfection assay (Fig. 5). The effects on the reporter activity are shown as fold induction as in Fig. 4. Every compound tested showed ligand activity on transactivation, nearing saturation at 500 nm while showing only marginal increase at 5 lm. Among these, JH III, which is one of the native JHs of Drosophila, was found to be the most effective over the range of concentrations tested. Of note is that JH III was conspicuously active in the range of 5–50 nm, whereas the other JHs or JHA showed only Fig. 4. Ligand specificity of gene activation by MET. The transfec- tion assay was carried out as described in Fig. 3 with pAcGAL4– DBD-Met or an empty pAc5.1 ⁄ V5-His A vector as expression con- structs. S2 cells were incubated with several different compounds indicated at a concentration of 5 l M. Activities are shown as fold induction by dividing the activity obtained with the fusion-expres- sing construct by that in negative controls using empty vectors (mean ± SD). The mean value obtained in ethanol controls is taken as unity. Fig. 3. MET regulates transcription in a JH-dependent manner. S2 cells were transfected with several different expression constructs (vector pAc5.1 ⁄ V5-His A, pAcMet, pAcGAL4–DBD, or pAcGAL4– DBD-Met) together with reporter (pG5luc) and coreporter (pRL-tk) constructs. After incubation in either the presence or absence of 5 l M JH III for 24 h, cells were harvested and subjected to dual luciferase assay. Luciferase activities are shown normalized to that of the coreporter (mean ± SD). Fig. 5. Dose–response curves for transactivation by MET with JHs and JHA. JH I (r), JH II (n), JH III (m), methoprene (x) were inclu- ded in the culture media in the range from 5 n M to 5 lM after trans- fecting S2 cells with the expression (pAcGAL4–DBD-Met or empty pAc5.1 ⁄ V5-His A), reporter (pG5luc) and coreporter (pRL-tk) con- structs. Fold induction was calculated as in Fig. 4. Characterization of Drosophila Met gene product K. Miura et al. 1172 FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS slight effects. The other native JH of Drosophila, JH- bisepoxide [34] was not tested. The induction activities are in the following order: JH III  JH II > JH I > methoprene. The most effective transcriptional activation produced by Drosophila MET with its native JH species further supports the putative role of MET as a JH-dependent transcription factor. We should mention here that JHs are highly sticky to glass or plastic surfaces [35] and would be adsorbed by pipette tips, test tubes or culture dishes. Thus, the effective concentrations of these compounds would be lower than the values indicated in Fig. 5. These data, thus, indicate that the threshold activity concentration of JHs is reasonably low in this transient transfection system. Localization of MET in S2 cells In the transfection assays described above, MET was fused to GAL4–DBD, which has a nuclear localization sequence. To test the subcellular localization of MET, we used a fusion to enhanced green fluorescent protein (EGFP), which does not have a nuclear localization sequence. S2 cells were transfected with the expression plasmid pAcMET–EGFP together with the reporter and coreporter constructs used above. After trans- fection, cells were incubated for 24 h in the presence or absence of JH III, then observed by Nomarski DIC (differential-interference contrast) or fluorescence microscopy (Fig. 6). In both cases, the fluorescence of the fusion proteins was seen in the nucleus. In these experiments the use of cultured cells allows for com- plete depletion of JH. These observations are consis- tent with the previous report in vivo [29] and rule out the ligand-dependent nuclear translocation reported for the Ahrs of vertebrates [36]. Then, how is JH transported to the nucleus? A process such as verteb- rate retinoid transport including cellular retinol-bind- ing protein [37] may be involved. Discussion From its identification as a Drosophila gene responsible for resistance to morphogenetic and toxic effects of JH and JHAs, the Met gene product has been implicated to have an involvement in JH reception. Previous works on Met do not contradict the hypothesis that MET may be a component of a JH-dependent tran- scriptional regulator complex. Direct evidence, how- ever, for the immediate interaction with JH and involvement in gene regulation is lacking. To test this hypothesis, we chose S2 cells as experimental material because the use of cultured cells would be advanta- geous for examining ligand-dependent gene regulation and JH responses in this cell line have been reported [38–40]. Our principal new contributions are: (a) demonstra- tion of direct, reversible binding of JH III to MET; (b) demonstration of its JH-dependent transactivation potential. The former was enabled by the use of cou- pled in vitro transcription and translation, as we had experienced difficulty in obtaining soluble preparations of full-length bHLH-PAS proteins from mosquitoes using prokaryotic expression systems (K. Miura, unpublished data). The binding of JH III by MET showed high affinity with a nanomolar K d value, and was competed away by an excess of cold JH III. In the present study, MET was tethered to a promo- ter by using the GAL4–DBD fusion ⁄ UAS reporter system. In this heterologous system, the MET fusion Fig. 6. Subcellular localization of MET in S2 cells. S2 cells were transfected with pAcMET–EGFP together with the reporter and coreporter constructs. After transfection for 5 h, cells were incuba- ted with either 0.5 l M JH III or ethanol for 24 h. Then, cells were fixed and observed by Nomarski DIC or fluorescence microscopy. Horizontal bars represent 10 lm. The results shown are representa- tive of two independent experiments. K. Miura et al. Characterization of Drosophila Met gene product FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS 1173 exhibited specific ligand-dependent activation of a reporter gene placed downstream of the UAS. The MET fusion responded only to JH or the JHA metho- prene while compounds that are structurally related but hormonally inactive elicited no response. Among compounds tested, JH III was the most effective lig- and, even at nanomolar concentrations, which is in accordance with its nature as one of Drosophila’s native JHs. The typical range of concentration for JH In insect haemolymph is 0.3–180 nm [41]. Further, the maximal JH titre in the Drosophila life cycle is 5–7 pmolÆg )1 wet weight [12], which would correspond to 25–35 nm in the haemolymph, assuming that haemolymph occupies one-fifth of the body weight. In view of these physiological JH titres, it is thus notable that in this study JH III was found to be overwhelm- ingly active in the physiological range of 5–50 nm over the other JHs or JHA. The ligand-dependent trans- activation profile exhibited by MET clearly rules out the possibility that it is simply a JH binder, like cyto- solic JH binding proteins, and suggests that it might play a role in JH signaling in vivo. Recently, Wozniak et al. [42] have reported the con- formational changes of recombinant Drosophila USP exposed to several different farnesoid compounds including natural JHs. They have shown that JH III and JH I at 100 lm elicit the conformational changes to a similar degree whereas JH II is the least effective. Furthermore, by using Drosophila white puparial bio- assay they have demonstrated that the biochemical dif- ferences in the three JHs mentioned above parallel the respective biological activity. For example, in preven- tion of adult emergence the 50% effective doses (ED 50 s) for JH I–III are 153, 678 and 143 pmolÆpupa- rium )1 , respectively. Another report describes that the ED 50 of methoprene is 5 pmolÆpuparium )1 in the same assay and that Met mutant flies are more resistant to another JHA, S31183, than the parental fly stock, sug- gesting the involvement of the Met locus in this resist- ance [43]. Based on these studies, the order of efficacy of these compounds in this bioassay seems to be metho- prene  JH III ‡ JH I > JH II. On the other hand, the order was JH III  JH II > JH I > methoprene in our transfection assay. Methoprene is the most effective in the former and the least effective in the latter. We do not find this surprising as methoprene is often highly active over naturally occurring JHs when applied topically. For example, the early trypsin gene of Aedes aegypti is upregulated by JH, and low doses of methoprene, but higher doses of its native JH III are required to restore its expression in the ligated abdomens [44]. The higher efficacy of methoprene in these bioassays may be due to its higher resistance to enzymatic degradation and possible higher penetration through the cuticle than natural JHs. Another point is that JH I has been shown to be more active than JH II in the white puparial assay [42] whereas JH II is more active in our transfection assay. The difference between these two studies is the concen- trations of JH used. Wozniak et al. [42] used supra- physiological concentrations in both biochemical and biological assays whereas we tested JHs or JHA at much lower range of concentrations. In our assay JH I and JH II were similarly much less effective than JH III in the physiological range (5–50 nm) while these differences were somewhat obscured at higher doses, although JH II was still more effective than JH I. At present we do not have data to explain this discrep- ancy. Possibly, there might be more than one pathway of JH signaling underlying in the white puparial bio- assay, one mediated by USP and another by MET. In the reporter assays, we noted that unliganded MET repressed the intrinsic activation function pos- sessed by GAL4–DBD. Although GAL4–DBD is believed to lack transactivation domains [33], it showed moderate transactivation potential in Dro- sophila S2 cells in this study. The nuclear localization of MET [29] was confirmed by our finding that the MET–EGFP fusion is concentrated in the nuclei of transfected S2 cells. In addition, GAL4–DBD has a nuclear localization sequence. Therefore, it is reason- able to consider that the GAL4–DBD fusion of MET sits on the UAS of the reporter construct even in the absence of ligand, and that the MET moiety is respon- sible for the observed repression. In the case of verteb- rate Ahr, a multimeric complex including hsp90 anchors the unliganded Ahr in the cytoplasm, thereby preventing its transactivation function [36]. Upon lig- and binding, Ahr translocates to the nucleus and forms a transcription factor complex with Arnt. In fact, the C-terminal portion of Ahr fused to GAL4–DBD has been shown to act as a constitutive activator of gene regulation [45]. Contrary to this, MET exists in the nucleus even in the absence of ligand. Upon ligand binding, it becomes a transcriptional activator. This resembles the ligand-dependent activation that has been shown in the activation function-2 of many nuc- lear hormone receptors [46,47], rather than the case of the vertebrate Ahr whose activation function is regula- ted by its subcellular localization. Two questions arise here as to whether MET func- tions as a homo- or heterodimer, and as to what DNA sequences are responsible for the binding of this tran- scriptional regulator complex. These questions are related since DNA-binding specificities of bHLH-PAS proteins are determined by their dimerization properties Characterization of Drosophila Met gene product K. Miura et al. 1174 FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS [48]. For example, the dioxin receptor complex Ahr ⁄ Arnt heterodimer binds to TNGCGTG [25]. Ahr recognizes the 5¢-half-site TNGC, while Arnt recogni- zes the 3¢-half-site GTG. Arnt is also capable of forming a homodimer that recognizes a consensus palindromic E-box sequence, CACGTG [48]. Dro- sophila Sim protein forms a heterodimer with Tango (a Drosophila Arnt-like protein) and binds to ACGTG core sequence [49]. Thus, DNA binding specificities of bHLH-PAS dimers are dependent upon the dimer con- figuration while Arnt or Tango always recognize the GTG motif. In the present study, we used the GAL4– DBD fusion of MET in transfection assays. Under these conditions MET is likely to behave as a homo- dimer because of its overexpression and because of dimerization interfaces provided by the GAL4–DBD moiety. Therefore, the natural dimerization partner and binding sequence of MET are unknown at present. Since the bHLH domain of MET shows relatively high similarity to vertebrate Arnts [19], the use of the con- sensus sequence CACGTG may be a good starting point to answer these questions. Based on the framework by Wilson and coworkers, our results have further supported the notion that MET may function as a JH-dependent transcription factor. In further studies, identification of its target genes will help elucidate its in vivo function. Molecular dissection of MET and structural studies may lead to the development of new biologically active JHA and new strategies for pest management. Experimental procedures JHs, JHA and related compounds JH I and JH II were obtained from SciTech. JH III was from Sigma. The JHA, methoprene was a gift from Otsuka Chemicals Co. Ltd. Squalene, farnesol and geraniol were from Sigma. Farnesyl acetate was from Aldrich. cDNA cloning of Met Total RNA was isolated from S2 cells as described previ- ously [50]. First-strand cDNA was synthesized by Super- script reverse transcriptase II (Invitrogen) with oligo dT primer, and used as a template for RT–PCR. The cDNA containing a full ORF of Met was amplified by 30 cycles of PCR using a proofreading polymerase (long and accurate Taq polymerase, Takara) with the primer pair based on the published sequence [19]: 5¢-GCCGAATTCCAACATGGC AGCACCAGAGACGGG-3¢;5¢-GCCTCTAGATCATCG CAGCGTGCTGGTCAG-3¢. The amplified products were purified, digested and subcloned into EcoRI and XbaI sites of pBluescript II (Stratagene), and the identity of the cDNA clone was confirmed by sequencing. Binding assay The DCC assay was carried out as described [51]. Full- length MET was prepared by a TNT T7 Quick for PCR DNA Kit (Promega). A cDNA template for the TNT reac- tion was prepared by PCR using the Met cDNA inserted downstream of the T7 promoter site of pBluescript II. After PCR, the cDNA fragments containing the T7 promoter were purified by a QIAquick PCR purification kit (Qiagen). The TNT reaction was carried out as follows: 20 lL lysate was programmed by 400 ng of the cDNA fragment in a total vol- ume of 25 lL, and the reaction mixture was kept at 30 °C for 90 min. A reaction in the presence of 35 S-methionine was performed in parallel, and the lysate was analysed by SDS ⁄ PAGE and autoradiography to confirm the production of a polypeptide with the expected size. The DCC assay used the TNT lysate as a protein source. Each reaction mixture included 25 lL of the programmed lysate, 74 lL of buffer C (20 mm Tris ⁄ HCl pH 7.9, 5 mm magnesium acetate, 1 mm EDTA, 1 mm dithiothreitol) and 1 lL of variable amounts of 3 H-labeled JH III (specific activity: 17.5 CiÆmmol )1 , PerkinElmer) in ethanol in a polyethylene glycol-coated glass tube. The mixture was incubated at 22 °C for 90 min. This was followed by the addition of 5% DCC suspension, gentle mixing for 2 min and centrifugation for 1 min. The supernatant was collected into a scintillation vial, decolo- rized overnight with 2 mL 30% H 2 O 2 , and counted by scin- tillation. Mock-programmed lysates were incubated with the corresponding amounts of 3 H-labeled JH III and processed in parallel with the programmed lysates, and the counts obtained were taken as nonspecific binding. Specific binding was obtained by subtracting the counts of mock-pro- grammed lysates from those of corresponding programmed lysate. The addition of esterase or protease inhibitors in the binding reaction mixture did not affect binding values (data not shown). Saturation curves were obtained, and K d values were calculated by the method of Scatchard [52]. Plasmids The plasmid pAcGAL4–DBD-Met, which expresses a fusion protein of MET possessing the yeast GAL4–DBD toward the N terminus, was constructed as follows: a full- length Met cDNA fragment having overhangs of EcoRV and XbaI sites was prepared by PCR and subcloned into pBIND vector (Promega); the cDNA fragment containing the fused ORF of GAL4–DBD and Met was amplified by PCR and subcloned into NotI and XbaI sites of pAc5.1 ⁄ V5-His A vector (Invitrogen). The location of the junction was confirmed by sequencing. A control plasmid pAc- GAL4–DBD was constructed by inserting the GAL4–DBD K. Miura et al. Characterization of Drosophila Met gene product FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS 1175 region of the pBIND vector into the pAc5.1 ⁄ V5-His A vector. Another control vector pAcMet was prepared by subcloning the full ORF of Met into the pAc5.1 ⁄ V5-His A vector. An empty pAc5.1 ⁄ V5-His A vector was used as a negative control. The reporter plasmid pG5luc, which con- tains five GAL4 binding sites (UAS) upstream of the firefly luciferase gene, was from Clontech. The coreporter plasmid pRL-tk, which expresses Renilla reniformis luciferase, was from Promega. The plasmid pAcMET–EGFP, which expresses a fusion protein of MET possessing the EGFP polypeptide in the C terminus, was constructed by transfer- ring the fused ORF from pEGFP-N1 vector (Clontech) to pAc5.1 ⁄ V5-His A vector. The in-frame nature of the junction was confirmed by sequencing. Cell culture and transfection Drosophila S2 cells [53,54] were cultured in Drosophila Serum-Free Medium (SFM, Invitrogen). The cells were see- ded at a density of 2.5 · 10 5 cells per well of 24-well plates one day before transfection. 2 lL of lipofectin (Invitrogen) per each well was mixed with 25 lL of SFM and incubated for 40 min at room temperature. One-hundred and fifty nanograms of DNA (50 ng each of expression, reporter and coreporter plasmids) per well was mixed with 25 lLof SFM, and this was combined with the lipofectin ⁄ SFM mix- ture and incubated for another 15 min. Then, this was mixed with 200 lL of SFM and overlaid onto S2 cells in each well. This was followed by 5 h incubation at 27 °C, and the transfection mixture was replaced by 250 lLof SFM either containing natural JH (JH I, JH II and JH III), JHA methoprene, related compounds (squalene, farnesol, geraniol and farnesyl acetate), or solvent ethanol. The cells were incubated at 27 °C for another 24 h, lysed and subjec- ted to the dual luciferase assay (Promega) in a luminometer (Turner Designs, Model TD-20 ⁄ 20). The reporter activity was shown as relative luciferase activity by normalizing the reporter activity to the coreporter activity. Where indicated, the effect of test compounds were shown as fold induction by dividing the reporter activity obtained with the pAc- GAL4–DBD-Met by that in negative controls using empty pAc vectors. The transfection assay was done at least three times independently in triplicate, and the reproducibility was confirmed. The values of relative luciferase activity in each transfection assay fluctuated a little, but tendency was always reproducible. In Results, representative data are shown in the respective figures. For the observation of the MET–EGFP fusion proteins, 1 · 10 6 of S2 cells were seeded on a 35-mm glass-bottomed dish (Matsunami Glass, #D110400) 1 day before transfec- tion. The pAcMET–EGFP was transfected together with the reporter and coreporter constructs as described above so as to mimic the transfection conditions of the reporter assays. 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Characterization of Drosophila Met gene product K. Miura et al. 1178 FEBS Journal 272 (2005) 1169–1178 ª 2005 FEBS . Characterization of the Drosophila Methoprene -tolerant gene product Juvenile hormone binding and ligand-dependent gene regulation Ken Miura,. sequence. Therefore, it is reason- able to consider that the GAL4–DBD fusion of MET sits on the UAS of the reporter construct even in the absence of ligand, and