Binding affinity of nonsteroidal ecdysone agonists against the ecdysone receptor complex determines the strength of their molting hormonal activity Chieka Minakuchi 1 , Yoshiaki Nakagawa 1 , Manabu Kamimura 2 and Hisashi Miyagawa 1 1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan; 2 National Institute of Agrobiological Sciences, Tsukuba, Japan N-tert-Butyl-N,N¢-dibenzoylhydrazine and its analogs are nonsteroidal ecdysone agonists that exhibit insect molting hormonal and larvicidal activities. The interaction mode of those ecdysone agonists with the heterodimer of the ecdy- sone receptor and ultraspiracle has not been fully elucidated. We expressed the ecdysone receptor B1 and the ultraspiracle of the lepidopteran, Chilo suppressalis,usinganin vitro transcription/translation system and confirmed, using gel- shift assays, that the proteins function as ecdysone receptors. We also analyzed their ligand-binding affinity. A potent ecdysteroid, ponasterone A, specifically bound to the ecdy- sone receptor with low affinity (K D ¼ 55 n M ), and the spe- cific binding was dramatically increased (K D ¼ 1.2 n M )in the presence of the ultraspiracle. For seven nonsteroidal ecdysone agonists and five ecdysteroids, the binding activity to the in vitro-translated ecdysone receptor–ultraspiracle complex was linearly correlated with the binding activity to the inherent receptor protein in the cell-free preparation of C. suppressalis integument. The binding to the ecdysone receptor–ultraspiracle complex for a series of compounds was highly correlated with their molting hormonal activity, indicating that the binding affinity of nonsteroidal ecdysone agonists to the ecdysone receptor–ultraspiracle complex primarily determines the strength of their molting hormonal activity. Keywords: ecdysone receptor; dibenzoylhydrazines; nuclear receptor; receptor binding; dissociation constant. Insect molting is triggered by the binding of 20-hydroxy- ecdysone (Fig. 1I) to its receptor protein. Significant effort has been made to purify the receptor proteins for 20-hydroxyecdysone [1–3], but the isolation and purification of these receptors from whole insects has, to date, been unsuccessful owing to their instability and low yield. How- ever, cDNAs for the ecdysone receptor (EcR) and the ultraspiracle (USP) have been cloned from Drosophila mel- anogaster [4–7], and it was later shown that EcR and USP constitute a heterodimer which functions as the receptor for 20-hydroxyecdysone [8–10]. It was also made clear that 20-hydroxyecdysone binds to EcR, and that USP is an essential partner for the binding of 20-hydroxyecdysone [10]. The 20-hydroxyecdysone–EcR–USP complex binds to the ecdysone response element (EcRE), located upstream of other genes that are involved in molting and metamorpho- sis, and regulates the transcription of those genes. Both EcR and USP belong to the nuclear receptor superfamily, which is generally composed of six distinct regions (A–F), and the predicted amino acid sequences of C (DNA binding) and E (ligand binding) regions are conserved among insects [11]. Little is known about the interactions between the EcR– USP complex and ligands, because the EcR 3-D structure has not yet been elucidated, while the crystal structures of USPs were recently published for D. melanogaster and Heliothis virescens [12,13]. N-tert-Butyl-N,N¢-dibenzoylhydrazine (RH-5849; Fig. 1II: X n ¼ Y n ¼ H) [14–16] and its analogs are nonsteroidal ecdysone agonists that, via binding to the EcR–USP complex, cause incomplete molting in insects leading to death. Among these nonsteroidal ecdysone agonists, tebufenozide (RH-5992; Fig. 1II: X n ¼ 3,5-CH 3 ,Y n ¼ 4-C 2 H 5 ), methoxyfenozide (RH-2485; Fig. 1II: X n ¼ 3,5- CH 3 ,Y n ¼ 2-CH 3 -3-OCH 3 ), halofenozide (RH-0345; Fig. 1II: X n ¼ H, Y n ¼ 4-Cl) and chromafenozide (ANS- 118; Fig. 1III) are currently on the market as safer insecticides with reduced mammalian toxicity [17–21]. Previously, we evaluated the larvicidal and molting hormonal activities of nonsteroidal ecdysone agonists in the lepidopteran rice stem borer, C. suppressalis [22–29]. In these studies, we analyzed the substituent effects of non- steroidal ecdysone agonists on the larvicidal and molting hormonal activities by using classical quantitative structure– activity relationship procedures [30] to identify the import- ant physicochemical properties for their biological activity. However, the binding activity of ecdysone agonists against the receptor proteins of C. suppressalis has not yet been investigated. Quantitative structure–activity relationship Correspondence to Y. Nakagawa, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. Fax: + 81 75 753 6123, Tel.: + 81 75 753 6117, E-mail: naka@kais.kyoto-u.ac.jp Abbreviations: ANS-118, chromafenozide; EC 50 , 50% effective concentration; EcR, ecdysone receptor; EcRE, ecdysone response element; IC 50 , 50% inhibitory concentration; pIC 50 , reciprocal logarithmic value of the IC 50 ; RH-0345, halofenozide; RH-2485, methoxyfenozide; RH-5849, N-tert-butyl-N,N¢-dibenzoylhydrazine; RH-5992, tebufenozide; USP, ultraspiracle. (Received 9 June 2003, revised 30 July 2003, accepted 21 August 2003) Eur. J. Biochem. 270, 4095–4104 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03801.x studies for the receptor-binding activity would be useful to elucidate their mode of action. For D. melanogaster, three EcR isoforms (EcR-A, EcR- B1, EcR-B2) and one USP isoform have been cloned [4–7]. Subsequently, EcR and USP genes were cloned from several insects [11]. Recently we also cloned cDNAs for EcR-A, EcR-B1 and USP from C. suppressalis [31,32]. In this study, we prepared EcR-B1 and USP for C. suppressalis by an in vitro transcription/translation reaction to analyze their functions. We also evaluated the binding activity of a number of ecdysone agonists against the expressed EcR– USP complex to obtain information on the ligand–receptor interactions. Experimental procedures Chemicals Ponasterone A was purchased from Invitrogen Corp. (Carlsbad, CA, USA), and ecdysone and 20-hydroxyecdy- sone were from Sigma Chemical Co. (St Louis, MO, USA). Chromafenozide was a gift from Sankyo Agro and Nippon Kayaku Co. Ltd. Other ecdysteroids and nonsteroidal ecdysone agonists were from our stock samples [23–26,28, 29,33–35]. 3 H-Labeled ponasterone A (150 CiÆmmol )1 )was purchased from American Radiolabeled Chemicals Inc. (St Louis, MO, USA). In vitro transcription/translation and gel mobility shift assay The full coding regions of two splicing variants of EcR-B1, encoding 547 aa or 542 aa (EcR-547aa and EcR-542aa, respectively), and USP, were cloned into pBluescript II vector (Stratagene, La Jolla, CA, USA), as described previously [31,32]. Coupled in vitro transcription/translation of these constructs was performed using a TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI, USA), under control of the T7 promoter, according to the manufacturer’s instructions. Gel mobility shift assays were conducted using in vitro- translated EcR-547aa, EcR-542aa, USP and the 32 P-labeled Pal1 EcRE (5¢-GATCTAGAGAGGTCAATGACCTCG TCC-3¢) [36] as a probe, as previously reported [31,32]. Briefly, in vitro-translated proteins were incubated in 20 m M modified Hepes (pH 7.5) buffer on ice for 30 min, then 1 ng of 32 P-labeled EcRE probe was added. The mixture was incubated for another 30 min at 25 °C. The proteins were separated by electrophoresis on a nondenaturing polyacryl- amide gel and analyzed using a BAS-2000 bioimaging analyzer (Fuji Photo Film Co., Ltd, Tokyo, Japan). Ligand binding to in vitro -translated EcR and USP proteins Ligand-binding assays were performed according to pub- lished methods [37,38]. Briefly, 4 lLofin vitro-translated EcR and/or USP was placed in a siliconized tube (BM Equipment Co. Ltd, Tokyo, Japan) in low-salt buffer (20 m M Hepes, 20 m M NaCl, 20% glycerol, 1 m M EDTA, 1m M 2-mercaptoethanol, pH 7.9, containing 1 lgÆmL )1 aprotinin, 1 lgÆmL )1 pepstatin and 1 lgÆmL )1 leupeptin) with 3 H-labeled ponasterone A (25 000 d.p.m., final con- centration 5 n M ) and a test compound. To estimate the nonspecific binding, a 500-fold excess of unlabeled pona- sterone A was added to the incubation mixture. The total volume of the incubation mixture was 16 lL. The final concentration of solvent (ethanol or dimethylsulfoxide) in the incubation mixture was less than 1%, which did not affect the ligand–receptor binding. After a 60-min incubation at 25 °C, sample tubes were transferred to ice, and the reaction mixture was filtered immediately through nitrocellulose membrane NC45 (Schleicher & Schuell, Einbeck, Germany) with the aid of a vacuum filtration apparatus (KGS-25 microanalysis holder; Advantec Toyo, Tokyo, Japan), as described below. The incubation mixture was transferred to the filtration apparatus with NC45 membrane, loading 1 mL of ice-cold washing buffer (i.e. low salt buffer with 10% glycerol and no protease inhibitors). The whole solution on the membrane was immediately filtered by applying vacuum. After washing the membrane three times with 1.5 mL of ice-cold washing buffer, the radioactivity collected on the membrane was measured in Aquasol-2 (PerkinElmer Life Sciences, Wellesley, MA, USA) using a liquid scintillation counter LSC-1000 (Aloka Co., Ltd, Tokyo, Japan). Ligand binding to the cell-free preparation from C. suppressalis integument Rice stem borer larvae were reared on rice seedlings at 28 °C under a long-day photoperiod (16-h light : 8-h dark). Integuments of six larvae in the wandering stage were collected and sonicated in 6 mL of EcR40 buffer (40 m M KCl, 25 m M Hepes, pH 7.0, 10% glycerol, 1 m M EDTA, 1m M dithiothreitol, 10 m M Na 2 S 2 O 5 , 500 l M phenyl- methanesulfonyl fluoride, 1 l M leupeptin and 1 l M pepst- atin) according to a previously reported protocol [39–41]. The sonication was performed using an ultrasonic disruptor UR-200P (Tomy Seiko, Tokyo, Japan) at an output of 4 (80 W), with eight cycles of 5-s pulses under cold conditions. Hard cuticle, which remains in the homogenate, was removed by use of forceps. The protein concentration of this cell-free preparation was determined to be 1.0 mgÆmL )1 by the Bradford method [42]. The cell-free preparation of C. suppressalis integument (300 lL) was placed in a disposable glass tube (12 · 75 mm) containing 1 lL of a dimethylsulfoxide or ethanol solution of each compound and 45 lLofEcR40 buffer. After incubating the tubes for 5–10 min on ice, 4 lL Fig. 1. Structures of ecdysone agonists. (I) 20-Hydroxyecdysone, (II) dibenzoylhydrazine analogs, (III) chromafenozide. 4096 C. Minakuchi et al. (Eur. J. Biochem. 270) Ó FEBS 2003 of an ethanol solution of 3 H-labeled ponasterone A (1.0 · 10 5 d.p.m., final concentration 1.0 n M ) was added to each tube, and the mixture was incubated for 30 min at 25 °C. The reaction mixture was then filtered rapidly through a Whatman GF/F glass-fiber filter presoaked with 0.1% polyethylenimine, and the filter was washed three times with 2 mL of water. The radioactivity collected on the filter was measured in Aquasol-2 using a liquid scintillation counter (Aloka LSC-1000). Data analyses The dissociation constant, K D , was evaluated from the saturation curve for radioligand binding using nonlinear regression analysis in the GRAPHPAD PRISM program (GraphPad Software, San Diego, CA, USA). The K D value was also calculated from Scatchard plots. From the concentration–response curve for the binding of 3 H-labeled ponasterone A, the 50% inhibitory concentration (IC 50 ) was evaluated by probit transformation [43,44] for each compound. pIC 50 (the reciprocal logarithmic value of the IC 50 ) was used as an index of the binding activity. Results Gel mobility shift assay of in vitro -translated protein As shown in Fig. 2, none of EcR-547aa, EcR-542aa or USP alone bound to the Pal1 probe (lanes 1, 2 and 3). However, both EcR-547aa and EcR-542aa bound to the Pal1 probe in the presence of USP (lanes 4 and 5). Although the density of the bands was slightly different between EcR-547aa–USP (lane 4) and EcR-542aa–USP (lane 5), this may be caused by differences in protein concentration. Specific binding of ponasterone A to in vitro -translated protein Ponasterone A bound to EcR-547aa specifically, but did not bind to USP (Fig. 3). When EcR-547aa and USP were mixed, specific binding of ponasterone A to EcR-547aa increased dramatically. Similar results were observed using EcR-542aa instead of EcR-547aa [EcR-542 alone, 203 ± 6 d.p.m. (control) vs. 111 ± 15 d.p.m. (with pona- sterone A); EcR-542aa–USP, 754 ± 19 d.p.m. (control) vs. 105 ± 4 d.p.m. (with ponasterone A)]. The amount of nonspecific binding of ponasterone A to each protein (Fig. 3, lanes 2, 4 and 6) was equal to the radioactivity captured in the nitrocellulose membrane without proteins (data not shown). Specific binding to the co-expressed EcR- 547aa–USP was equivalent to that to the mixture of EcR- 547aa and USP expressed in the separate tubes (data not shown). Dissociation constant ( K D ) for the binding of ponasterone A to receptor proteins As stated above, 3 H-labeled ponasterone A bound specific- ally to EcR alone or to the EcR–USP complex. In order to examine the kinetics for the ligand–receptor binding, we calculated the dissociation constants (K D ) of ponasterone A against three different in vitro-translated proteins: EcR- 547aa; EcR-547aa–USP; and EcR-542aa–USP. As shown in Fig. 4C,E, ponasterone A bound to EcR-547aa–USP and EcR-542aa–USP with high affinity. The K D values of ponasterone A to these complexes were 1.2 n M and 1.0 n M , respectively. These values are comparable to those for in vitro-translated EcR–USP of D. melanogaster (0.9 n M ) [10] and Bombyx mori (1.1 n M ) [45]. On the other hand, the K D value for EcR-547aa protein alone was 55 n M (Fig. 4A). The K D value for EcR-542aa alone was not measured in this study. The K D value of ponasterone A to inherent receptor proteins in the cell-free preparation of C. suppressalis integument was determined to be 6.9 n M by nonlinear regression analysis (Fig. 5A). Scatchard plots for the binding of ponasterone A to expressed proteins (Fig. 4) and inherent receptor proteins (Fig. 5) were linear, indicating only one type of binding site. The K D values evaluated from Scatchard plots (Figs 4B,D,F and 5B) were similar to those from the nonlinear regression model (Figs 4A,C,E and 5A). Receptor-binding activity of ecdysone agonists pIC 50 values of ecdysone agonists (ponasterone A and tebufenozide) were very similar between EcR-547aa–USP and EcR-542aa–USP, and each value was highly reprodu- cible with only a small standard deviation (Table 1). In the Fig. 2. Binding of the ecdysone receptor–ultraspiracle (EcR–USP) complex to the ecdysone response element (EcRE). In vitro-translated EcR-547aa, EcR-542aa and USP were incubated with 32 P-labeled Pal1 EcRE, and then analyzed following electrophoresis on a nondena- turing polyacrylamide gel. Ó FEBS 2003 Receptor binding affinity of ecdysone agonists (Eur. J. Biochem. 270) 4097 following binding assay, EcR-547aa–USP was used, and the activity data were obtained from each single binding assay. Binding activities of a series of ecdysone agonists did not vary markedly between in vitro-translated EcR-547aa–USP and the cell-free preparation (Table 2). As shown in Table 2, some of the 3,5-dimethylbenzoyl analogs, such as tebufenozide, methoxyfenozide and chromafenozide, bound to the EcR–USP complex with very high affinity, being  200-fold higher than that of 20-hydroxyecdysone. By introducing a C 2 H 5 group at the para-position of the B-ring moiety, the binding activity against the EcR–USP complex was enhanced 30 times (no. 1 vs. no. 2; see Table 2 for a description of the compounds represented by the numbers), while a Cl atom was not as effective as a C 2 H 5 group (no. 1 vs. no. 3; Table 2). With respect to the A-ring moiety, introduction of a Cl atom at the ortho-position and methyl groups at both of the meta-positions enhanced the activity sixfold (no. 1 vs. no. 4; Table 2) and eightfold (no. 2 vs. no. 6; Table 2), respectively. Three commercially available insecticides, having a 3,5-dimethyl substitution pattern at the A-ring moiety (no. 6, no. 7, no. 9; Table 2), are very potent irrespective of the substitution pattern of the B-ring moiety. However, by replacing the C 2 H 5 group at the para- position of tebufenozide with an n-C 4 H 9 group, the activity decreased 10-fold (no. 6 vs. no. 8; Table 2). Among the ecdysteroids, the order of the binding activity was ponasterone A > 20-hydroxyecdysone ‡ cyasterone > makisterone A > ecdysone, against both the in vitro-translated EcR–USP complex and the cell-free preparation. Ponasterone A (no. 10; Table 2) showed a high binding activity against the in vitro-translated EcR– USP complex, but it was  10-fold less potent than chromafenozide and about sixfold less potent than tebufenozide and methoxyfenozide. The binding activities of 20-hydroxyecdysone (no. 11; Table 2) and ecdysone (no. 15; Table 2) against the in vitro-translated EcR–USP and inherent receptor proteins were 1/26 and 1/1000– 1/2000 lower than that of ponasterone A, being compar- able to their molting hormonal activities against C. sup- pressalis [25]. Discussion In a previous study, we cloned two cDNA variants from C. suppressalis [31]; these variants encoded EcR-547aa and EcR-542aa, with the 15-bp difference in the D region located between the C (DNA binding) and E (ligand binding) regions. The presence of two homologous EcR splicing variants with a 15-bp difference in the D region has also been reported in Manduca sexta [46], but the functional difference between these two variants has not yet been investigated. Perera et al. observed no ligand binding in the mutated EcR of the Lepidoptera Choristoneura fumiferana, in which the D region was completely deleted [47]. Recently, Grebe et al. suggested that the C-terminal part of the D region of D. melanogaster EcR contributes to the ligand binding and the dimerization with USP, even though the N-terminal part is not essential for ligand binding [48]. In this study, we showed that the lack of five amino acids (LECLQ) in the D region of EcR did not affect the ligand– receptor binding, the heterodimerization of EcR and USP, or the binding of the EcR–USP heterodimer towards EcRE (Figs 2 and 4 and Table 1). As previously reported, this sequence is not located in the C-terminus of the D region of CsEcR, but in the middle part [31]. Although the role of these five amino acids is still unknown, the amino acids in the middle part of the D region probably do not affect the ligand–receptor binding. Our results, demonstrating that ponasterone A specifi- cally bound to EcR but not to USP, and that the specific binding of ponasterone A to EcR was remarkably enhanced by adding USP, are consistent with those observed for D. melanogaster EcR and USP proteins [10,48]. In the case of Chironomus tentans, the specific binding of ponaster- one A was not observed for either EcR or USP, whereas specific binding was observed for the EcR–USP complex [38,49]. Recently, Grebe and co-workers showed that the binding of ponasterone A to D. melanogaster EcR is increased by 10-fold in the presence of D. melanogaster USP [38]. They also suggested that the binding ability of EcR to ligands in the absence of USP might be species specific [48]. In this study, we have shown that the binding of ponasterone A to C. suppressalis EcR is increased by eightfold in the presence of C. suppressalis USP (Fig. 3), which is consistent with the report by Grebe et al.on D. melanogaster. We have clearly shown that the binding affinity of ponasterone A to C. suppressalis EcR (K D ¼ 55 n M ) was also enhanced 50-fold by adding USP (K D ¼ 1.2 n M ). These results indicate that allosteric inter- action between EcR and USP would change the confor- mation of the ligand-binding pocket of EcR. The K D value of ponasterone A for the expressed C. suppressalis EcR– USP complex (K D ¼ 1.2 n M ) was not far from that for the inherent receptor proteins in the cell-free preparation of C. suppressalis integument (6.9 n M ). It is to be expected that Fig. 3. Binding of ponasterone A (PoA) to the in vit ro-translated ecdy- sone receptor (EcR)-547aa (lanes 1 and 2), ultraspiracle (USP) (lanes 3 and 4), and a mixture of EcR-547aa and USP (lanes 5 and 6). In vitro- translated EcR-547aa and/or USP were incubated with 3 H-labeled PoA (5 n M ), in the presence or absence of excess PoA, and filtered through a nitrocellulose membrane. The radioactivity collected in the filter was counted using a liquid scintillation counter. T, total binding; N, nonspecific binding. The vertical bars show the standard deviation of three replications. *P <0.01(Student’st-test). 4098 C. Minakuchi et al. (Eur. J. Biochem. 270) Ó FEBS 2003 these K D values vary slightly because the cell-free prepar- ation contains various proteins that can affect binding. The binding activity of ecdysone agonists to the inherent receptor proteins was highly correlated (r ¼ 0.98) with that measured against the in vitro-expressed EcR–USP complex (Fig. 6). We therefore conclude that the EcR–USP complex expressed in vitro in this study is a functional and useful material for using in binding assays of ecdysone agonists. In a previous study, we evaluated the molting hormonal activity of different ecdysteroids and nonsteroidal ecdysone agonists by measuring the induction of chitin synthesis in the cultured integument of C. suppressalis larvae; the 50% effective concentration (EC 50 ) for the induction of the chitin synthesis in the cultured integument of C. suppressalis larvae was determined from the concentration–response curve [25,27–29,50]. As shown in Fig. 7, the binding activity (pIC 50 ) is highly correlated with the molting hormonal activity (pEC 50 ), indicating that the strength of the hormo- nal activity of ecdysone agonists is primarily determined at the step of their binding to the EcR–USP complex. Fig. 4. Binding of ponasterone A (PoA) to the in vitro-translated ecdysone receptor (EcR)-547aa (A, B), EcR-547aa–ultraspiracle (USP) (C, D), and EcR-542aa–USP (E, F). In vitro-translated EcR and/or USP were incubated with different concentrations of 3 H-labeled PoA, in the presence or absence of excess PoA, and filtered through a nitrocellulose membrane. The radioactivity collected in the filter was counted using a liquid scintillation counter. Saturation radioligand-binding data (A, C, E) and Scatchard plots (B, D, F) are shown. Ó FEBS 2003 Receptor binding affinity of ecdysone agonists (Eur. J. Biochem. 270) 4099 Furthermore, we reported previously that the compounds possessing high molting hormonal activity are potent insecticides for a series of dibenzoylhydrazine analogs [28,29]. We therefore concluded that, regarding nonsteroidal ecdysone agonists, the binding activity to the EcR–USP complex results in potent larvicidal activity as well as potent molting hormonal activity. On the other hand, regarding ecdysteroids, the binding activity to EcR–USP is not correlated to larvicidal activity: topical application of 20-hydroxyecdysone at 52 nmol resulted in no mortality of C. suppressalis (Y. Nakagawa, Kyoto, Japan, unpub- lished data). The topically applied ecdysteroids could not easily permeate insect epidermis because of their low hydrophilicity, or ingested ecdysteroids would be easily metabolized and excreted from the insect body. It has been reported that tomato moth larvae are able to feed on a diet containing 400 p.p.m. 20-hydroxyecdysone without any adverse effects on growth and development, while ingestion of nonsteroidal ecdysone agonists, such as RH-5849 and tebufenozide, induces a premature and lethal molt, indica- ting that the ingested 20-hydroxyecdysone was metabolized and rapidly excreted [51]. Although it has been shown that nonsteroidal ecdysone agonists would also be metabolized and excreted to some extent [52,53], we assumed that the metabolism and excretion of nonsteroidal ecdysone agonists in C. suppressalis would be less significant because piperonyl butoxide, an inhibitor of oxidative metabolism, was used to measure the larvicidal activity [23,24]. In this study, the binding activity of ecdysone agonists was highly correlated with the molting hormonal activity measured in C. suppressalis integument (Fig. 7). We had expected that the physicochemical properties (such as hydrophobicity) of compounds might affect their uptake into the target cells. However, no physicochemical proper- ties were taken into consideration for the correlation of the binding activity to the molting hormonal activity of these compounds. In fact, the partition coefficient (P) between 1-octanol and water, an index of hydrophobicity, varied by 7 000 000-fold among the compounds tested, as listed in Table 2. We therefore concluded that hydrophobicity does not affect compound cellular uptake in the C. suppressalis integument. Fig. 5. Binding of ponasterone A (PoA) to the inherent receptor proteins in the cell-free preparation of Chilo suppressalis integument. The cell-free preparations were incubated with different concentrations of 3 H-labeled PoA, in the presence or absence of excess PoA, and filtered through a glass- fiber filter (GF/F). The radioactivity collected in the filter was measured using a liquid scintillation counter. (A) Saturation radioligand-binding data. (B) Scatchard plot. Table 1. Binding activity [reciprocal logarithmic value of the 50% inhibitory concentration (pIC 50 )] of ponasterone A (PoA) and tebufenozide against in vitr o-translated ecdysone receptor EcR-547aa–ultraspiracle (USP) or EcR-542aa–USP. The mean value ± SD of two duplicate experiments is shown. 4100 C. Minakuchi et al. (Eur. J. Biochem. 270) Ó FEBS 2003 It is of great interest how the EcR interacts with nonsteroidal ecdysone agonists, such as dibenzoylhydrazine analogs, whose structures are totally different from that of 20-hydroxyecdysone. Wurtz and co-workers constructed the ligand-binding domain of Chir. tentans EcR, in which ligand- binding domains of the retinoic acid and vitamin D receptors were used as templates, and predicted the binding modes of 20-hydroxyecdysone or unsubstituted RH-5849 to EcR [54]. Kumar and co-workers also modeled the ligand-binding domain of Chor. fumiferana EcR [55]. They performed point mutation analysis on the Chor. fumiferana EcR to identify some of the amino acid residues essential for ligand binding (ponasterone A and methoxyfenozide). Grebe and co-workers created mutants of the D. melanogaster EcR by site-directed mutagenesis and elucidated the function of amino acid residues involved in the ligand binding to EcR and the heterodimerization to USP [48]. Even though these homology-modeling and point-mutation studies help to clarify the interaction mode of ecdysone agonists to EcR– USP, the detailed mechanism remains unknown. In conclusion, we functionally expressed EcR and USP of C. suppresalis in vitro, and showed that the dissociation constant (K D ) of ponasterone A to EcR was enhanced  50-fold by the addition of USP. The K D value of ponasterone A to the EcR–USP complex was determined to be  1n M , which is consistent with such values reported for other insect EcR–USPs. The binding activity of ecdysone agonists to the inherent receptor proteins in the Fig. 6. Relationship between the binding activities of the in vitro-translated and inherent receptors. The binding activity [reciprocal logarithmic value of the 50% inhibitory con- centration (pIC 50 )] of ecdysone agonists against in vitro-translated ecdysone receptor (EcR)-547aa–ultraspiracle (USP) and the binding activity (pIC 50 ) against inherent receptor proteins from Chilo suppressalis integument. Table 2. Binding activities [reciprocal logarithmic value of the 50% inhibitory concentration (pIC 50 )] of ecdysone agonists against the in vitro -translated ecdysone receptor–ultraspiracle (EcR–USP) complex and inherent receptor proteins from Chilo suppressalis integument. a Mean ± SD. Values in parentheses indicate the number of replications. ND, not determined. b Single data. c Experimentally measured [23,24]. d Estimated empirically [23,24]. e From Table 1. f Calculated using the CLOGP method [56]. g From [32]. Ó FEBS 2003 Receptor binding affinity of ecdysone agonists (Eur. J. Biochem. 270) 4101 cell-free preparation of C. suppressalis integument was highly correlated with that of the in vitro-expressed EcR– USP complex. These results suggest that the EcR–USP complex expressed in vitro in this study is useful for binding assays of ecdysone agonists. The binding activity of a number of steroidal and nonsteroidal ecdysone agonists was linearly correlated to their molting hormonal activity with a high correlation coefficient. Thus, we conclude that the binding affinity of nonsteroidal ecdysone agonists to the EcR–USP complex primarily determines the strength of their biological activities. Acknowledgements We are thankful to Dr Craig Wheelock of the University of California Davis for carefully reviewing this manuscript. 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