Báo cáo khoa học: Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori pdf

Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori Mohamed Elmogy, Masafumi Iwami and Sho Sakurai Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakumamachi, Japan Nongenomic action of an insect steroid hormone, 20-hy- droxyecdysone (20E), has been implicated in several 20E- dependent events including the programmed cell death of Bombyx anterior silk glands (ASGs), but no information i s available for the mode of the action. We provide evidence for a putative membrane receptor located in t he plasma mem- brane of the ASGs. Membrane fractions prepared from the ASGs exhibit high binding activity to [ 3 H]ponasterone A (PonA). The m embrane fractions did not contain c onven- tional ecdysone receptor as revealed b y Western blot analysis using antibody raised against Bombyx ecdysone receptor A (EcR-A). The binding activity was not solubilized with 1 M NaCl or 0.05% (w/v) MEGA-8, indicating that the binding sites were l ocalized in the m embrane. Differential s olubili- zation and temperature-induced phase separation in Triton X-114 showed that the binding sites might be integrated membrane proteins. These results indicated that the binding sites are located in plasma membrane proteins, which we putatively referred t o as membrane ecdysone recep- tor (mEcR). The mEcR e xhibited saturable binding for [ 3 H]PonA (K d ¼ 17.3 n M , B max ¼ 0.82 pmolÆmg )1 pro- tein). Association and dissociation kinetics revealed that [ 3 H]PonA associated with and dissociated from mEcR within minutes. The combined results support the existence of a p lasmalemmal ecdysteroid receptor, w hich may act in concert with the conventional EcR i n various 20E-depend- ent d evelopmental events. Keywords: ecdysone ago nist; ecdysone receptor; kinetics; nongenomic; ponasterone A. Steroids elicit various physiological responses, particularly those involving the genomic aspects of action, in which they modulate gene transcription by interacting with intracellular nuclear receptors that serve as ligand- dependent transcription factors [1]. In addition to the genomic steroid actions, increasing evidence of rapid, nongenomic steroid effects has been demonstrated for virtually all groups of steroids [2]. Ecdysone, an insect steroid hormone synthesized by prothoracic glands, is essential for inducing the molecular and cellular events t hat lead to molting and metamorphosis in insects and crusta ceans [ 3–5]. 20- Hydroxyecdysone (20E), the biologically active form of ecdysone, binds to a functional nuclear ecdysone receptor consisting of an ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle (USP), and thereby controls the transcriptional activity of target genes [ 6]. I n addition, a nongenomic action of 20E has been supposed for decades. 20E increases the cellular c AMP level in the prothoracic glands of Manduca sexta [7] and in the f at body of Mamestra brassicae [8]. 20E rapidly reduces the excitatory potentials a t n euromuscular junctions in amplitude w ithin minutes in both the crayfish [9] and Drosophila [10]. These responses to 20E fail to fit the classical genomic model, and a ppear instead to r ely on mechanisms involving membrane receptors and second messenge rs. Nevertheless, the pre sence of mem brane recep- tors remains speculative. 20E is the primary factor i nducing programmed cell death (PCD) of l arval tissues at pupal metamorphosis [11–13]. The anterior silk gland (ASG) is a larval-specific tissue, which is destined to die shortly after pupation, and enters t he process of PCD in response to the high hemolymph ecdysteroid concentration that induces pupal metamorphosis [14]. In t he PCD of ASGs induced by 20E in vitro, the gene expression required for completion of PCD is accomplished during t he first 8 h of 20E challenge, but withdrawal of 20E before 30 h of the culture interferes with t he PCD sequence [14]. If the genomic theory of steroid action is applicable to 20E- induced PCD, 20E challenge for 8 h should be sufficient for execution of PCD. This implies that t he effects o f 20E during the period between 8 a nd 30 h are no t accompanied by gene expression but rather are mediated by a non- genomic pathway, probably through a membrane-bound receptor. If this is the case, ASG plasma membranes may contain high-affinity binding sites for ecdysteroid. The present study reports for the first time the presence of such sites in the membranes of insect cells and the biochemical Correspondence to S. Sakurai, D ivision of B iological Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakumamachi, Kanazawa 920-1192, Japan. Fax: +76 2646250, Tel.: + 76 2646255, E-mail: ssakurai@kenroku.kanazawa-u.ac.jp Abbreviations: ASG, anterior silk gland; 20E, 20-hydroxyecdysone; EcR, ecdysone receptor; mEcR, membrane ecdysone receptor; PCD, programmed cell death; PonA, ponasterone A (25-deoxy- 20-hydroxyecdysone); USP, ultraspiracle; ECL, enhanced chemoluminescence detection. (Received 1 9 April 2004, revised 3 June 2004, accepted 8 June 2004) Eur. J. Biochem. 271, 3171–3179 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04249.x characterization of the putative membrane ecdysteroid receptor. Materials and methods Animals and ASGs Silkworm, Bombyx mori (Kinshu · Showa F1 hybrid), were reared on an artificial diet (Silkmate, Nihon-Nosan- Kogyo, Yokohama, Japan) at 25 °C under 12-h light/12-h dark cycle [15]. ASGs were dissected on the day of gut purge [14] and cultured separately in 0.3 mL Grace’s insect culture m edium ( Gibco B RL) at 2 5 °Cfor18h with 20E followed b y a culture i n a hormone-free medium for a further 12 h [14]. Because preliminary experiments showed that the binding activity in the membrane fractions prepared from the cultured A SGs was higher than that from the freshly dissected ASGs, we mainly used such ASGs unless mentioned otherwise. Chemicals Ponasterone A (PonA, 25-deoxy-20-hydroxyecdysone) and 20E were from Sigma and nonsteroid ecdysone agonists [methoxyfenozide (RH-2485), tebufenozide (RH-5992), RH-5849] were gifts f rom Y. Nakagawa, Kyoto University, Japan. Ecdysteroids and the agonists were d issolved in ethanol and stored at )20 °C until use. [ 3 H]PonA (200 Ci Æmmol )1 )and[ 14 C]methoxyinulin (2.4 mCiÆmmol )1 ) were from PerkinElmer Life Sciences. Triton X-114 and butylatedhydroxytoluene were from Sigma. To remove any Triton X-114-insoluble materials, T riton X-114 (10 mL) was added with 8 mg of butylatedhydroxytoluene and 190 mL 20 m M potassium phosphate buffer pH 7.5 con- taining 0.15 M KCl [16]. The mixture was cooled to near 0 °C, centrifuged at 3000 g for 10 min to remove any insoluble material, and then the condensed detergent was incubated for 20 h at 35 °C. The purified detergent ( lower phase) was stored at room temperature. Preparation of membrane fraction Freshly dissected or cultured ASGs were w ashed three times with insect Ringe r’s solution (130 m M NaCl, 4.7 m M KCl, 1.9 m M CaCl 2 ). All subsequent procedures were performed at 4 °C. ASGs were homogenized in seven v ols binding assay buffer (20 m M Tris/HCl pH 7.0, 2 m M EDTA, 1 m M phenylmethylsulphonyl fluoride, 3 lgÆmL )1 pepstatin A , 3 lgÆmL )1 leupeptin) using a motor-driven, loose-fitting glass-plastic homogenizer at 1000 r.p.m. for 1 m in. After centrifugation a t 1000 g for 10 min, t he pellet w as suspen- ded in the buffer and centrifuged at 1500 g for 10 min. The pellet was again s uspended in t he buffer a nd centrifuged at 1800 g for 1 0 min. The resulting p ellet was resuspended i n the buffer, homogenized again using HG30 homogenizer (Hitachi) on ice, and centrifuged at 1000 g for 10 min. T he supernatant was centrifuged at 8000 g for 10 min, and the resulting supernatant was centrifuged at 105 000 g for 5 h. The pellet w as suspended i n the buffer, frozen with liquid nitrogen, a nd stor ed at )80 °C until use. Protein amounts were measured using a D C protein assay kit (Bio-Rad) with BSA as standard. Preparation of nuclear extracts Nuclear extract was m ainly pre pared according t o Wu [17] with a minor modification. Briefly, dissected ASGs were washed once i n 100 m M phosphate buffer pH 7.9 with 100 m M NaCl and homogenized on ice with two vols 10 m M Hepes pH 7.9 with 10 m M KCl, 0.3 M sucrose, 1.5 m M MgCl 2 ,0.1m M EDTA, 0.5 m M 2-mercaptoethanol and protease inhibitors cocktail (Complete; Roche Diagno- sis), by 12 strokes in a Dounce tissue grinder (1 mL; Wheaton, Millville, NJ, USA). The suspension was centri- fuged for 8 m in at 1600 g at 4 °C. The resulting pellet was resuspended in two vols 10 m M Hepes pH 7.9 containing 0.4 M NaCl, 5% ( v/v) glycerol, 1.5 m M MgCl 2 ,0.1m M EDTA, 0.5 m M dithiothreitol and 0.5 m M phenyl- methanesulfonyl fluoride, and 5 M NaClwasaddedtoyield a final concentration of 0.4 M NaCl. The suspension was incubated at 4 °C under gentle shaking for 30 min and subsequently centrifuged at 2 °C a t 105 000 g for 6 0 min. The r esulting supernatant was dialysed for 4 h using a dialysis tube with 14 000 Da cut-off size (Wako Pure Chemical Industries) against 1000 vols 20 m M Hepes pH 7.9 containing 20 m M NaCl, 20% (v/v) glycerol, 1 m M EDTA and 0.5 m M 2-mercaptoethanol and the protease inhibitor cocktail. The buffer was changed once after 2 h dialysis time. The sample was clarified by 10 min centrifugation at 10 000 g at 4 °C, and t he supernatant was supplemented with the protease inhibitors. SDS/PAGE and Western blot analysis SDS/PAGE was performed according to Laemmli [18] using 12% polyacrylamide gel. The samples in reducing loading buffe r were h eated in a boiling water bath for 10 min. The gel was stained with Coomassie brilliant blue. For Western blot analysis, the blotting membrane was agitated in Tris-buffered saline ( NaCl/Tris: 25 m M Tris/ HCl, pH 7.4, 3 m M KCl, 136 m M NaCl) containing 5 % (w/v) nonfat milk for 2 h and then incubated with a rabbit antibody raised against the EcR-A-specific region of EcR (a gift from H. Fujiwara, University of Tokyo) at 4 °C overnight. After washing with NaCl/Tris, the membrane was incubated with horseradish peroxidase-conjugated protein A in the f resh NaCl/Tris containing 5% (w/v) nonfat milk at 4 °C for 2 h. Visualization of the immuno- blot was carried out using the enhanced chemoluminescence detection (ECL) system according to the manufacturer’s instructions and exposed to Hyperfilm ECL (Amersham Pharmacia Biotech). Binding assay Specific binding of PonA was assayed by an ultracentri- fugation method [19] adapted to the measurement of ecdysteroid membrane receptors. Membrane fractions (100 lL reaction mixture containing 100 lgprotein)were incubated with [ 3 H]PonA in 0.5 lL of the buffer solution containing 0.05 lCiÆlL )1 [ 14 C]methoxyinulin. [ 14 C]Meth- oxyinulin was added to estimate the degree of contaminated [ 3 H]PonA in the precipitate after centrifugation, that originated from th e incubation medium. The mixture w as incubated at 2 5 °C for 10 min unless mentioned o therwise. 3172 M. Elmogy et al. (Eur. J. Biochem. 271) Ó FEBS 2004 After incubation, the mixture was c entrifuged at 100 000 g for 1 5 min at 2 °C. The supernatant was discarded and the insides of the tubes were rinsed w ith 100 lL of the buffer. Radioactivity in the pellet w as measured using a Beckman LS-700 counter with a dual-label program (Beckman- Coulter). Saturation analysis was performed to determine receptor number per mg protein and binding affinity to PonA. Membrane fractions were equilibrate d with increas- ing concentrations of [ 3 H]PonA in the absence or presence of a 1000-fold excess of unlabelled PonA. For association kinetics, membrane fractions were incubated with 25 n M [ 3 H]PonA (± excess unlabelled PonA) for periods of time ranging from 1 to 40 min. Dissociation kinetics were determined by equilibrating membrane fractions with 25 n M [ 3 H]PonA for 10 min followed by the addition of 25 l M unlabelled PonA. In the competition assay, mem- brane fractions were incubated with 25 n M [ 3 H]PonA in the presence of increasing concentrations of unlabelled ecdy- steroids and ecdysone agonists. A modified dextran-coated active charcoal method [19] was used for phase partitioning samples in Triton X-114. Topological localization of the binding sites To exam ine whether PonA binding sites are located in peripheral proteins that are not integrated to the lipid bilayers or integral membrane proteins, topological local- ization study of the binding sites in the membranes was performed a ccording to Kerkhoff et al. [20,21]. The m em- brane suspensions (5 mgÆmL )1 protein) were treated with a solution of high ionic strength (binding assay buffer containing 1 M NaCl) for 60 min at 4 °C. The mixtures were centrifuged at 105 000 g for 60 min at 4 °C to obtain supernatant (S1) and pellet (P1). The p ellet P 1 was resuspended in the binding assay buffer to a final protein concentration of 20 mgÆmL )1 , and an aliquot was stored at )80 °C for the binding assay. The remaining suspensions weredilutedto5mgÆmL )1 protein, treated with 0.05% (v/v) octanoyl-N-methylglycamide (MEGA-8; Wako Pure Chemical Industries) at 4 °Cfor60minwithconstant stirring, and then centrifuged at 105 000 g for 60 min at 4 °C. The resulting pellet (P2) was re-suspended in the binding assay buffer to a final protein concentration of 20 mgÆmL )1 and stored at )80 °C. The supernatants, S1 and S2, were dialysed against the binding assay buffer overnight at 4 °C. Differential solubilization and temperature-induced phase separation in Triton X-114: three phase system Integral membrane proteins are classified into two categor- ies, i.e., proteins that covalently attached to the lipid bilayers and those that a re anchored in the bilayers [16]. Phase partitioning in Triton X-114 is a quick me thod to determine which category t he mEcR belongs to. The procedure used is a modification of t he method of Pryde & Philips [22] a nd Hooper & Bashir [23]. Triton X-114 was precondensed before use [24]. Purified Triton X-114 solutions with different c oncentrations (0.5–3%) were added t o t he ASG membrane fractions (final concentration, 10 mgÆmL )1 )in 10 m M phosphate buffer pH 7.4 containing 150 m M KCl, vigorously mixed immediately for 1–2 s, and placed on ice for 1 h followed by centrifugation at 100 000 g for 1 h at 0 °C. The detergent insoluble pellet was washed with the buffer and resuspended in the buffer prior to assaying the binding activity. The supernatant was overlaid on a cushion of buffere d 6% sucrose, incubated at 30 °C for 10 min and centrifuged at 3000 g for 5 min in a swing-rotor. The lower phase was a detergent phase that was directly subjected to the b inding ass ay. The upper aqueous phase was t ransferred to a tube, and fresh Triton X-114 was added to a final concentration of 0.5% (v/v). After mixing and incubating on ice for 1 h, the mixture was overlaid on the s ame sucrose cushion, kept at 30 °C f or 10 min, and centrifuged at 3000 g for 5 min in a swing-rotor. The resulting upper aqueous phase was transferred to a t ube to which fresh Triton X-114 with the same starting concentration (0.5–3%) was added. The sample was mixed, kept on ice a nd then at 30 °Cfor 10 min. After centrifugation at 3000 g for 5 min, the supernatant was used as a final aqueous phase. The three phases (detergent-insoluble pellet, detergent phase and aqueous phase) were assayed for the b inding activities, and the activities were expressed as a percentage of the total activity in all three phases. Data analysis Experimental data were analysed using ORIGIN software (OriginLab, Northampton, MA, USA). Saturation bind ing curves were fitted and analysed using equations built into GRAPHPAD PRISM TM 3.02 (GraphPad Software, San Diego, CA, USA) according to Swillens [25]. Results Biochemical characterization of [3H]PonA binding We first performed biochemical characterization of the binding sites u sing ASG membrane f ractions. The optimal protein concentration for the b inding assay was determined using 25 n M [ 3 H]PonA and increasing amounts of proteins in individual incubations. The percentage of specific b inding increased in a protein concentration-dependent manner within the range of 25–150 lgÆmL )1 (Fig. 1A). Because the specific binding at 100 lgÆmL )1 protein was appr oximately 60% of the maximum value at 200 lgÆmL )1 , the protein concentration of 100 lgÆmL )1 was used in the following binding assays. The optimum pH was 7.0 at 25 °C (Fig. 1 B). The binding was temperature dependent, with optimum binding at 37 °C and no binding at 60 ° C (Fig.1C).BecauseBombyx larvae were reared at 25 °Cin our laboratory, we selected the incubation temperature o f 25 °C, although the specific binding at 25 °C was approxi- mately half of that at 37 °C. Based on those results, we u sed the a ssay c onditions in which 100 lL of binding assay buffer (pH 7.0) containing 100 lgofmembraneproteins was incubated at 25 °Cwith25 n M [ 3 H]PonA, except for the saturation analysis. Western blot analysis To confirm that the specific binding in the membrane fraction was not brought about by contamination of conventional nuclear EcR, membrane fractions that showed Ó FEBS 2004 Membrane ecdysone receptor (Eur. J. Biochem. 271) 3173 specific binding activity to [ 3 H]PonA were subjected to Western blot analysis using an antibody raised against EcR-A (Fig. 2). Although insect tissues contain two EcR isoforms, EcR-A and EcR-B1, EcR-A isoform is predominantly expressed in the ASGs at pupation in Bombyx [26], and ther efore we used anti-EcR-A s erum for the Western blotting. As samples con taining the EcRs, total lysate and nuclear extract prepared from freshly dissected ASGs were used. I n the total lysate and nuclear extract, a single immunoreactive s ignal band a t 57 kDa, an approxi- mate molecular mass of Bombyx EcRs, was found (Fig. 2 B). By c ontrast, no i mmunoreactive signals were found in the membrane fractions of either the freshly dissected ASGs or the ASGs cultured with 20E for 30 h. These results indicated that the specific binding activity in the membrane fractions was not caused by contamination of nuclear receptors. Association and dissociation kinetics The a ssociation kinetics of the membrane fractions showed that PonA became associated with the membranes very rapidly as the steady state w as attained within 10 min (Fig. 3 A). The observed association constant ( K obs )was 0.9 ± 0.2Æmin )1 . The dissociation of PonA from its b inding sites was measured by adding an excess amount of unlabelled PonA after equilibration with 25 n M [ 3 H]PonA Fig. 1. Binding of [ 3 H]PonA to memb rane fractions of ASGs. (A) Protein amount-dependence of specific [ 3 H]PonA binding. The mem- brane fractions (100 lL) with different prote in c oncentrat ions we re incubated for 10 min at 25 °Cwith25n M [ 3 H]PonA without or with a 1000-fold molar excess of i nert PonA. Each data point is m ean ± SD (n ¼ 3).(B)OptimalpHfor[ 3 H]PonA binding. Membrane fract ions containing 100 lg protein in 100 lL buffer w ere incubated with 25 n M [ 3 H]PonA at various pH. Other conditions were the same as for (A). (C) Temperatu re d ependen ce of [ 3 H]PonA binding. Mem brane frac- tions c ontaining 100 lgproteinin100lL buff er (pH 7) were incu- batedwith25n M [ 3 H]PonA at vario us temperatures. j, t otal binding; d, specific binding; s, nonspecific binding. Fig. 2. Membrane fractions are free o f conventional nuc lear EcR. Commassie brilliant blue-stained S DS/PAGE ( 12% acrylamide gel) (A) an d Western blotting for the identical gel using anti-EcR-A s erum (1 : 100) as a primary antibody and horseradish peroxidase-conju- gated protein A (1 : 1000) as a secondary antibody (B). Lanes 1, total lysate; lane 2, membrane f raction; lane 3, nuclear extract. S am ples for lane 1–3 were prepared from freshly dissected ASGs. Lane 4, mem- brane fraction prepared from the ASGs that were cu ltured in the sa me conditions as th ose used for binding e xperiments. Twenty m icrograms ofproteinwereusedineachlane. 3174 M. Elmogy et al. (Eur. J. Biochem. 271) Ó FEBS 2004 for 10 min (Fig. 3 B). The dissociation of PonA from the membranes occurred within 10 s with a dissociation constant (K off )of2.3±0.5min )1 . The calculated associ- ation rate constant (K on )was13.3· 10 7 M )1 Æmin )1 ,andthe estimated dissociation c onstant at equilibrium (K d )was 17.5 n M . Saturation analysis A saturation analysis of specific binding at 25 °Cwas performed by incubating the membrane fractions with increasing concentrations of [ 3 H]PonA, then subjecting the binding data to Scatchard analysis (Fig. 4). The analysis showed the presence of a single high-affinity binding site in each molecule, with an apparent K d and B max of 17.3 n M and 0.82 pmolÆmg )1 protein, respectively. This K d value was i n g ood accordance with the estimated K d of 17.5 n M derived from the kinetic constants. Topological localization of the binding sites We examined whether the PonA binding sites are located in integral membrane proteins or peripheral proteins that are not integrated to the lipid bilayers. The membranes were treated with N aCl to e xamine if t he binding sites were located on p roteins that simply associate with the lipid bilayers or other scaffold proteins (Fig. 5). After treatment with 1 M NaCl, the binding activity was found only in the P1 fraction, indicating that the proteins responsible for the binding are not peripheral membrane proteins. Then, the P1 fraction was treated with a detergent, MEGA-8, as prelim- inary e xperiments with eight detergents had shown t hat only MEGA-8 at low concentration did not solubilize integral membrane proteins but merely fragmented membranes. After t reatment with 0.05 % MEGA-8, the activity was recovered from P 2 fraction a nd little a ctivity was found in the s upernatant. Thus, the binding sites might be on integral membrane proteins. Phase partitioning in the detergent Triton X-114 Integral membrane proteins are generally classified into two categories, proteins that are anchored in the lipid bilaye rs through a transmembrane sequence(s) and those that are covalently attached to the bilayers [16]. To examine the mode of association of the binding sites with the plasma membranes, the membrane fractions were subjected to Fig. 3. Kinetics of association (A) and dissociation (B) of [ 3 H]PonA binding to ASG memb ranes . Association kinetics: membranes were incubated with 25 n M [ 3 H]PonA for various times without or with a 1000-fold excess o f inert P onA . K on ¼ 13.3 · 10 7 M )1 Æmin )1 .Disso- ciation kinetics: membranes w ere incubated with 25 n M [ 3 H]PonA for 10 m i n a t 25 °C a nd th en added with a 10 00 -fold excess of unlabelled PonA to initiate dissociation of [ 3 H]PonA. Inset represents linear regression analysis of the data. K off ¼ 2.3±0.5min )1 .Eachdata point is the mean ± SD (n ¼ 3). Fig. 4. PonA saturation analysis of ASG membranes. Membrane preparations (100 lg p rotein in 100 lL b uffe r) were i ncubated with increasing concentrations of [ 3 H]PonA at 25 °C for 10 min without or with a 1000-fold e xcess of unlabelled PonA. T he data were fitted by nonlinear regression analysis. I nset is Scatchard analyses o f t he bin ding data. K d ¼ 17.3 n M ; B max ¼ 0.82 pmolÆmg )1 protein. Each data point is the mean ± SD (n ¼ 3). Ó FEBS 2004 Membrane ecdysone receptor (Eur. J. Biochem. 271) 3175 differential solubilization and temperature-induced phase separation in Triton X-114 (Fig. 6 ). In the absence of detergent the binding activity was found only in the pellet after the first centrifugation step. On increasing the concentration of Triton X-114, the binding activity was recovered predominantly in the detergent-rich phase. The binding in the detergent-insoluble pellet decreased in a complementary manner, and only a low activity was found in the aqueous phase. As the concentration of Triton X-114 was increased from 0.5 to 3%, results were mostly t he same as that at 0.5%. These results indicate that the binding sites are neither on a polypeptide(s) that merely a ssociates to the membrane bilayers nor are they covalently associated with the membrane proteins; rather they are on an integral membrane protein(s) that may be anchored in the mem- brane by a transmembrane sequence. Displacement studies Binding affinities of ecdysteroids and nonsteroidal ecdysone agonists to the membrane bindin g sites were determined by incubating the membrane fractions with 25 n M [ 3 H]PonA in the p resence of increasing amounts of unlabelled ecdyster- oids and agonists (Fig. 7). The estimated 50% inhibitory concentration (IC 50 )for50%displacementof[ 3 H]PonA was 6.92 · 10 )7 M for PonA and 2.63 · 10 )7 M for 20E, showing that the affinity for 20E was approximately 2.6 times higher than that for PonA. The IC 50 values for three nonsteroid ago nists, RH-5849, tebufenozide (RH-5992) and methoxyfenozide (RH-2485) were much lower than those for PonA and 20E. Comparison of individual values of pIC 50 , reciprocal logarithm of the concentration that provides a 50% inhibition of [ 3 H]PonA binding, as well as relative activities to 20-hydroxyecdysone (Table 1) showed that the binding activity was in the order 20E > PonA >> methoxyfenozide > tebufenozide > RH-5849. Fig. 6. Effects of Triton X-114 concentration on the solubilization and phase separation of ASG membranes. ASG m embrane fractions were subjected to differential s olubilization and temp erature-induced phase separation at the indicated concentrations of Triton X-114. The resulting three phases, detergent-insoluble pellet (j), detergent-rich phase (d) a nd aqueous ph ase (s), were assayed for binding activity. Each data point is a m ean of duplicate determinations. Fig. 7. Inhibitory activities of e cdysteroids and e cdysone ag onists against the [ 3 H]PonA binding. Membrane fractions (100 lg protein in 100 lL buffer) were incubated f or 10 min a t 2 5 °C with increasing concentra- tions of unlabelled PonA (d), 20E ( s), methoxyfenozide ( RH -2485; m), tebufenozide (RH-5992; n) and RH-5849 (h) in the presence of 25 n M [ 3 H]PonA. Each data point is the m ean ± SD (n ¼ 3). Fig. 5. Topological localization of the binding sites in the ASG mem- branes. Themembranefractions(5mgÆmL )1 ) w ere treated with the binding assay buffer containing 1 M NaCl for 60 min at 4 °Cand centrifuged at 105 000 g for 60 min at 4 °C.Thepellet(P1)was resuspended in a ssay b uffer cont aining 0.05% MEGA-8. After i ncu- bation for 60 m in at 4 °C, the mixture was c entrifuged at 105 000 g for 60 min at 4 °C, and the pellet (P2) was resuspended in the assay buffer. The dialyzed supernatants ( S1, S2) an d the resuspended pellets (P1, P2) were incubated with 25 n M [ 3 H]PonA under standard assay condi- tions. Binding activity is r elative to that in t he crude e xtract (C) w ith that designated as 100. 3176 M. Elmogy et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Discussion The present study describes for the fi rst time evidence for t he presence of a putative receptor for ecdysteroid in tissue membranes and its biochemical characterization in an insect. The membrane receptor exhibits a specific and saturable binding for [ 3 H]PonA with a K d of 17.3 · 10 )9 M . This value is physiologically relevant to the prevailing hemolymph concentrations of 20E (ranging between 10 )7 and 10 )6 M ) in the prepupal period when PCD is triggered in vivo [14]. The association and dissociation kinetics indicated that PonA association with and dissociation from its binding sites were rapid, which is characteristic of the binding of several natural compounds to their membrane receptors [27,28]. The saturation curve indicates the pres- ence of a single high-affinity binding site and an apparent maximal number of binding sites of 0.82 p molÆmg )1 protein. The obtained K d value is supported by the good accordance with the estimated dissociation rate constant at equilibrium (K d ¼ 17 .5 · 10 )9 M ). Rapid effects o f steroids a re triggered by the intracellular signalling cascade, in which membrane -binding sites for some steroids have been linked to the conventional nuclear receptors. In the nongenomic action of estrogen, estrogen receptor a couples with the regulatory subunit of the lipid kinase PI3K to trigger the rapid e ffects of estradiol [29]. The nongenomic action of progesterone is also mediated by the conventional progesterone receptor that interacts with Src to trigger the mitogen-activated protein kinase cascade [30]. The binding affinities o f those steroid membrane receptors are orders of magnitude lower than those of nuclear receptors [31]. In ecdysone receptors, the K d value of the in vitro translated EcR/USP heterodimer for PonA is 0.9 n M in Drosophila [32] and 1.1 n M in Bombyx [33]. Thus, K d for EcR is in the nanomolar range. By contrast, the K d of PonA for mEcR is s ignificantly higher (lower affinity) t han those values. This result is in accordance with the fact that the binding affinity of mammalian steroids to conventional nuclear receptor is higher t han that to the same receptor that mediates nongenomic action. However, Western blot a na- lysis using antibody raised against EcR-A indicates t hat the binding activity in the ASG membrane fraction is not due to EcR. Accordingly, the put ative mEcR appears to differ from the conventional EcR. A second line of evidence to support the existence of a membrane receptor is that the binding affinity of PonA is less than that o f 20E. Th e b inding affinity of PonA to the nuclear receptor complex of EcR/USP i s one to two orders of magnitude higher than that of 20E [34]. In the inherent receptor c omplex of the rice stem borer Chilo suppressalis, binding affinity for PonA is 26-fold h igher t han t hat f or 20E [35], and nuclear extracts of Dr osophila Kc-H cells exhibit high binding affinity for PonA with a K d of 3.4 n M , w hile K d for 20E is 24 0 n M , 70 times lower than that for PonA [36]. Similarly, the affinity of PonA to tick EcR is 28-fold higher than that of 20E [37]. By contrast, the competition assay using the ASG membrane fractions shows that the binding affinity for PonA is one-fourth of that for 20E and that the values for nonsteroidal ecdysone agonists are much lower than 20E, which totally differs from the binding character- istics of the conven tional EcR (Table 1). Finally, the topological studies indicated the presence of mEcR. The effects of solutions of high ionic strength and detergents have been used to establish the topological localization of several microsomal enzymes involved in phospholipid and triglyceride metabolism [20]. Using a similar approach, the present study revealed a distinct binding activity in the membrane. The differential solubi- lization and temperature-induced phase separation in Tri- ton X-114 (three phase system) gave additional evidence for the presence of mEcR. The particular advantage of Triton X-114 is that i ts micelles aggregate on w arming from 0 °C, eventually separating ou t into a second phase when temperature is raised above 2 0 °C ( the s o called c loud point). Therefore, integral membrane proteins solubilized at 0–4 °C tend to be partitioned preferentially into the detergent-rich phase at the cloud point [16]. When t he porcine kidney microvillar membranes are subjected to the three phase system, t he ectoenzymes with a covalently attached glycosyl-phosphatidyinositol membrane anchor are recovered i n t he detergent insoluble pellet, while those anchored by transmembrane spanning polypeptide are recovered in the detergent-rich phase [23]. Similarly, the majority of the integral membrane proteins in adrenal chromaffin granules migrate into a detergent-rich phase, and an aqueous phase contains the insoluble, hydrophilic proteins [22]. We found most of the binding activities for [ 3 H]PonA in the detergent-rich phase, indicating that the mEcR is an integral membrane protein which might be anchored in the membrane by transmembrane sequence of hydrophobic amino acids. Several mammalian steroid hormones have been demon- strated to exert rapid effects on cells by interacting with specific re ceptors present on the cell s urface [2,29]. Effects of 20E that may have physiological relevance to membrane receptors have been described i n insect tissues. I n wing epidermis of Hyalopora gloveri pupae, 20E stimulates adenylyl cyclase activity within 15 min of exposure to the hormone in vitro [7]. Similarly, cAMP levels in the ASGs increases significantly within 1 min after a 20E challenge (unpublished data). The rapid increase in the cAMP level indicates a nongenomic action of 20E, and a membrane receptor may mediate the increase in the cAMP level. Recently, a membrane progestin recep tor has been des- cribed as a s even-transmembrane receptor coupling to a Gi protein [28]. The putative mEcR could mediate the rapid Table 1. Binding activities of ecdysteroids and nonsteroidal ecdysone agonists against t he membrane binding site s and comparison with the conventional nuclear receptor complex. pCI 50 ( M ) reciprocal logarith- mic value of the 50% inhibition. RA activities relative to that of 20-hydrox yecd yso ne. Compounds mEcR nEcR a pCI 50 ( M ) RA pCI 50 ( M )RA 20-Hydroxyecdysone 6.58 1 6.70 1 Ponasterone A 6.16 0.38 8.12 26.4 Methoxyfenozide (RH-2485) 5.01 0.027 9.05 224 Tubefenozide (RH-5992) 4.86 0.019 9.07 234 RH-5849 4.56 0.0096 6.88 1.51 a Binding activities against inherent receptor complex of nuclear EcR (nEcR) and USP from Chilo suppressalis integuments [34]. Ó FEBS 2004 Membrane ecdysone receptor (Eur. J. Biochem. 271) 3177 increase in cAMP levels in ASG cells, although further studies are necessary to determine whether the me mbrane receptor identified in the present study is involved in the activation of adenylyl cyclase and in distinct physiological responses to 20E in the Bombyx ASG. In conclusion, our study indicates, at a biochemical level and for the first time, t hat ecdysteroids m ay act t hrough a membrane receptor in addition to the conventional nuclear receptor. By furnishing new i nsights into t he functional properties o f t wo classes of insect ecdysone receptors, these findings are expected to pave the way for the understanding of ecdysone action on insect development. Acknowledgements We express our sincere gratitude to Drs Michiyasu Yoshikuni and Yoshitaka Nagahama of National Institute for Basic Biology f or their valuable comments for establishing the binding assay. We are also thankful to Dr Haruhiko Fujiwara of the University of Tokyo for the gift of anti-EcR-A serum and Dr Yoshiaki Nakagawa of Kyoto University for the gift of nonsteroidal e cdysone agonists. This work was supported by a JSPS Research Grant (No. 1 4360033 ) to S.S. References 1. Beato, M. & Klug, J. (2000) Steroid hormone receptors: An update. Hum Reprod. Update 6, 236–255. 2. Lo ¨ sel, R. & W ehling, M. (2003) Nongenomic actions of steroid hormones. Natur e Rev. 4, 46–56. 3. Gilbert, L.I., Rybczynski, R. & T obe, S. (1996) Endocrine c ascade in insect metamorphosis. In Metamorphosis: Post-Embryonic Reprogramming of Gene E xpression in Amphibian and Insect C ells. (Gilbert, L.I., Tata, J. & Atkison, P., eds), pp. 59–107. Acad emic Press, San Diego, CA. 4. Henrich, V.C., Rybczynski, R. & Gilbert, L.I. (1999) Peptide hormones, steroid hormones, and p uffs: mechanisms and models in insect development. Vitam. Horm. 55, 73–125. 5. Chen, C., Gu, S. & Chow, Y. (2001) A denylate cyclase in pro- thoracic glands during the last larval instar of silkworm, Bombyx mori. Insect Biochem. Mol. Biol. 31, 659–664. 6. Riddiford, L.M., Cherbas, P. & Tr uman, J.W. (2000) Ecdysone receptors and their biological actions. Vitam. Horm. 60, 1–73. 7. Applebaum, S.W. & Gilbert, L.I. (1972) Stimulation of adenyl cyclase in pupal wing epidermis by b-ecdyso ne. Dev. Biol. 27, 165– 175. 8. Sass, M., Csikos, G., K omuves, L. & Kovacs, J. (1983) Cyclic AMP in t he fat body of Mamestra brassicae du ring t he l ast instar and its possible involvement in the cellular autophagocytosis induced by 20-hydroxyecdysone. Gen. Comp. End ocrino l. 50, 116– 123. 9. Cooper, R.L. & Ruffner, M.E. (1998) Depression of synaptic efficacy at intermolt in crayfish neuromuscular junctions by 20- hydroxyecdysone, a molting hormone. J. Neurophysiol. 79, 1931–1941. 10. Ruffner,M.E.,Cromarty,S.I.&Cooper,R.L.(1999)Depression of synaptic efficacy in high- and low-outp ut Drosophila neuro- muscular junctions by the molting h ormone (20-HE). J. Neuro- physiol. 81, 7 88–794. 11. Lockshin, R .A. & Williams, C.M. (1965) Programmed cell death – III. Neural control of the breakdo wn of t he intersegmental mus- cles of silkmoths. J. Insect Physiol. 11, 601–610. 12. Ozeki, K. (1968) Experimental studies on the regression of the vertebrate glands of the earwig Anisolabis maritime,during metamorphosis. Scientific Paper of the College of General Edu- cation, University of Tokyo, 18, 1 99–219. 13. Streichert, L.C., Pierce, J.T., Nelson, J.A. & Weeks, J.C. (1997) Steroid hormones act directly to trigger segment-specific pro- grammed cell death of identified motoneurons in vitr o. Dev. Biol. 183, 95–107. 14. Terashima, J., Yasuhara, N., Iwami, M. & Sakurai, S. (2000) Programmed cell death triggered by insect steroid hormone, 20-hydroxyecdysone, in the anterior s ilk gland of the silkworm, Bombyx mori. Dev. Ge ne Evol. 210, 545–558. 15. Sakurai, S. (1984) Temporal organization of endocrine events underlying larval-pupal metamorphosis in t he silkworm, Bombyx mori. J. Insect Physiol. 30, 6 57–664. 16. Findly, J.P.C. (1989) Purificatio n of membrane proteins. In Pro- tein Purification M ethods; A Practical Approach (Harris, E.L.V. & Angal, S, eds), pp. 59–82. IRL Press, Oxford. 17. Wu, C. (1984) Activating protein factor binds in vitro to upstream control sequences in heat shock gene chromatin. Nature 311,81– 84. 18. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680– 685. 19. Yoshikuni, M., Shibata, N. & Nagahama, Y. (1993) Specific binding of [ 3 H]17a,20b- dihydroxy-4-pregnen-3- one to oocyte cortices of rainbow trout (Oncorhynchus mykiss). Fish Physiol. Biochem. 11, 15–24. 20. Kerkhoff, C., Gehring, L., Habben, K., Resch, K. & Laever. V. (1996) Identification of two different lysophosphatidyl choline: acyl-CoA acyltransferase (LAT) in pig spleen w ith putative dis- ctinct top ological l ocalization. Bioc hem. Biophys. Acta 1 302, 249– 256. 21. Kerkhoff, C., Tru ¨ mbach,B.,Gehring,L.,Habben,K.,Schmitz, G. & Kaever, V. (2000) Solubilization, partial purification and photolabelling o f the integral membrane protein lysopho spho- lip i c: acyl-CoA acyltransferase (LAT). Eur. J. Biochem. 267, 6339–6345. 22. Pryde, J.G. & P hillips, J.H. (1986) Fra ctionation of m embrane proteins by temperature-induced phase separation in Triton X-114. Application to subcellular fractions of the adrenal me dulla. J. Biochem. 233, 525–533. 23. Hooper, N.M. & Bashir, A. (1991) Glycosyl-phosphatidylinositol- anchored membrane prote ins can be distinguished fro m transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114. J. Bioc hem. 280, 745–751. 24. Bordier, C. (1981) Phase separation of integral membrane prote ins in Triton X-114 solution. J. Biol. Chem. 256, 1604–1607. 25. Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: pra ctical aid for computer analysis. Mol. Pharmacol. 47, 1197–1203. 26. Kamimura, M., Tom ita, S ., Kiuchi, M. & Fujiwara, H. (1997) Tissue-specific a nd stage-specific expression of two silkworm ecdysone receptor isoforms – ecdysteroid-dependent transcriptio n in cultured anterior silk glands. Eur. J. Biochem. 248, 786–793. 27.Yoshikuni,M.,Matsushita,H.,Shibata,N.&Nagahama,Y. (1994) Purification and characterization of 17a,20b-dihydroxy-4- pregnen-3-one binding protein from. plasma of rainbow trout, Oncorhynchus mykiss. General Comp. Endocrinol. 96, 189–196. 28. Zhu, Y., Rice, C.D., P ang, Y ., P ace, M. & Thomas, P. (2003) Cloning, expression, and characterization of a membrane pro- gestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. Proc. Natl Acad. Sci. USA 10 0 , 2231– 2236. 29. Simoncini, T. & Genazzani, A.R. ( 2003) Nongenomic actions of sex steroid hormones. Eur. J. Endocrinol. 148, 281–292. 30. Boonyaratanakornkit, V., Sc ott, M.P., Ribon, V., S herman, L., Anderson, S.M., Maller, J.L., Miller, W.T. & Edwards, D.P. (2001) Progesterone receptor contains a proline-rich motif that 3178 M. Elmogy et al. (Eur. J. Biochem. 271) Ó FEBS 2004 directly interacts with SH3 domains and activates c-Src family tyrosine kinases. Mol. Cell 8, 269–280. 31. Falkenstein,E.,Tillmann,H.,Christ,M.,Feuring,M.&Wehling, M. (2000) Multiple actions of steroid hormones – A focus on rapid, nongenomic effects. Pharmacol. Rev. 52, 513–555. 32. Yao, T P., Forman, B.M., Jiang, Z., Cherbas, L., Chen, J D., McKeown, M., Cherbas, P . & Evans, R.M. (1993) Fu nctional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature 366, 476–479. 33. Swevers, L., C herbas, L., Cherbas, P. & Iatrou, K. (1996) Bombyx EcR (BmEcR) and Bombyx US P (BmCF1) c om bine to f orm a functional ecdysone re cepto r. Insect Biochem. Mol. Biol. 26 ,217– 221. 34. Bidmon, H.J. & Slite, T.J. (1990) The ecdysteroid receptor. Invert. Report Dev. 18, 13–27. 35. Minakuchi, C., Nakagawa, Y., Kamimura, M. & Miyagawa, H. (2003) B inding affinity of nonsteroidal ecdysone agonists against the ecd ysone r eceptor complex determines the strength of their molting hormone activity. Eur. J. Biochem. 270, 4095–4104. 36. Sage, B.A., Tanis, M.A. & O’Connor, J.D. (1982) Characteriza- tion o f ecdysteroid recept ors in cytosol and naive nuclear pre pa- rations of Drosophila Kc cells. J. Biol. Chem. 25 7, 6373–6379. 37. Mao, H. & Kaufman, W.R. (1998) DNA binding properties of the ecdysteroid receptor in the s alivary g land of the female ixodid tick, Amblyomma hebraeum. Insect Biochem. Mol. Biol. 28, 947–957. Ó FEBS 2004 Membrane ecdysone receptor (Eur. J. Biochem. 271) 3179 . Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori Mohamed Elmogy, Masafumi Iwami and Sho Sakurai Division of Life Sciences, Graduate School of Natural. determined using 25 n M [ 3 H]PonA and increasing amounts of proteins in individual incubations. The percentage of specific b inding increased in a protein concentration-dependent manner within the range of. only in the pellet after the first centrifugation step. On increasing the concentration of Triton X-114, the binding activity was recovered predominantly in the detergent-rich phase. The binding in