FEBS Letters 583 (2009) 655–660

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Suppression of the ecdysteroid-triggered growth arrest by a novel Drosophilamembrane steroid binding protein

Ikuko Fujii-Taira a,b,c, Shinji Yamaguchi a,c, Ryoko Iijima b,c, Shunji Natori b, Koichi J. Homma a,c,*a Faculty of Pharmaceutical Sciences, Teikyo University, Sagamihara, Kanagawa 229-0195, Japanb Natori Special Laboratory, RIKEN, Wako-shi, Saitama 351-0198, Japanc Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 16 September 2008Revised 11 December 2008Accepted 29 December 2008Available online 20 January 2009

Edited by Lukas Huber

Keywords:EcdysteroidProgesterone receptor membranecomponentGrowth arrestMetamorphosisDrosophila

0014-5793/$34.00 � 2009 Federation of European Biodoi:10.1016/j.febslet.2008.12.056

Abbreviations: Dm_MSBP, Drosophila membrane sextracellular signal-regulated kinase; EcR, ecdysone rereceptor membrane component; TMR, transmembrandomain; DmDopEcR, Drosophila melanogaster dopamHE, 20-hydroxyecdysone; S2 cell, Drosophila Schneintronic gene

* Corresponding author. Address: Faculty of PharUniversity, Sagamihara, Kanagawa 229-0195, Japan. F

E-mail addresses: homma-kj@umin.ac.jp, hommaHomma).

Ecdysteroid is a crucial steroid hormone in insects, especially during metamorphosis. Here, we showthat the Drosophila membrane steroid binding protein (Dm_MSBP) is a novel structural homolog ofthe vertebrate membrane-bound receptor component for progesterone. Dm_MSBP exhibited bind-ing affinity to ecdysone when expressed on the cell surface of Drosophila S2 cells. In S2 cells, the sta-ble overexpression of Dm_MSBP suppressed the growth arrest triggered by 20-hydroxyecdysone andprevented the temporal activation of extracellular signal-regulated kinase proteins. These resultssuggest that Dm_MSBP is a membranous suppressor to ecdysteroid and blocks the signaling by bind-ing it in extracellular fluid.� 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction tein synthesis [8]. For example, the progesterone receptor mem-

Ecdysteroid is a steroid hormone that is involved in multiplecellular responses during development, especially in the metamor-phic reorganization in insects [1]. The ecdysone receptor (EcR), oneof the nuclear receptor superfamily, is a functional nuclear recep-tor for ecdysteroid. The EcR gene encodes three isoforms of theEcR protein in Drosophila. The differences in their expression pat-terns produce the variation of in vivo cellular responses [2]. EcR re-sides in the cell nucleus, and is thus unable to directly mediaterapid non-genomic effects which occur in the cytoplasmic mem-brane [3–7].

In vertebrates, it has been reported that some non-genomic cel-lular responses to steroid hormones are initiated through plasmamembrane receptors, and these responses do not require new pro-

chemical Societies. Published by E

teroid binding protein; ERK,ceptor; PGRMC, progesteronee region; SBD, steroid bindingine/ecdysteroid receptor; 20-ider’s cell line 2; VIG, vasa

maceutical Sciences, Teikyoax: +81 42 685 3738.kj@pharm.teikyo-u.ac.jp (K.J.

brane component (PGRMC) has been purified as a membraneprotein that binds to progesterone [9]. PGRMC family proteins havea single transmembrane region (TMR) and a steroid binding do-main (SBD). The overexpression study of rat PGRMC has providedevidence of resistance to apoptosis by progesterone in the cellswhich do not express a classical nuclear receptor for progesterone[10,11], suggesting that rat PGRMC mediates a non-genomicresponse.

Apart from in vertebrates, non-genomic response pathwaysmediate some rapid actions of steroid hormones in invertebrates[3,12], and the membranous receptor for ecdysteroid has been sug-gested to play important developmental roles. For example, theDrosophila melanogaster dopamine/ecdysteroid receptor (DmDo-pEcR) has been identified as a G-protein coupled receptor, whichshowed the binding activity for ecdysteroid [12]. DmDopEcR trans-mits catechol signals, and ecdysteroid acts as its antagonist. SinceDmDopEcR expression was not detected in pupal stages, thereseemed to be another signaling cue for ecdysteroid mediated bymembrane receptors during insect metamorphosis.

Here, we identify the Drosophila membrane steroid binding pro-tein (Dm_MSBP) gene as a PGRMC homolog of Drosophila. WhenDm_MSBP cDNA was introduced in Drosophila cells, the proteinwas expressed on the cell membrane and exhibited binding affinityto ecdysone. The stable overexpression of Dm_MSBP suppressed

lsevier B.V. All rights reserved.

mailto:homma-kj@umin.ac.jpmailto:hommakj@pharm.teikyo-u.ac.jphttp://www.FEBSLetters.org

656 I. Fujii-Taira et al. / FEBS Letters 583 (2009) 655–660

cellular responses triggered by 20-hydroxyecdysone (20-HE).Dm_MSBP may thus be a modulator for ecdysteroid signaling.

2. Materials and methods

2.1. cDNA cloning

EST-based DNA probe fragments (Supplementary Fig. 1A) wereamplified from a Drosophila embryo cDNA library (Stratagene) byPCR. Primers used were 50-GGATTCCAAAGTGGGTGCAGATCC-30

(sense) and 50-CATTTCCCATTCCCGCACAGAGTCC-30 (antisense).Using plaque hybridization, two clones were isolated from2.2 � 105 clones of a Drosophila embryo cDNA library, and theirnucleotide sequences were determined.

2.2. Sequence analysis

The transmembrane region was predicted using the SOSUI [13](http://bp.nuap.nagoya-u.ac.jp/sosui/) and the TransmembraneHidden Markov Model (TMHMM) [14] (http://www.cbs.dtu.dk/ser-vices/TMHMM/) programs. The multiple sequence alignmentswere carried out with the ClustalW program at http://clu-stalw.ddbj.nig.ac.jp. The phylogenetic tree was constructed onthe basis of amino acid difference (p-distance) by the neighbour-joining method.

2.3. Cells

The Drosophila Schneider’s cell line 2 (S2 cell) was cultured at25�C in Schneider’s Drosophila medium (GIBCO) supplementedwith 10% (v/v) heat-inactivated fetal calf serum, 100 lg/ml strep-tomycin, and 100 U/ml penicillin. 20-HE (SIGMA) was dissolvedin sterile milliQ water and added directly to the culture mediumto produce the final concentration. The transient expression exper-iments were performed as described previously [15]. To establishthe stable Dm_MSBP transfectants, we used pIB/V5 vector (Invitro-gen). The cells were selected through cultivation with Blasticidin–HCl (Invitrogen) and cloned by limiting dilution [16] after transfec-tion of pIB-Dm_MSBP construct.

2.4. Ecdysone binding assay

To prepare membrane fractions, the cells were homogenized inphosphate-buffered saline containing protease inhibitors by glasshomogenizer and centrifuged (1000�g, 10 min). The resultingsupernatants were centrifuged (100000�g, 30 min), and the pel-lets were used as membrane fractions. The binding assay was per-formed according to the method of Meyer et al. [9] with somemodifications. In brief, the membrane fractions were solubilizedby 20 mM 3-[(3-cholamidopropyl)dimethylammonio]propanesul-fonic acid with 10 mg/ml bovine serum albumin. They were incu-bated in the presence of 10 nM a-[23, 24-3H(N)]-ecdysone (NEN)at 25�C for 1 h, then collected on a Whatman GF/C glass filter. Afterwashing with cold Tris–buffered saline, the radioactivities remain-ing on the filters were measured using a liquid scintillationcounter.

2.5. Measurement of cell growth

The S2 cells were suspended at a density of 1 � 105 cells/ml inthe culture medium, and cultured for 2 days in the presence or ab-sence of 20-HE. Growth of the S2 cells was assessed by manual cellcounting using the hemocytometer as well as the AlamarBlue as-say. The AlamarBlue assay is the method to measure the level of

cellular metabolism using the AlamarBlue reagent. In brief, 10%volume of AlamarBlue (TREK Diagnostic Systems, Cleveland, OH)was added to the culture medium, and the change in the color ofthe culture medium was monitored by measuring the absorbanceat 570 nm and 610 nm. In this assay, the reducing activity gener-ated by the proliferating cells changed the color of AlamarBlue.To confirm the correlation between cell number and metabolicactivity, calibration curves were constructed in each experiment.

2.6. Immunoblotting and immunofluorescence

Immunoblotting and immunofluorescence staining were per-formed, as described previously [15]. Anti-Dm_MSBP rabbit anti-bodies were raised against the peptides corresponding to theamino acid residues 182–197 and 203–218 of Dm_MSBP, and usedfor immunoblotting and immunofluorescence staining, respec-tively. These two antibodies are both suitable for immunofluores-cent staining and immunoblotting. However, the antibodyagainst the peptide corresponding to the amino acid residues203–218 of Dm_MSBP worked better for immunoblotting and theother (antibody against amino acid 182–197) worked better forimmunocytochemistry. Each antibody was affinity-purified withthe recombinant Dm_MSBP produced by Escherichia coli. The anti-body against b-galactosidase protein was purchased from Prome-ga. The extracellular signal-regulated kinase (ERK) proteins weredetected according to the method of Gabay et al. [17]. To preparethe samples for the short 20-HE-exposure experiments, cells werebriefly washed by ice-cold PBS and lysed immediately in a RIPAbuffer (50 mM Tris–HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxy-cholate, 30 mM NaF, 150 mM NaCl, 5 mM ethylenediaminetetra-acetic acid, 1 mM ethyleneglycol bis(2-aminoethylether)tetraacetic acid, 5 mM sodium diphosphate, 1 mM orthovanadate,1 mM dithiothreitol, 10 lg/ml leupeptin, 10 lg/ml pepstatin A,10 lg/ml aprotinin, 10 lg/ml E64, 0.1 mM PMSF). They were thenfrozen by liquid N2. ERK and phospho-ERK antisera were purchasedfrom SIGMA.

3. Results

3.1. Cloning and characterization of Dm_MSBP cDNA

Database analyses have indicated that there is an EST clonewhich has a similarity to vertebrate PGRMC (Genbank AI297635).We designed a 502-bp probe based on this EST clone (Supplemen-tary Fig. 1), and obtained two cDNA clones by plaque hybridization.One of them completely matched to one locus, symbolized asCG9066 in Drosophila genome (Genbank AB290904). The otherclone had a 68-nt deletion in 50-UTR region (GenbankAB290905). This deletion started and ended with a 2-bp consensussequence (GT at the 50-end and AG at the 30-end, see Supplemen-tary Fig. 1A), suggesting that the clone appeared to be splicing var-iant from the original single gene (Supplementary Fig. 1A). Theycoded an identical protein which showed 43% homology to thatof the human PGRMC1 overall. The region between amino acid res-idues 81 and 179 showed similarities with the SBD of the humanPGRMC1 and the human PGRMC2 of 61% and 66%, respectively(Fig. 1A). Based on the BLAST searches, Dm_MSBP is the only genewhich shows a significant similarity to both Hs_PGRMC1 andHs_PGRMC2 in the Drosophila genome. This is true for other insectsas well. The BLAST searches showed that other insects, Culex quin-quefasciatus (southern house mosquito), Aedes aegypti, Anophelesgambiae and Bombyx mori, had one ortholog gene of Dm_MSBP.From these characteristics, we concluded that Dm_MSBP is a mem-ber of the PGRMC family, hence its name, D. melanogaster mem-brane steroid binding protein (Dm_MSBP).

http://bp.nuap.nagoya-u.ac.jp/sosui/http://www.cbs.dtu.dk/services/TMHMM/http://www.cbs.dtu.dk/services/TMHMM/http://clustalw.ddbj.nig.ac.jphttp://clustalw.ddbj.nig.ac.jp

Fig. 1. Comparison of amino acid sequences between Dm_MSBP and PGRMCs. (A) Multiple alignment of Dm_MSBP and PGRMCs sequences. The predicted TMR is indicatedwith a thick horizontal line above the sequence alignment. The SBD (ProDom ID PD006731) of PGRMC is boxed, and the amino acid residues matching to Dm_MSBP areshaded. The open arrowhead indicates the Asp residue corresponding to Asp120 in rat PGRMC1. (B) A phylogenetic tree of PGRMCs based on amino acid sequences isgenerated by the neighbour-joining method.

Fig. 2. Expression of Dm_MSBP during metamorphosis. Extracts of Drosophilawhole bodies were subjected to immunoblotting using the anti-Dm_MSBPantibody. The positions of the Dm_MSBP monomer and presumed dimer areindicated by the closed arrowhead and the open arrowhead, respectively.

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In rat PGRMC1, Asp120 is essential for binding to the progester-one [10]. This residue was also conserved in Dm_MSBP (Fig. 1A,open arrowhead). The sequence analysis using SOSUI and TMHMMprograms revealed that this protein had a single TMR (Fig. 1A). Inphylogenetic terms, Dm_MSBP is located intermediate to PGRMC1and 2 (Fig. 1B). Dm_MSBP may play roles similar to those of thetwo PGRMCs in vertebrates.

The Dm_MSBP mRNA was ubiquitously expressed in larval andadult tissues (Supplementary Fig. 1), but was found in increasedamounts of the Dm_MSBP protein in both the prepupal and earlypupal stages (Fig. 2). Focusing on the prepupal stage, an immuno-reactive protein with a molecular mass of 65 kDa was detected, be-sides the monomer of Dm_MSBP (Fig. 2, open arrowhead). Ingeneral, dimers can be separated under reducing condition. Butthe vertebrate PGRMC1 has been reported to form the homodimereven in reducing condition, suggesting the homodimer was linkedcovalently [9,18]. Dm_MSBP also may thus form a homodimer, and

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this dimerization may be involved in the function of Dm_MSBP atthe beginning of metamorphosis.

3.2. Cellular localization of Dm_MSBP and its binding affinity toecdysone

To determine whether in fact Dm_MSBP is a membrane protein,we used the Drosophila S2 cell, an embryonically-derived cell line.The immunoblotting showed that the Dm_MSBP was detectable inS2 cells at low levels, but increased greatly following the introduc-tion of Dm_MSBP cDNA (Fig. 3A). We then examined the cellularlocalization of Dm_MSBP by immunostaining under non-fixativeconditions using an antibody against the Dm_MSBP C-terminal re-

Fig. 3. Expression of Dm_MSBP on the cell surface and its binding affinity to ecdysuntransfected cells were subjected to immunoblotting using an anti-Dm_MSBP antibodexpressing cells (left) and the b-galactosidase-expressing cells (right) were stained witStrongly stained cells are indicated by arrowheads. Bars indicate 10 lm. (C) Ecdysone bDm_MSBP-overexpressing and empty vector-transfected cells. Their binding affinitdeterminations. *P < 0.05 (Student’s t-test).

Fig. 4. Suppression of 20-HE-triggered cell responses by Dm_MSBP. (A) The inhibitory effwere manually counted after 2 days of culture in the presence of various concentrations oin the clonal cells were examined using immunoblotting. (C) Suppression of the 20-HE-tri0.1 lM 20-HE were compared. Data represent mean ± S.E.M. of three determinationphosphorylated ERK proteins in untransfected S2 cells (D), and Dm_MSBP-expressing cell10, and 60 min, respectively.

gion. As a result, those cells expressing Dm_MSBP were stronglystained with the anti-Dm_MSBP antibody. But cells expressingthe cytosolic b-galactosidase protein were not stained with theanti-b-galactosidase antibody under the same experimental condi-tions (Fig. 3B). Both the anti-Dm_MSBP antibody and the anti-b-galactosidase antibody reacted to the transformant, respectively,when the cells were fixed and permeabilized (data not shown).These results indicate that Dm_MSBP localizes on the cell surface,and exposes its C-terminus extracellularly.

The fact that Dm_MSBP shows high sequence homology to theSBD of PGRMC (Fig. 1), suggests that ecdysone may be a ligand ofDm_MSBP, because ecdysone is a typical insect steroid hormone.Therefore, we tested whether Dm_MSBP has an affinity to an

one. (A) Immunoblotting of Dm_MSBP cDNA-transfected cells. Transfectants andy. (B) Immunofluorescence staining under non-fixative conditions. The Dm_MSBP-h the anti-Dm_MSBP antibody and the anti-b-galactosidase antibody, respectively.inding assay on the membrane fraction. Membrane fractions were prepared from

ies to [3H]-ecdysone were measured. Data represents mean ± S.E.M. of three

ect of 20-HE on cell proliferation. The cells were suspended and the numbers of cellsf 20-HE. (B) The clonal cells highly expressing Dm_MSBP. The amounts of Dm_MSBPggered growth arrest by Dm_MSBP. Growth of the clonal cell lines in the presence ofs. *P < 0.05, **P < 0.01 (Student’s t-test). (D and E) Changes in the quantity of

s (E) by a short-period stimulation of 20-HE. Cells were exposed to 20-HE for 0, 2, 5,

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[3H]-ecdysone by performing a binding assay using the membranefraction. As a result, the overexpression of Dm_MSBP elevated thebinding ability to ecdysone (Fig. 3C), suggesting that ecdysone isindeed a ligand of Dm_MSBP. The Kd value of the Dm_MSBP pro-tein was calculated to be about 300 nM. This value is very similarto that of porcine 28-kDa PGRMC1 [9], indicating that the bindingto ecdysteroids is substantial enough to attenuate 20-HE signaling,and is physiologically meaningful.

3.3. Suppression of 20-HE-triggered cell growth arrest of S2 cells

We examined the involvement of Dm_MSBP in ecdysteroid sig-naling using Dm_MSBP-overexpressing cells. It has been reportedthat 20-HE can induce G2 arrest [19] and apoptosis [20] of S2 cells.Therefore, we examined the effect of 20-HE on S2 cell growth reg-ulation. When S2 cells were cultured in the presence of 20-HE for 2days, the growth was clearly inhibited by 20-HE in a dose-depen-dent manner (Fig. 4A, Supplementary Fig. 2A). In addition, the cellstreated with 20-HE were stained by calcein-staining (Supplemen-tary Fig. 2B), indicating that the cells remained viable. There is a re-port which suggests that growth arrest following the exposure ofS2 cells to 20-HE was based on the genomic response. In the paper,it is shown that the depletion of the ecdysone nuclear receptor byRNAi suppressed the G2 arrest of S2 cells [19]. We also observedthe G2 arrest of S2 cells needed day-scale cell culture longer than24 h in the presence of 20-HE. These findings suggest that the G2arrest of S2 cells is required for the ecdysone signaling through agenomic pathway. After washing away the 20-HE, they started toreproliferate (Supplementary Fig. 2C). These results show that20-HE-triggered growth arrest is reversible.

Next, we examined whether Dm_MSBP is involved in thegrowth arrest induced by 20-HE. For this, we established clonal celllines of high-expression of Dm_MSBP (Fig. 4B), and compared theirgrowth in the presence of 0.1 lM of 20-HE. As the expression levelsof Dm_MSBP increased, the growth arrest by 20-HE was sup-pressed (Fig. 4C). This suggests that Dm_MSBP has a competitiveeffect against 20-HE on S2 cell growth in a long-term genomicpathway. We also focused on short-term non-genomic effects byanalyzing the intracellular signaling. There is significant evidencethat progesterone signaling non-genomically activates ERK path-way in mammals [21–23]. Based on this evidence, we expectedthat ecdysteroid might be effective on ERK activity in insects. Forthis, we examined ERK activity in 20-HE-exposed cells and foundthat phosphorylated ERK levels increased 2–5 min after the addi-tion of 20-HE, but decreased again 10 min post-stimulation(Fig. 4D). Conversely, the clonal cell line with highest expressionof Dm_MSBP (clone E1) did not activate ERK but rather diminishedit, following 20-HE stimulation (Fig. 4E). These results stronglysuggest that Dm_MSBP down-regulates ecdysteroid signaling in arapid non-genomic manner.

4. Discussion

We have identified Dm_MSBP in Drosophila which is a singlegene similar to vertebrate PGRMC1 and 2, suggesting thatDm_MSBP is the ortholog of vertebrate PGRMC. To our knowledge,this is the first report of an insect membrane ecdysone-bindingprotein being expressed during pupal stages. Considering the ori-entation of Dm_MSBP on the cell surface, Dm_MSBP captures theecdysone in the extracellular fluid and competes with its signaling.

Our results suggest that Dm_MSBP negatively regulates theecdysteroid signaling pathway on the cell surface. Since the cyto-solic region of Dm_MSBP is very short and has no structural fea-tures of a signal transducer, it is reasonable to speculate thatDm_MSBP does not transmit the ecdysteroid signals directly. We

hypothesize that the function of Dm_MSBP is to capture ecdyster-oid on the cell surface and passively attenuate the intracellular sig-naling of ecdysteroid. But we do not exclude the possibility thatother cytosolic protein(s) bind to the cytosolic region of Dm_MSBPand thus actively down-regulate the ecdysteroid signaling.Whether Dm_MSBP passively attenuates or actively down-regu-lates ecdysteroid signaling will be tested by expressing the extra-cellular portion of Dm_MSBP together with transmembrane andcytoplasmic domains from another unrelated protein. If the effectsof Dm_MSBP do not involve active down-regulation of signalingthrough the MSBP cytoplasmic domain, then these hybrid proteinsshould inhibit ecdysone signaling in a manner similar to normalDm_MSBP. In vertebrates, PGRMC interacts with plasminogen acti-vator inhibitor RNA binding protein-1 [10], which is known as ahomolog of Drosophila vasa intronic gene (VIG), a member ofRNA-induced silencing complex [24]. It is known that the expres-sion of microRNAs are regulated by ecdysteroid in Drosophila[25]. If Dm_MSBP interacts with VIG, the interaction may affectthe processing and function of microRNA.

The expression patterns of Dm_MSBP provide some cues to itsphysiological function. The quantity of Dm_MSBP increased inthe early stages of metamorphosis, and the up-regulatedDm_MSBP expression may suppress excessive ecdysteroidal sig-naling and contribute to cell proliferation of some populations ex-posed to high concentrations of ecdysteroid. According to thegenome-wide gene expression profile in the Gene Expression Data-base of Berkeley Drosophila Genome Project [26], the gene locus ofDm_MSBP, CG9066, is expressed in several tissues such as the ringgland and embryonic corpus allatum in embryos at stage 13–16.The concentration of ecdysteroid also peaks during the embryonicstage, in addition to the commencement of the pupal stage in Dro-sophila [1]. Gene expression of CG9066 (We named as Dm_MSBP),increases in mbn2 cells following LPS-treatment [27], and down-regulates the in adult ovary after mating [28], implying thatDm_MSBP is involved in immune responses, reproduction as wellas the process of the metamorphosis. Thus Dm_MSBP may be aregulator of ecdysteroid signaling in a wide range of physiologicalevents. The establishment of the Dm_MSBP mutant fly and RNAi-expressing transgenic flies targeted to Dm_MSBP [29] will allowfor further investigation of the various functions of Dm_MSBP.

Acknowledgments

This work was supported by a Grant-in-Aid from the SpecialPostdoctoral Researchers Program of RIKEN (I.F.-T.), Scientific Re-search on Priority Areas (Molecular Brain Science) from the Minis-try of Education, Culture, Sports, Science and Technology of Japan(K.J.H.), Scientific Research from the Japan Society for the Promo-tion of Science (K.J.H.), the Ministry of Education, Culture, Sports,Science and Technology of Japan (S.Y.), the Naito Foundation(K.J.H.), the Japan Foundation of Applied Enzymology (K.J.H.), theUehara Memorial Foundation (S.Y.) and, the Sagawa Foundationfor Promotion of Cancer Research (S.Y.).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.febslet.2008.12.056.

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Suppression of the ecdysteroid-triggered growth arrest by a novel Drosophila membrane steroid binding proteinIntroductionMaterials and methodscDNA cloningSequence analysisCellsEcdysone binding assayMeasurement of cell growthImmunoblotting and immunofluorescence

ResultsCloning and characterization of Dm_MSBP cDNACellular localization of Dm_MSBP and its binding affinity to ecdysoneSuppression of 20-HE-triggered cell growth arrest of S2 cells

DiscussionAcknowledgmentsSupplementary dataReferences