RESEARCH ARTICLE Open Access Structural diversity and ......The major insect ecdysteroid, 20-hydroxyecdysone (20E), binds directly to a heterodimeric transcription factor comprising

RESEARCH ARTICLE Open Access

Structural diversity and evolution of theN-terminal isoform-specific region of ecdysonereceptor-A and -B1 isoforms in insectsTakayuki Watanabe*, Hideaki Takeuchi, Takeo Kubo*

Abstract

Background: The ecdysone receptor (EcR) regulates various cellular responses to ecdysteroids during insectdevelopment. Insects have multiple EcR isoforms with different N-terminal A/B domains that contain the isoform-specific activation function (AF)-1 region. Although distinct physiologic functions of the EcR isoforms have beencharacterized in higher holometabolous insects, they remain unclear in basal direct-developing insects, in whichonly A isoform has been identified. To examine the structural basis of the EcR isoform-specific AF-1 regions, weperformed a comprehensive structural comparison of the isoform-specific region of the EcR-A and -B1 isoforms ininsects.

Results: The EcR isoforms were newly identified in 51 species of insects and non-insect arthropods, includingdirect-developing ametabolous and hemimetabolous insects. The comprehensive structural comparison revealedthat the isoform-specific region of each EcR isoform contained evolutionally conserved microdomain structures andinsect subgroup-specific structural modifications. The A isoform-specific region generally contained four conservedmicrodomains, including the SUMOylation motif and the nuclear localization signal, whereas the B1 isoform-specificregion contained three conserved microdomains, including an acidic activator domain-like motif. In addition, theEcR-B1 isoform of holometabolous insects had a novel microdomain at the N-terminal end.

Conclusions: Given that the nuclear receptor AF-1 is involved in cofactor recruitment and transcriptionalregulation, the microdomain structures identified in the isoform-specific A/B domains might function as signaturemotifs and/or as targets for cofactor proteins that play essential roles in the EcR isoform-specific AF-1 regions.Moreover, the novel microdomain in the isoform-specific region of the holometabolous insect EcR-B1 isoformsuggests that the holometabolous insect EcR-B1 acquired additional transcriptional regulation mechanisms.

BackgroundsMorphogenetic events during insect development are trig-gered by ecdysteroids. The major insect ecdysteroid, 20-hydroxyecdysone (20E), binds directly to a heterodimerictranscription factor comprising two nuclear receptors, theecdysone receptor (EcR) and the ultraspiracle, to activate acomplex transcriptional cascade [1]. In the holometabo-lous insects, there are two or three EcR isoforms (A andB1 isoforms; Drosophila has an additional B2 isoform) thatare produced from a single genetic locus by differentialpromoter usage and alternative splicing [2]. The EcR iso-forms have a common C-terminal region that includes the

DNA binding domain and the ligand binding domain (theC-F domains), but also have isoform-specific regions inthe N-terminal A/B domain. In the holometabolousinsects, each EcR isoform govern distinct steroid-stimu-lated responses during metamorphosis. In Drosophila, theEcR-A isoform is predominantly expressed in the prolif-erative tissues during metamorphosis, and is required foradult-specific developmental processes. In contrast, theEcR-B isoforms (B1 and B2 isoforms) are expressed in thelarva-specific tissues, and are involved in 20E-triggered lar-val tissue remodeling during metamorphosis [2-7]. In thered flour beetle Tribolium castaneum, the EcR-A isoformhas a dominant role in transduction of the ecdysteroidaction by regulating the expression of 20E-response genes[8]. Only the A isoform has been identified in basal direct-

* Correspondence: [email protected]; [email protected] of Biological Sciences, Graduate School of Science, TheUniversity of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

Watanabe et al. BMC Evolutionary Biology 2010, 10:40http://www.biomedcentral.com/1471-2148/10/40

© 2010 Watanabe et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

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developing (ametabolous and hemimetabolous) insects[9,10], therefore, it is not known whether direct-develop-ing insects have multiple EcR isoforms with distinct phy-siologic functions.In addition to the differential expression patterns and

the distinct physiologic functions during development,EcR isoforms have differential transcriptional regulationmechanisms. The A/B domain of the nuclear receptorsgenerally contains the ligand-independent activationfunction (AF)-1 region [11]. Several studies of the AF-1function of Drosophila EcR isoforms have revealed thatthe A/B domain of Drosophila EcR-B isoforms havestrong transactivation activity, whereas that of the A iso-form has weaker transactivation activity [12]. The AF-1of Drosophila EcR-B1 mainly locates in the N-terminalregion (amino acid residues 1-53), whose sequence isconsiderably conserved among higher holometabolousinsects such as flies, mosquitoes, and moths [13].Although structural differences in the AF-1 regionamong the EcR isoforms result in differential transcrip-tional regulation, the structural basis and molecularmechanisms underlying the isoform-specific AF-1 func-tions remain obscure.In the present study, to examine the detailed struc-

tural characteristics of the isoform-specific region ofeach EcR isoform, we performed a comprehensive struc-tural comparison of the isoform-specific regions of theinsect EcR-A and -B1 isoforms. First, we newly identi-fied EcR isoforms in 51 insect and non-insect arthropodspecies, focusing mainly on the basal direct-developinginsects. Then, we compared the deduced amino acidsequences of the isoform-specific region of the EcR-Aand -B1 isoforms (45 and 76 species, respectively).Sequence comparison revealed that the isoform-specificregion of each EcR isoform contains several conservedmicrodomain structures, some of which were evolution-ally acquired or modified. Interestingly, the isoform-spe-cific region of the holometabolous insect EcR-B1isoform contained a highly conserved (K/R)RRW motifat the N-terminus, which was lacking in basal direct-developing insects. Given that the nuclear receptor AF-1regions provide an interaction interface for co-regulatoryproteins, the conserved microdomain structures in theEcR isoform-specific AF-1 regions might play essentialroles in transcriptional regulation. Moreover, the holo-metabolous insect EcR-B1 might have novel transcrip-tional regulation mechanisms that are mediated by thenewly acquired (K/R)RRW motif.

ResultsCloning of cDNA fragments encoding the A/B domain ofthe EcR-A and -B1 isoformsThe EcR gene encodes two or three isoforms in theholometabolous insects (A, B1 and B2 isoforms), but

only A isoform was previously identified in the direct-developing insects. In the present study, to perform acomprehensive sequence comparison of the isoform-spe-cific region of the EcR-A and -B1 isoforms amonginsects and non-insect arthropods, we obtained cDNAfragments encoding the A/B domains of the EcR-A and-B1 isoforms from 18 and 51 arthropod species, respec-tively, by using reverse transcription polymerase chainreaction (see Additional file 1, Table S1 and Additionalfile 2, Table S2). We newly identified the EcR-A isoformin the direct-developing insects, including Paleoptera (1species), Polyneoptera (2 species), and Paraneoptera (8species). For a detailed analysis, we also identified theEcR-A isoform in 5 holometabolous insect species andone non-insect arthropod species. Although the EcR-B1isoform was identified in many holometabolous insectsand several non-insect arthropods, it has not been iden-tified in basal hemimetabolous and ametabolous insects.Therefore, we newly identified the EcR-B1 isoform inbasal insects, including Apterygota (2 species), Paleop-tera (2 species), Polyneoptera (8 species), and Paraneop-tera (12 species). In addition, we identified the EcR-B1isoform in 25 holometabolous insect species and twonon-insect arthropod species. To our knowledge, this isthe first report of the identification of the EcR-B1 iso-form in the direct-developing insects. In the direct-developing insects, the EcR isoforms were cloned fromthe whole body of the nymph and adult, and isolatedtissues, including the nymphal wing pad, fat body, testis,and ovaries (data not shown), suggesting that the EcRisoforms are broadly distributed in the direct-developinginsects.

The A isoform-specific region was classified into fivestructural typesWe compared the deduced amino acid sequences of theisoform-specific region in the A/B domain of the EcR-Aisoform identified from 45 arthropod species (5 non-insect arthropod species [Crustacea and Chelicerata], 15direct-developing insect species [1 paleopteran species, 4polyneopteran species, and 9 paraneopteran species] and26 Endopterygota species). GenBank accession numbersof the EcR-A isoforms were listed in Additional file 1,Table S1.General structural features of the A isoform-specific regionThe C-terminal region of the A isoform-specific regioncontained a conserved Ser/Thr-rich region, which wasformally referred to as the “A-box” [10,14]. In the N-terminal region of the A isoform-specific region, wefound two conserved microdomains: the SUMOylationmotif and the nuclear localization signal (NLS). In addi-tion, we found conserved (D/E)(D/E)W residuesbetween the NLS and the C-terminal A-box. TheSUMOylation motif in the A isoform-specific region


Page 2 of 17

contained a canonical consensus sequence for theSUMOylation (ΨKxE, where Ψ represents a large hydro-phobic amino acid) [15]. The NLS of the A isoform-spe-cific region contained a consensus sequence of themonopartite NLS [K(K/R)x(K/R)] [16].Comprehensive structural comparison revealed several

subgroup-specific structural modifications. Based on thestructural similarity, we classified the isoform-specificregion of the EcR-A isoform into five structural types.Type-1 A isoform-specific region (Figure 1)The type-1 A isoform-specific region contained theSUMOylation motif, the NLS, the (D/E)(D/E)W resi-dues, and the A-box. The consensus sequence of the A-box of the type-1 A isoform-specific region wasNGxSPSxxSSYDxxYSP. The EcR-A isoform with thetype-1 A isoform-specific region was identified in non-insect arthropods.Type-2 A isoform-specific region (Figure 2)The type-2 A isoform-specific region contained theSUMOylation motif, the NLS, the (D/E)(D/E)W resi-dues, and the A-box. In addition, the type-2 A isoform-specific region generally contained two conserved N-terminal sequences (DLKHE and ΨAYRG, where Ψrepresents a large hydrophobic amino acid). The con-sensus sequence of the A-box of the type-2 A isoform-specific region was NGYSSP(M/L)SSGSYDPYSP. TheEcR-A isoform with the type-2 A isoform-specific regionwas identified in the direct-developing insects except forHeteroptera (Order Hemiptera) and three holometabo-lous orders (Coleoptera, Trichoptera, and Lepidoptera).The EcR-A isoforms identified from two psicopteraninsects (Pediculus humanus corporis and Liposcelis sp.)

did not contain the DLKHE sequence at their N-termini.The EcR-A isoforms identified from several polyneop-teran insects (Blattella germanica [Order Dictyoptera],Locusta migratoria [Order Orthoptera], and Anisolabismaritima [Order Dermaptera]), and non-neopteraninsects (Ephemera strigata [Order Ephemeroptera]) didnot contain the ΨAYRG sequence at their N-termini.The EcR-A isoforms identified in Lepidoptera lacked theNLS.Type-3 A isoform-specific region (Figure 3)The type-3 A isoform-specific region contained the (D/E)(D/E)W residues and the A-box. The type-3 Aisoform-specific region lacked the N-terminal regioncontaining the SUMOylation motif and the NLS. Theconsensus sequence of the A-box of the type-3 A iso-form-specific region was NGxPSPTMSSMSYDPYSP.The EcR-A isoform with the type-3 A isoform-specificregion was identified in Heteroptera (OrderHemiptera).Type-4 A isoform-specific region (Figure 4)The type-4 A isoform-specific region contained theSUMOylation motif, the NLS, the (D/E)(D/E)W resi-dues, and the A-box. In addition, the type-4 A isoform-specific region contained D(T/S)S repeats in the N-ter-minus. The consensus sequence of the A-box of thetype-4 A isoform-specific region was NGYASPMS(T/S)GSYDPYSP. The EcR-A isoform with the type-4 A iso-form-specific region was identified in Hymenoptera andMecoptera. The NLS of the EcR-A isoforms identifiedfrom several hymenopteran insects (Apis mellifera, Phei-dole megacephala, and Camponotus japonicus) wasmodified (consensus; KxxR).

17512196111110

14610480

9495

* ** : :: *:.**SPLKRSRHININNNNNNSSSSAMEE---WLPSPSPSKRIR---------------QDDAGSWIASP-PPKRVR---------------QDDAGAWISSPSPSKRTK---------------QED-GTWLPSPSPNKRIR---------------QED-GTWIPSP

Daphnia magnaOrnithodoros moubata

Amblyomma americanumNephila clavata

Liocheles australasiae

4495709075

3082577862

::* *** . ** IIKTEPRNSDSPTTTIVKVEPRLP-SPCVSVVKVEPRLP-SPCVGIVKMEPRLP-SPCN-IVKVEPRLP-SPCLN

258161134143151

232142121127133

* ** : * **: : *. : NGYSP-SPMSTGSYEPPF-SPGGGGGKLG-NGLSPAS-CS--SYD-TY-SP-----RGPC-GLSPVSNCS--SYD-TY-SP-----RGPCNGHSP-T--S--SYD-AYCSS-----KD--NGLSPVSMVS--SYDP-Y-SP-----KG--

SUMO A-boxNLS + (D/E)(D/E)W

SUMO NLS (D/E)(D/E)W A-box

Daphnia magna

Ornithodoros moubata

Amblyomma americanum

Nephila clavata

Liocheles australasiae

SUMO

NLS

(D/E)(D/E)W

A-box

50 amino acids

Chelicerata

Crustacea

Figure 1 Type-1 A isoform-specific region. The type-1 A isoform-specific region contained four conserved microdomains. (A) Schematicrepresentation of the type-1 A isoform-specific regions. Positions of the SUMOylation motif, the NLS, the (D/E)(D/E) residues, and the A-box areindicated by colored boxes. (B) The alignment and Sequence Logo representation of the conserved microdomains of the type-1 A isoform-specific region. The amino acid residues are represented in the default color scheme of ClustalX. Asterisks indicate identical residues and colonsindicate similar residues.


Page 3 of 17

Type-5 A isoform-specific region (Figure 5)The type-5 A isoform-specific region contained theSUMOylation motif, the NLS, the (D/E)(D/E)W resi-dues, and the A-box. In addition, the type-5 A isoform-specific region contained YRLN residues at the N-termi-nus. The consensus sequence of the A-box of the type-5A isoform-specific region was NGYASPMSAGSYD-PYSPNG. The EcR-A isoform with the type-5 A iso-form-specific region was identified in Diptera. The EcR-A isoforms identified from several species in genus Dro-sophila (D. melanogaster, D. simulans, and D. yakuba)did not contain the YRLN sequence at their N-termini.

The B1 isoform-specific region was classified into sixstructural typesWe compared the deduced amino acid sequences of theisoform-specific region in the A/B domain of theEcR-B1 isoforms identified from 76 arthropod species(4 non-insect arthropod species [Crustacea, Myriapoda,and Chelicerata], 24 direct-developing insectspecies [2 apteryogote species, 2 Paleopteran species,8 polyneopteran species, and 12 paraneopteran species]and 48 Endopterygota species). GenBank accessionnumbers of the EcR-B1 isoforms were listed in Addi-tional file 2, Table S2.

Stenopsyche marmorata

Tribolium castaneum

Leptinotarsa decemlineata

Graptopsaltria nigrofuscata

Bothrogonia ferruginea

Aphrophora pectoralis

Acyrthosiphon pisum

Pediculus humanus corporis

Liposcelis sp.

Blattella germanica

Locusta migratoria

Anisolabis maritima

Nemoura sp.

Ephemera strigata

Bombyx mori

Choristoneura fumiferana

Omphisa fuscidentalis

Chilo suppressalis

Manduca sexta

50 amino acids

Lepidoptera

Trichoptera

Coleoptera

Polyneoptera

Hemiptera(without Heteroptera)

Psocoptera

Dictyoptera

Orthoptera

Dermaptera

Plecoptera

Ephemeroptera

Paraneoptera

Endopterygota

ΨAYRGΨAYRG

DLKHE SUMOSUMO

NLSNLS

(D/E)(D/E)W(D/E)(D/E)W

A-boxA-box

7875767577121151161145111103168114115150121106158119

59565756591021321421259183133959513110174139100

* .* ** :*: .**.**** SN--------------GY-----SSP-LSS-GSYGPYSP-NGNN--------------GY-----SSP-LSS-GSYGPYSP-NGNN--------------GY-----SSP-LSS-SSYGPYSP-NGNN--------------GY-----SSP-LSS-SSYGPYSP-NGNN--------------GY-----SSP-LSS-GSYGPYSP-N-SN--------------GY-----SSP-MSS-GSYDPYSP-NGSN--------------GY-----SSP-MSS-GSYDPYSP-NGSN--------------GY-----SSP-MSS-GSYDPYSP-NGSN--------------GY-----SSPTMSS-GSYDPYSP-NGSN--------------GY-----SSPTMSS-GSYDPYSP-NGSN--------------GY-----SSPTMSS-GSYDPYSP-NESNGGGGGGGGGGGGGGGYN----TSP-MST-NSYDPYSPMSGSN--------------GY-----SSP-MSS-GSYDPYSP-NGSN--------------GY-----SSPTMSS-GSYDPYSP-NGSN--------------GY-----SSP-MSS-GSYDPYSP-NGPN--------------GYA-----SP-LSSGGSYDPYSP-GGTNNNNNNST-------SYAGGGAPSP-LSSGGSYDPYSP-TGNN--------------GY-----SSP-MSS-GSYDPYSP-NGSN--------------GY-----SSP-LSS-GSYDPYSP-NG

4341424243801111179567551177272741077210963

3634353536669810280544110358585993619545

: -----------D--W-MAGGAA-----------D--W-MAGGAG-----------E--W-MAGATA-----------Y--W-MAGVVG-----------D--W-MAGGAAVKRPR--TD--D--W-LAAASPSKRQR--TD--D--W-LSSPS-ATK-RQMTE--D--F-MSSPSPNKRPR--TD--E--WPLSSPSPTKR-R--VD--D--W-MSSPSPTKRQR--TD--D--W-LSSPSPVKRLR--PD--D--W-LAVNSPSKRPR--TD--D--W-LSPPSPNKRSR--TD--D--W-LSPPSPAKRAR--LDS-D--W-LSSPGSNKRPR--PD--D--W-LSSPSPAKRLRPSTE--ES-W-------NKRTR--PD--D--W-LSAVSPTKRARLSSEQQEGGW-L--PSP

Bombyx moriChoristoneura fumiferana

Omphisa fuscidentalisChilo suppressalis

Manduca sextaStenopsych marmorata

Tribolium castaneumLeptinotarsa decemlineata

Graptopsaltria nigrofuscataBothrogonia ferruginea

Aphrophora pectoralisAcyrthosiphon pisum

Pediculus humanus corporisLiposcelis sp.

Blattella germanicaLocusta migratoria

Anisolabis maritimaNemoura sp.

Ephemera strigata

NLS + (D/E)(D/E)W A-box

NLS (D/E)(D/E)W A-box

Bombyx moriChoristoneura fumiferana

Omphisa fuscidentalisChilo suppressalis

Manduca sextaStenopsych marmorata

Tribolium castaneumLeptinotarsa decemlineata

Graptopsaltria nigrofuscataBothrogonia ferruginea

Aphrophora pectoralisAcyrthosiphon pisum

Pediculus humanus corporisLiposcelis sp.

Blattella germanicaLocusta migratoria

Anisolabis maritimaNemoura sp.

Ephemera strigata

1111111111111111111

11111111111421241514637781930261422

-------------MEL-K-HE---------------------VAY-RG----------------MDL-K-HE---------------------VAY-RG----------------MDL-K-HE---------------------VAY-RG----------------MDL-K-HE---------------------VAY-RG----------------MDL-K-HE---------------------VAY-RG----------------MDL-KQ-E--------------------LL-F-RPGPAMTAICS--GNLGSMDI-K-HE--------------------MI-Y-R----MTTIHSITSHLGSMDI-K-HE--------------------MI-Y-RD----------------MDL-KQHD--------------------SL-YNR-SGG-------------MDL-K-HE--------------------LL-YGR-SGT-------------MDL-K-HD------------------------------------------MMDQ-K-CDGGGGGVAAAAAGIGGGGVGGLMSYNRGRGG------------------------------------------M-F-RGGAS------------------------------------------M-YQRGTST-------------MDL-K-HE-LGGLGGGGASGNC-----------------------------MELFRGADPLA-VSVPLALPLPGHASPASAA--------------------MD-----EDM--LAAPAA-----TATVAGAAAAVASAT-------------MDLKRQ----------------------MM-Y-RGGVQ-------------MDL-K-HEEGL-LRLSDPGDGPGPT-------------

25242424253138403535214834334139

4936

16161515162227292424144123223428

3925

:* QVKAEPGVSH---QVKAEPGV-H---QVKAEPGVST---QVKAEPGVSH---QVKAEPGVSH---VVKAEPD--HGG-LVKSEPQL-HSPCLVKSEPQL-FTSNYIKSEPRL-HSPVIIKSEPRL-HSPPLIKSEP---HG--IIK--PR---SPAQVKSEPRV-HSPCQVKSEPRV-HSPCVVKTEPRE-----SVKTEPRV-HSPC

AVKAE-RV-QSPCLVKTEPRT-MSPC

-------------

DLKHE + ΨAYRG SUMO

DLKHE ΨAYRG SUMO

Figure 2 Type-2 A isoform-specific region. The type-2 A isoform-specific region contained four conserved microdomains and two conservedN-terminal sequences (DLKHE and ΨAYRG). (A) Schematic representation of the type-2 A isoform-specific regions. Positions of the two N-terminal sequences, the SUMOylation motif, the NLS, the (D/E)(D/E) residues, and the A-box are indicated by colored boxes. (B) The alignmentand Sequence Logo representation of the N-terminal region and the conserved microdomains of the type-2 A isoform-specific region. Theamino acid residues are represented in the default color scheme of ClustalX. Asterisks indicate identical residues and colons indicate similarresidues.


Page 4 of 17

General structural features of the B1 isoform-specific regionThe N-terminal region of the B1 isoform-specific regionessentially contained three microdomains: the S-richmotif, the SP residues, and the DL-rich motif. The S-rich motif was composed of acidic residues (D and E)and neutral polar residues (S and T), and contained aconserved core EV(T/S)SS sequence. The DL-rich motifwas composed of repeats of acidic residues and aro-matic/bulky hydrophobic residues (W, A, F, I, L, M, andV). The region between the S-rich and the DL-richmotifs generally contains conserved SP residues. In addi-tion to these microdomains, the isoform-specific regionof the holometabolous insect EcR-B1 isoform containedan additional conserved (K/R)RRW motif at the N-terminus.Comprehensive structural comparison revealed several

subgroup-specific structural modifications. Based on thestructural similarity, we classified the isoform-specificregion of the EcR-B1 isoform into seven structuraltypes.

1 39-MCEDSRR--DEWSVGGG-----Q-NG---CPSPTMSSNSYDPYSPASKIG

(D/E)(D/E)W A-box

50 amino acids

Corythucha marmorata

Physopelta gutta

Pachygrontha antennata

Heteroptera(Order Heteroptera)

(D/E)(D/E)W A-box

Corythucha marmorataPhysopelta gutta


1

(D/E)(D/E)W + A-box

50 * *: :* *:* * ** * ** ***************::*:*MMGEE-KRTEDDWMVTGGSPRTYQANGGAGFPSPTMSSNSYDPYSPSSKLG

1 39-MCEESRR--DEWTVGGG-----Q-NG---CPSPTMSSNSYDPYSPATKIG

Figure 3 Type-3 A isoform-specific region. The type-3 A isoform-specific region contained two conserved microdomains. (A)Schematic representation of the type-3 A isoform-specific regions.Positions of the (D/E)(D/E) residues and the A-box are indicated bycolored boxes. (B) The alignment and Sequence Logorepresentation of the full-length of the type-3 A isoform-specificregions. The amino acid residues are represented in the defaultcolor scheme of ClustalX. Asterisks indicate identical residues andcolons indicate similar residues.

96122797258148

85112696248135

.* * ** *:.GKGAARSDD---WLANK-TARSDD---WLANK-TTRPDD---WLANK-RPRTDD---WLANK-RPRPDD---WLTNK-RLRIDDTSPWVV

NLS + (D/E)(D/E)W

175191143171164191

151167119147140170

.* *:: ** :: ***.:** : .NSNNGYA--SPMSTSSYDPYSPNSKIAVSNNGYA--SPMSTGSYDPYSPNGKIGVSNNGYA--SPMSTGSYDPYSPNGKIGPSNNGYA--SPMSSGSYDPYSPNGKIGPNNNGYA--SPMSSGSYDPYSPNGKIGLSNMGHTTLSP-TS-SYDSFSP--R-G

A-box

Pheidole megacephalaApis mellifera

Camponotus japonicusNasonia vitripennis

Urocerus antennatusPanorpa pryeri

NLS (D/E)(D/E)W A-box

Pheidole megacephalaApis mellifera

Camponotus japonicusNasonia vitripennis

Urocerus antennatusPanorpa pryeri

4912

1717729

11

1111

:*** : ------------------------------------MDTS-DSSMDTTN----MGDVVLAAPSRLRRAINLSTAMTTTAKTETTTITLSMDTSGDSNMDTDN----------------------------------------MDTSGDSSLDTANRPLQ------------------------------------MDTSQQQQQQQQQQQDP------------------------------------MDTS-EGP-------------------------------------MSSLSIGKVDTSANGLSNHLSATLN

D(T/S)S repeats

D(T/S)S repeats

377031201084

:*.* * LLVKAETPEHLLVKAERPDNLTIKAERPDHGLVKAEAPEQARVKVETPEHSIVKVE-PR-

SUMO

467940291990

SUMO

Apis mellifera

Pheidole megacephala

Camponotus japonicus

Nasonia vitripennis

Urocerus antennatus

Panorpa pryeri

Hymenoptera

50 amino acids

Mecoptera

(D/E)(D/E)W

A-box

SUMO

NLSD(T/S)S repeats

Figure 4 Type-4 A isoform-specific region. The type-4 A isoform-specific region contained four conserved microdomains and conserved N-terminal D(T/S)S repeats. (A) Schematic representation of the type-4 A isoform-specific regions. Positions of the D(T/S)S repeats, the SUMOylationmotif, the NLS, the (D/E)(D/E) residues, and the A-box are indicated by colored boxes. (B) The alignment and Sequence Logo representation ofthe N-terminal region and the conserved microdomains of the type-4 A isoform-specific region. The amino acid residues are represented in thedefault color scheme of ClustalX. Asterisks indicate identical residues and colons indicate similar residues.


Page 5 of 17

Type-1 B1 isoform-specific region (Figure 6)The type-1 B1 isoform-specific region contained threeconserved microdomains (the S-rich motif, the SP resi-dues, and the DL-rich motif). The consensus sequencesof the S-rich motif and the DL-rich motif of the type-1B1 isoform-specific region were GDExSxEVSSSS andDIGEVDLDFWDLDL, respectively. The N-terminalregion and the region between each two conservedmicrodomains varied in length and sequence. The EcR-B1 isoform with the type-1 B1 isoform-specific regionwas identified in non-insect arthropods and non-neop-teran insects.Moreover, the EcR isoforms identified from the filarial

nematode Brugia malayi [EcR-A and -C isoforms (Gen-Bank accession numbers; ABQ28713 and ABQ28714)]contained the S-rich motif-like sequence and the DL-rich motif in the N-terminal A/B domain (amino acidresidues 1-153, both isoforms contained the commonA/B domain; Figure 6C).Type-2 B1 isoform-specific region (Figure 7)The type-2 B1 isoform-specific region contained threeconserved microdomains (the S-rich motif, the SP resi-dues, and the DL-rich motif). In addition, the type-2B1 isoform-specific region generally contained a

partially conserved TxxΨW sequence at the N-termi-nus. The consensus sequences of the S-rich motif andthe DL-rich motif of the type-2 B1 isoform-specificregion were GxESSPEVTSSS and DIGEVDLEFWDLDL,respectively. The EcR-B1 isoform with the type-2 B1isoform-specific region was identified in Polyneopteraand Paraneoptera except for Heteroptera (OrderHemiptera).Type-2’ B1 isoform-specific region (Figure 8)The type-2’ B1 isoform-specific region structurallyresembled to the type-2 B1 isoform-specific region, butthe motif structures were modified. The consensussequences of the S-rich motif and the DL-rich motif ofthe type-2’ B1 isoform-specific region wereEDxxxQVSSS and EVDLELWDLGL, respectively. More-over, the region between the S-rich motif and the DL-rich motif were shortened. In some cases, these twomotifs were completely fused (e.g. EcR-B1 isoforms ofPhysopelta gutta and Pachygrontha antennata). Like thetype-2 B1 isoform-specific region, the type-2’ B1 iso-form-specific region contained the N-terminal TxxΨWsequence. The EcR-B1 isoform with the type-2’ B1 iso-form-specific region was identified in Heteroptera(Order Hemiptera).

196186212226258268206208190253249

SUMO NLS + (D/E)(D/E)W

195174175165195205237247185187169232228

*****.* ****.***.*NMSNGYASPMSAGSYDPYSPTGNMSNGYASPMSAGSYDPYSPTGNMSNGYASPMSAGSYDPYSPTG-MSNGYAS---EGSYDAYSPNGNMSNGYASPMSAGSYDPYSPNGNMSNGYASPMSAGSYDPYSPNGNMSNGYASPMSAGSYDPYSPNGNMSNGYASPMSAGSYDPYSPNGNMSNGYASPMSAGSYDPYSPNGNMSNGYPSPMSAGSYDPYSPNGTMSNGYSSPMSTGSYDPYSPNGTMSNGYSSPLSSGSYDPYSPNG

8787781111191491459799

140140

7777681011091391368789

129129

:* **:. *. AIKIEPMP-DVIAIKIEPMP-DVITIKIEPMP-DVITIKIEPMA-DVITIKTEPMP-DVIAIKTEPMP-DVI-IKTEPMP-DVITIKIEPMP-DVITIKIEPMP-DVI

AVKVEPLPMDTGAVKVEPLPSDTG

------------

Drosophila melanogusterDrosophila simulans

Drosophila yakubaDrosophila ananassaeDrosophila grimshawi

Drosophila mojavensisDrosophila virilis

Drosophila persimilisDrosophila pseudoobscura pseudoobscura

Ceratitis capitataAedes aegypti

Anopheles gambiae

8879999771999

. ------------MLTTSGQQ------------MLTTSGQQ------------MLTSEL-Q-----------MYRLNAIQQ-----------MYRLNATQQ-----------MYRLNATQQ-----------MYRLNATQQ-----------MYRLNAT-------------MYRLNAT--MNNGNNNNNIIALTVN-TLP-----------MYRLNIVST-----------MYRVNIVST

111111111111

149150140174180212222160162142205201

134135125159165197207145147125191187

** * : *:*:*:PNKRPRLDVT---EDWMSTPNKRPRLDVT---EDWMSTPNKRPRIDVT---EDWMSTPNKRARLEVS---EDWMSAPTKRARLDAP---EDWMSTPNKRARLDAP---EDWMSTPNKRARLDAP---EDWMSTPSKRPRLDAP---EDWMSTPSKRPRLDAP---EDWMSTS-KRMRLECPPSNEEWISTSGKR-RLE---SNEEWISSGGKR-RHE---SNEEWISS

A-boxYRLN

YRLN SUMO NLS (D/E)(D/E)W A-box

Drosophila melanoguster

Drosophila yakuba

Drosophila simulans

Drosophila ananassae

Drosophila grimshawi

Drosophila mojavensis

Drosophila virilis

Drosophila persimilis

Drosophila pseudoobscura pseudoobscura

Ceratitis capitata

Aedes aegypti

Anopheles gambiae

NLS

(D/E)(D/E)W

A-box

SUMO

YRLN

50 amino acids

Diptera

Figure 5 Type-5 A isoform-specific region. The type-5 A isoform-specific region contained four conserved microdomains and conserved N-terminal YRLN residues. (A) Schematic representation of the type-5 A isoform-specific regions. Positions of the YRLN residues, the SUMOylationmotif, the NLS, the (D/E)(D/E) residues, and the A-box are indicated by colored boxes. (B) The alignment and Sequence Logo representation ofthe N-terminal region and the conserved microdomains of the type-5 A isoform-specific region. The amino acid residues are represented in thedefault color scheme of ClustalX. Asterisks indicate identical residues and colons indicate similar residues.


Page 6 of 17

Sympetrum infuscatumEphemera strigata

114

1630

3341

S-rich SP + DL-rich� � � � � � � � � � �

50 amino acids

Modi ed S-rich DL-rich

. *: :* ATVTYHELPPLTSW

DEGSXEV--SSS

S-rich consensus

(Type 1)

4 18

DL-rich consensus

(Type 1)

*.LS

:*: *:* * ** RTDV-EID-DNFWAATSF

LDIGEVDLD-FWDLD--

93 110� � � � � � � � � � �

Modi ed S-rich DL-rich

ThysanuraCtenolepisma villosa

Celuca pugilator

Daphnia magna

Thereuopoda clunifera

Nephila clavata

50 amino acids

SPS-rich

Crustacea

Chelicerata

Myriapoda

Apterygota

Insecta

DL-rich

Sympetrum infuscatum

Ephemera strigata

Thermobia domestica

Ephemeroptera

OdonataBasal Pteryogota

S-rich

: . ****: MQ-LEDEGDEEVSSTTPMQRLGAESSPEVSSSSPMQ-MGDEGSLEVSSSSPMQ-MGDEGSPEVSSSSPST-MGDESCSEVSSSSPMT-MGDHG--EVSSSSSMT-MGDVGSPEVSSSQRRS-LGDESSPEVSSSNI

SP

5765

Thermobia domestica 5 20 26 49

DL-rich

** .: **.****::****:*SPQ--PVLSSSSDIAEVDLDFWDLDLNSPN--PTGSSSSDIGEVDLDLWDLDLNSPV--PSLASS-DIGEVDLEFWDLDLNSPV--PSLASS-DIGEVDLEFWDLDLNSPP--TSLASS-DIGEVDLDFWDLDLNSPSDEHSLPSS-DIGEVDLDLWDLDINSPV--RSLVSTPDISEVDLEFWDLDINSPV--PSLAPS-DIGEVDLEFWDLDIN

Ctenolepisma villosaCeluca pugilatorDaphnia magna

Thereuopoda cluniferaNephila clavata

5261158

2041141673

26631392287

498616446110

Figure 6 Type-1 B1 isoform-specific region. The type-1 B1 isoform-specific region contains three conserved microdomains. (A) Schematicrepresentation of the type-1 B1 isoform-specific regions. Positions of the S-rich motif, the SP residues, and the DL-rich motif are indicated bycolored boxes. (B) The alignment and Sequence Logo representation of the conserved microdomains found in the type-1 B1 isoform-specificregion. The amino acid residues are represented in the default color scheme of ClustalX. Asterisks indicate identical residues and colons indicatesimilar residues. (C) The N-terminal region of Brugia malayi EcR contained the S-rich motif-like sequence (modified S-rich motif) and the DL-richmotif. Positions of the modified S-rich motif and the DL-rich motif are indicated in the schematic representation of the N-terminal region of B.malayi EcR (amino acid residues 1-151). The alignment of the microdomains found in B. malayi with their corresponding microdomains in thetype-1 B1 isoform-specific region reveals their sequence similarity. The amino acid residues are represented in the default color scheme ofClustalX. Asterisks indicate identical residues and colons indicate similar residues.

Achetus domesticus

Locusta migratoria

Anisolabis maritima

Nemoura sp.

50 amino acids

TxxΨW

S-rich

SP

DL-rich

Paraneoptera

PolyneopteraOrthoptera

Dermaptera

Plecoptera

Gryllus bimaculatus

Bothrogonia ferruginea

Franklinothrips vespiformis

Liposcelis sp.

Tenodera angustipennis

Reticulitermes speratus

Periplaneta fuliginosa

Oncotympana maculaticollis

Aphrophora pectoralis

Acyrthosiphon pisum

Thysanoptera

Hemiptera(without Heteroptera)

Psocoptera

Dictyoptera

Graptopsaltria nigrofuscata

Graptopsaltria nigrofuscataOncotympana maculaticollis

11111111111

1414

1818

31 49 73

TxxΨW S-rich SP + DL-rich

TxxΨW

----------MTTSIWL-RPGGIPI-----------MTTSIWL-RPGGIPI-----------MTASIWV-RPGGLPV-----------MTT-IWI-RPGIIPM---------------M-L-RLASQNDG---------MMTG-I-L-ARGGCPV-----------MKK-L-ITDRDMTDLY-----------------------MQY----------MSF-MWVSDKH--LQF----------MTS-IWIPS-----------------MTS-VWACGAGGVLGL----------MTS-VWACGAGGVLGLMERGMSVVSAMS--MWSRAGAGVL-L----------MAT--WVAN-----------------MTSTVWARF-------

S-rich

. .**:** MGDESSPEVTSSSAMGDESSPEVTSSSAMGDEGSPEVTSSSA--EDSSSEVTSSSA----SSSEVTSSSS--PESSPEVTSSSS-AEESSPEVTSSCGSGE-GSPEVTSSSGMGDEGSPEVTSSCSMGDEGSPEVTSSCSMGDESSPEVTSSGLMGDESSPEVTSSGLEGCDGSPEVSSSS-DG--SSSEVTSSSLDG-QGVPEVTSSSA

SP DL-rich

::: *** ISPA--HSLGSSDIGEV-DLEFW-DLDLNISPA--HSLGSSDIGEV-DLEFW-DLDLNISPA--HSLGSSDIGEV-DLEFW-DLDLNMSPP--TSLGSSDIDEVVNIDLF-DLDL-NSPM------TRES-----FEFLQDLDDSISP--EHSLGSPDMGGV-DMEFW-DLDQT-------SN--SDFRDV-DLELW-DLDVNISPA--HSLGSSDIGEV-DLEFW-DLDLNISPA--HSHGSSDIGDV-AIEFW-DLDLDISPA--HSLGSSDIGEV-DLEFW-DLDLNISPA--HSLGSSDIGEV-DLDLF-DLDINISPA--HSLGSSDIGEV-DLDLF-DLDINLSPA--HSLTSS-IGEV-DIEFW-DLDINLSPSIASSNA-PDLGEV-YLEFW-DLDLNISPA--HSLGSSDIGEV-DLEFW-DLDLN

Aphrophora pectoralisBothrogonia ferruginea

Acyrthosiphon pisumFranklinothrips vespiformis

Liposcelis sp.Tenodera angustipennisReticulitermes speratus

Periplaneta fuliginosaGryllus bimaculatus

111

Achetus domesticusLocusta migratoria

Anisolabis maritimaNemoura sp. 1

1413101314313815152379

21191421342938312530341619

3134302332464151443843462731

4949454953494976587882484237

7373696677667310082102106716761

Figure 7 Type-2 B1 isoform-specific region. The type-2 B1 isoform-specific region contained three conserved microdomains and the N-terminal TxxΨW sequence. (A) Schematic representation of the type-2 B1 isoform-specific regions. Positions of the TxxΨW sequence, the S-richmotif, the SP residues, and the DL-rich motif are indicated by colored boxes. (B) The alignment and Sequence Logo representation of the N-terminal region and the conserved microdomains of the type-2 B1 isoform-specific region. The amino acid residues are represented in thedefault color scheme of ClustalX. Asterisks indicate identical residues and colons indicate similar residues.


Page 7 of 17

Type-3 B1 isoform-specific region (Figure 9)The type-3 B1 isoform-specific region contained fourconserved microdomains [the (K/R)RRW motif, the S-rich motif, the SP residues, and the DL-rich motif]. Theconsensus sequences of the S-rich motif and the DL-rich motif of the type-3 B1 isoform-specific region wereEESSSEVTSSS and DIGDVDLEFWDLDL, respectively.The EcR-B1 isoform with the type-3 B1 isoform-specificregion was identified in Coleoptera, Strepsiptera, Neu-roptera, Megaroptera, and Raphidioptera.Type-4 B1 isoform-specific region (Figure 10)The type-4 B1 isoform-specific region contained fourconserved microdomains [the (K/R)RRW motif, the S-rich motif, the SP residues, and the DL-rich motif]. Inaddition, the type-4 B1 isoform-specific region containeda conserved LQTVPRVPVAGV sequence just N-termin-ally adjacent to the (K/R)RRW motif. The consensussequences of the S-rich motif and the DL-rich motif ofthe type-4 B1 isoform-specific region were ESSPEVSSSand EDLQLWDLDL, respectively. The EcR-B1 isoformwith the type-4 B1 isoform-specific region was identifiedin the aculeate Hymenoptera and horntails (Family Siri-cidae, Order Hymenoptera). The EcR-B1 isoform identi-fied from two vespine wasps (Vespa mandarinia andAnterhynchium flavomarginatum) contained a S/H-richinsertion between the (K/R)RRW and the S-rich motifs.Type-5 B1 isoform-specific region (Figure 11)The type-5 B1 isoform-specific region structurallyresembled to the type-4 B1 isoform-specific region, but

contained an additional N-terminal extension region.The consensus sequences of the conserved N-terminalsequence, the S-rich motif and the DL-rich motif of thetype-5 B1 isoform-specific region were LAVPRVP-VAGV, ESSPEVSSS, and EDLQLWDLDL, respectively.The EcR-B1 isoform with the type-5 B1 isoform-specificregion was identified in sawflies, the basal lineage ofHymenoptera. Moreover, the isoform-specific region ofthe EcR-B1 isoform identified in Panorpa pryeri (OrderMecoptera) contained an N-terminal extension regionsimilar to that of the type-5 B1 isoform-specific region.The P. pryeri EcR-B1 isoform contained the S-rich motif(amino acid residues 75-83; ESSPEVSSS) and the DL-rich motif (amino acid residues 99-112;ELGEVDLDFWDLDI), which were structurally similarto the S-rich motif of the type-4 B1 isoform-specificregion and the DL-rich motif of type-3 B1 isoform-spe-cific region, respectively.Type-6 B1 isoform-specific region (Figure 12)The type-6 B1 isoform-specific region contained con-served microdomains [the (K/R)RRW motif, the S-richmotif, the SP residues, and the DL-rich motif]. The con-sensus sequence of the S-rich motif of the type-6 B1 iso-form-specific region was EESSSEVTSSS. Although theDL-rich motif of type-6 B1 isoform-specific region wascomposed of acidic residues and bulky hydrophobic resi-dues, its sequence was highly modified [consensus; (D/E)Y(C/G)(E/D)LWxxxxD)]. The EcR-B1 isoform withthe type-6 B1 isoform-specific region was identified inDiptera, Lepidoptera, and Trichoptera.

Phylogenetic evolution of motif structures in the EcRisoform-specific regions in insectsBased on the phylogenetic relationship of insect orders,we constructed evolution models for the structural evo-lution of the isoform-specific region of the EcR isoformsin insects (Figure 13). The structural modifications inthe A isoform-specific region accompanied by insectevolution are represented in Figure 13A. The A iso-form-specific region of non-insect arthropods (type-1 Aisoform-specific region) was composed of four con-served microdomains [the SUMOylation motif, the NLS,the (D/E)(D/E)W residues, and the A-box]. The EcR-Aisoform of direct developing insects acquired two N-terminal sequences (DLKHE and ΨAYRG) (type-2 Aisoform-specific region), which were conserved amongmost direct-developing insect groups except for Hetero-ptera, whose EcR-A isoform lacked the N-terminal halfof the isoform-specific region including the SUMOyla-tion motif and the NLS (type-3 A isoform-specificregion). The two N-terminal sequences and four con-served microdomains were also conserved among mostholometabolous insect groups except for Hymenoptera,Mecoptera and Diptera. The isoform-specific region of

323233

11

Physopelta guttaPachygrontha antennata

Corythucha marmorata 4911

Orius strigicollis

TxxΨW + S-rich + SP + DL-rich

Appasus japonicus

Corythucha marmorata

50 amino acids

TxxΨW

S-rich

SP

DL-rich

Heteroptera(Order Heteroptera)

Physopelta gutta


Orius strigicollis

TxxΨW S-rich SP DL-rich

:*: * * :: **:**. * *****:**---------MTAMWVPR-FGQVSR-ED----QVSSSS---EV-DLELWDLG---------MTAMWVPR-FGQVSR-ED----QVSSSS---EV-DLELWDLG------------MWV-RGY-AM-R-EEG-CDQVTSSCSPG-VEDLELWDLGMRSVKNGSNEVMMWV-RGYAAM-RGEDGCCDQVSSSCSPGVVDDLELWDLG---------MTTLWL-RAGGG--R-DETSCDQVSSSCSPGQV-DLELWELGAppasus japonicus 371

Figure 8 Type-2’ B1 isoform-specific region. The type-2’ B1isoform-specific region contained three conserved microdomainsand the N-terminal TxxΨW sequence. (A) Schematic representationof the type-2’ B1 isoform-specific regions. Positions of the TxxΨWsequence, the S-rich motif, the SP residues, and the DL-rich motifare indicated by colored boxes. (B) The alignment and SequenceLogo representation of the N-terminal region of the type-2’ B1isoform-specific region. The amino acid residues are represented inthe default color scheme of ClustalX. Asterisks indicate identicalresidues and colons indicate similar residues.


Page 8 of 17

***** ------MKRRWAG------MKRRWNG------MKRRWSG------MKRRWS-------MKRRWTG------MKRRWSGMYTFSKMKRRWTG------MKRRWSG

Anthonomus grandisLeptinotarsa decemlineata

Tenebrio molitorTribolium castaneum

Pseudoxenos iwataiHagenomyia micans

Protohermes grandisInocellia japonica

11111111

777677137

2013161216192516

:.*.**:*** VDSSAEVTSSSSSEESSSEVTSSST-EESSSEVTSSSTTEESSSEVTSSS--EESSSEVSSSSTSDESSPEVTSSS--DESSPEVTSSST-DEGSPEVTSSS--

3224282228293626

3928322738354132

:*. ::*.:: *:.:. :* ***:*::SPA------NSLVST-DLGEA-DLEFWDLDLNSPA------NSLASA-DIGDV-DLEFWDLDLNSPA------NSLAST-DIGDV-DLEFWDLDLNSPA------NSLAST-DIGDV-DLEFWDLDLNTPVTHTNNYQTLATT-DISDV-ELAFWDIDMNSPA------NSLAST-DIGDV-DLEFWDLDLNSPA------NSLASTTDIGDVVDLEFWDLDLQSPA------NSLAST-DIGDV-DLEFWDLDIN

6251555067586655

(K/R)RRW S-rich SP + DL-rich

(K/R)RRW S-rich SP DL-rich

Tenebrio molitor

Anthonomus grandis

Leptinotarsa decemlineata

Tribolium castaneum

Pseudoxenos iwatai

Hagenomyia micans

Protohermes grandis

Inocellia japonica

50 amino acids

(K/R)RRW

S-rich

SP

DL-rich

Coleoptera

Strepsiptera

Megaroptera

Neuroptera

Raphidioptera

Neuropterida

Figure 9 Type-3 B1 isoform-specific region. The type-4 B1 isoform-specific region contained four conserved microdomains. (A) Schematicrepresentation of the type-4 B1 isoform-specific regions. Positions of the (K/R)RRW motif, the S-rich motif, the SP residues, and the DL-rich motifare indicated by colored boxes. (B) The alignment and Sequence Logo representation of the conserved microdomains of the type-4 B1 isoform-specific region. The amino acid residues are represented in the default color scheme of ClustalX. Asterisks indicate identical residues and colonsindicate similar residues.

Apis melliferaBombus hypocrita

Xylocopa appendiculataOsmia cornifrons

Coelioxys fenestrataAmmophila infestaVespa mandarinia

Anterhynchium flavomarginatumScolia oculata

Anospilus sp.Amblyjoppa sp.

Eretmocerus eremicusNasonia vitripennis

Urocerus antennatus

** : **:*:**::***. MLQTV-PRVPVAGIKRRWG-MLQTV-PRVPVAGVKRRWG-MLQTM-PRVPVAGVRRRWG-MLQTV-PRVPVAGVRRRWG-MLQTV-PRVPIAGVRRRWG-MLQTV-PRVPVAGVRRRWGTMLQTV-PRVPVAGVRRRWGGMLQTV-PRVPVAGVRRRWG-MLQTM-PRVPVAGVRRRWG-

MLQTM-PRVPVAGVRRRWGTMLQTM-PRVPVAGVRRRWG-ML-A-QPRLPVAGVKRRWASMLQTMQPRVPVAGVKRRWGGMLQTV-PRVPMAGVKRRWGG

111111111

11111

181818181819191818

1918182019

252525252528624225

2525272824

** * .****: :*. LQ------E-SSPEVSSSGVLSPPPLQ------E-SSPEVSSSGVLSPPPLQ------E-SSPEVSSSGVLSPPPLQ------E-SSPEVSSSGVLSPPLLQ------E-SSPEVSSSGVLSPPPLQ------E-SSPEVSSSGVLSPPPLQ------E-SCPEVSSSGVLSPPPLQ------E-SSPEVSSSGVLSPPPLQ------ETSSPEVSSSVVLSPPP

LQ------E-SSPEVSSSGVLSP--LQ------E-SSPEVSSSGVLSPPPLQPSPAHGE---SEVSSSGMLSPPPLQ------E-SSSEVSSSGVLSPPPLQ------E-SSPEVSSTGVLSPPP

424242424245795943

4042484541

5757575757611068361

5558846555

* *******: * EE-----LQLWDLDLH-RGEE-----LQLWDLDLH-RGEE-----LQLWDLDLH-RGEE-----LQLWDLDLH-RGEE-----LQLWDLDLH-RGED-----LQLWDLDLQ-RGED-----LQLWDLDLH-RGED-----LQLWDLDLH-RGED-----LQLWDLDLH-RG

ED-----LQLWDLDLH-RGED-----LQLWDLDLQ-RGEGSPGNGAQLWDLDLHGRQED-----PQLWDLDLQPRQED-----LQLWDLDLQ-RG

6969696969731189573

Pheidole megacephala MLQTM-PRIPVAGVRRRWS-1 18 25 LQ------E-SSPEVSSSDTFSS-- 40 53 ED-----LQLWDLDLH-RG 6567701027867

LQTVPRVPVAGV

LQTVPRVPVAGV + (K/R)RRW S-rich + SP DL-rich

(K/R)RRW S-rich SP DL-rich

Apoidea

Apis mellifera

Bombus hypocrita

Xylocopa appendiculata

Osmia cornifrons

Coelioxys fenestrata

Ammophila infesta

Vespa mandarinia

Anterhynchium flavomarginatum

Scolia oculata

Pheidole megacephala

Campsomeris schulthessi

Anospilus sp.

Amblyjoppa sp.

Eretmocerus eremicus

Nasonia vitripennis

Urocerus antennatus

50 amino acids

SP

DL-richLQTVPRVPVAGV

(K/R)RRW

S-rich

Suborder Apocrita(Order Hymenoptera)

Ichneumonoidea

Vespoidea

Campsomeris schulthessi MLQTM-PRVPVAGVRRRWG-1 18 24 LQ------ETSSPEVSSSEVLSPPA 42 61 ED-----LQLWDLDLH-RG 73

Chalcidoidea

Siricoidea

Aculeata

Suborder Symphyta(Order Hymenoptera)

Figure 10 Type-4 B1 isoform-specific region. The type-4 B1 isoform-specific region contained four conserved microdomains and the N-terminal LQTVPRVPVAGV sequence. (A) Schematic representation of the type-4 B1 isoform-specific regions. Positions of the LQTVPRVPVAGVsequence, the (K/R)RRW motif, the S-rich motif, the SP residues, and the DL-rich motif are indicated by colored boxes. (B) The alignment andSequence Logo representation of the N-terminal region and the conserved microdomains of the type-4 B1 isoform-specific region. The aminoacid residues are represented in the default color scheme of ClustalX. Asterisks indicate identical residues and colons indicate similar residues.


Page 9 of 17

the hymenopteran/mecopteran EcR-A isoform and thedipteran EcR-A isoform lacked the two N-terminalsequences, but contained the subgroup-specificsequences: The A isoform-specific region of the hyme-nopteran/mecopteran EcR-A isoform (type-4 A isoform-specific region) contained the D(S/T)S repeats, and thatof the dipteran EcR-A isoform generally contained theYRLN sequence at the N-terminus. In addition,although the isoform-specific region of the lepidopteranEcR-A isoform structurally classified as the type-2 A iso-form-specific region, it lacked the NLS.The structural modifications in the B1 isoform-specific

region that accompanies insect evolution are repre-sented in Figure 13B. All types of the B1 isoform-speci-fic region contained three conserved microdomains: theS-rich motif, the SP residues, and the DL-rich motif (theB1 isoform-specific region of several heteropteraninsects did not contain the SP residues). The DL-richmotif of the type-6 B1 isoform-specific region was struc-turally modified and showed a limited sequence similar-ity with the corresponding motif of other insects.Interestingly, although Mecoptera is thought to be a sis-ter taxon of Diptera [17], the P. pryeri EcR-B1 isoformhad a DL-rich motif quite similar to the correspondingmotif of the type-3 B1 isoform-specific region.In addition to the three conserved microdomains, the

isoform-specific region of the EcR-B1 isoform contained

subgroup-specific N-terminal sequences. The EcR-B1isoform of polyneopteran and paraneopteran insects(types 2 and 2’ B1 isoform-specific region) generally con-tained the N-terminal TxxΨW sequence. In the holome-tabolous insects, the TxxΨW sequence was replaced withthe (K/R)RRW motif, a novel conserved microdomain atthe N-terminal end of the holometabolous insect EcR-B1(types 3-6 B1 isoform-specific region). The hymenop-teran/mecopteran EcR-B1 isoform contained the N-term-inal LQTVPRVPVAGV (or LAVPRVPVAGV) sequencejust N-terminally adjacent to the (K/R)RRW motif (types4 and 5 B1 isoform-specific region). Moreover, the EcR-B1 isoform of the basal hymenopteran insects (sawflies)and the mecopteran insect had the N-terminal extensionregion (type 5 B1 isoform-specific region).

DiscussionGenerally, the nuclear receptor A/B domain contains theligand-independent AF-1 transactivation domain that isinvolved in the interaction with transcriptional cofactors[18,19], and some nuclear receptors have multiple iso-forms with different A/B domains that exhibit isoform-specific transcriptional regulation and physiological func-tions [20-23]. In the present study, we performed a struc-tural analysis of the isoform-specific A/B domain of theEcR-A and -B1 isoforms. Our data revealed the presenceof the conserved microdomain structures as well as the

Cimbex femoratusArge similis

Allantus luctiferTenthredo fagiPanorpa pryeri

:. * . :. * :**** *:* ::* MLVT-CPSVGSRLV-LVAKDCTLGGTVAS-DPSNH-----------------KVQEIAEMLAVPRV--------------------PVAGVKRRWGGMLGSLFP-GGSSVAALVAANGSAIEDI-SEEPTTATTTTNPPPKTPPPMLVVKVDE-GEMIAVPRV--------------------PVAGVKRRWG-MLVT-WPSGGSGLV-LLAKDSGL-DTLESEDTGSD-----------------KTGETAEMLAVPRV--------------------PVAGVKRRWGGMLVT-WPSGGSSLV-LVAKDSTFQ----SSDPSSD-----------------KIGETAEMLAVPRV--------------------PVAGVKRRWGG------------MVIKKAR-AVFPDDFMNEESM-------------------KLGPC----SVPRVKKEAAESSSRHASSSSGSLQPIA--RKRA--

11111

5773575457

N-terminal extension + LAVPRVPVAGV+ (K/R)RRW

N-terminal extension LAVPRVPVAGV (K/R)RRW

6278625975

********* ESSPEVSSSGESSPEVSSSGESSPEVSSSGESSPEVSSSGESSPEVSSSQ

7187716884

7389737089

:** * .: * : *:.:****:: LSPPPNSLPAVPECAMNAEDLQLWDLDLQ-RGLSPPPHFHPAVPENAVHAEDLHLWDLDLQ-RGLSPPPNFHPATPECAVNTEDLQLWDLDLQ-RGLSPPPNFHPSTPECAVNADDIQFWDLDLQQRGISPVPSLNSS--E--LGEVDLDFWDLDIN---

103119103101113

Cimbex femoratusArge similis

Allantus luctiferTenthredo fagiPanorpa pryeri

S-rich SP + DL-rich

S-rich SP DL-rich

Cimbex femoratus

Arge similis

Allantus luctifer

Tenthredo fagi

Panorpa pryeri

50 amino acids

Sawflies(Order Hymenoptera, suborder Symphyta )

Mecoptera

S-rich

SP

DL-rich

N-terminal extension

LAVPRVPVAGV

(K/R)RRW

Figure 11 Type-5 B1 isoform-specific region. The type-5 B1 isoform-specific region contained four conserved microdomains, the N-terminalLAVPRVPVAGV sequence, and the N-terminal extension region. (A) Schematic representation of the type-5 B1 isoform-specific regions. Positionsof the N-terminal extension, the LAVPRVPVAGV sequence, the (K/R)RRW motif, the S-rich motif, the SP residues, and the DL-rich motif areindicated by colored boxes. (B) The alignment and Sequence Logo representation of the N-terminal region and the conserved microdomains ofthe type-5 B1 isoform-specific region. The amino acid residues are represented in the default color scheme of ClustalX. Asterisks indicateidentical residues and colons indicate similar residues.


Page 10 of 17

insect subgroup-specific structural variations in the iso-form-specific region in the A/B domain of each EcR iso-form. The conserved microdomain structures in the EcRisoform-specific regions might be the fundamental struc-tural basis for the EcR isoform-specific AF-1 functions.The A isoform-specific region generally contained the

SUMOylation motif, the NLS, the (D/E)(D/E)W resi-dues, and the A-box. Among these structural motifs, the(D/E)(D/E)W residues and the A-box were highly con-served in the C-terminal half of the A isoform-specificregion across arthropod species, suggesting that thesetwo microdomains are the essential structures for the Aisoform-specific AF-1. Moreover, the NLS was con-served among all species except for Heteroptera andLepidoptera. The NLS in the isoform-specific region ofDrosophila EcR-A isoform is involved in nucleo-cyto-plasmic shuttling in mammalian cells [24]. Our datasuggest that the isoform-specific region-mediated

nuclear localization mechanism of the EcR-A isoform isconserved among most species.The A/B domain of Drosophila EcR-A isoform exhi-

bits weak transactivation activity in Drosophila Kc167cells [12], whereas it exhibits transcriptional inhibitionactivity in HeLa cells [13]. In the red flour beetle T. cas-taneum, the EcR-A isoform initiates ecdysone responsesby regulating the expression of downstream 20E-response genes, indicating that the EcR-A isoform func-tions as a transactivating factor. In the N-terminal halfof the A isoform-specific region, we found a conservedSUMOylation motif. SUMOylation is a reversible post-translational modification involved in various cellularprocesses, such as nucleo-cytoplasmic shuttling andtranscriptional regulation [25-27]. SUMOylation of tran-scription factors generally results in the repression oftranscriptional activation activity [28]. The high level ofconservation of the SUMOylation motif in the A

Chilo suppressalisManduca sexta

Stenopsych marmorata

Drosophila melanogasterDrosophila simulans

Drosophila yakubaDrosophila ananassaeDrosophila grimshawi

Drosophila mojavensisDrosophila virilis

Drosophila persimilisDrosophila pseudoobscura pseudoobscura

Calliphora vicinaLucilia cuprina

Ceratitis capitataAedes albopictus

Bombyx moriSpodoptera frugiperdaOmphisa fuscidentalis

1111111111111111111

27272727272727272830302930876728282728

:**..* * * * : MSPSSLDSHDYCDQDLWLCGNEMSPSSLDSHDYCDQDLWLCGNEMSPSSLDSHDYCDQELWLCGNEMSPSSLDSHDYCDQELWLCGNDMSPSSLDSHDYGEPEMWLCGNDMSPSPLDSPDYGDQGSWLCGNDMSPSSLDSHDYGEQELWLCGNDMSPSSLDSHDYCDQDLWLCGNDMSPSSLDSHDYCDQDLWLCGNDMSPSSLDSPVYGDQDMWLC-NDMSPSSLDSPVYGDQEMWLC-NDLSPSPLDSPVYGDQDLW--YD-MSPNSLGSPNYDELELWSSYEDMSPESLASPEYRALELW-SYDDMSPESLASPEYGGLELW-GYEDMSPESLASPEYSNLELW-TYEDMSPESLASPEYAGLELW-GYDDMSPESLASPEYGGLELW-SYDEMSPESLGSPEYGEIELW-GYDE

37373737

3840403736977738383737

37373737

58585858

59606055571179758585757

58585858

*:*** *** * ::: ::**:**:*** -------------------------------------------------------------MKRRWSNNGGF--MRLPEESSSEVTSSSN-------------------------------------------------------------MKRRWSNNGGF--MRLPEESSSEVTSSSN-------------------------------------------------------------MKRRWSNNGGF--MRLPEESSSEVTSSSN-------------------------------------------------------------MKRRWSNNGGF--MRLPEESSSEVTSSSN-------------------------------------------------------------MKRRWSNNGGF--LRIQEESSSEVTSSSN-------------------------------------------------------------MKRRWSNNGGF--LRIHEESSSEVTSSSN-------------------------------------------------------------MKRRWSNNGGF--LRIQEESSSEVTSSSN-------------------------------------------------------------MKRRWSNNGGF--LRVPEESSSEVTSSSN------------------------------------------------------------MMKRRWSNNGGF--LRVPEESSSEVTSSSN------------------------------------------------------------MMKRRWSNNGGFAALKMLEESSSEVTSSSN------------------------------------------------------------MMKRRWSNNGGFAALKMLEESSSEVTSSSN------------------------------------------------------------MMKRRWSNNGGF-ALRMLEESSAEVSSSSN------------------------------------------------------------MMKRRWSNNGGFTALRMLDDSSSEVTSSSAMRVENVDNVSFALNGRADEWCMSVETRLDSLVREKSEVKAYVGGCPS-VITDAGAYDALFDMRRRWSNNGGF-PLRMLEESSSEVTSSS----------------------MSIESRLDSLVRGKSEVKAFTGGCPSALV-DTGACDTLADMRRRWYNNGGFQTLRMLEESSSEVTSSS--------------------------------------------------------------MRRRWSNNGGFQTLRMLEESSSEVTSSS--------------------------------------------------------------MRRRWSNNGGFQTLRMLEESSSEVTSSS--------------------------------------------------------------MRRRWSNNGCF-PLRMFEESSSEVTSSS--------------------------------------------------------------MKRRWSNNGGFQTLRMLEDSSAEVSSSS-

(K/R)RRW + S-rich SP + Modi ed DL-rich

(K/R)RRW S-rich SP Modi ed DL-rich

50 amino acids

Drosophila melanogaster

Drosophila yakuba

Drosophila simulans

Drosophila ananassae

Drosophila grimshawi

Drosophila mojavensis

Drosophila virilis

Drosophila persimilis

Drosophila pseudoobscura pseudoobscura

Calliphora vicina

Lucilia cuprina

Ceratitis capitata

Aedes albopictus

Bombyx mori

Spodoptera frugiperda

Omphisa fuscidentalis

Chilo suppressalis

Manduca sexta

Stenopsych marmorata

Lepidoptera

Diptera

Trichoptera

SP

Modi ed DL-rich

(K/R)RRW

S-rich

Figure 12 Type-6 B1 isoform-specific region. The type-6 B1 isoform-specific region contained four conserved microdomains. The DL-richmotif was structurally modified in the type-6 B1 isoform-specific region (termed as modified DL-rich motif). (A) Schematic representation of thetype-6 B1 isoform-specific regions. Positions of the (K/R)RRW motif, the S-rich motif, the SP residues, and the modified DL-rich motif are indicatedby colored boxes. (B) The alignment and Sequence Logo representation of the conserved microdomains of the type-6 B1 isoform-specific region.The amino acid residues are represented in the default color scheme of ClustalX. Asterisks indicate identical residues and colons indicate similarresidues.


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Figure 13 Structural diversity of the EcR isoform-specific region in insects. (A) Evolution model for the structural modification in the Aisoform-specific region in insects. (B) Evolution model for the structural modification in the B1 isoform-specific region in insects. Schematicrepresentation shows the phylogenetic relationship of insect subgroups with the structural types of the isoform-specific region. Conservedmicrodomains are indicated by colored boxes.


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isoform-specific region suggests that it is functionallyimportant. SUMOylation of the A isoform-specificregion might contribute to the cell-type/organism-dependent transcriptional regulation mediated by the A/B domain of the EcR-A isoform.All types of the B1 isoform-specific region contained

the S-rich and the DL-rich conserved motifs. The iso-form-specific region of the holometabolous insect EcR-B1 isoform (types 3-6 B1 isoform-specific region) con-tained an additional (K/R)RRW motif at the N-terminus.In Drosophila, strong transactivation activity wasmapped at the N-terminal end of the A/B domain ofthe EcR-B1 isoform (amino acid residues 1-53) [13],which includes all three conserved motifs. Moreover,the S-rich motif-like sequences and the DL-rich motifare found in the A/B domain of the ecdysone receptoridentified from the filarial nematode B. malayi. Thehigh level of conservation of the S-rich and DL-richmotifs across ecdysozoan species suggests that thesemotifs are the essential structures for the B1 isoform-specific AF-1 function.The length and sequences of the linker regions

between microdomains, as well as the distribution ofmicrodomains in the isoform-specific region variedacross species. For example, the B1 isoform-specificregion of the non-insect arthropod EcR-B1 isoforms(type-1 B1 isoform-specific region) contained a long lin-ker region between the S-rich and the DL-rich motifs(e.g., Daphnia magna EcR-B1 contained an 137-aminoacid linker region). In the holometabolous insects, thethree conserved motifs [the (K/R)RRW, the S-rich, andthe DL-rich motifs] were contained in the N-terminalhalf of the B1 isoform-specific region, and the linkerregions between the motifs were shortened (e.g. type-6B1 isoform-specific region). The isoform-specific regionof the EcR-B1 isoform identified in vespoid wasps con-tained a long S/H-rich insertion between the holometa-bolous insect-specific (K/R)RRW motif and the S-richmotif. These data suggest that the conserved microdo-mains in the EcR isoform-specific regions are structu-rally, and even functionally, independent of each other.In vitro and in silico structural analysis for the A/B

domain of the Drosophila EcR isoforms revealed thatthe N-terminal region of the Drosophila EcR-B1 iso-form-specific region, which contains the conservedmicrodomains, is structurally disordered [29]. Theintrinsically disordered AF-1 region was found in manyother nuclear receptors, such as the androgen receptorand the glucocorticoid receptor [30-34]. The intrinsicdisordered regions are found in a lot of proteinsinvolved in protein-protein interactions [35,36] and thatthe nuclear receptor AF-1 region is involved in cofactorrecruitment and transcriptional regulation. Thus, theconserved microdomains found in the EcR isoform-

specific region might function as signature motifs and/or target sites for the EcR isoform-specific transcriptioncofactors.The A/B domain of the Drosophila EcR-B1 isoform

activates transcription even in yeast and mammaliancells [13,37,38], therefore it is likely that the AF-1 regionof the EcR-B1 isoform activates transcription throughmechanisms conserved among eukaryotic organisms.Among the conserved microdomains contained in theDrosophila EcR-B1 isoform-specific region, the DL-richmotif, which comprises tandem repeats of acidic resi-dues (D and E) and aromatic/bulky hydrophobic resi-dues (W, A, F, I, L, M, and V), was structurally similarto the acidic activation domain (Figure 14A). Acidicactivation domains are found in viral and cellular tran-scription factors such as VP16, GAL4 and p53 [39-42],as well as in the AF-1 region of several nuclear

Direct-developing insect EcR-B1 AF-1

Co-regulatory proteins

essential for EcR-B1 AF-1

Direct-developing insect EcR-B1 AF-1

Holometabolous insect-specific

Co-regulatory protein

Holometabolous insect EcR-B1 AF-1

(K/R)R

RW

S-ric

h

DL-

rich

DL

-ric

h

S-rich

447477

437466

* :D--ALD--DG--ALDMAD

DIGEVDL-DDIGEVDL-E---EVDL-EDIGDVDL-E---E-DL-Q---E-DL-Q

: :: F-DLDMF-EFEQ

FWDLDLFWDLDLLWDLGLFWDLDLLWDLDLLWDLDL

Type 1 Type 2 Type 2' Type 3 Type 4 Type 5

DL-rich motif

consensus

Acidic activator

domains of VP16

Figure 14 Hypothetical model of the B1 isoform-specific regionmediated transcriptional regulation. (A) Sequence comparisonbetween two acidic activator domains of herpes virus protein VP16and the consensus sequences of the DL-rich motif of the EcR-B1isoform. Two acidic activation domains of VP16 (GenBank accessionnumber; NP_044650) were aligned with the consensus sequences ofthe DL-rich motifs of the type 1-5 B1 isoform-specific regions. TheDL-rich motif of EcR-B1 isoform and the VP16 acidic activationdomains comprise tandem repeats of acidic residues and aromatic/bulky hydrophobic residues. The amino acid residues arerepresented in the default color scheme of ClustalX. Asterisksindicate identical residues and colons indicate similar residues. (B)Schematic model of the action of the EcR-B1 isoform-specific AF-1.The S-rich and DL-rich motifs might interact with co-regulatoryproteins essential for B1 isoform-specific AF-1. The holometabolousinsect EcR-B1 isoform might have acquired a novel interactionpartner(s) in the holometabolous insect-specific (K/R)RRW motif.


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receptors [43,44]. Further studies, such as protein-pro-tein interaction studies, will shed light on the transcrip-tional regulation mechanism of the EcR-B1 isoform-specific AF-1.In the present study, we discovered that the N-termini

of holometabolous insect EcR-B1 isoforms contain anovel conserved (K/R)RRW motif, which is lacking inthe direct-developing insects. The (K/R)RRW motif wasfound in the N-terminal region of the EcR-B1 isoformidentified in Hymenoptera, the most basal lineage ofholometabolous insects, suggesting that the (K/R)RRWmotif was acquired in association with the evolution ofholometabolous insects. Strikingly high conservation ofthe (K/R)RRW motif across holometabolous insect spe-cies suggests its importance in the AF-1 function of theholometabolous insect EcR-B1 isoform. Taken together,we hypothesize that the holometabolous insect EcR-B1isoform acquired a novel transcriptional regulationmechanism that is mediated by the (K/R)RRW motif(Figure 14B).

ConclusionsA comprehensive structural comparison of the N-term-inal isoform-specific region in the A/B domain of theEcR-A and -B1 isoforms in insects revealed severalmicrodomain structures that are conserved acrossinsects or acquired/modified in a specific-subgroup ofinsects. These microdomains might be the essentialstructural basis for the EcR isoform-specific AF-1 trans-activation functions by serving as binding sites for co-regulatory proteins or as post-translational modificationsites. The novel (K/R)RRW motif in the isoform-specificregion of the holometabolous insect EcR-B1 isoformmight provide additional interaction sites for co-regula-tory proteins and mediate the holometabolous insect-specific regulation of the B1 isoform-specific AF-1 trans-activation function. Identification of binding partners ofthe EcR isoform-specific regions and structural analysisof the interaction interface will provide further insightinto the evolutionary importance of the structural modi-fications of the EcR isoform-specific AF-1 regions.

MethodsAnimalsWe collected 51 species of insects and non-insectarthropods for EcR cDNA cloning. Some of them werecollected in the field and others were purchased. Theircollection sites are listed in Additional file 3, Table S3.Franklinothrips vespiformis, Orius strigicollis and Eret-mocerus eremicus were purchased from Arysta Life-Science (Tokyo, Japan). Colonies of Apis mellifera werepurchased from Kumagaya bee farm (Saitama, Japan).Pupae of Osmia cornifrons were purchased from MasonBee Research Institute (Sendai, Miyagi, Japan). Pupae of

Nasonia vitripennis were purchased from Nosan cor-poration (Tsukuba, Ibaraki, Japan). Gryllus bimaculatus,Acheta domestica and Thermobia domestica were pur-chased from local pet stores.

RNA isolation and reverse transcriptionTissues were dissected in ice-cold phosphate-bufferedsaline (138 mM NaCl, 3 mM Na2HPO4, 10 mMNaH2PO4, pH 7.4), and immediately homogenized inTRIzol reagent (Invitrogen, Carlsbad, CA) or stored inRNA later reagent (Ambion, Austin, TX) at -20°C untiluse. Total RNA was isolated with TRIzol reagent andtreated with DNase I (TaKaRa, Shiga, Japan) at 37°C for1 h. For standard reverse transcription-polymerase chainreaction (RT-PCR) experiments, total RNA (1 μg) wasreverse transcribed using Superscript III reverse tran-scriptase (Invitrogen) with a 3’ rapid amplification of thecDNA end (3’ RACE) adapter packaged in the First-Choice RLM-RACE kit as a primer. For 5’ RACE, totalRNA was modified using the FirstChoice RLM-RACEkit and reverse transcribed using Superscript III reversetranscriptase with a random decamer as a primer.

Cloning of cDNA fragments that encode the A/B and Cdomains of EcR isoformsFirst, we cloned cDNA fragments encoding the Cdomain and the C-terminal region of the A/B domain ofEcR isoforms by RT-PCR. Then, we performed 5’ RACEusing the FirstChoice RLM-RACE kit (Ambion) todetermine translation initiation site of the gene. Finally,we cloned cDNA fragments encoding the full length ofthe A/B domain of EcR isoforms. The obtained cDNAsequences were registered with GenBank. Detailedexperimental procedures are described below.Generally, we performed nested or semi-nested PCR

to amplify a desired cDNA fragment in the RT-PCR andthe 5’ RACE experiments. First, PCR was performedusing either KOD-plus polymerase (ToYoBo, Osaka,Japan) or TaKaRa LA Taq (TaKaRa), and nested PCRwas performed using either TaKaRa Ex Taq (TaKaRa)or TaKaRa LA Taq. The amplified DNA fragmentswere subcloned into the plasmid vector pGEM-T easy(Promega, Madison, WI), and the nucleotide sequenceswere determined using ABI PRISM 3100 Genetic Analy-zer (Applied Biosystems, Foster City, CA). Thesequences of the degenerate/consensus primers used forthe partial cDNA cloning are listed in Additional file 3,Table S3. The sequences of the primers used to amplifya cDNA encoding the full-length of the A/B domain ofEcR isoforms are listed in Additional file 4, Table S2.Cloning of cDNA fragments encoding the EcR C domainTo obtain the partial cDNA fragments encoding theecdysone receptor (EcR) C domain, we performedreverse transcription polymerase chain reaction (RT-


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PCR) with primers designed on the basis of the highlyconserved amino acid residues among the insect EcR Cdomain (EELCLVCGD and TCEGCKGFF for forwarddegenerate primers, MYMRRKCQE and VGMRAECVfor reverse degenerate primers).Cloning of cDNA fragments encoding the C-terminal regionof the A/B domain of EcR-B1 isoform from insects and non-insect arthropods expect for Hemiptera, Hymenoptera, andTrichopteraTo obtain the cDNA fragment encoding the C-terminalregion of the A/B domain of EcR-B1 isoform frominsects and non-insect arthropods other than Hemipteraand Hymenoptera, we performed RT-PCR with a for-ward consensus primer designed on the basis of theconserved nucleotide sequence among three Coleop-teran EcR-B1s (GenBank accession numbers: CAL25731,CAA72296, and BAD99297) and two Crustacean EcRs(GenBank accession numbers: AAC33432 andBAF49033). The reverse gene-specific primers weredesigned on the basis of the obtained cDNA sequenceencoding the C domain. To determine the translationinitiation site, we performed 5’ RACE with gene-specificprimers designed on the basis of the obtained partialcDNA encoding the A/B domain of EcR-B1 isoform.Cloning of cDNA fragments encoding the C-terminal regionof the A/B domain of EcR-B1 isoform from HymenopteraWe designed an alternative forward primer on thebasis of the consensus nucleotide sequence conservedamong three hymenopteran insects. Briefly, we per-formed a genomic tBlastn search with an amino acidsequence of the Pheidole megacephala EcR-B1 isoform(GenBank accession number: BAE47510) against thegenomic DNA sequences of Nasonia vitripennis andApis mellifera to predict the DNA sequences encodingthe A/B domain of EcR-B1 isoform. We then designedtwo forward consensus primers on the basis of theconserved nucleotide sequence encoding the A/Bdomain of hymenopteran insect EcR-B1 isoform. Wesimultaneously designed a reverse consensus primer onthe basis of the conserved nucleotide sequence encod-ing the C domain of EcR. To obtain the partial cDNAfragment encoding the C-terminal region of the A/Bdomain of EcR-B1 isoform from Hymenoptera, we per-formed RT-PCR with the forward and reverse consen-sus primers.Cloning of cDNA fragments encoding the C-terminal regionof the A/B domain of EcR-B1 isoform from Hemiptera andTrichopteraBecause the forward degenerate primer described abovedid not work for hemipteran and trichopteran EcR-B1gene, we performed 5’ RACE with reverse gene-specificprimers designed on the basis of the obtained cDNAsequence encoding the C domain of EcR.

Cloning of cDNA fragments encoding the C-terminal regionof the A/B domain of EcR-A isoformTo obtain the cDNA fragment encoding the A/Bdomain of EcR-A isoform from arthropods other thanHymenoptera, we performed 5’ RACE with gene-specificprimers designed on the basis of the obtained partialcDNA sequence encoding the C domain of EcR.To obtain cDNA fragment encoding EcR-A isoform

from hymenopteran insects, we first designed a forwardconsensus primer encoding the C-terminal region of theA isoform-specific region. The forward consensus pri-mer for hymenopteran EcR-A isoform was designed onthe basis of the conserved nucleotide sequences of threehymenopteran EcR-A isoforms (GenBank accessionnumbers: XP_001602885, XP_394760, BAF79665, andBAE47509). We then performed RT-PCR with the for-ward and reverse consensus primers to obtain the par-tial cDNA encoding the C-terminal of the A/B domainof EcR-A isoform.5’ RACETo determine a translation initiation site of the gene, weperformed 5’ RACE using the FirstChoice RLM-RACEkit (Ambion) according to the manufacturer’s protocol.The reverse gene-specific primers were designed basedon the obtained cDNA encoding the isoform-specificregion of EcR isoforms.Cloning of cDNA fragments encoding the full-length of theA/B domain of EcR isoformsTo obtain a cDNA fragment encoding the full-length A/B domain of EcR isoforms, we designed a forward gene-specific primer at the 5’ untranslated region (UTR) ofthe gene. Reverse gene specific primers were designedon the basis of the obtained cDNA corresponding to theC domain of EcR. To clone A. mellifera EcR isoforms,we designed a reverse gene-specific primer at the 3’UTR of the gene on the basis of the predicted full-length cDNA encoding EcR-A isoform (GenBank acces-sion number: XM_394760).

Sequence comparisonGenBank accession numbers of EcR-A and -B1 isoformsused for sequence comparison are listed in Additionalfile 1, Table S1 and Additional file 2, Table S2, respec-tively. The deduced amino acid sequences of the iso-form-specific region of EcR-A and -B1 isoforms werealigned with the ClustalX 2.0 program [45] or MAFFT6.0 program [46], and then manually adjusted. Predic-tion of potential SUMOylation sites was performedusing SUMOsp 2.0 program [47]. The Sequence Logosand the consensus sequences were generated using theGeneious 4.8 program [48] to represent sequence con-servation. All alignment files were supplemented inFASTA format (Additional file 5).


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Additional file 1: Table S1. Taxa used in the structural comparison ofthe EcR-A isoform-specific region. These files can be viewed with:CLUSTAL X.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2148-10-40-S1.PDF ]

Additional file 2: Table S2. Taxa used in the structural comparison ofthe EcR-B1 isoform-specific region. These files can be viewed with:CLUSTAL X.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2148-10-40-S2.PDF ]

Additional file 3: Table S3. Degenerate/consensus primers used forcDNA cloning. These files can be viewed with: CLUSTAL X.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2148-10-40-S3.PDF ]

Additional file 4: Table S4. Gene-specific primers used for cDNAcloning and the collection sites of animals. These files can be viewedwith: CLUSTAL X.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2148-10-40-S4.PDF ]

Additional file 5: Sequence alignments of the isoform-specificregions of the EcR-A and -B1 isoforms. Aligned sequences of the fivetypes of the A isoform-specific regions and seven types of the B1isoform-specific regions. These files can be viewed with: CLUSTAL X.Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2148-10-40-S5.ZIP ]

AcknowledgementsThis work was supported by the Program for Promotion of Basic ResearchActivities for Innovative Bioscience (PROBRAIN) and Grant-in-Aid from theMinistry of Education, Science, Sports.

Authors’ contributionsTW conceived of the study, collected specimen, carried out the molecularcloning studies and the sequence alignment and drafted the manuscript. TKhelped to draft the manuscript. TH advised on the manuscript. All authorsread and approved the final manuscript.

Received: 21 August 2009Accepted: 12 February 2010 Published: 12 February 2010

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