Báo cáo khoa học: Molecular cloning of the ecdysone receptor and the retinoid X receptor from the scorpion Liocheles australasiae pot

Abbreviations EcR, ecdysone receptor; EcRE, ecdysone response element; 20E, 20-hydroxyecdysone; LaEcR, Liocheles australasiae ecdysone receptor; LaEcR-A, Liocheles australasiae ecdysone

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the retinoid X receptor from the scorpion

Liocheles australasiae

Yoshiaki Nakagawa, Atsushi Sakai, Fumie Magata, Takehiko Ogura, Masahiro Miyashita

and Hisashi Miyagawa

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

The largest phylum in the animal kingdom, the

Arthropoda, is subdivided into two subphyla – the

Mandibulata and the Chelicerata; the former includes

the classes Insecta and Crustacea; and the latter

includes the class Arachnida, which contains the

scor-pions, ticks and spiders among others Scorpions are

ancient arachnids that originated some 420 million years ago during the Silurian period (Paleozoic era) The evolutionary relationship between the various groups is shown in the form of a phylogenetic tree of Arthropoda in Fig 1 To date, some 1600 scorpion species in 14 families have been identiﬁed and they are

Keywords

ecdysone receptor (EcR); Liocheles

australasiae; retinoid X receptor (RXR);

scorpion; ultraspiracle (USP)

Correspondence

Y Nakagawa, Division of Applied Life

Sciences, Graduate School of Agriculture,

Kyoto University, Kyoto 606-8502, Japan

Fax: +81 75 7536123

Tel: +81 75 7536117

E-mail: naka@kais.kyoto-u.ac.jp

(Received 13 June 2007, revised 9 October

2007, accepted 11 October 2007)

doi:10.1111/j.1742-4658.2007.06139.x

cDNAs of the ecdysone receptor and the retinoid X receptor were cloned from the Japanese scorpion Liocheles australasiae, and the amino acid sequences were deduced The full-length cDNA sequences of the L austra-lasiae ecdysone receptor and the L australasiae retinoid X receptor were

2881 and 1977 bp in length, respectively, and the open reading frames encoded proteins of 560 and 414 amino acids The amino acid sequence of the L australasiae ecdysone receptor was similar to that of the ecdysone receptor-A of the soft tick, Ornithodoros moubata (68%) and to that of the ecdysone receptor-A1 of the lone star tick, Amblyomma americanum (66%), but showed lower similarity to the ecdysone receptors of Orthoptera and Coleoptera (53–57%) The primary sequence of the ligand-binding region

of the L australasiae ecdysone receptor was highly homologous to that of ticks (85–86%) The amino acid sequence of the L australasiae retinoid X receptor was also homologous to the amino acid sequence of ultraspiracles

of ticks (63%) and insects belonging to the orders Orthoptera and Coleop-tera (60–64%) The identity of both the L australasiae ecdysone receptor and the L australasiae retinoid X receptor to their lepidopteran and dip-teran orthologs was less than 50% The cDNAs of both the L australasiae ecdysone receptor (L australasiae ecdysone receptor-A) and the L austra-lasiaeretinoid X receptor were successfully translated in vitro using a rabbit reticulocyte lysate system An ecdysone analog, ponasterone A, bound to

L australasiaeecdysone receptor-A (KD¼ 4.2 nm), but not to L australa-siae retinoid X receptor The L australasiae retinoid X receptor did not enhance the binding of ponasterone A to L australasiae ecdysone

receptor-A, although L australasiae retinoid X receptor was necessary for the bind-ing of L australasiae ecdysone receptor-A to ecdysone response elements

Abbreviations

EcR, ecdysone receptor; EcRE, ecdysone response element; 20E, 20-hydroxyecdysone; LaEcR, Liocheles australasiae ecdysone receptor; LaEcR-A, Liocheles australasiae ecdysone receptor A-isoform; LaRXR, Liocheles australasiae retinoid X receptor; PonA, ponasterone A; RXR, retinoid X receptor; USP, ultraspiracle.

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represented around the world [1,2] Although scorpions

molt like insects and crustaceans, the hormonal

regula-tion of the molting process and details of the molting

mechanism are not clear In insects, the physiology of

molting and metamorphosis has been intensively

stud-ied and the role of the molting hormone,

20-hydroxy-ecdysone (20E), at the molecular level has been well

established 20E is the ligand that binds to a

hetero-dimeric receptor complex made up of two proteins, the

ecdysone receptor (EcR) and the retinoid X receptor

(RXR) homolog ultraspiracle (USP) This complex,

upon binding to the ecdysone response element

(EcRE), transactivates the various genes involved in

the molting process [3,4] On the other hand, in

crusta-ceans, 20E has an inhibitory role, unlike its

stimula-tory role in insects [5] To date, about 30 EcRs and

USPs (or RXRs) have been characterized primarily in

insects, along with several in other arthropod species

(http://www.ncbi.nlm.nih.gov/) It is generally thought

that RXR orthologs of Lepidoptera and Diptera are

USPs, although other arthropods have RXRs, based

upon their sequence homologies These USPs and RXRs

have similar roles In Orthoptera, it was shown that the

RXR can be replaced with the USP of other insects

[6,7] EcRs, USPs and RXRs are members of the steroid

and thyroid hormone receptor superfamily and their

sequences consist of regions referred to as A⁄ B

(transactivation domain), C (DNA-binding domain),

D (hinge region) and E⁄ F (ligand- or hormone-binding domain) [8,9] The X-ray crystal structures of the ligand-binding domains of EcR, USPs and RXRs have been resolved in a few insects [10–13], and the binding of ponasterone A (PonA) to EcR has been shown [12,13] Previously, we determined the cDNA sequences of the EcRs and the USPs (RXRs) of Chilo suppressalis [14] and Leptinotarsa decemlineata [15] Dissociation constants of the binding of PonA to these receptors have been determined using an in vitro translated EcR⁄ USP (RXR) heterodimer, as well as other crude molting hormone receptor proteins [16–18] The afﬁn-ity of PonA for EcR is dramatically enhanced in the presence of USP [14,15] We also measured the activity

of various ecdysone agonists by measuring their bind-ing ability to in vitro translated EcR⁄ USP heterodi-mers [14,15,19] and found that the ligand-binding afﬁnity to the receptor is affected by the structure of EcR [20] Therefore, the elucidation of EcR and USP (RXR) structures is important for understanding the molecular mechanism of the action of 20E

In this study, we report the cloning of cDNAs for EcR and RXR from an ancient terrestrial arachnid, the Japanese scorpion Liocheles australasiae as the ini-tial step towards understanding the molting process in this species We also studied the binding of a molting hormone analog, PonA, to the in vitro translated receptor proteins – L australasiae EcR (LaEcR) and

L australasiae RXR (LaRXR) – as well as to the ecdysone response element (EcRE), and the results are presented here

Results

cDNA cloning of LaEcR and LaRXR

A 379-bp fragment was ampliﬁed by RT-PCR using degenerate primers (Table 1) designed from the highly

Arthropoda

Chelicerata Arachinida Scorpiomorpha Acaromorpha

Mandibulata Insecta Crustacea

Fig 1 Phylogeny of Arthropoda.

Table 1 Degenerate primers used in this study a

a

N means a mixture of A, T, G and C In the same way, D (A, G, T), H (A, C, T), K (G, T), M (A, C), R (A, G), S (C, G), W (A, T) and Y (C, T) means a mixture of deoxynucleoside.

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conserved regions of the DNA- and ligand-binding

domains of several insect EcRs, and the nucleotide

sequence was converted to an amino acid sequence

The deduced amino acid sequence from the PCR

prod-uct was similar to the corresponding EcR region of

ar-thropods Subsequently, we determined the full length

of the cDNA sequence by 5¢-RACE and 3¢-RACE By

combining the sequences of the PCR fragments, we

were able to establish the full length of the cDNA

sequence as 2861 bp The longest ORF encoded 539

amino acids A blast search (http://www.ncbi.nlm

nih.gov/BLAST/) showed that the deduced amino acid

sequence was analogous to the EcR-A of the soft tick

Ornithodoros moubata(accession number: AB191193.1)

as shown in Table 2 Therefore, we decided that this

sequence represented the LaEcR A-isoform

(LaEcR-A) In a similar manner, we cloned the full length

1977-bp cDNA sequence, and deduced the 410-amino

acid sequence from the cDNA sequence We decided

that this sequence corresponded to the LaRXR These

sequences have been submitted to DDBJ⁄ EMBL ⁄

GenBank under the accession numbers AB297929

(LaEcR-A) and AB297930 (LaRXR) The amino acid

sequence alignment indicated that this EcR

polypep-tide included the entire A⁄ B (1–187), C (188–253),

D (254–317), E (318–536) and F (537–539) regions (numbers in parentheses indicate the ﬁrst and last amino acids of the primary sequence of the proteins) The F-region, which exists in the Drosophila EcR and other mammalian nuclear receptors, was very small (three amino acids: IQE) in LaEcR LaRXR is also constructed from A⁄ B (1–87), C (88–153), D (154–182) and E (183–410) regions The C-regions of EcRs and USPs are highly conserved However, other regions, particularly the N-terminal parts of USP⁄ RXR, vary The alignments of amino acid sequences of the A⁄ B and

E regions of LaEcR-A and LaRXR with those of other arthropods are shown in Fig 2

We compared the deduced amino acid sequences of LaEcR-A and LaRXR with those of EcRs and USPs (RXRs) from other species (Tables 2 and 3) LaEcR-A

is most similar to the EcR-A of O moubata (68%), and LaRXR is most similar to the RXR of Locusta migratoria (64%) The identity of LaRXR with RXRs

of other arthropods such as Orthoptera and Coleop-tera is relatively high (> 60%), but less than 50% when compared with the USP sequences from Lepi-doptera and Diptera Interestingly, the identity of LaRXR to the RXRa of Homo sapiens is relatively high (63%) We also compared A⁄ B, C, D and E

Table 2 Identities of amino acid sequences of EcR-A isoforms

against that of LaEcR-A (%).

Species

Length (amino acids)

Identity against LaEcR-A (%)a

A ⁄ B region C region D region E region Total Ornithodoros

moubata b

Amblyomma

americanum c

Blattella germanica d 570 26 100 48 66 54

Locusta migratoriae 541 25 98 48 67 53

Tribolium castaneum f 549 26 100 48 68 54

Leptinotarsa

decemlineatag

Drosophila

melanogaster k

Chironomus tentans l 536 23 89 41 55 43

Chilo suppressalis o 518 23 89 36 54 44

a Identity values were not calculated for the F regions of EcRs

because most of them are too short for sequence comparison.

b Accession number AB191193.1 c Ref [22] d Ref [39] e Ref.

[40] f Accession number AM295015.1 g Ref [15] h Accession

number AJ251542.1.iRef [41].jRef [42].kRef [43].lRef [44].

m Ref [45] n Ref [46] o Ref [33].

Table 3 Identities of amino acid sequences of USPs (RXRs) against that of LaRXR (%).

Species

Length (amino acids)

Identity against LaRXR (%)a

A ⁄ B region C region D region E region Total Amblyomma

americanum b

Blattella germanica c 436 28 89 75 69 63 Locusta migratoria d 411 28 89 75 71 64 Tribolium castaneum e 407 28 91 75 64 61 Leptinotarsa

decemlineata f

Drosophila melanogaster j 508 28 91 31 46 48 Chironomus tentansk 552 32 89 34 40 44

Chilo suppressalis n 410 31 92 45 43 45

a Identity values were not calculated for the F regions of EcRs because most of them were too short for sequence comparison.

b Accession number AF305213.1 c Ref [7] d Ref [47] e Ref accession number AM295015.1.fRef [15].gRef accession num-ber AJ251542.1 h Ref [48] i Ref [49] j Ref [50] k Ref [44] l Ref [51] m Ref [52] n Ref [21] o Ref [53].

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regions of EcRs and USPs (RXRs) among several

spe-cies It showed that the C region of EcRs is highly

conserved among all species (89–100%), but the amino

acid sequences of E regions varied among the species The sequence of the E region of LaEcR-A is highly analogous to that of O moubata EcR (OmEcR; 86%)

51 167 119 132

42 62 78 74 3 52 120 79 28 10

125 134 141 113

53

56 51 49

158 234 290 186 198

187 196 178 179

117

114 115 112

L australasiae EcR-A

O moubata EcR-A

A americanum EcR-A1

B germanica EcR-A

L migratoria EcR-A

T castaneaum EcR-A

L decemlineata EcR-A

T molitor EcR-A

A mellifera EcR-A

A aegypti EcR-A

D meelanogaster EcR-A

C tentans EcR-A

M sexta EcR-A

B mori EcR-A

C suppressalis EcR-A

O moubata EcR-A

B germanica EcR-A

T molitor EcR-A

A mellifera EcR-A

A aegypti EcR-A

C tentans EcR-A

M sexta EcR-A

B mori EcR-A

O moubata EcR-A

B germanica EcR-A

T molitor EcR-A

A mellifera EcR-A

A aegypti EcR-A

C tentans EcR-A

M sexta EcR-A

B mori EcR-A

A

435 456 465 442 374 566 518 436 395

O moubata EcR-A

B germanica EcR-A

T molitor EcR-A

A mellifera EcR-A

A aegypti EcR-A

C tentans EcR-A

M sexta EcR-A

B mori EcR-A

B

535 563 556 536 559 562 670 511 495

O moubata EcR-A

B germanica EcR-A

T molitor EcR-A

A mellifera EcR-A

A aegypti EcR-A

C tentans EcR-A

M sexta EcR-A

B mori EcR-A

Fig 2 Alignment of the primary sequences of (A) A ⁄ B regions of EcRs, (B) E regions of EcRs, (C) A ⁄ B regions of USPs and RXRs, and (D)

E regions of USPs and RXRs Alignments were performed using the CLC FREE WORKBENCH 4.0.1 (CLC bio A ⁄ S) In the alignment figure (C) the amino acid residues that correspond to those important for the binding of PonA to the EcR of H virescens are boxed The arrow head indi-cates the 396th amino acid of LaEcR-A, which is unique to LaEcR-A.

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and Amblyomma americunum EcR (AmaEcR; 85%),

and moderately analogous to those of Orthoptera and

Coleoptera (65–69%) The identity of the A⁄ B regions

of EcRs and USPs (RXRs) are not as high as the

iden-tity for the C and E regions (< 41%)

In vitro translation of LaEcR-A and LaRXR LaEcR-A and LaRXR were translated using an

in vitrotranscription⁄ translation kit (rabbit reticulocyte lysate), with 35S-labelled methionine ([35S]Met), and

L australasiae RXR

H sapiens RXR

C suppressalis USP

B mori USP

M sexta USP1

C tentans USP

D melanogaster USP

A aegypti USP-A1

A mellifera RXR

T moritor RXR

L decemlineata RXR

T castaneum RXR

L migratoria RXR

B germanica RXR1

A americanum RXR1

52 50 55 51 31 44 73 48 118 76 76 23

H sapiens RXR

B mori USP

M sexta USP1

C tentans USP

A aegypti USP-A1

A mellifera RXR

T moritor RXR

T castaneum RXR

L migratoria RXR

B germanica RXR1

A americanum RXR1

87 79 94 85 67 109 137 103 196 113 137 59

C

H sapiens RXR

B mori USP

M sexta USP1

C tentans USP

A aegypti USP-A1

A mellifera RXR

T moritor RXR

T castaneum RXR

L migratoria RXR

B germanica RXR1

A americanum RXR1

268 258 292 264 241 265 333 339 401 305 251

H sapiens RXR

B mori USP

M sexta USP1

C tentans USP

A aegypti USP-A1

A mellifera RXR

T moritor RXR

T castaneum RXR

L migratoria RXR

B germanica RXR1

A americanum RXR1

369 358 393 365 342 363 444 459 512 413 361 419

D

H sapiens RXR

B mori USP

M sexta USP1

C tentans USP

A aegypti USP-A1

A mellifera RXR

T moritor RXR

T castaneum RXR

L migratoria RXR

B germanica RXR1

A americanum RXR1

410 436 411 407 408 427 484 552 461 462 462

Fig 2 (Continued).

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subjected to SDS⁄ PAGE (Fig 3) The molecular

masses of LaEcR-A and LaRXR were estimated to be

63 and 51 kDa, respectively, from the band shifts in

electrophoresis, and they were consistent with the

values (60.8 kDa for LaEcR-A and 46.3 kDa for

LaRXR) calculated from the amino acid sequences

The extra bands of lower molecular mass are probably

degradation products of the full-length proteins

Specific binding of PonA to an in vitro translated

protein

We measured the binding afﬁnity of ligands for the

in vitro translated receptor proteins (LaEcR-A and

LaRXR) using3H-labelled ponasterone A ([3H] PonA)

The speciﬁc binding of in vitro-translated LaEcR-A and

LaEcR-A⁄ LaRXR proteins to PonA was calculated as

the difference between the total binding and nonspeciﬁc

binding, as previously reported [14,15] As shown in

Fig 4, PonA bound to LaEcR-A, but not to LaRXR

The speciﬁc binding of LaEcR-A was not increased in

the presence of LaRXR These results are in contrast to

the insect receptors where the speciﬁc binding of PonA

to EcR was markedly increased in the presence of USP

(RXR) [14,15]

In further experiments, the dissociation equilibrium

constant, KD, for the binding of PonA to LaEcR-A

alone and to the LaEcR-A⁄ LaRXR heterodimer, was

calculated from the saturation curve of speciﬁc binding

using a nonlinear model (Fig 5) The KD values of

LaEcR-A and LaEcR-A⁄ LaRXR were determined to

be 4.2 and 3.2 nm, respectively, and the difference between these KDvalues was not signiﬁcant

Gel mobility shift assay of LaEcR and LaRXR Binding of LaEcR-A and LaRXR to EcRE was tested

by the gel mobility shift assay We had previously shown that EcR⁄ USP (RXR) bound to pal1 and hsp27 EcRE [15,21] We also found in this study that the LaEcR-A⁄ LaRXR heterodimer bound to these seq-uences, as shown in Fig 6 LaEcR-A alone did not bind

to pal1 and hsp27 in the absence of LaRXR PonA did not signiﬁcantly affect the binding of the LaEcR-A⁄ LaRXR heterodimer or of LaEcR-A alone to both pal1 and hsp27 LaRXR alone did not bind to pal1 and hsp27 Our results are similar to those reported for

L decemlineata EcR (LdEcR)⁄ L decemlineata USP (LdUSP) [15]

Discussion

We have successfully cloned cDNAs for EcR-A and RXR from L australasiae using a PCR protocol that

we had standardized for our earlier studies [14,15] Deduced amino acid sequences of EcR and RXR of

L australasiae were homologous to those from ticks that are also arachnids and a member of the subphylum Chelicerata (Fig 1) Even though three EcR isoforms [22] and two USP (RXR) isoforms [23] were found for

A americanum, only a single pair of cDNAs for EcR and RXR could be ampliﬁed in L australasiae by using our method We could not isolate LaEcR B-isoforms

LaEcR-A

LaRXR

148kDa

98kDa

64kDa

Free [ 35 S] methonine 36kDa

LaRXR 51kDa

LaEcR-A 63kDa 50kDa

22kDa

Fig 3 SDS ⁄ PAGE of in vitro translated LaEcR-A and LaRXR

pro-teins pET-23a(+) vector (lane1), LaEcR (lane 2), LaRXR (lane 3) and

LaEcR ⁄ LaRXR (lane 4) were incubated with [ 35 S]Met The + and )

signs indicate the presence and absence, respectively, of

corre-sponding proteins In vitro translation of proteins was conducted

using a TNT T7 Quick Coupled Transcription ⁄ Translation System

(Promega), according to the manufacturer’s protocol.

6000

3 H] PonA binding (dpm)

4000

2000

0

N

−

LaEcR-A LaRXR −

Fig 4 Binding of ponasterone A to the in vitro-translated LaEcR-A and LaRXR The radioactivity of the precipitate collected in the filter was measured using a liquid scintillation counter In vitro-translated LaEcR-A and LaRXR were incubated with [ 3 H]PonA in the presence

or absence of excess unlabeled PonA T, total binding; N, nonspe-cific binding; + and – indicate the presence and absence, respec-tively, of corresponding proteins The vertical bars show the standard deviation of three replicates.

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from L australasiae It is well known that amino acid

sequences of the A⁄ B region from EcRs and USPs

(RXRs) are diverse However, sequences of A⁄ B regions

from EcR-As were relatively conserved among species

in the same order (Fig 2A) The A⁄ B region of nuclear

receptors is thought to be the transactivation domain

There may be a speciﬁc transactivation system that is

common in the same taxonomic order of arthropods

The A⁄ B regions of USPs (RXRs) were moderately

sim-ilar among insects, as shown in Fig 2C Because the

A⁄ B regions of USPs (RXRs) are shorter than those of

EcRs, it appears that the sequence similarity among

A⁄ B regions of all insect USPs (RXRs) is higher than

that of EcRs (Fig 2A,C) However, the identity among

RXR A⁄ B regions is low, except in the C-terminal area (Fig 2C) In mammalian RXRs, AF-1 ligand-indepen-dent activation of transcription activity mediated by the

A⁄ B region through its phosphorylation was reported [24,25] It is known that some protein kinases have pro-line-directed function Therefore, it is interesting that the amino acid residues at the regions of USPs (RXRs) showing identity are prolines

We also compared the amino acid sequences of the

E region of EcR-As (Fig 2B), and those of USPs and RXRs (Fig 2D) The E regions of EcRs were consider-ably conserved among all species This suggests that the EcR⁄ USP (RXR) system regulates the development of

L australasiae with 20E On the other hand, the USP (RXR) sequences were diverse compared with EcR sequences, although some parts of the sequence were conserved The E regions of nuclear receptors are also thought to be involved in transactivation The con-served sequences among the E regions of USPs (RXRs) may be related to regulation of the transcription The similarity of LaEcR-A and LaRXR with other EcRs and USPs (or RXRs) were compared (Table 2) The identity of LaEcR-A and LaRXR to those of archinids was highest, followed by those to Orthoptera (Blattodea) and Coleoptera, as well as Crustacea The C-region sequences of 14 EcRs were also highly con-served among several species, as shown in Table 2 In the C region, there are two zinc ﬁnger regions contain-ing a P-box and a D-box, which are important for DNA recognition [26] The P-box of LaEcR is 100% identical to that of other EcRs as well as USPs (RXRs) The D-box is 100% identical to that of crabs, ticks and orthopteran insects, and is also highly homologous to that of Coleoptera (100% to Tenebrio molitor, 80% to

L decemlineata) However, it shows only 40% identity with the D-boxes of Lepidoptera and Diptera Ortho-ptera is geologically one of the oldest orders in Insecta,

LaEcR-A/LaRXR

Concentration (nM)

K D = 3.2 n M

LaEcR-A

K D = 4.2 n M

3H] PonA binding (dpm)

3H] PonA binding (dpm) 0 1000 2000

3000 4000

3000 2000 1000 0

Concentration (nM)

Fig 5 The affinity of PonA for (A) LaEcR-A and (B) LaEcR-A ⁄ LaRXR In vitro translated proteins were incubated with various concentrations

of [ 3 H]PonA Specific binding was determined at the various [ 3 H]PonA concentrations to derive the curves as the difference of the radioactiv-ity in the presence and absence of nonradioactive PonA (10 l M ) The K D values of PonA to LaEcR-A alone and to LaEcR-A ⁄ LaRXR hetero-dimer were evaluated by nonlinear regression using PRISM software (Graphpad Software Inc.).

LaEcR-A

LaRXR

PonA

pET-23a(+)

+ +

-+

+ +

-+ + +

-+ +

-+

+ +

-+ + +

-Bound

Free

probe

Fig 6 Binding of LaEcR-A and LaEcR-A ⁄ LaUSP to the ecdysone

response element (EcRE) In vitro translated proteins were

incubated with 32 P-labelled hsp27 or pal1 and then analyzed on a

nondenaturating polyacrylamide gel.

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originating in the Carboniferous period (Paleozoic era)

and Coleoptera appeared later in the lower Permian

period (Paleozoic era) Diptera appeared still later in

the Permian period, while Lepidoptera appeared even

later than that, during the Jurassic period (Mesozoic

era) The result obtained in this study is consistent with

the phylogenetic relationship

The E region of LaEcR-A is most similar to that of

OmEcR (86%) Although the E region of LaEcR-A is

very similar to those of insect EcR-As, the similarity

of LaEcR-A to archnid EcR-A is deﬁnitely high, as

shown in Table 2 It is thought that the E-region

sequence is very important in determining the binding

afﬁnity of EcR to ligand molecules [19] Therefore, the

difference of EcR E-region structures between arachnid

and insect is related to the recognition of the structure

of ligand molecule by EcRs LaEcR-A may have

unique ligand selectivity compared with insect EcRs

As shown in Fig 6, LaEcR-A alone binds strongly to

PonA, and LaRXR does not enhance the binding This

is different from the case of EcRs and USPs (RXRs)

of insects, and such a unique characteristic may be

dependent on the E-region structure of LaEcR-A

Because the nuclear receptor proteins are often used

as the gene switch, the ligand-binding afﬁnity of

LaEcR-A, which is not enhanced by LaRXR, is

expected to be interesting Ecdysone and its agonists,

together with their receptors, are present only in

arthropods and are relatively nontoxic to plants and

mammals Also, plant steroid hormones, such as

bras-sinolide and castasterone, and the mammalian

steroi-dal hormone, estradiol, do not bind to ecdysone

receptor [27,28] Therefore, the ecdysone–receptor

complex can be safely used for studying various

aspects of genetic engineering in plants and

mam-mals [4] For example, the Choristoneura fumiferna

EcR (CfEcR)⁄ Locusta migratoria RXR (LmRXR)

cas-sette, together with luciferase as a reporter gene placed

under the GAL4 response element and the )46 34S

minimal promoter, was successfully turned on by an

ecdysone agonist, resulting in the expression of the

luciferase gene in plants and protoplasts [29]

Further-more, this cassette regulated the expression of a

Super-man-like single zinc ﬁnger protein 11 (ZFP11) in both

Arabidopsis and transgenic tobacco plants [30] In

addition, the EcR gene switch was successfully tested

in a mammalian cell system [31] The unique

character-istics of LaEcR-A and LaRXR may precisely control

gene regulation and contribute to various studies such

as functional genomics, gene therapy, therapeutic

pro-tein production and tissue engineering

Although LaRXR is required for the strong binding

of LaEcR-A to EcRE, it has no effect on the binding

of PonA to LaEcR-A Because the main role of recep-tors is to activate the particular gene responding to the ligand binding, it is generally thought that the hetero-dimerization of receptor proteins is required for the ligand binding However, this study indicates that the heterodimerization between USP (RXR) and EcR may

be more important for the DNA binding than for ligand binding

The taxonomic similarity among different species of arthropods was examined by constructing phylogenetic trees using clc free workbench 4.0.1 (CLC bio A⁄ S, Aarhus, Denmark) for full-length sequences of EcR and USP (RXR) (Fig 7) EcR and RXR of scorpions are similar to those of crabs and ticks, and are placed

in a different group separate from the insects The

‘USP’ of L australasiae was deduced from a PCR product obtained using degenerate primers designed on the basis of the C region of insect USPs, but it turned out to be closer to RXR and not USP Therefore, it was designated as LaRXR Interestingly, human RXR

is also highly homologous to LaRXR (63%) Because

it is known that mammalian RXRs have a couple of functions, LaRXR may work alone rather than in a EcR⁄ RXR heterodimer system

Previously, we reported the speciﬁc binding of PonA

to in vitro translated EcR and EcR⁄ USP heterodimers

of a lepidopteran C suppressalis [14] and a coleopteran

L decemlineata[15] In these species, the speciﬁc bind-ing of PonA to EcR was signiﬁcantly enhanced in the presence of USP The heterodimerizing effect of USP on ligand–receptor binding is common to the EcR⁄ USP heterodimers of insects However, as reported in this study, the binding of PonA to

LaEcR-A is not affected by the addition of LaRXR in L aus-tralasiae The KD value (4.2 nm) for the binding

of PonA to LaEcR-A is comparable to that for the binding of EcR⁄ USP heterodimers such as C suppres-salis EcR (CsEcR)⁄ C suppressalis USP (CsUSP) (KD

1.2 nm) [14], L decemlineata (LdEcR)⁄ L decemlineata (LdUSP) (KD 2.8 nm) [32], and D melanogaster EcR (DmEcR)⁄ D melanogaster USP (DmUSP) (KD

0.85 nm) [15] The KDvalues for the binding of PonA

to CsEcR alone and to LdEcR alone were 55 and

73 nm, respectively, which are signiﬁcantly larger (lower afﬁnity) than for LaEcR alone Recently, an X-ray crystal structure of the EcR ligand-binding domain⁄ USP ligand-binding domain of Heliothis vires-cens with PonA was solved In the analysis of the EcR⁄ ligand-binding domain ⁄ PonA complex, amino acid residues of H virescens EcR (HvEcR), which are important for the binding with PonA, were shown Most of these residues were conserved in LaEcR-A, with the exception of 396T of LaEcR-A (Fig 2) The

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corresponding residues of other EcR-As were

lipo-philic This difference may affect the strong binding

afﬁnity of LaEcR-A alone to PonA

Even though EcR and USP have been characterized

in a tick, A americanum, the molting mechanism in

the subphylum Chelicerata, which includes the scorpi-ons, ticks and spiders, is not completely understood The presence of EcR and USP homologs in scorpions suggests that the molting is regulated by ecdysteroids Unlike insects there is no cooperative interaction

A mellifera EcR-A

M sexta EcR-A

P megacephala EcR-A

T castaneum EcR-A

B germanica EcR-A

T molitor EcR-A

B mori EcR-A

A aegypti EcR-A

O moubata EcR-A

D magna EcR-A1

D melanogaster EcR-A

C tentans EcR-A

100 100 100

100

100 99

82 100

100 90 100 100

90 63 100

A americanum RXR1

M musculus RXRα1

D magna RXR

A mellifera RXR

B mori USP

M sexta USP1

S depilis RXR

T castaneum RXR

A aegypti USP-A1

L migratoria RXR

B germanica RXR1

100

C pugilator RXR

G lateralis RXRα

H sapiens RXRα

Xenos pecki RXR

100

95 100

100

100 66

71

31 19

53

T molitor RXR

100 100

100 99

C tentans USP

A

B

Fig 7 Phylogenetic tree constructed using the primary sequences of (A) EcRs and (B) USPs (RXRs) References for sequences are shown

in Tables 2 and 3 unless noted otherwise Other EcRs and RXRs were obtained either from references or from the NCBI website EcR-A of Pheidole megacephala (AB194765.1); EcR-A1 of Daphnia magna (AB274820.1); RXR of Xenos pecki [34], Daphnia magna [35], Celuca pugila-tor [36] and Gecarcinus lateralis [37]; and RXRa1 of Mus musculus [38] and Scaptotrigona depilis (DQ190542.1) Unrooted neighbour-joining (NJ) trees were prepared using CLC Free Workbench 4.0.1 (CLC bio A ⁄ S) A bootstrap value is attached to each branch, and the value is a measure of the confidence in this branch The number of replicates in the bootstrap analysis is adjusted to 100.

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between EcR and RXR in terms of binding to PonA

in L australasiae, although LaRXR is needed for the

binding of LaEcR-A to EcRE If LaEcR-A functions

alone as a receptor protein, another appropriate EcRE,

different from pal1 and hsp27, may be required for the

binding of LaEcR-A

In conclusion, cDNAs of EcR and RXR were

success-fully cloned from the Japanese scorpion L australasiae

and the deduced amino acid sequences were similar to

their counterparts in the tick A americanum Among

insect species, orthopteran insects such as L migratoria

and Blattella germanica were more similar to L

austra-lasiae, in terms of molting hormone receptor proteins,

than lepidopteran and dipteran insects, which are

phylo-genetically younger An ecdysone agonist, PonA,

speciﬁ-cally bound to the in vitro translated LaEcR-A alone

with high afﬁnity, and this PonA⁄ LaEcR-A binding was

not enhanced in the presence of RXR The dissociation

constant, KD, for the binding of PonA to LaEcR-A was

determined to be 4.2 nm, which was similar to that for

insect EcR⁄ RXR(USP) heterodimers

Experimental procedures

Chemicals

Tritiated PonA ([3H]PonA, 150 CiÆmmol)1) was purchased

from American Radiolabeled Chemicals Inc (St Louis,

MO, USA) PonA was from Invitrogen Corp (Carlsbad,

CA, USA)

Isolation of RNA from L australasiae

The scorpions, L australasiae, were collected on Ishigaki

Island located at the southern end of the Ryukyu island

chain in Japan A scorpion whole body (0.37 g) was frozen

in liquid nitrogen and transferred to a glass homogenizer,

then homogenized in 0.5 mL of TRIzol (Gibco BRL,

Grand Island, NY, USA) Total RNA was isolated using

an acid guanidinium thiocyanate⁄ phenol ⁄ chloroform

method described previously [14,15] The concentrations

and purity of RNA were determined by spectrophotometry

Poly (A)-rich RNA was puriﬁed from the total RNA using

an mRNA Puriﬁcation Kit (Amersham Bioscience Corp.,

Piscataway, NJ, USA) for the RACE method The

concen-tration of RNA was determined using a UV spectrometer

Reverse transcription

cDNA was synthesized from total RNA by RT, using a

ReadyÆToÆGoTM T-Primed First-Strand Kit (Amersham

Bioscience Corp.) A total RNA solution (3 lL) prepared

from a whole scorpion was added and incubated for 10 min

at 65C, then immediately cooled on ice This RNA solu-tion was added to the ReadyÆToÆGoTM T-Primed First-Strand Kit, which was prewarmed to 37C, and incubated for 5 min at 37C After mixing gently with a pipette, the reaction mixture was incubated for 60 min at 37C to obtain the ﬁrst-strand cDNA

PCR using degenerate primers The ﬁrst-strand cDNA prepared from RNA was ampliﬁed

by PCR using the degenerate primers listed in Table 1 Three forward and three reverse degenerate primers were designed for LaEcR based on amino acid sequences con-served in the C and E regions of other EcRs (Table 3) and are identical to those used for cDNA cloning of the EcR

of L decemlineata [15] The ﬁrst PCR was performed using EcR-F1 and EcR-R1 (94C ⁄ 2 min; 35 cycles of

92C ⁄ 1 min, 48 C ⁄ 1 min, 72C ⁄ 1 min; and 72C ⁄ 10 min) To conduct the second and third PCRs (nested PCR), EcR-F2⁄ R2 and EcR-F3 ⁄ R3 were used for PCR at 52 C and 46C, instead of 48 C, for annealing The presence of the cDNA product was resolved by agarose gel electropho-resis Other PCR protocols used are identical to those we previously reported [15,21,33] The degenerate primers RXR-F1 and RXR-R1 (Table 1) were used for the first PCR of cDNA of RXR, and the RXR-F2 and RXR-R1 primers were used for the second PCR (nested PCR) To confirm unidentified sequences of the 3¢-terminus after the stop codon, we performed another PCR by designing new primers (RXR-F3 and RXR-R3) The annealing tempera-ture was set as 48C and 46 C, respectively

RACE Poly (A)-rich RNA was subjected to 5¢- and 3¢-RACE with

a SMARTTM RACE cDNA ampliﬁcation kit (Clontech, Palo Alto, CA, USA) For both EcR and RXR, two reverse primers for 5¢-RACE, and two forward primers for 3¢-RACE, were designed (Table 1) The 5¢-RACE for EcR was performed by PCR with the primer EcR-RR1, and the 3¢-RACE for EcR was performed with the primer EcR-RF1, according to the manufacturer’s instructions Both the 5¢-RACE and the 3¢-RACE were followed by a nested PCR using EcR-RR2 (annealing temperature: 66C) and EcR-RF2 (66C) primers, respectively In the same way, the 5¢-RACE for RXR was executed with RXR-RR1, and the 3¢-RACE for RXR was executed with RXR-RF1 Each RACE reaction was followed by a nested PCR using RXR-RR2 (68C) and RXR-RF2 (68 C), respectively

DNA sequencing and sequence analysis PCR products were puriﬁed by agarose gel electrophoresis and cloned into the pGEM-T Easy vector (Promega,

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