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

Molecular cloning of the ecdysone receptor and
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:
(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-
lasiae retinoid X receptor were successfully translated in vitro using a rabbit
reticulocyte lysate system. An ecdysone analog, ponasterone A, bound to
L. australasiae ecdysone receptor-A (K
D
¼ 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.
FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS 6191
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
( 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
Acaromor
p
ha
Mandibulata
Insecta
Crustacea
Fig. 1. Phylogeny of Arthropoda.
Table 1. Degenerate primers used in this study.
a
LaEcR LaRXR
Primers for PCR F1 WSNGGNTAYCAYTAYAAYGC F1 ATHTGYGGNGAYMGNGC
F2 GARGGNTGYAARGGNTTYTT F2 GGNAARCAYTAYGGNGTNTA
F3 TGMGNMGNAARTGYCARGARTG F3 GATTCAGATCCCGACCATAAAGA
R1 TCNSWRAADATNRCNAYNGC R1 TCYTCYTGNACNGCYTC
R2 CATCATNACYTCNSWNSWNSWNGC R2 CAYTTYTGRTANCKRCARTA
R3 AAYTCNACDATNARYTGNACNGT R3 GCAAGCTGGAAAAGAGTAATGTGAC
Priners for 5¢-RACE RR1 AGACTCCCGTTTGATGGCACACTG RR1 ATACTGGCAGCGATTCCTTTGAC
RR2 GCATTCCGACACTGAGGCACTTTT RR2 AGCCTTTACAACCTTCACAGC
Primers for 3¢-RACE RF1 GAAAAAGTGCCTCAGTGTCGGAATG RF1 ATAGCTGTGAAGGTTGTAAAGG

RF2 CAGTGTGCCATCAAACGGGAGTCTA RF2 GACAAACGTCAAAGGAATCG
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.
Molting hormone receptors of a scorpion Y. Nakagawa et al.
6192 FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 (.
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
567 41 98 56 86 68
Amblyomma
americanum
c
560 38 98 50 85 66
Blattella germanica
d
570 26 100 48 66 54
Locusta migratoria
e
541 25 98 48 67 53
Tribolium castaneum
f
549 26 100 48 68 54
Leptinotarsa

decemlineata
g
565 25 94 47 67 53
Tenebrio molitor
h
481 27 100 47 65 57
Apis mellifera
i
567 20 98 42 69 52
Aedes aegypti
j
776 26 88 42 60 48
Drosophila
melanogaster
k
849 25 88 39 58 47
Chironomus tentans
l
536 23 89 41 55 43
Manduca sexta
m
568 19 89 33 54 42
Bombyx mori
n
515 27 89 25 54 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.
i
Ref. [41].
j
Ref. [42].
k
Ref. [43].
l
Ref. [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
400 20 92 75 71 63
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
384 30 89 75 59 60
Tenebrio molitor
g
408 28 91 75 64 61
Apis mellifera
h
427 32 91 71 67 60
Aedes aegypti
i
484 31 89 38 44 46
Drosophila melanogaster
j
508 28 91 31 46 48
Chironomus tentans
k
552 32 89 34 40 44
Manduca sexta
l
461 26 91 45 43 45
Bombyx mori
m
462 28 89 50 40 45
Chilo suppressalis
n
410 31 92 45 43 45
Homo sapiens
o

462 20 88 83 73 63
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.
f
Ref. [15].
g
Ref. 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].
Y. Nakagawa et al. Molting hormone receptors of a scorpion
FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS 6193
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
223
119
132
42
62
39
78
55
74
77
3
52
120
79
28

10
10
10
125
134
114
141
113
122
53
56
51
49
158
234
290
186
198
187
196
178
207
179
187
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
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
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
A
435
463
456
465
436
442
458
374
462
566
518
407
436
391
395
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
B
535
563
556
565
536
543
559
475
562
670
622
511
540
495
499

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
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.
Molting hormone receptors of a scorpion Y. Nakagawa et al.
6194 FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 vitro transcription ⁄ translation kit (rabbit reticulocyte

lysate), with
35
S-labelled methionine ([
35
S]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
48
31
44
73
80
48

118
76
77
76
23
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
87
79
94
87
85
67
86
109
137
103

196
112
113
137
59
C
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
268
258
292
268
264
241
265
284
333

339
401
304
305
251
318
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
369
358
393
369
365
342
363
385
444

459
512
413
414
361
419
D
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
410
400
436
411
407
384
408
427

484
508
552
461
462
410
462
Fig. 2. (Continued).
Y. Nakagawa et al. Molting hormone receptors of a scorpion
FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS 6195
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) using
3
H-labelled ponasterone A ([
3
H] 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, K
D
, 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 K
D
values of
LaEcR-A and LaEcR-A ⁄ LaRXR were determined to
be 4.2 and 3.2 nm, respectively, and the difference
between these K
D
values 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
NNNTTT
−
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.
Molting hormone receptors of a scorpion Y. Nakagawa et al.
6196 FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS
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
02030
Concentration (nM)
K
D
= 3.2 nM

LaEcR-A
K
D
= 4.2 nM
[
3
H] PonA binding (dpm)
[
3
H] PonA binding (dpm)
0
1000
2000
3000
4000
0 10 20 30
3000
2000
1000
0
Concentration (nM)
AB
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.).
hsp27
pal1
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.
Y. Nakagawa et al. Molting hormone receptors of a scorpion
FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS 6197
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 K
D
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) (K
D
1.2 nm) [14], L. decemlineata (LdEcR) ⁄ L. decemlineata
(LdUSP) (K
D
2.8 nm) [32], and D. melanogaster
EcR (DmEcR) ⁄ D. melanogaster USP (DmUSP) (K
D
0.85 nm) [15]. The K
D
values 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
Molting hormone receptors of a scorpion Y. Nakagawa et al.
6198 FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS

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
L. migratoria EcR-A
B.
germanica
EcR-A
L. decemlineata EcR-A
T. molitor EcR-A
B. mori EcR-A
A. aegypti EcR-A
C. suppressalis EcR-A
L. australasiae EcR-A
A. americanum EcR-A1
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
100 100
90
90
63
100
L. australasiae RXR
A. americanum
RXR1
M. musculus
RXRα1
D. magna RXR
A. mellifera RXR
B.
mori USP
M. sexta USP1
D. melanogaster USP
S.
depilis RXR
T. castaneum RXR
A. aegypti USP-A1

L. migratoria RXR
B. germanica
RXR1
L. decemlineata RXR
100
C. pugilator RXR
G. latera
lis RXRα
H. sapiens
RXRα
Xenos
pecki
RXR
100
100
95
100
100
100
100
100
66
71
31
19
53
C. suppressalis USP
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 RXRa1ofMus 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.
Y. Nakagawa et al. Molting hormone receptors of a scorpion
FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS 6199
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, K
D
, 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 ([
3
H]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 TRIzolÒ (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
ReadyÆToÆGo
TM
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 65 °C, then immediately cooled on ice. This RNA solu-
tion was added to the ReadyÆToÆGo
TM
T-Primed First-
Strand Kit, which was prewarmed to 37 °C, and incubated
for 5 min at 37 °C. After mixing gently with a pipette, the
reaction mixture was incubated for 60 min at 37 °Cto
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 (94 °C ⁄ 2 min; 35 cycles of
92 °C ⁄ 1 min, 48 °C ⁄ 1 min, 72 °C ⁄ 1 min; and 72 °C ⁄ 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 46 °C, 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 48 °C and 46 °C, respectively.
RACE
Poly (A)-rich RNA was subjected to 5¢- and 3¢-RACE with
a SMART
TM
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: 66 °C)
and EcR-RF2 (66 °C) 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 (68 °C) 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,
Molting hormone receptors of a scorpion Y. Nakagawa et al.

6200 FEBS Journal 274 (2007) 6191–6203 ª 2007 The Authors Journal compilation ª 2007 FEBS
Madison, WI, USA). The sequencing of cDNA fragments
was carried out by Shimadzu Corp. (Kyoto, Japan).
Sequences were analyzed using genetyx -win version 5.1.0
(Software Development Co., Tokyo, Japan).
In vitro transcription ⁄ translation and gel mobility
shift assay
Full-length DNA fragments for the ORFs of LaEcR-A and
LaRXR were amplified by PCR and cloned into the
pET-23a(+) vector (Novagen, Darmstadt, Germany).
Using these constructs, LaEcR-A and LaRXR proteins
were prepared in vitro using T
N
TÒ T7 Coupled Reticulo-
cyte Lysate Systems (Promega) according to the manu-
facturer’s instructions. To confirm a successful in vitro
protein translation, [
35
S]Met (> 30 TBqÆmmol
)1
; Institute
of Isotopes Co., Budapest, Hungary) was added to the
mixture as a marker. The in vitro translated proteins were
separated in a 10% SDS polyacrylamide gel and analyzed
using the BAS-2000 Bioimaging Analyzer (FUJIFILM,
Tokyo, Japan) (Fig. 3).
A gel mobility shift assay was conducted according
to our previously reported method [15,33]. Two labeled
EcREs – [
32

P]hsp27 and [
32
P]pal1 – were prepared from
synthetic oligomers; (5¢-GATCTAGAGAGGTCAATGAC
CTCGTCC-3¢ and 3¢-ATCTCTCCAGTTACTGGAGCA
GGTCTAG-3¢ for pal1) and (5¢-GATCGACAAGGGT
TCAATGCACTTGTC-3¢ and 3¢-CTGTTCCCAAGTTA
CGTGAACAGCTAG-5¢ for hsp27) were purchased from
Invitrogen Corp. and [
32
P]dCTP (111 TBqÆmm
-1
; Institute
of Isotopes Co.) as well as using Megaprime
TM
DNA as
the labeling system (Amersham Bioscience Corp.). In vitro
translated LaEcR and LaRXR were incubated with
[
32
P]hsp27 or [
32
P]pal1 and analyzed on a nondenaturing
polyacrylamide gel. After drying the gel, the radioactivity
in the bands was detected in BAS2500 Bioimaging Analyzer
(FUJIFILM).
Ligand-binding assay
The ligand-binding assay was performed as described pre-
viously [14]. Briefly, in vitro-translated LaEcR-A and ⁄ or
LaRXR proteins were incubated with [

3
H]PonA
(5.55 TBqÆmmol
)1
) for 90 min at 25 °C. A 1000-fold excess
of unlabeled PonA was added to measure nonspecific bind-
ing, and total binding was obtained for the treatment with a
carrier. After incubation, the reaction mixtures were filtered
through GF-75 glass filters (0.30 lm; Advantec, Dublin, CA,
USA). Filters were washed and transferred to a vial contain-
ing 3 mL of Aquasol-2 (Perkin-Elmer Inc., Wellesley, MA,
USA) to measure the radioactivity in a Aloka LSC-1000
liquid scintillation counter (Aloka Co., Ltd, Tokyo, Japan).
Specific binding was determined at various [
3
H]PonA
concentrations to derive the curves (Fig. 5) as the difference
between the radioactivity in the presence and absence of
nonradioactive PonA (4 lm). The K
D
values of PonA to
LaEcR-A alone and to the LaEcR-A ⁄ LaRXR heterodimer
were evaluated by nonlinear regression analysis using prism
software (Graphpad Software Inc., San Diego, CA, USA).
Calculation of sequence homology
For the bioinformatics study, we used the ‘GenomeNet
Computation Service’ provided by the Kyoto University
Bioinformatics Center ( Sequence
similarity search was performed using the ‘blast’ program,
and the sequence alignment and the calculation of homol-

ogy were performed with ‘clustalw’ (ome.
jp/) using default parameters.
Acknowledgements
We sincerely thank Dr Arthur Retnakaran for the
careful review of this manuscript, and Drs Tadafumi
Nakata and Ken Nakamura of Japan International
Research Center for Agricultural Sciences for their
help in capturing scorpions. Part of this study was per-
formed in the RI center of Kyoto University. The
study was supported, in part, by the 21st century COE
program for Innovative Food and Environmental
Studies Pioneered by Entomomimetic Sciences, from
the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
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