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

Presence of membrane ecdysone receptor in the anterior silk gland
of the silkworm
Bombyx mori
Mohamed Elmogy, Masafumi Iwami and Sho Sakurai
Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakumamachi, Japan
Nongenomic action of an insect steroid hormone, 20-hy-
droxyecdysone (20E), has been implicated in several 20E-
dependent events including the programmed cell death of
Bombyx anterior silk glands (ASGs), but no information i s
available for the mode of the action. We provide evidence for
a putative membrane receptor located in t he plasma mem-
brane of the ASGs. Membrane fractions prepared from the
ASGs exhibit high binding activity to [
3
H]ponasterone A
(PonA). The m embrane fractions did not contain c onven-
tional ecdysone receptor as revealed b y Western blot analysis
using antibody raised against Bombyx ecdysone receptor A
(EcR-A). The binding activity was not solubilized with 1
M
NaCl or 0.05% (w/v) MEGA-8, indicating that the binding
sites were l ocalized in the m embrane. Differential s olubili-
zation and temperature-induced phase separation in Triton
X-114 showed that the binding sites might be integrated
membrane proteins. These results indicated that the binding
sites are located in plasma membrane proteins, which
we putatively referred t o as membrane ecdysone recep-
tor (mEcR). The mEcR e xhibited saturable binding for
[
3
H]PonA (K

d
¼ 17.3 n
M
, B
max
¼ 0.82 pmolÆmg
)1
pro-
tein). Association and dissociation kinetics revealed that
[
3
H]PonA associated with and dissociated from mEcR
within minutes. The combined results support the existence
of a p lasmalemmal ecdysteroid receptor, w hich may act in
concert with the conventional EcR i n various 20E-depend-
ent d evelopmental events.
Keywords: ecdysone ago nist; ecdysone receptor; kinetics;
nongenomic; ponasterone A.
Steroids elicit various physiological responses, particularly
those involving the genomic aspects of action, in which
they modulate gene transcription by interacting with
intracellular nuclear receptors that serve as ligand-
dependent transcription factors [1]. In addition to the
genomic steroid actions, increasing evidence of rapid,
nongenomic steroid effects has been demonstrated for
virtually all groups of steroids [2].
Ecdysone, an insect steroid hormone synthesized by
prothoracic glands, is essential for inducing the molecular
and cellular events t hat lead to molting and metamorphosis
in insects and crusta ceans [ 3–5]. 20- Hydroxyecdysone (20E),

the biologically active form of ecdysone, binds to a
functional nuclear ecdysone receptor consisting of an
ecdysone receptor (EcR) and its heterodimeric partner,
ultraspiracle (USP), and thereby controls the transcriptional
activity of target genes [ 6]. I n addition, a nongenomic action
of 20E has been supposed for decades. 20E increases the
cellular c AMP level in the prothoracic glands of Manduca
sexta [7] and in the f at body of Mamestra brassicae [8]. 20E
rapidly reduces the excitatory potentials a t n euromuscular
junctions in amplitude w ithin minutes in both the crayfish
[9] and Drosophila [10]. These responses to 20E fail to fit the
classical genomic model, and a ppear instead to r ely on
mechanisms involving membrane receptors and second
messenge rs. Nevertheless, the pre sence of mem brane recep-
tors remains speculative.
20E is the primary factor i nducing programmed cell death
(PCD) of l arval tissues at pupal metamorphosis [11–13]. The
anterior silk gland (ASG) is a larval-specific tissue, which is
destined to die shortly after pupation, and enters t he process
of PCD in response to the high hemolymph ecdysteroid
concentration that induces pupal metamorphosis [14]. In t he
PCD of ASGs induced by 20E in vitro, the gene expression
required for completion of PCD is accomplished during t he
first 8 h of 20E challenge, but withdrawal of 20E before
30 h of the culture interferes with t he PCD sequence [14]. If
the genomic theory of steroid action is applicable to 20E-
induced PCD, 20E challenge for 8 h should be sufficient for
execution of PCD. This implies that t he effects o f 20E
during the period between 8 a nd 30 h are no t accompanied
by gene expression but rather are mediated by a non-

genomic pathway, probably through a membrane-bound
receptor. If this is the case, ASG plasma membranes may
contain high-affinity binding sites for ecdysteroid. The
present study reports for the first time the presence of such
sites in the membranes of insect cells and the biochemical
Correspondence to S. Sakurai, D ivision of B iological Science,
Graduate School of Natural Science and Technology, Kanazawa
University, Kakumamachi, Kanazawa 920-1192, Japan.
Fax: +76 2646250, Tel.: + 76 2646255,
E-mail:
Abbreviations: ASG, anterior silk gland; 20E, 20-hydroxyecdysone;
EcR, ecdysone receptor; mEcR, membrane ecdysone receptor;
PCD, programmed cell death; PonA, ponasterone A (25-deoxy-
20-hydroxyecdysone); USP, ultraspiracle; ECL,
enhanced chemoluminescence detection.
(Received 1 9 April 2004, revised 3 June 2004,
accepted 8 June 2004)
Eur. J. Biochem. 271, 3171–3179 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04249.x
characterization of the putative membrane ecdysteroid
receptor.
Materials and methods
Animals and ASGs
Silkworm, Bombyx mori (Kinshu · Showa F1 hybrid),
were reared on an artificial diet (Silkmate, Nihon-Nosan-
Kogyo, Yokohama, Japan) at 25 °C under 12-h light/12-h
dark cycle [15]. ASGs were dissected on the day of gut
purge [14] and cultured separately in 0.3 mL Grace’s
insect culture m edium ( Gibco B RL) at 2 5 °Cfor18h
with 20E followed b y a culture i n a hormone-free medium
for a further 12 h [14]. Because preliminary experiments

showed that the binding activity in the membrane
fractions prepared from the cultured A SGs was higher
than that from the freshly dissected ASGs, we mainly
used such ASGs unless mentioned otherwise.
Chemicals
Ponasterone A (PonA, 25-deoxy-20-hydroxyecdysone) and
20E were from Sigma and nonsteroid ecdysone agonists
[methoxyfenozide (RH-2485), tebufenozide (RH-5992),
RH-5849] were gifts f rom Y. Nakagawa, Kyoto University,
Japan. Ecdysteroids and the agonists were d issolved in
ethanol and stored at )20 °C until use. [
3
H]PonA
(200 Ci Æmmol
)1
)and[
14
C]methoxyinulin (2.4 mCiÆmmol
)1
)
were from PerkinElmer Life Sciences. Triton X-114 and
butylatedhydroxytoluene were from Sigma. To remove any
Triton X-114-insoluble materials, T riton X-114 (10 mL)
was added with 8 mg of butylatedhydroxytoluene and
190 mL 20 m
M
potassium phosphate buffer pH 7.5 con-
taining 0.15
M
KCl [16]. The mixture was cooled to near

0 °C, centrifuged at 3000 g for 10 min to remove any
insoluble material, and then the condensed detergent was
incubated for 20 h at 35 °C. The purified detergent ( lower
phase) was stored at room temperature.
Preparation of membrane fraction
Freshly dissected or cultured ASGs were w ashed three times
with insect Ringe r’s solution (130 m
M
NaCl, 4.7 m
M
KCl,
1.9 m
M
CaCl
2
). All subsequent procedures were performed
at 4 °C. ASGs were homogenized in seven v ols binding
assay buffer (20 m
M
Tris/HCl pH 7.0, 2 m
M
EDTA, 1 m
M
phenylmethylsulphonyl fluoride, 3 lgÆmL
)1
pepstatin A ,
3 lgÆmL
)1
leupeptin) using a motor-driven, loose-fitting
glass-plastic homogenizer at 1000 r.p.m. for 1 m in. After

centrifugation a t 1000 g for 10 min, t he pellet w as suspen-
ded in the buffer and centrifuged at 1500 g for 10 min. The
pellet was again s uspended in t he buffer a nd centrifuged at
1800 g for 1 0 min. The resulting p ellet was resuspended i n
the buffer, homogenized again using HG30 homogenizer
(Hitachi) on ice, and centrifuged at 1000 g for 10 min. T he
supernatant was centrifuged at 8000 g for 10 min, and the
resulting supernatant was centrifuged at 105 000 g for 5 h.
The pellet w as suspended i n the buffer, frozen with liquid
nitrogen, a nd stor ed at )80 °C until use. Protein amounts
were measured using a D C protein assay kit (Bio-Rad) with
BSA as standard.
Preparation of nuclear extracts
Nuclear extract was m ainly pre pared according t o Wu [17]
with a minor modification. Briefly, dissected ASGs were
washed once i n 100 m
M
phosphate buffer pH 7.9 with
100 m
M
NaCl and homogenized on ice with two vols
10 m
M
Hepes pH 7.9 with 10 m
M
KCl, 0.3
M
sucrose,
1.5 m
M

MgCl
2
,0.1m
M
EDTA, 0.5 m
M
2-mercaptoethanol
and protease inhibitors cocktail (Complete; Roche Diagno-
sis), by 12 strokes in a Dounce tissue grinder (1 mL;
Wheaton, Millville, NJ, USA). The suspension was centri-
fuged for 8 m in at 1600 g at 4 °C. The resulting pellet was
resuspended in two vols 10 m
M
Hepes pH 7.9 containing
0.4
M
NaCl, 5% ( v/v) glycerol, 1.5 m
M
MgCl
2
,0.1m
M
EDTA, 0.5 m
M
dithiothreitol and 0.5 m
M
phenyl-
methanesulfonyl fluoride, and 5
M
NaClwasaddedtoyield

a final concentration of 0.4
M
NaCl. The suspension was
incubated at 4 °C under gentle shaking for 30 min and
subsequently centrifuged at 2 °C a t 105 000 g for 6 0 min.
The r esulting supernatant was dialysed for 4 h using a
dialysis tube with 14 000 Da cut-off size (Wako Pure
Chemical Industries) against 1000 vols 20 m
M
Hepes
pH 7.9 containing 20 m
M
NaCl, 20% (v/v) glycerol, 1 m
M
EDTA and 0.5 m
M
2-mercaptoethanol and the protease
inhibitor cocktail. The buffer was changed once after 2 h
dialysis time. The sample was clarified by 10 min
centrifugation at 10 000 g at 4 °C, and t he supernatant
was supplemented with the protease inhibitors.
SDS/PAGE and Western blot analysis
SDS/PAGE was performed according to Laemmli [18]
using 12% polyacrylamide gel. The samples in reducing
loading buffe r were h eated in a boiling water bath for
10 min. The gel was stained with Coomassie brilliant blue.
For Western blot analysis, the blotting membrane was
agitated in Tris-buffered saline ( NaCl/Tris: 25 m
M
Tris/

HCl, pH 7.4, 3 m
M
KCl, 136 m
M
NaCl) containing 5 % (w/v)
nonfat milk for 2 h and then incubated with a rabbit
antibody raised against the EcR-A-specific region of EcR
(a gift from H. Fujiwara, University of Tokyo) at 4 °C
overnight. After washing with NaCl/Tris, the membrane
was incubated with horseradish peroxidase-conjugated
protein A in the f resh NaCl/Tris containing 5% (w/v)
nonfat milk at 4 °C for 2 h. Visualization of the immuno-
blot was carried out using the enhanced chemoluminescence
detection (ECL) system according to the manufacturer’s
instructions and exposed to Hyperfilm ECL (Amersham
Pharmacia Biotech).
Binding assay
Specific binding of PonA was assayed by an ultracentri-
fugation method [19] adapted to the measurement of
ecdysteroid membrane receptors. Membrane fractions
(100 lL reaction mixture containing 100 lgprotein)were
incubated with [
3
H]PonA in 0.5 lL of the buffer solution
containing 0.05 lCiÆlL
)1
[
14
C]methoxyinulin. [
14

C]Meth-
oxyinulin was added to estimate the degree of contaminated
[
3
H]PonA in the precipitate after centrifugation, that
originated from th e incubation medium. The mixture w as
incubated at 2 5 °C for 10 min unless mentioned o therwise.
3172 M. Elmogy et al. (Eur. J. Biochem. 271) Ó FEBS 2004
After incubation, the mixture was c entrifuged at 100 000 g
for 1 5 min at 2 °C. The supernatant was discarded and the
insides of the tubes were rinsed w ith 100 lL of the buffer.
Radioactivity in the pellet w as measured using a Beckman
LS-700 counter with a dual-label program (Beckman-
Coulter). Saturation analysis was performed to determine
receptor number per mg protein and binding affinity to
PonA. Membrane fractions were equilibrate d with increas-
ing concentrations of [
3
H]PonA in the absence or presence
of a 1000-fold excess of unlabelled PonA. For association
kinetics, membrane fractions were incubated with 25 n
M
[
3
H]PonA (± excess unlabelled PonA) for periods of time
ranging from 1 to 40 min. Dissociation kinetics were
determined by equilibrating membrane fractions with
25 n
M
[

3
H]PonA for 10 min followed by the addition of
25 l
M
unlabelled PonA. In the competition assay, mem-
brane fractions were incubated with 25 n
M
[
3
H]PonA in the
presence of increasing concentrations of unlabelled ecdy-
steroids and ecdysone agonists. A modified dextran-coated
active charcoal method [19] was used for phase partitioning
samples in Triton X-114.
Topological localization of the binding sites
To exam ine whether PonA binding sites are located in
peripheral proteins that are not integrated to the lipid
bilayers or integral membrane proteins, topological local-
ization study of the binding sites in the membranes was
performed a ccording to Kerkhoff et al. [20,21]. The m em-
brane suspensions (5 mgÆmL
)1
protein) were treated with
a solution of high ionic strength (binding assay buffer
containing 1
M
NaCl) for 60 min at 4 °C. The mixtures
were centrifuged at 105 000 g for 60 min at 4 °C to obtain
supernatant (S1) and pellet (P1). The p ellet P 1 was
resuspended in the binding assay buffer to a final protein

concentration of 20 mgÆmL
)1
, and an aliquot was stored at
)80 °C for the binding assay. The remaining suspensions
weredilutedto5mgÆmL
)1
protein, treated with 0.05% (v/v)
octanoyl-N-methylglycamide (MEGA-8; Wako Pure
Chemical Industries) at 4 °Cfor60minwithconstant
stirring, and then centrifuged at 105 000 g for 60 min at
4 °C. The resulting pellet (P2) was re-suspended in the
binding assay buffer to a final protein concentration of
20 mgÆmL
)1
and stored at )80 °C. The supernatants, S1
and S2, were dialysed against the binding assay buffer
overnight at 4 °C.
Differential solubilization and temperature-induced
phase separation in Triton X-114: three phase system
Integral membrane proteins are classified into two categor-
ies, i.e., proteins that covalently attached to the lipid bilayers
and those that a re anchored in the bilayers [16]. Phase
partitioning in Triton X-114 is a quick me thod to determine
which category t he mEcR belongs to. The procedure used is
a modification of t he method of Pryde & Philips [22] a nd
Hooper & Bashir [23]. Triton X-114 was precondensed
before use [24]. Purified Triton X-114 solutions with
different c oncentrations (0.5–3%) were added t o t he ASG
membrane fractions (final concentration, 10 mgÆmL
)1

)in
10 m
M
phosphate buffer pH 7.4 containing 150 m
M
KCl,
vigorously mixed immediately for 1–2 s, and placed on ice
for 1 h followed by centrifugation at 100 000 g for 1 h at
0 °C. The detergent insoluble pellet was washed with the
buffer and resuspended in the buffer prior to assaying the
binding activity. The supernatant was overlaid on a cushion
of buffere d 6% sucrose, incubated at 30 °C for 10 min and
centrifuged at 3000 g for 5 min in a swing-rotor. The lower
phase was a detergent phase that was directly subjected to
the b inding ass ay. The upper aqueous phase was t ransferred
to a tube, and fresh Triton X-114 was added to a final
concentration of 0.5% (v/v). After mixing and incubating
on ice for 1 h, the mixture was overlaid on the s ame sucrose
cushion, kept at 30 °C f or 10 min, and centrifuged at 3000 g
for 5 min in a swing-rotor. The resulting upper aqueous
phase was transferred to a t ube to which fresh Triton X-114
with the same starting concentration (0.5–3%) was added.
The sample was mixed, kept on ice a nd then at 30 °Cfor
10 min. After centrifugation at 3000 g for 5 min, the
supernatant was used as a final aqueous phase. The three
phases (detergent-insoluble pellet, detergent phase and
aqueous phase) were assayed for the b inding activities,
and the activities were expressed as a percentage of the total
activity in all three phases.
Data analysis

Experimental data were analysed using
ORIGIN
software
(OriginLab, Northampton, MA, USA). Saturation bind ing
curves were fitted and analysed using equations built into
GRAPHPAD PRISM
TM
3.02 (GraphPad Software, San Diego,
CA, USA) according to Swillens [25].
Results
Biochemical characterization of [3H]PonA binding
We first performed biochemical characterization of the
binding sites u sing ASG membrane f ractions. The optimal
protein concentration for the b inding assay was determined
using 25 n
M
[
3
H]PonA and increasing amounts of proteins
in individual incubations. The percentage of specific b inding
increased in a protein concentration-dependent manner
within the range of 25–150 lgÆmL
)1
(Fig. 1A). Because the
specific binding at 100 lgÆmL
)1
protein was appr oximately
60% of the maximum value at 200 lgÆmL
)1
, the protein

concentration of 100 lgÆmL
)1
was used in the following
binding assays. The optimum pH was 7.0 at 25 °C
(Fig. 1 B). The binding was temperature dependent, with
optimum binding at 37 °C and no binding at 60 °
C
(Fig.1C).BecauseBombyx larvae were reared at 25 °Cin
our laboratory, we selected the incubation temperature o f
25 °C, although the specific binding at 25 °C was approxi-
mately half of that at 37 °C. Based on those results, we u sed
the a ssay c onditions in which 100 lL of binding assay
buffer (pH 7.0) containing 100 lgofmembraneproteins
was incubated at 25 °Cwith25 n
M
[
3
H]PonA, except for the
saturation analysis.
Western blot analysis
To confirm that the specific binding in the membrane
fraction was not brought about by contamination of
conventional nuclear EcR, membrane fractions that showed
Ó FEBS 2004 Membrane ecdysone receptor (Eur. J. Biochem. 271) 3173
specific binding activity to [
3
H]PonA were subjected to
Western blot analysis using an antibody raised against
EcR-A (Fig. 2). Although insect tissues contain two
EcR isoforms, EcR-A and EcR-B1, EcR-A isoform is

predominantly expressed in the ASGs at pupation in
Bombyx [26], and ther efore we used anti-EcR-A s erum for
the Western blotting. As samples con taining the EcRs, total
lysate and nuclear extract prepared from freshly dissected
ASGs were used. I n the total lysate and nuclear extract, a
single immunoreactive s ignal band a t 57 kDa, an approxi-
mate molecular mass of Bombyx EcRs, was found
(Fig. 2 B). By c ontrast, no i mmunoreactive signals were
found in the membrane fractions of either the freshly
dissected ASGs or the ASGs cultured with 20E for 30 h.
These results indicated that the specific binding activity in
the membrane fractions was not caused by contamination
of nuclear receptors.
Association and dissociation kinetics
The a ssociation kinetics of the membrane fractions showed
that PonA became associated with the membranes very
rapidly as the steady state w as attained within 10 min
(Fig. 3 A). The observed association constant ( K
obs
)was
0.9 ± 0.2Æmin
)1
. The dissociation of PonA from its b inding
sites was measured by adding an excess amount of
unlabelled PonA after equilibration with 25 n
M
[
3
H]PonA
Fig. 1. Binding of [

3
H]PonA to memb rane fractions of ASGs. (A)
Protein amount-dependence of specific [
3
H]PonA binding. The mem-
brane fractions (100 lL) with different prote in c oncentrat ions we re
incubated for 10 min at 25 °Cwith25n
M
[
3
H]PonA without or with a
1000-fold molar excess of i nert PonA. Each data point is m ean ± SD
(n ¼ 3).(B)OptimalpHfor[
3
H]PonA binding. Membrane fract ions
containing 100 lg protein in 100 lL buffer w ere incubated with 25 n
M
[
3
H]PonA at various pH. Other conditions were the same as for (A).
(C) Temperatu re d ependen ce of [
3
H]PonA binding. Mem brane frac-
tions c ontaining 100 lgproteinin100lL buff er (pH 7) were incu-
batedwith25n
M
[
3
H]PonA at vario us temperatures. j, t otal binding;
d, specific binding; s, nonspecific binding.

Fig. 2. Membrane fractions are free o f conventional nuc lear EcR.
Commassie brilliant blue-stained S DS/PAGE ( 12% acrylamide gel)
(A) an d Western blotting for the identical gel using anti-EcR-A s erum
(1 : 100) as a primary antibody and horseradish peroxidase-conju-
gated protein A (1 : 1000) as a secondary antibody (B). Lanes 1, total
lysate; lane 2, membrane f raction; lane 3, nuclear extract. S am ples for
lane 1–3 were prepared from freshly dissected ASGs. Lane 4, mem-
brane fraction prepared from the ASGs that were cu ltured in the sa me
conditions as th ose used for binding e xperiments. Twenty m icrograms
ofproteinwereusedineachlane.
3174 M. Elmogy et al. (Eur. J. Biochem. 271) Ó FEBS 2004
for 10 min (Fig. 3 B). The dissociation of PonA from the
membranes occurred within 10 s with a dissociation
constant (K
off
)of2.3±0.5min
)1
. The calculated associ-
ation rate constant (K
on
)was13.3· 10
7
M
)1
Æmin
)1
,andthe
estimated dissociation c onstant at equilibrium (K
d
)was

17.5 n
M
.
Saturation analysis
A saturation analysis of specific binding at 25 °Cwas
performed by incubating the membrane fractions with
increasing concentrations of [
3
H]PonA, then subjecting the
binding data to Scatchard analysis (Fig. 4). The analysis
showed the presence of a single high-affinity binding site in
each molecule, with an apparent K
d
and B
max
of 17.3 n
M
and 0.82 pmolÆmg
)1
protein, respectively. This K
d
value
was i n g ood accordance with the estimated K
d
of 17.5 n
M
derived from the kinetic constants.
Topological localization of the binding sites
We examined whether the PonA binding sites are located in
integral membrane proteins or peripheral proteins that are

not integrated to the lipid bilayers. The membranes were
treated with N aCl to e xamine if t he binding sites were
located on p roteins that simply associate with the lipid
bilayers or other scaffold proteins (Fig. 5). After treatment
with 1
M
NaCl, the binding activity was found only in the
P1 fraction, indicating that the proteins responsible for the
binding are not peripheral membrane proteins. Then, the P1
fraction was treated with a detergent, MEGA-8, as prelim-
inary e xperiments with eight detergents had shown t hat only
MEGA-8 at low concentration did not solubilize integral
membrane proteins but merely fragmented membranes.
After t reatment with 0.05 % MEGA-8, the activity was
recovered from P 2 fraction a nd little a ctivity was found in
the s upernatant. Thus, the binding sites might be on integral
membrane proteins.
Phase partitioning in the detergent Triton X-114
Integral membrane proteins are generally classified into two
categories, proteins that are anchored in the lipid bilaye rs
through a transmembrane sequence(s) and those that are
covalently attached to the bilayers [16]. To examine the
mode of association of the binding sites with the plasma
membranes, the membrane fractions were subjected to
Fig. 3. Kinetics of association (A) and dissociation (B) of [
3
H]PonA
binding to ASG memb ranes . Association kinetics: membranes were
incubated with 25 n
M

[
3
H]PonA for various times without or with a
1000-fold excess o f inert P onA . K
on
¼ 13.3 · 10
7
M
)1
Æmin
)1
.Disso-
ciation kinetics: membranes w ere incubated with 25 n
M
[
3
H]PonA for
10 m i n a t 25 °C a nd th en added with a 10 00 -fold excess of unlabelled
PonA to initiate dissociation of [
3
H]PonA. Inset represents linear
regression analysis of the data. K
off
¼ 2.3±0.5min
)1
.Eachdata
point is the mean ± SD (n ¼ 3).
Fig. 4. PonA saturation analysis of ASG membranes. Membrane
preparations (100 lg p rotein in 100 lL b uffe r) were i ncubated with
increasing concentrations of [

3
H]PonA at 25 °C for 10 min without or
with a 1000-fold e xcess of unlabelled PonA. T he data were fitted by
nonlinear regression analysis. I nset is Scatchard analyses o f t he bin ding
data. K
d
¼ 17.3 n
M
; B
max
¼ 0.82 pmolÆmg
)1
protein. Each data point
is the mean ± SD (n ¼ 3).
Ó FEBS 2004 Membrane ecdysone receptor (Eur. J. Biochem. 271) 3175
differential solubilization and temperature-induced phase
separation in Triton X-114 (Fig. 6 ). In the absence of
detergent the binding activity was found only in the pellet
after the first centrifugation step. On increasing the
concentration of Triton X-114, the binding activity was
recovered predominantly in the detergent-rich phase. The
binding in the detergent-insoluble pellet decreased in a
complementary manner, and only a low activity was found
in the aqueous phase. As the concentration of Triton X-114
was increased from 0.5 to 3%, results were mostly t he same
as that at 0.5%. These results indicate that the binding sites
are neither on a polypeptide(s) that merely a ssociates to the
membrane bilayers nor are they covalently associated with
the membrane proteins; rather they are on an integral
membrane protein(s) that may be anchored in the mem-

brane by a transmembrane sequence.
Displacement studies
Binding affinities of ecdysteroids and nonsteroidal ecdysone
agonists to the membrane bindin g sites were determined by
incubating the membrane fractions with 25 n
M
[
3
H]PonA in
the p resence of increasing amounts of unlabelled ecdyster-
oids and agonists (Fig. 7). The estimated 50% inhibitory
concentration (IC
50
)for50%displacementof[
3
H]PonA
was 6.92 · 10
)7
M
for PonA and 2.63 · 10
)7
M
for 20E,
showing that the affinity for 20E was approximately 2.6
times higher than that for PonA. The IC
50
values for three
nonsteroid ago nists, RH-5849, tebufenozide (RH-5992) and
methoxyfenozide (RH-2485) were much lower than those
for PonA and 20E. Comparison of individual values of

pIC
50
, reciprocal logarithm of the concentration that
provides a 50% inhibition of [
3
H]PonA binding, as well as
relative activities to 20-hydroxyecdysone (Table 1) showed
that the binding activity was in the order 20E >
PonA >> methoxyfenozide > tebufenozide > RH-5849.
Fig. 6. Effects of Triton X-114 concentration on the solubilization and
phase separation of ASG membranes. ASG m embrane fractions were
subjected to differential s olubilization and temp erature-induced phase
separation at the indicated concentrations of Triton X-114. The
resulting three phases, detergent-insoluble pellet (j), detergent-rich
phase (d) a nd aqueous ph ase (s), were assayed for binding activity.
Each data point is a m ean of duplicate determinations.
Fig. 7. Inhibitory activities of e cdysteroids and e cdysone ag onists against
the [
3
H]PonA binding. Membrane fractions (100 lg protein in 100 lL
buffer) were incubated f or 10 min a t 2 5 °C with increasing concentra-
tions of unlabelled PonA (d), 20E ( s), methoxyfenozide ( RH -2485; m),
tebufenozide (RH-5992; n) and RH-5849 (h) in the presence of 25 n
M
[
3
H]PonA. Each data point is the m ean ± SD (n ¼ 3).
Fig. 5. Topological localization of the binding sites in the ASG mem-
branes. Themembranefractions(5mgÆmL
)1

) w ere treated with the
binding assay buffer containing 1
M
NaCl for 60 min at 4 °Cand
centrifuged at 105 000 g for 60 min at 4 °C.Thepellet(P1)was
resuspended in a ssay b uffer cont aining 0.05% MEGA-8. After i ncu-
bation for 60 m in at 4 °C, the mixture was c entrifuged at 105 000 g for
60 min at 4 °C, and the pellet (P2) was resuspended in the assay buffer.
The dialyzed supernatants ( S1, S2) an d the resuspended pellets (P1, P2)
were incubated with 25 n
M
[
3
H]PonA under standard assay condi-
tions. Binding activity is r elative to that in t he crude e xtract (C) w ith
that designated as 100.
3176 M. Elmogy et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
The present study describes for the fi rst time evidence for t he
presence of a putative receptor for ecdysteroid in tissue
membranes and its biochemical characterization in an
insect. The membrane receptor exhibits a specific and
saturable binding for [
3
H]PonA with a K
d
of 17.3 · 10
)9
M
.

This value is physiologically relevant to the prevailing
hemolymph concentrations of 20E (ranging between 10
)7
and 10
)6
M
) in the prepupal period when PCD is triggered
in vivo [14]. The association and dissociation kinetics
indicated that PonA association with and dissociation from
its binding sites were rapid, which is characteristic of the
binding of several natural compounds to their membrane
receptors [27,28]. The saturation curve indicates the pres-
ence of a single high-affinity binding site and an apparent
maximal number of binding sites of 0.82 p molÆmg
)1
protein. The obtained K
d
value is supported by the good
accordance with the estimated dissociation rate constant at
equilibrium (K
d
¼ 17 .5 · 10
)9
M
).
Rapid effects o f steroids a re triggered by the intracellular
signalling cascade, in which membrane -binding sites for
some steroids have been linked to the conventional nuclear
receptors. In the nongenomic action of estrogen, estrogen
receptor a couples with the regulatory subunit of the lipid

kinase PI3K to trigger the rapid e ffects of estradiol [29]. The
nongenomic action of progesterone is also mediated by the
conventional progesterone receptor that interacts with Src
to trigger the mitogen-activated protein kinase cascade [30].
The binding affinities o f those steroid membrane receptors
are orders of magnitude lower than those of nuclear
receptors [31]. In ecdysone receptors, the K
d
value of the
in vitro translated EcR/USP heterodimer for PonA is 0.9 n
M
in Drosophila [32] and 1.1 n
M
in Bombyx [33]. Thus, K
d
for
EcR is in the nanomolar range. By contrast, the K
d
of PonA
for mEcR is s ignificantly higher (lower affinity) t han those
values. This result is in accordance with the fact that the
binding affinity of mammalian steroids to conventional
nuclear receptor is higher t han that to the same receptor that
mediates nongenomic action. However, Western blot a na-
lysis using antibody raised against EcR-A indicates t hat the
binding activity in the ASG membrane fraction is not due to
EcR. Accordingly, the put ative mEcR appears to differ
from the conventional EcR.
A second line of evidence to support the existence of a
membrane receptor is that the binding affinity of PonA is

less than that o f 20E. Th e b inding affinity of PonA to the
nuclear receptor complex of EcR/USP i s one to two orders
of magnitude higher than that of 20E [34]. In the inherent
receptor c omplex of the rice stem borer Chilo suppressalis,
binding affinity for PonA is 26-fold h igher t han t hat f or 20E
[35], and nuclear extracts of Dr osophila Kc-H cells exhibit
high binding affinity for PonA with a K
d
of 3.4 n
M
, w hile K
d
for 20E is 24 0 n
M
, 70 times lower than that for PonA [36].
Similarly, the affinity of PonA to tick EcR is 28-fold higher
than that of 20E [37]. By contrast, the competition assay
using the ASG membrane fractions shows that the binding
affinity for PonA is one-fourth of that for 20E and that the
values for nonsteroidal ecdysone agonists are much lower
than 20E, which totally differs from the binding character-
istics of the conven tional EcR (Table 1).
Finally, the topological studies indicated the presence of
mEcR. The effects of solutions of high ionic strength and
detergents have been used to establish the topological
localization of several microsomal enzymes involved in
phospholipid and triglyceride metabolism [20]. Using a
similar approach, the present study revealed a distinct
binding activity in the membrane. The differential solubi-
lization and temperature-induced phase separation in Tri-

ton X-114 (three phase system) gave additional evidence for
the presence of mEcR. The particular advantage of Triton
X-114 is that i ts micelles aggregate on w arming from 0 °C,
eventually separating ou t into a second phase when
temperature is raised above 2 0 °C ( the s o called c loud
point). Therefore, integral membrane proteins solubilized at
0–4 °C tend to be partitioned preferentially into the
detergent-rich phase at the cloud point [16]. When t he
porcine kidney microvillar membranes are subjected to
the three phase system, t he ectoenzymes with a covalently
attached glycosyl-phosphatidyinositol membrane anchor
are recovered i n t he detergent insoluble pellet, while those
anchored by transmembrane spanning polypeptide are
recovered in the detergent-rich phase [23]. Similarly, the
majority of the integral membrane proteins in adrenal
chromaffin granules migrate into a detergent-rich phase,
and an aqueous phase contains the insoluble, hydrophilic
proteins [22]. We found most of the binding activities for
[
3
H]PonA in the detergent-rich phase, indicating that the
mEcR is an integral membrane protein which might be
anchored in the membrane by transmembrane sequence of
hydrophobic amino acids.
Several mammalian steroid hormones have been demon-
strated to exert rapid effects on cells by interacting with
specific re ceptors present on the cell s urface [2,29]. Effects of
20E that may have physiological relevance to membrane
receptors have been described i n insect tissues. I n wing
epidermis of Hyalopora gloveri pupae, 20E stimulates

adenylyl cyclase activity within 15 min of exposure to the
hormone in vitro [7]. Similarly, cAMP levels in the ASGs
increases significantly within 1 min after a 20E challenge
(unpublished data). The rapid increase in the cAMP level
indicates a nongenomic action of 20E, and a membrane
receptor may mediate the increase in the cAMP level.
Recently, a membrane progestin recep tor has been des-
cribed as a s even-transmembrane receptor coupling to a Gi
protein [28]. The putative mEcR could mediate the rapid
Table 1. Binding activities of ecdysteroids and nonsteroidal ecdysone
agonists against t he membrane binding site s and comparison with the
conventional nuclear receptor complex. pCI
50
(
M
) reciprocal logarith-
mic value of the 50% inhibition. RA activities relative to that of
20-hydrox yecd yso ne.
Compounds
mEcR nEcR
a
pCI
50
(
M
) RA pCI
50
(
M
)RA

20-Hydroxyecdysone 6.58 1 6.70 1
Ponasterone A 6.16 0.38 8.12 26.4
Methoxyfenozide (RH-2485) 5.01 0.027 9.05 224
Tubefenozide (RH-5992) 4.86 0.019 9.07 234
RH-5849 4.56 0.0096 6.88 1.51
a
Binding activities against inherent receptor complex of nuclear
EcR (nEcR) and USP from Chilo suppressalis integuments [34].
Ó FEBS 2004 Membrane ecdysone receptor (Eur. J. Biochem. 271) 3177
increase in cAMP levels in ASG cells, although further
studies are necessary to determine whether the me mbrane
receptor identified in the present study is involved in the
activation of adenylyl cyclase and in distinct physiological
responses to 20E in the Bombyx ASG.
In conclusion, our study indicates, at a biochemical level
and for the first time, t hat ecdysteroids m ay act t hrough a
membrane receptor in addition to the conventional nuclear
receptor. By furnishing new i nsights into t he functional
properties o f t wo classes of insect ecdysone receptors, these
findings are expected to pave the way for the understanding
of ecdysone action on insect development.
Acknowledgements
We express our sincere gratitude to Drs Michiyasu Yoshikuni and
Yoshitaka Nagahama of National Institute for Basic Biology f or their
valuable comments for establishing the binding assay. We are also
thankful to Dr Haruhiko Fujiwara of the University of Tokyo for the
gift of anti-EcR-A serum and Dr Yoshiaki Nakagawa of Kyoto
University for the gift of nonsteroidal e cdysone agonists. This work was
supported by a JSPS Research Grant (No. 1 4360033 ) to S.S.
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