Differential solubili-zation and temperature-induced phase separation in Triton X-114 showed that the binding sites might be integrated membrane proteins.. Binding assay Specific binding o
Trang 1Presence of membrane ecdysone receptor in the anterior silk gland
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
Bombyxanterior silk glands (ASGs), but no information is
available for the mode of the action We provide evidence for
a putative membrane receptor located in the plasma
mem-brane of the ASGs Memmem-brane fractions prepared from the
ASGs exhibit high binding activity to [3H]ponasterone A
(PonA) The membrane fractions did not contain
conven-tional ecdysone receptor as revealed by Western blot analysis
using antibody raised against Bombyx ecdysone receptor A
(EcR-A) The binding activity was not solubilized with 1M
NaCl or 0.05% (w/v) MEGA-8, indicating that the binding
sites were localized in the membrane Differential
solubili-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 to as membrane ecdysone recep-tor (mEcR) The mEcR exhibited saturable binding for [3H]PonA (Kd¼ 17.3 nM, Bmax¼ 0.82 pmolÆmg)1 pro-tein) Association and dissociation kinetics revealed that [3H]PonA associated with and dissociated from mEcR within minutes The combined results support the existence
of a plasmalemmal ecdysteroid receptor, which may act in concert with the conventional EcR in various 20E-depend-ent developm20E-depend-ental ev20E-depend-ents
Keywords: ecdysone agonist; 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 that lead to molting and metamorphosis
in insects and crustaceans [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] In addition, a nongenomic action
of 20E has been supposed for decades 20E increases the cellular cAMP level in the prothoracic glands of Manduca sexta[7] and in the fat body of Mamestra brassicae [8] 20E rapidly reduces the excitatory potentials at neuromuscular junctions in amplitude within minutes in both the crayfish [9] and Drosophila [10] These responses to 20E fail to fit the classical genomic model, and appear instead to rely on mechanisms involving membrane receptors and second messengers Nevertheless, the presence of membrane recep-tors remains speculative
20E is the primary factor inducing programmed cell death (PCD) of larval 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 the process
of PCD in response to the high hemolymph ecdysteroid concentration that induces pupal metamorphosis [14] In the PCD of ASGs induced by 20E in vitro, the gene expression required for completion of PCD is accomplished during the first 8 h of 20E challenge, but withdrawal of 20E before
30 h of the culture interferes with the 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 the effects of 20E during the period between 8 and 30 h are not 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, Division of Biological Science,
Graduate School of Natural Science and Technology, Kanazawa
University, Kakumamachi, Kanazawa 920-1192, Japan.
Fax: +76 2646250, Tel.: + 76 2646255,
E-mail: ssakurai@kenroku.kanazawa-u.ac.jp
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 19 April 2004, revised 3 June 2004,
accepted 8 June 2004)
Trang 2characterization 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 25C 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 medium (Gibco BRL) at 25C for 18 h
with 20E followed by a culture in 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 ASGs 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 from Y Nakagawa, Kyoto University,
Japan Ecdysteroids and the agonists were dissolved in
ethanol and stored at )20 C until use [3H]PonA
(200 CiÆmmol)1) and [14C]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, Triton X-114 (10 mL)
was added with 8 mg of butylatedhydroxytoluene and
190 mL 20 mM potassium phosphate buffer pH 7.5
con-taining 0.15MKCl [16] The mixture was cooled to near
0C, centrifuged at 3000 g for 10 min to remove any
insoluble material, and then the condensed detergent was
incubated for 20 h at 35C The purified detergent (lower
phase) was stored at room temperature
Preparation of membrane fraction
Freshly dissected or cultured ASGs were washed three times
with insect Ringer’s solution (130 mMNaCl, 4.7 mMKCl,
1.9 mMCaCl2) All subsequent procedures were performed
at 4C ASGs were homogenized in seven vols binding
assay buffer (20 mMTris/HCl pH 7.0, 2 mMEDTA, 1 mM
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 min After
centrifugation at 1000 g for 10 min, the pellet was
suspen-ded in the buffer and centrifuged at 1500 g for 10 min The
pellet was again suspended in the buffer and centrifuged at
1800 g for 10 min The resulting pellet was resuspended in
the buffer, homogenized again using HG30 homogenizer
(Hitachi) on ice, and centrifuged at 1000 g for 10 min The
supernatant was centrifuged at 8000 g for 10 min, and the
resulting supernatant was centrifuged at 105 000 g for 5 h
The pellet was suspended in the buffer, frozen with liquid
nitrogen, and stored at)80 C until use Protein amounts
were measured using a DC protein assay kit (Bio-Rad) with
BSA as standard
Preparation of nuclear extracts Nuclear extract was mainly prepared according to Wu [17] with a minor modification Briefly, dissected ASGs were washed once in 100 mM phosphate buffer pH 7.9 with
100 mM NaCl and homogenized on ice with two vols
10 mM Hepes pH 7.9 with 10 mM KCl, 0.3M sucrose, 1.5 mMMgCl2, 0.1 mMEDTA, 0.5 mM2-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 min at 1600 g at 4C The resulting pellet was resuspended in two vols 10 mM Hepes pH 7.9 containing 0.4M NaCl, 5% (v/v) glycerol, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol and 0.5 mM phenyl-methanesulfonyl fluoride, and 5MNaCl was added to yield
a final concentration of 0.4M NaCl The suspension was incubated at 4C under gentle shaking for 30 min and subsequently centrifuged at 2C at 105 000 g for 60 min The resulting 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 mM Hepes
pH 7.9 containing 20 mMNaCl, 20% (v/v) glycerol, 1 mM EDTA and 0.5 mM 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 4C, and the 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 buffer were heated 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 mM Tris/ HCl, pH 7.4, 3 mMKCl, 136 mMNaCl) 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 4C overnight After washing with NaCl/Tris, the membrane was incubated with horseradish peroxidase-conjugated protein A in the fresh NaCl/Tris containing 5% (w/v) nonfat milk at 4C 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 lg protein) were incubated with [3H]PonA in 0.5 lL of the buffer solution containing 0.05 lCiÆlL)1 [14C]methoxyinulin [14 C]Meth-oxyinulin was added to estimate the degree of contaminated [3H]PonA in the precipitate after centrifugation, that originated from the incubation medium The mixture was incubated at 25C for 10 min unless mentioned otherwise
Trang 3After incubation, the mixture was centrifuged at 100 000 g
for 15 min at 2C The supernatant was discarded and the
insides of the tubes were rinsed with 100 lL of the buffer
Radioactivity in the pellet was 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 equilibrated with
increas-ing concentrations of [3H]PonA in the absence or presence
of a 1000-fold excess of unlabelled PonA For association
kinetics, membrane fractions were incubated with 25 nM
[3H]PonA (± excess unlabelled PonA) for periods of time
ranging from 1 to 40 min Dissociation kinetics were
determined by equilibrating membrane fractions with
25 nM [3H]PonA for 10 min followed by the addition of
25 lM unlabelled PonA In the competition assay,
mem-brane fractions were incubated with 25 nM[3H]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 examine 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 according to Kerkhoff et al [20,21] The
mem-brane suspensions (5 mgÆmL)1protein) were treated with
a solution of high ionic strength (binding assay buffer
containing 1M NaCl) for 60 min at 4C The mixtures
were centrifuged at 105 000 g for 60 min at 4C to obtain
supernatant (S1) and pellet (P1) The pellet P1 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
were diluted to 5 mgÆmL)1protein, treated with 0.05% (v/v)
octanoyl-N-methylglycamide (MEGA-8; Wako Pure
Chemical Industries) at 4C for 60 min with constant
stirring, and then centrifuged at 105 000 g for 60 min at
4C The resulting pellet (P2) was re-suspended in the
binding assay buffer to a final protein concentration of
20 mgÆmL)1and stored at )80 C The supernatants, S1
and S2, were dialysed against the binding assay buffer
overnight at 4C
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 are anchored in the bilayers [16] Phase
partitioning in Triton X-114 is a quick method to determine
which category the mEcR belongs to The procedure used is
a modification of the method of Pryde & Philips [22] and
Hooper & Bashir [23] Triton X-114 was precondensed
before use [24] Purified Triton X-114 solutions with
different concentrations (0.5–3%) were added to the ASG
membrane fractions (final concentration, 10 mgÆmL)1) in
10 mMphosphate buffer pH 7.4 containing 150 mMKCl,
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
0C 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 buffered 6% sucrose, incubated at 30C 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 binding assay The upper aqueous phase was transferred
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 same sucrose cushion, kept at 30C for 10 min, and centrifuged at 3000 g for 5 min in a swing-rotor The resulting upper aqueous phase was transferred to a tube to which fresh Triton X-114 with the same starting concentration (0.5–3%) was added The sample was mixed, kept on ice and then at 30C for
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 binding 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 binding curves were fitted and analysed using equations built into GRAPHPAD PRISMTM3.02 (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 using ASG membrane fractions The optimal protein concentration for the binding assay was determined using 25 nM[3H]PonA and increasing amounts of proteins
in individual incubations The percentage of specific binding increased in a protein concentration-dependent manner within the range of 25–150 lgÆmL)1(Fig 1A) Because the specific binding at 100 lgÆmL)1protein was approximately 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 25C (Fig 1B) The binding was temperature dependent, with optimum binding at 37C and no binding at 60 C (Fig 1C) Because Bombyx larvae were reared at 25C in our laboratory, we selected the incubation temperature of
25C, although the specific binding at 25 C was approxi-mately half of that at 37C Based on those results, we used the assay conditions in which 100 lL of binding assay buffer (pH 7.0) containing 100 lg of membrane proteins was incubated at 25C with 25 nM[3H]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
Trang 4specific binding activity to [3H]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 therefore we used anti-EcR-A serum for the Western blotting As samples containing the EcRs, total lysate and nuclear extract prepared from freshly dissected ASGs were used In the total lysate and nuclear extract, a single immunoreactive signal band at 57 kDa, an approxi-mate molecular mass of Bombyx EcRs, was found (Fig 2B) By contrast, no immunoreactive 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 association kinetics of the membrane fractions showed that PonA became associated with the membranes very rapidly as the steady state was attained within 10 min (Fig 3A) The observed association constant (Kobs) was 0.9 ± 0.2Æmin)1 The dissociation of PonA from its binding sites was measured by adding an excess amount of unlabelled PonA after equilibration with 25 n [3H]PonA
Fig 1 Binding of [ 3 H]PonA to membrane fractions of ASGs (A)
Protein amount-dependence of specific [ 3 H]PonA binding The
mem-brane fractions (100 lL) with different protein concentrations were
incubated for 10 min at 25 C with 25 n M [3H]PonA without or with a
1000-fold molar excess of inert PonA Each data point is mean ± SD
(n ¼ 3) (B) Optimal pH for [ 3 H]PonA binding Membrane fractions
containing 100 lg protein in 100 lL buffer were incubated with 25 n M
[3H]PonA at various pH Other conditions were the same as for (A).
(C) Temperature dependence of [ 3 H]PonA binding Membrane
frac-tions containing 100 lg protein in 100 lL buffer (pH 7) were
incu-bated with 25 n M [3H]PonA at various temperatures j, total binding;
d, specific binding; s, nonspecific binding.
Fig 2 Membrane fractions are free of conventional nuclear EcR Commassie brilliant blue-stained SDS/PAGE (12% acrylamide gel) (A) and Western blotting for the identical gel using anti-EcR-A serum (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 fraction; lane 3, nuclear extract Samples for lane 1–3 were prepared from freshly dissected ASGs Lane 4, mem-brane fraction prepared from the ASGs that were cultured in the same conditions as those used for binding experiments Twenty micrograms
of protein were used in each lane.
Trang 5for 10 min (Fig 3B) The dissociation of PonA from the
membranes occurred within 10 s with a dissociation
constant (Koff) of 2.3 ± 0.5 min)1 The calculated
associ-ation rate constant (Kon) was 13.3· 107
M )1Æmin)1, and the estimated dissociation constant at equilibrium (Kd) was
17.5 nM
Saturation analysis
A saturation analysis of specific binding at 25C was
performed by incubating the membrane fractions with
increasing concentrations of [3H]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 Kdand Bmaxof 17.3 nM and 0.82 pmolÆmg)1 protein, respectively This Kd value was in good accordance with the estimated Kdof 17.5 nM 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 NaCl to examine if the binding sites were located on proteins that simply associate with the lipid bilayers or other scaffold proteins (Fig 5) After treatment with 1MNaCl, 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 experiments with eight detergents had shown that only MEGA-8 at low concentration did not solubilize integral membrane proteins but merely fragmented membranes After treatment with 0.05% MEGA-8, the activity was recovered from P2 fraction and little activity was found in the supernatant 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 bilayers 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 membranes Association kinetics: membranes were
incubated with 25 n M [ 3 H]PonA for various times without or with a
1000-fold excess of inert PonA K on ¼ 13.3 · 10 7
M )1 Æmin)1 Disso-ciation kinetics: membranes were incubated with 25 n M [3H]PonA for
10 min at 25 C and then added with a 1000-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 ± 0.5 min)1 Each data
point is the mean ± SD (n ¼ 3).
Fig 4 PonA saturation analysis of ASG membranes Membrane preparations (100 lg protein in 100 lL buffer) were incubated with increasing concentrations of [ 3 H]PonA at 25 C for 10 min without or with a 1000-fold excess of unlabelled PonA The data were fitted by nonlinear regression analysis Inset is Scatchard analyses of the binding data K d ¼ 17.3 n M ; B max ¼ 0.82 pmolÆmg)1protein Each data point
is the mean ± SD (n ¼ 3).
Trang 6differential 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 the same
as that at 0.5% These results indicate that the binding sites are neither on a polypeptide(s) that merely associates 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 transmemmem-brane sequence
Displacement studies Binding affinities of ecdysteroids and nonsteroidal ecdysone agonists to the membrane binding sites were determined by incubating the membrane fractions with 25 nM[3H]PonA in the presence of increasing amounts of unlabelled ecdyster-oids and agonists (Fig 7) The estimated 50% inhibitory concentration (IC50) for 50% displacement of [3H]PonA was 6.92· 10)7M for PonA and 2.63· 10)7M for 20E, showing that the affinity for 20E was approximately 2.6 times higher than that for PonA The IC50values for three nonsteroid agonists, RH-5849, tebufenozide (RH-5992) and methoxyfenozide (RH-2485) were much lower than those for PonA and 20E Comparison of individual values of pIC50, reciprocal logarithm of the concentration that provides a 50% inhibition of [3H]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 membrane fractions were
subjected to differential solubilization and temperature-induced phase
separation at the indicated concentrations of Triton X-114 The
resulting three phases, detergent-insoluble pellet (j), detergent-rich
phase (d) and aqueous phase (s), were assayed for binding activity.
Each data point is a mean of duplicate determinations.
Fig 7 Inhibitory activities of ecdysteroids and ecdysone agonists against the [ 3 H]PonA binding Membrane fractions (100 lg protein in 100 lL buffer) were incubated for 10 min at 25 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 [3H]PonA Each data point is the mean ± SD (n ¼ 3).
Fig 5 Topological localization of the binding sites in the ASG
mem-branes The membrane fractions (5 mgÆmL)1) were treated with the
binding assay buffer containing 1 M NaCl for 60 min at 4 C and
centrifuged at 105 000 g for 60 min at 4 C The pellet (P1) was
resuspended in assay buffer containing 0.05% MEGA-8 After
incu-bation for 60 min at 4 C, the mixture was centrifuged 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) and the resuspended pellets (P1, P2)
were incubated with 25 n M [3H]PonA under standard assay
condi-tions Binding activity is relative to that in the crude extract (C) with
that designated as 100.
Trang 7The present study describes for the first time evidence for the
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 [3H]PonA with a Kdof 17.3· 10)9M
This value is physiologically relevant to the prevailing
hemolymph concentrations of 20E (ranging between 10)7
and 10)6M) 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 pmolÆmg)1
protein The obtained Kdvalue is supported by the good
accordance with the estimated dissociation rate constant at
equilibrium (Kd¼ 17.5 · 10)9M)
Rapid effects of steroids are 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 effects 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 of those steroid membrane receptors
are orders of magnitude lower than those of nuclear
receptors [31] In ecdysone receptors, the Kdvalue of the
in vitrotranslated EcR/USP heterodimer for PonA is 0.9 nM
in Drosophila [32] and 1.1 nMin Bombyx [33] Thus, Kdfor
EcR is in the nanomolar range By contrast, the Kdof PonA
for mEcR is significantly higher (lower affinity) than those
values This result is in accordance with the fact that the
binding affinity of mammalian steroids to conventional
nuclear receptor is higher than that to the same receptor that
mediates nongenomic action However, Western blot
ana-lysis using antibody raised against EcR-A indicates that the
binding activity in the ASG membrane fraction is not due to
EcR Accordingly, the putative 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 of 20E The binding affinity of PonA to the nuclear receptor complex of EcR/USP is one to two orders
of magnitude higher than that of 20E [34] In the inherent receptor complex of the rice stem borer Chilo suppressalis, binding affinity for PonA is 26-fold higher than that for 20E [35], and nuclear extracts of Drosophila Kc-H cells exhibit high binding affinity for PonA with a Kdof 3.4 nM, while Kd for 20E is 240 nM, 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 conventional 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 its micelles aggregate on warming from 0C, eventually separating out into a second phase when temperature is raised above 20C (the so called cloud point) Therefore, integral membrane proteins solubilized at 0–4C tend to be partitioned preferentially into the detergent-rich phase at the cloud point [16] When the porcine kidney microvillar membranes are subjected to the three phase system, the ectoenzymes with a covalently attached glycosyl-phosphatidyinositol membrane anchor are recovered in the 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 [3H]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 receptors present on the cell surface [2,29] Effects of 20E that may have physiological relevance to membrane receptors have been described in insect tissues In 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 receptor has been des-cribed as a seven-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 the membrane binding sites 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-hydroxyecdysone.
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].
Trang 8increase in cAMP levels in ASG cells, although further
studies are necessary to determine whether the membrane
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, that ecdysteroids may act through a
membrane receptor in addition to the conventional nuclear
receptor By furnishing new insights into the functional
properties of two 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 for 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 ecdysone agonists This work was
supported by a JSPS Research Grant (No 14360033) to S.S.
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