Ecdysteroid receptors in a tumorous blood cell line of Drosophila melanogaster.

Archives of Insect Biochemistry and Physiology 2:295-317 (1985)
Ecdysteroid Receptors in a Tumorous Blood
Cell Line of Drosophila melanogaster
Laurence Dinan
lnstitut fiir Zoologie der TechnischenHochschule Damstadt, Schnittspuhnstrusse 10, 0-6100
Dumstadt and lnstitut fiir Zoologie 111, Universitat Dusseldorf, Universitatsstrasse1, D-4000
Dusseldorf 7, Federal Republic of Germany
The tumorous Drosophila melanogaster blood cell line BII has been studied
for evidence for the presence of ecdysteroid receptors. The 13H]ponasterone
A (pon A)* used in this study has been extensively purified, and the location
of the tritium in the molecule has been partially determined. Bll cells do not
metabolise ecdysteroids. Intact cells demonstrate a considerable specific
uptake of [3H]pon A which is saturable, apparently showing two specific
components: a very high affinity component (KD = 0.3 nM) and a high affinity
component (KD = 2 nM). The specific binding of [3H]pon A t o whole cells is
compatible with unlabelled ecdysteroids, but not with mammalian steroid
hormones. The association rate constant (kJ for [3H]pon A was determined
t o b e 3 x 107M-’min-’ at 2I0C, while the dissociation rate constant (kd) for
the specifically bound [3H]pon A was found to be 4.4 x 10-3/min. Together,
the kinetic rate constants yield a value of 0.15 n M for the KD.
The receptors have been partially characterised in a cell-free extract
prepared by sonification of the cells. The optimum p H for extraction and
hormone binding i s 8.2. Scatchard plots of binding data indicate that the cell-
*Abbreviations: BSA = bovine serum albumin; DCC = dextran-coated charcoal; DTE =
dithioerythreitol; EDTA = sodium ethylenediamine tetraacetate; HEPES = 4(2-hydroxyethyl)I-piperazineethane sulphonic acid; HPLC = high-performance liquid chromatography; hptlc
= high-performance thin-layer chromatography; H-R complex = hormone-receptor complex;
k, = association rate constant; kd = dissociation rate constant; KA = equilibrium association
constant; KD = equilibrium dissociation constant; MS = mass spectrometry; NEM = Nethylmaleimide; pon A = ponasterone A; Rf = distance migrated relative to solvent front;
RIA = radioimmunoassay; t% = half-life; tlc = thin-layer chromatography; tris =
tris(hydroxymethy1)methylamine.
Acknowledgments: The work reported in this paper was supported by the Deutsche Forschungsgemeinschaft with grants to Professor Emmerich and Professor Spindler. During the
major part of this work (in Darmstadt), the author was the recipient of a Royal Society
Overseas Fellowship, which he gratefully acknowledges. He should also like to thank Professor Ernmerich and Professor Spindler for providing facilities, Professor Spindler, Drs. G .
Gellissen and M. Londershausen for their comments on the manuscript, Dr. H.H. Rees for
the MS of poststerone, and Professor E. Gateff for providing the BII cell line. The RIA was
kindly performed by M. Schwab. The synthesis of [3H]ponasteroneA was funded by the
Deutsche Forschungsgemeinschaft.
Laurence Dinan is now at the Department of Biological Sciences, University of Exeter, Exeter,
Devon, United Kingdom; address reprint requests there.
Received July 19,1984; accepted December 20,1984.
0 1985 Alan R. Liss, Inc.
296
Dinan
free extract also contains two high affinity specific binding components (KD
= 0.1 n M and KD = 1 nM). The high affinity binders are macromolecular, as
shown by chromatography on Sephadex (2-25, and are susceptible to protease
digestion, heat, and treatment with N-ethylmaleimide. Sucrose density
centrifugation of the labelled receptor shows one peak at approximately 6s.
The stability of the receptor preparation has been studied and conditions
have been empirically determined (10% w/v sucrose, 25 mM dithioerythreitol,
and I0 m M citrate), whereby the binding capacity of the unlabelled receptor
is stable for at least 8 weeks if frozen at - 2 O O C .
Key words: ecdysteroids, steroid hormone receptors, Drosophila cell line, haemocytes
INTRODUCTION
Persuasive evidence for the existence of steroid hormone receptors in
insects has only been recently obtained [1,2].All subsequent work on steroid
receptors in insect systems has concentrated on one species, Drosophila melanogaster, using imaginal discs [2],salivary glands [3], and the K, cell line [1,461. Cell lines offer several advantages for the study of steroid receptors. It is
relatively easy to obtain large quantities of homogeneous material, and also,
the cell line contains no endogenous hormone (if the cells themselves do not
produce the hormone). A large number of DrosophiZa cell lines exist; several
of these are known to respond to ecdysteroids [7l and are, therefore, good
candidates for studies on ecdysteroid receptors. However, almost all these
cell lines have been derived from dissociated embryos, and it is not known
which, if any, in vivo cell type is represented by the cell line.
Two Drosophila cell lines of defined origin do, however, exist. They were
derived from tumorous blood cell mutants [8]. Mutant larvae develop normally until the third instar when the normal increase in the number of
haemocytes that occurs in wild-type larvae becomes uncontrolled in the
mutants, resulting in the production of between 20- and 200-fold more
haemocytes. Additionally, these mutant blood cells cannot recognise self
from non-self; they digest not only the histolysing larval tissues, but also all
other tissues that should be carried over into the pupal and adult stages [8].
In cell culture the blood cells respond to the presence of ecdysteroids by
increased phagocytosis and lysis, which results in a declining cell density
and an enlargement of the remaining cells [8].
In this report, the initial results concerning the demonstration of the
specific uptake and binding of ecdysteroids by one of these two tumorous
blood cell lines are presented. Since this is the first report using this batch of
radiolabelled ponasterone A* for receptor studies, the purification and characterisation are reported in some detail.
*Trivial names: Ecdysone = 20, 3f3,14or, 22R, 25-pentahydroxy-5-cholest-7-en-6-one; 20-hydroxyecdysone = 2&3& 14a,20R, 22R, 25-hexahydroxy-5cholest-7-end-one; ponasterone A = 20,
3f3,14a, 20R, 22R-pentahydroxy-5-cholest-7-en-6-one.
Ecdysteroid Receptors
297
MATERIALS AND METHODS
Preparation and Purification of [3H]Ponasterone A
The synthesis of [3H]pon A was carried out as a collaborative project by
the research groups of Professor Emmerich (Technische Hochschule, Darmstadt), Professor Koolman (Philipps-Universitat, Marburg), and Professor
Spindler (Universitat Dusseldorf).
20-Hydroxyecdysone (Fig. 1, 11) was dehydrated by the method of Hein(IV). However,
rich [9] to yield 25-deoxy-26-dehydro-20-hydroxyecdysone
reverse-phase HPLC separation of the product showed that it consisted of
two components. NMR and MS indicate that the product probably contained
24-dehydro-25-deoxy-20-hydroxyecdysone
(V) in addition to the expected
product. This mixture (35 mg) was tritiated with tritium gas by NEN (Dreieich,
FRG). The crude ponasterone A was purified by column chromatography on
silicic acid to a specific radioactivity of 13.2 Cilmmol. However, the reactants
and products of the tritiation possess almost identical Rf values on tlc, and
they would not have been separated under the conditions used. This separation was achieved by reverse-phase HPLC (Column: Waters Partisil ODs,
25 cm X 4.6 mm i.d. Solvent: methanollwater, 6:4.Flow rate: 0.5 mllmin.
Retention times: pon A, 19.5 min; starting compounds, 15.5 and 16.5 min).
The column had previously been calibrated by the injection of known quantities of a standard solution of pon A, and it was therefore possible to
calculate the specific radioactivity of the purified [3H]pon A (three determinations: 182, 182, and 186 Cilmmol). Radioassay indicated that the recovered
reactants were not radioactive.
Hptlc of the purified [3H]pon A (Merck Kieselgel 60 F254 plates; solvent,
CH2C12/EtOH,9:1, run three times) showed that it contained a 12% radioactive impurity of the same specific radioactivity, which was slightly less polar
than pon A. This impurity was separated by normal-phase HPLC (Column:
Merck Lichrosorb Si 60, 5 pm, 25 cm x 4.6 mm i.d. Solvent: CH2C12l
isopropanollH20, 250:50:3. Flow rate: 0.5 mllmin. Retention times: pon A, 21
R
HHO o d 0?
R=.
I.
IV .
Fig. 1. Ecdysteroid structures. I, Ponasterone A; II, 20-hydroxyecdysone; Ill, ecdysone; IV, 25deoxy-26-dehydro-20-hydroxyecdysone; V, 24-dehydro-25-deoxy-20-hydroxyecdysone; VI,
poststerone.
Dinan
298
min; impurity, 17.5 min.). The highly purified [3H]pon A was stored in
methanollbenzene (9:l) at 4°C.
Stability of [3H]Pon A
The radiochemical purity of the [3H]pon A was tested regularly by hptlc
and reverse-phase HPLC. As an example, the results obtained 2 years after
the synthesis of the [3H]pon A are shown in Figure 2. Hptlc of the crude
[3H]pon A preparation reveals little degradation after this time. In contrast,
reverse-phase HPLC shows that only approximately 50% of the radioactivity
is still in the form of pon A. Fortunately, all the radioactive degradation
products elute from the reverse-phase column before pon A, and this system
can be used to remove the degradation products.
Determination of the Location of 3H in the Purified [3H]Pon A
The very high specific radioactivity of the purified [3H]pon A indicates
that an average of six tritium atoms has been introduced per molecule of pon
A during the reductive titiation. It was therefore of interest to determine
where those tritium atoms are located. Since the unreacted starting compounds recovered after the tritiation were not radioactive, this would suggest
that the tritium atoms are all located in the side-chain (ie, no nonspecific
tritiation has taken place). This was shown to be the case by the chemical
side-chain cleavage between C-20 and C-22 of the [3H]ponA with pyridinium
chlorochromate according to the method of Nali and Rees (personal communication). Carrier pon A (2 mg) was mixed with [3H]p~nA (3 x lo5 dpm),
dissolved in 150 pl anhydrous pyridine, and cooled to - 20°C for 30 min.
Pyridinium chlorochromate (3 mg) was added to the cold solution and the
mixture was stirred at room temperature for 3.5 h. The mixture was partiB
-
1
t
f
PON A
7
6
4-
z .
0
I2m
1
0
2
4
6
'
MIGRATION DISTANCE
8
'
[CM]
OJ
0
15
20
ELUTION TIME
[MINI
5
10
Fig. 2. Radiochemical purity of [3H]pon A 2 years after its synthesis. A small aliquot was
removed from the stock solution and unlabelled pon A was added as carrier. The radiochemical purity was then tested by A) hptlc (solvent; CH,CI,/MeOH, 41 v/v) and B) reverse-phase
HPLC (solvent; MeOH/H,O, 1:l v/v. Flow rate; 1 ml/min, isocratic elution). The shoulder on
the pon A peak in the hptlc scan i s the radioactive impurity, which can be separated by
normal-phase HPLC. The arrows show the positions of standard ponasterone A (pon A) and
20-hydroxyecdysone (0). SF, solvent front; 0, origin.
Ecdysteroid Receptors
299
tioned between 5 ml each of countersaturated butanol and water. The aqueous
phase was further extracted with 5 ml butanol, and the combined butanol
phases were washed twice with 5 ml water. After rotary evaporation, the
residue from the butanol phases was adsorbed onto Celite and applied to a
silica column according to the method of Dinan and Rees [lo]. The 30%
methanol in chloroform fraction from the column (containing free ecdysteroids) contained only 6% of the original radioactivity, and analysis by reversephase and normal-phase HPLC showed that the main UV-absorbing compound in this fraction co-chromatographed with poststerone (VI). The identity of this peak was further demonstrated by electron-impact mass
spectrometry: mlz 362 YO, M+), mlz 344 (6%, Mf -H20), mlz 250 (57%),
mlz 55 (100%). The overall yield of poststerone was 54%, and radioassay of
the poststerone eluted from HPLC showed that at least 97% of the tritium
had been lost during the side-chain cleavage.
Purification of [3H]Ecdysone and Preparation of [3H]20-Hydroxyecdysone
[23,24-3H]ecdysone (68 Cilmmol; a gift of the Deutsche Forschungsgemeinschaft) was purified on a 4 g silica column (Merck Kieselgel 60, 70-230
mesh) and eluted sequentially with 40 ml each of 5%, 6%, 8%, lo%, and 15%
methanol in chloroform. Five millilitre fractions were collected and analysed
by tlc and radioscanning. Fractions containing pure [3H]ecdysone were
pooled.
A portion of the purified [3H]ecdysonewas converted to [3H]20-hydroxyecdysone by incubation with Malpighian tubules from Locustu rnigruforb
according to Feyereisen et a1 [ll].The products were extracted with methanol, partitioned between butanol and water, applied to a 4 g silica column,
and eluted with 40 ml each of 5%, looh, 12%, 15%, and 20% methanol in
chloroform. Fractions (5 ml) were collected and tested by tlc for ecdysone
and 20-hydroxyecdysone. Fractions containing both were re-chromatographed on a second silica column, and fractions containing radiochemically
pure [3H]20-hydroxyecdysone were combined with those from the first column. The specific radioactivity of the [3H]20-hydroxyecdysoneproduced was
approximately 40 Cilmmol.
Purification of Unlabelled Ecdysteroids
Pon A was purified by reverse-phase and normal-phase HPLC as described for [3H]pon A. Ecdysone and 20-hydroxyecdysone were purchased
from Simes, Milan, and purified by reverse-phase HPLC. Stock solutions
were made by dissolving the purified ecdysteroids in methanol and determining the absorbance at 242 nm ( E = 12,000 1lmoUcm).
Growth and Isolation of Cells
The BII cell line (derived from 1 [2]mbn mutant larvae of D. rnelanoguster)
was obtained from E. Gateff (Freiburg, FRG). They were grown in Schneider’s medium supplemented with 10% foetal calf serum as described previously [12]. Cells were isolated by gently shaking the T-flasks or roller bottles
300
Dinan
in which they were growing. The cell suspension was then centrifuged (5
min at 350g) in order to pellet the cells.
Radioimmunoassay
BII cells (2 x lo7 cells) and the medium (10 ml) in which they had grown
were extracted with 70% aqueous methanol, and the extracts were tested for
using
RIA-positive material according to the method of Spindler et a1 [B],
the antiserum ICT-1, which has a detection limit of 10-20 pg. No RIA-positive
response was detectable in the cells or in the medium.
Uptake of [3H]Pon A Into BII Cells
Cells (3 x lo7) were incubated in 0.5 ml medium with 0.4nM [3H]pon A
(with or without 50 nM unlabelled pon A) at 7°C or 22°C for various times.
Cells were then cooled on ice and diluted with 8 ml cold medium. After 5
min, the cells were pelleted (2,0008 for 5 min at 0°C). Aliquots of the
supernatant were radioassayed. The cell pellet, after resuspension in 1 ml
distilled water, was also radioassayed.
Association kinetics were determined with a mini-assay in order to conserve radioligand. The labelled hormone (2.7 x lo6 dpm in 10 pl ethanol/
benzene, 9:1, with or without a 200-fold excess of unlabelled pon A in
methanol) was added to 2-ml Eppendorf vials and carefully evaporated under
a stream of nitrogen. The hormone was dissolved in 100 pl Schneider's
medium and then brought to the required temperature for the experiment
(21°C or lT).The cell suspension (1ml, 1.7 x 10' cellslml medium) was
also brought to the required temperature and added to the hormone solution,
and the vials were quickly vortexed. Aliquots (50 11.) were removed at regular
intervals, placed in ice-cooled Eppendorf vials (2 ml), and diluted immediately with 2 ml ice-cold Schneider's medium. After 5 min, the cells were
pelleted (1min in a Hettich Mikroliter centrifuge at 4°C). The supernatant
was removed by suction and the pellet was washed with a further 2 ml cold
medium. The cell pellet was then resuspended in 100 pl distilled water and
radioassayed after the addition of 2 ml Aqualuma. The maximal specific
binding was determined by incubating an aliquot of the cell suspension with
the hormone under the above conditions for 2 h at 21°C and determining the
amount of hormone taken up as described above.
The saturability of the uptake of pon A was determined by incubating 3 x
lo7 cells in 0.5 ml medium with increasing concentrations of [3H]p~n
A for 3
h at 22°C in the presence or absence of a 100-fold excess of unlabelled pon
A. The uptake was determined as described above, and the results were
plotted according to Scatchard [14].
Dissociation of [3H]Pon A From BII Cells
The method of Beckers et a1 [5] was used. Cells (1.7 x 108/0.8ml medium)
were incubated with 3 nM [3H]pon A (with or without a 200-fold excess of
unlabelled pon A) for 3 h at 22°C. The cells were diluted with cold medium
and pelleted at 0°C. The medium was removed and replaced with medium
(22"C, 0.5 ml; total volume = 0.8 ml) containing unlabelled pon A at the
Ecdysteroid Receptors
301
same concentration as the [3H]pon A which had not been taken up, or at a
200-fold excess over the initial [3H]pon A concentration. At known time
points thereafter, aliquots (25 pl) were removed, cooled on ice, and diluted
with cold medium (1 ml). The cells were pelleted, and the radioactivity
associated with the cell pellet was determined by radioassay.
Sonification of the Cells and Preparation of the Cell-Free Extract
The cell pellet was resuspended in buffer (75 mM TrislHCl, pH 8, or 10
mh4 HEPES buffer, pH 8, both containing 160 pglml aprotinin) to a density
of 10' cellslml and sonified at 4°C with one 15s burst (Branson Sonic Power
Co., Danbury, CT, USA; setting 1, 40 W). The homogenate was centrifuged
for 30 min at 4°C and 20,OOOg, and the supernatant was used for receptor
studies.
Charcoal Assays
Specifically bound [3H]pon A was determined by adsorption of free pon
A to dextran-coated charcoal. In the standard assay, 100 pl of the sonification
preparation was incubated with 100 pl t3H]pon A (with or without a 200-fold
excess of unlabelled pon A) in buffer for 2 h at 25°C. At the end of the
incubation, the assay tubes were cooled in ice water, and charcoal suspension
(50 pl) was added. The tubes were quickly vortexed and then left to stand at
1°C for 5 min before the charcoal was pelleted (Hettich Mikrohter centrifuge;
2 min at 4°C). An aliquot of the supernatant (125 p1) was removed for
radioassay.
Two charcoal suspensions have been used during the course of this work.
During the initial part of the work, the cells were sonified in Tris buffer and
a charcoal suspension (A: 0.5 g acid-washed Norit A charcoal
0.05 g T60
Dextranl20 ml Tris buffer) was added. In the later experiments, the cells were
sonified in HEPES buffer, and a more concentrated charcoal suspension (B:
2.5 g acid-washed Norit A charcoal + 0.25 g T60 Dextranl20 ml HEPES
buffer) was used, as it was found that this removed a large portion of the
nonspecifically bound radioactivity but did not affect the level of specifically
bound radioactivity. Specific binding is expressed as the difference between
the dpm bound in the absence and presence of excess unlabelled pon A.
+
Gel-Filtration Chromatography
Gel-filtration chromatography was performed at 2°C on a column of Sephadex G-25 Superfine (Pharmacia, Uppsala, Sweden; 7 x 2 cm i.d.), equilibrated with 75 mh4 TrislHCl (pH 8 + 25 mM NaC1). The flow rate was 0.75
mllmin, and the total separation time was 15 min.
Sucrose Gradient Centrifugation
Sucrose gradient determination of the sedimentation coefficient of the
receptor was performed on 10% to 40% sucrose gradients (16 ml in 75 mh4
TrislHCl buffer, pH8) in a Sorvall TV-865B vertical rotor (60,000 rpm, 4 h at
1°C). The sonification preparation was incubated with t3H]pon A (12 nh4 f
302
Dinan
200-fold excess unlabelled pon A) for 2 h at 25°C. Unbound hormone was
then removed by charcoal treatment. Samples (0.5 ml; approximately 2 mg
protein) were applied to the pre-cooled gradients and overlayered with 0.5
ml silicon oil. After the centrifugation, the gradient was separated into 10
drop ( - 450 pl) fractions and these were radioassayed. The S-value was
determined by comparison with known standards: BSA (4.35)and cytochrome C (1.9s).The sucrose concentration of the fractions was determined
with a refractometer (Schmidt and Haensch, Berlin, FRG).
Protein Determination
Protein was determined by the method of Bradford [15] using BSA (Sigma
Chemie, Munich, FRG) as standard.
Radioassay
Samples were radioassayed in 6-mI mini-vials after the addition of Aqualuma (Baker Chemikalien, Gross-Gerau, FRG). Cell pellets were first resuspended in 1 ml distilled water before addition of the scintillant. When the
mini-assay was used, cell pellets in Eppendorf vials were resuspended in 100
pl distilled water before the addition of 2 ml Aqualuma to the Eppendorf
vial.
Radioactivity was measured in a Packard Tri-Carb 460 CD scintillation
spectrometer. Where data are presented in dpm, corrections were automatically made for background, counting efficiency, and chemical quenching.
RESULTS
Metabolism of Ecdysteroids by BII Cells
After a 4-h incubation of BII cells with [3H]pon A at 22"C, the radiolabelled
compounds present in the cells and in the medium were analysed by hptlc,
normal-phase HPLC, and reverse-phase HPLC. No metabolism could be
detected by any of the separation techniques used.
Since the uptake of [3H]ecdysoneor [3H]20-hydroxyecdysoneinto BII cells
is low, cells and medium were extracted together with 6 h incubation at 22°C
with the labelled hormone. Hptlc analyses showed that there was no conversion of the labelled ecdysone to 20-hydroxyecdysone or any other metabolite.
Similarly, no metabolism of [3H]20-hydroxyecdysonecould be detected.
Saturability of the Uptake of [3H]Pon A by BII Cells
Preliminary experiments to determine the time-course of the specific uptake of [3H]pon A (0.4 nM) by BII cells showed that equilibrium was reached
between 90 and 120 min at 22"C, and after 9 h at 7°C. Comparison of the
uptake of [3Hlpon A with that of [14C]inulin (which cannot enter cells)
showed a large amount of tritium associated with the cell pellet after 75-min
incubation at 22"C, but a very low level of 14C. On washing the cell pellet at
0°C to 4°C with medium, most of the 14Cradioactivity was removed from
the cells, whereas the amount of specifically bound 3H remained constant,
thus showing that the r3H]pon A is not just loosely adsorbed to the cells.
Ecdysteroid Receptors
303
The dependence of the specific uptake on pon A concentration is shown
in Figure 3. When these data are plotted according to Scatchard [14], a plot
with two components is obtained (Fig. 3A). The point of inflexion corresponds to the point at which the high specific activity [3H]ponA was diluted
with unlabelled pon A to reach the required concentration. The experiment
was repeated, but this time an extra series of assay tubes was added in which
the required concentration of pon A was obtained with [3H]pon A alone (Fig.
3C, D).
Association Kinetics
The association kinetics of [3H]ponA (6.75 nM) have been studied at 21°C
and 1°C (Fig. 4A,B), and they show second order kinetics (Fig. 4, insets). At
21°C the association rate constant, determined from the slope in Fi
(inset), is 2.6 x 1O7M-'min-', while at 1"C, k, is 9.6 X 105M-'min- . 4A
gure
Dissociation of [3H]Pon A From BII Cells
Cells which had been equilibrated in the presence of 3 nM [3H]pon A were
isolated and resuspended in the same volume of medium containing unlabelled pon A either at the same concentration as the unbound [3H]pon A [5]
or at 200 times the initial [3H]pon A concentration (ie, 0.6 pM). The latter
experiment was performed as a check on the former experiment because it
Q
2
LL
\
Bound
[pM]
Bound
[pM]
Fig. 3. Dependence of binding of pon A by intact cells on the hormone concentration at
22OC. Cells (3 x 107/0.5ml) were incubated for 3 h in the presence of increasing concentrations of 13H]pon A in the presence or absence of a 100-fold excess of unlabelled pon A to
correct for nonspecific binding. [3H]pon A concentrations were obtained by using increasing
amounts of radiolabelled ligand (180 Cilrnmol, 0 ) or by adding increasing amounts of
unlabelled pon A to a constant amount of [3H]pon A (B). Only the data for the specific
component of the binding are shown. B and D show plots of the bound pon A vs the included
concentration of pon A. A and C give the corresponding Scatchard plots. The dotted lines in
C are the resolution of the Scatchard plot, where the total binding was determined with
undiluted (3H]ponA into two linear components.
304
Dinan
“1
1
-
1
1
I
2
2 3 4 5
MINUTES
6
7
8
3
4
5
6
Incubation Time
I
9
7
6
[minl
9
120
I
I
5
I
s
10 15 20 2 5 30
MINUTES
.
10
is
35
do
3’0 i s 40
20 2 5
Incubation Time
[mini
Fig. 4. Determination of the association rate constant for [3H]pon A (6.75 nM) with intact BII
cells at A) 21OC and B) 1OC. The uptake was measured as described in Materials and Methods.
nonspecific binding (m); specific binding (0).
The data for the
Total dpm bound by cells (0);
specific binding show second-order kinetics (insets). The gradients were determined by linear
regression (r = A, 0.964; €3, 0.982). [R,],
initial receptor concentration; [H,], initial free
hormone concentration; [R], free receptor concentration at stated time point; [HI, free hormone concentration at stated time point.
was considered that the unlabelled pon A concentration may not have been
sufficient to compete with the labelled pon A as it dissociated from the cells,
owing to the lower affinity of the cells for unlabelled pon A than for the
labelled pon A used in this study.
The dissociation of [3H]pon A from BII cells obtained at 20°C after the
replacement of the unbound [3H]pon A by a 200-fold excess of unlabelled
pon A is shown in Figure 5. There are two main components to the dissociation: a rapid initial dissociation (kd = 8 x 10-2/min)owing to the dissociation
of the nonspecifically bound t3HIpon A, and a slower component owing to
the dissociation of the specifically bound C3H]pon A. The dissociation rate
constant for this slower component is 4.4 x 10P3/min.The value obtained
when the unbound [3H]pon A was replaced with the same concentration of
unlabelled pon A was 3.9 x 10P3/min(data not shown).
Competition of the Uptake of [3H]Pon A With Other Ecdysteroids
Aliquots of 1.7 x lo7 cells in 100 pl medium were incubated for 4 h at 22°C
with 2.5 nh4 [3H]pon A in the presence of various concentrations of other
Ecdysteroid Receptors
305
I
20
40
60
Incubation Time
80
100
120
140
[minl
Fig. 5. Dissociation of [3H]pon A from BII cells. Cells were incubated with [3H]pon A (3 nM)
with or without a 200-fold excess of unlabelled pon A for 3 h at 21OC. The cells were then
isolated and resuspended in medium containing 0.5 KM unlabelled hormone. Aliquots (25 PI)
were removed at fixed time points, diluted with ice-cold medium, centrifuged, and the cell
pellet was radioassayed. Total binding (0);nonspecific binding (m); specific binding (0).
The
inset shows the data for the specific binding plotted as a pseudo-first order reaction.
unlabelled steroids. The amount of radioactivity associated with the cells was
determined as described for the association kinetics and is expressed as a
percentage of that obtained when the cells were incubated with [3H]pon A
alone (Fig. 6 ) . Of the steroids tested, unlabelled pon A (50% competition at 2
nM) was the most effective competitor, followed by 20-hydroxyecdysone
(50% competition at 200 nM). The vertebrate steroid hormones, oestradiol,
corticosterone, and hydrocortisone, do not compete for the binding, even at
0.1 mM (oestradiol)or 1mM (corticosterone and hydrocortisone).
Sonification as a Means of Releasing the High-Affinity Binding Sites From
BII Cells
After sonification of the cell suspension and centrifugation of the cell
debris, the resulting supernatant was tested for its ability to specifically bind
[3H]pon A. Preliminary experiments showed that during incubation of the
prelabelled preparation with charcoal at 1"C, the amount of specifically
bound radioactivity remained constant between 5 and 15 min and that the
optimal number of sonifications was one 15-s burst at 40 W. The extent of
specific binding, as measured by the charcoal assay, is proportional to the
amount of receptor preparation added. The time-course of the uptake of
[3H]pon A at 22°C is similar to that obtained with whole cells, equilibrium
being obtained after 60-120 min (data not shown).
The optimum pH for the extraction and binding of the receptor was
determined by resuspending the cells in buffers of different pH values before
306
Dinan
Log [Steroid]
Fig. 6. Competition of the binding of [3H]pon A by unlabelled steroids. Cells were incubated
with radioligand (2.5 nM) for 4 h at 22OC in the presence of increasing concentrations of
P-oestradiol (A);
unlabelled steroids. Pon A (0);20-hydroxyecdysone (*);ecdysone (0);
corticosterone (W); hydrocortisone (A).The cells were diluted with ice-cold medium and
pelleted. The radioactivity in the cell pellet was determined and the specifically bound dpm
are expressed as a percentage of the specific binding obtained when no unlabelled steroid is
present.
sonification (Fig. 7A). [3H]Pon A was added to the supernatant after centrifugation in the buffer of appropriate pH, but the charcoal suspension was
added in TrislHCl buffer (pH 8). A preliminary experiment showed that
charcoal binds free pon A equally well at all pH values between 6.2 and 9.0.
The maximum specific binding is obtained at pH 8.2. The effect of various
salts on the specific binding is shown in Figure 7B.
The dependence of the extent of binding on the hormone concentration is
given in Figure 8. These results were obtained by incubating the sonification
preparation with increasing amounts of [3H]pon A (180 Ciimmol, with or
without a 200-fold excess of unlabelled pon A). Comparison of the results
obtained here with those obtained by incubating the same batch of cells
(intact)with 9 nM [3H]ponA showed that 60% of the specific binding activity
was recovered in the supernatant after sonification.
Dissociation of [3H]Pon A From the Ecdysteroid Receptor
The receptor preparation was incubated for 2 h at 25°C with [3H]pon A (9
nM
200-fold excess unlabelled pon A) and then cooled to 1°C. Free
hormone was removed by adsorption to charcoal (assay B). The rate of
dissociation of the bound hormone from the receptor was determined by
incubating aliquots of the charcoal supernatant at the required temperature.
After known times, the aliquots were incubated at 1°C for 5 min, and then a
second charcoal assay was performed to adsorb the dissociated hormone. An
aliquot of the supernatant was radioassayed. The dissociation is pseudo-first
Ecdysteroid Receptors
307
1
(0
1
E
a
a
v)
C
.r-7
r
x
Y
E,
0
6.2 6 6 7.0 7 4 7 8 8.2 86 90
Salt Concentration
PH
[MI
Fig. 7. A) Effect of pH on the extractability and binding of the ecdysteroid receptor. BII cells
were resuspended in buffers (imidazole/HCI or Tris/HCI; 75 mM) of varying pH, sonified once,
and centrifuged. Aliquots of the supernatant were equilibrated with [3H]pon A in the appropriate buffer, and the binding was determined with DCC assay A. Total binding ( 0 ) nonspe;
cific binding (m); specific binding (0).6)Effect of salts on the specific binding. Aliquots (100
pl) of the cell extract were incubated for 3 h at 22OC with [3H]pon A (f100-fold excess of
unlabelled pon A) in the presence of various salts. NaCl (A);KCI (0);
MgCI2 (m); CaCI, (0);
MnCI, (0).
Results are expressed as a percentage of the specific binding obtained in 75 m M
Tris/HCI buffer (pH 8) without the addition of salt.
A
2501
9
5
Q
n
z
3
P
[3H-PON
A1
nM
BOUND
PM
Fig. 8. A) Saturation of ecdysteroid receptor with [3H]pon A. Aliquots of the cell extract (100
PI)were incubated with 100 pI of buffer containing increasing concentrations of [3H]pon A
(180 Ci/mmol; f200-fold excess of unlabelled pon A) for 3 h at 22OC. The amount of bound
hormone was determined with DCC assay 6. Total binding ( 0 ) ;nonspecific binding (m);
specific binding (0).
6)Data for the specific binding expressed according to Scatchard [14].
308
Dinan
order. The kd is 1 x 10-2/min at 25°C (tv, = 70 min) and 8 x 10-4/min at 1°C
(ty2= 15 h).
Gel-Filtration Chromatography of the Cell Extract
It is possible to demonstrate a specific binding to the cell extract by
chromatography on Sephadex G-25 (Fig. 9). Table 1gives the relative amount
of label found in the macromolecular fraction after treatment of the extract in
various ways, and also, it shows that the amount is considerably reduced by
addition of a 100-fold excess of unlabelled pon A, pronase, trypsin, heat, and
the addition of NEM or detergents.
Sucrose Density Gradient Centrifugation
The separation of the prelabelled receptor preparation on 10% to 40%
sucrose gradients (Fig. 10) shows one major peak of bound radioactivity at
an estimated sedimentation coefficient of 5.8s ( f O . l ; n = 5). This peak is
totally absent if the receptor preparation is incubated with [3H]pon A in the
presence of a 200-fold excess of unlabelled pon A.
Stability of the Receptor Preparation
The stability of the receptor in the presence or absence of hormone (9 nM)
was studied at 8°C and 25°C (Fig. 11).The receptor (in HEPES buffer +
1 2-
10-
10
20
30
Fraction Number
Fig. 9. Demostration of the macromolecular nature of the binding component in the sonification preparation by passage through a gel-filtration column. The preparation was incubated
or without ( 0 )a 100-fold excess of unlabelled pon A for 3 h at 22OC.
with [3H]pon A with (0)
Samples (0.25 ml) were then cooled on ice and separated at 2OC on a Sephadex C-25 Superfine
column (7 x 2 cm i.d.) and equilibrated with 75 m M Tris/HCI buffer (pH 8 + 25 mM NaCI;
flow rate, 0.75 mllmin). Fractions (10 drops) were collected and radioassayed.
Ecdysteroid Receptors
309
TABLE 1. Macromolecular Binding of 13H]Pon A to BII Cell Extract*
Percent of control
(cpm) in
macromolecular peak
Incubation conditions
r3H]pon A
f 100-fold excess unlabelled pon A
+ pronase 0.6 mglml
trypsin 0.4 mglml
+ RNase 0.4 mglml
+ DNase 0.4 mglml
+ heat 5 min at 90°C
+ NEM 40 mM
+ Triton X-100 0.8% added with pon A
+ NP-40 0.8% added with pon A
+ NP-40 0.8% added at end of incubation
100
2
18
41
70
85
+
2
2
3
2
100
*Radiolabelled pon A (50 pl; 8,000 cpm) was incubated with 150 p1 cell extract for 3 h at 20°C
in a total volume of 250 pl. Enzymes included in the incubations were pronase (ex Streptomyces;
Serva), trypsin (ex bovine pancreas; type 111, Sigma), DNase (ex bovine pancreas; Serva), and
RNase (ex bovine pancreas; Serva). The incubation mixture was cooled on ice and applied to
a Sephadex G-25 column as described in Materials and Methods.
r40
FRACTION
NUMBER
Fig. 10. Sucrose gradient density centrifugation of prelabelled BI I ecdysteroid receptor. T h e
receptor preparation was equilibrated with 9 nM (3H]pon A with (M) or without ( 0 )a 200fold excess of unlabelled pon A. The preparation was then cooled to IoC and treated with
charcoal to remove unbound hormone. Aliquots (0.5 mi) were applied to 10% to 40% sucrose
gradients (16 ml), overlayered with 0.5 ml silicon oil, and centrifuged at 60,000 rpm in a
vertical rotor for 4 h at 1OC. Gradients were fractionated (10 drops) from the bottom of the
gradient. Arrows mark the positions of standard proteins run in a parallel gradient.
aprotinin) is considerably more unstable at 8°C in the absence of hormone.
In the presence of hormone the specific binding is stable for at least 3 days,
whereas in the absence of hormone the specific binding decreases with a
half-life of approximately 3 days. At 25°C the decline in specific binding is
considerably more rapid, even in the presence of hormone (50% of initial
specific binding remains after 20 h).
310
Dinan
Y
U
En
v)
1
20
40
60
8b
INCUBATION TIME
160
120
[ HOURS]
Fig. 11. Stability of the B l l ecdysteroid receptor. The receptor preparation was incubated
with 9 n M [3H]ponA (f200-fold excess of unlabelled pon A) for 2 h at 25"C, and then aliquots
Alternatively, aliquots
were either allowed to continue at 25°C (A) or transferred to 8°C (0).
were incubated without hormone at 8OC (W); hormone was then added, followed by an
incubation for 2 h at 25°C. Specifically bound hormone was determined with the charcoal
assay, and results are expressed as a percentage of the specific binding obtained with freshly
prepared cell extract after 2 h incubation with [3H]ponA.
The ability of various compounds to improve the stability of the receptor
preparation (without hormone) was initially tested by incubating aliquots of
the preparation in the presence of the compounds for 3 days at 8°C. Hormone (9 nM k 200-fold excess of unlabelled pon A) was added, and the
assays were then incubated at 25°C for 2 h. The specifically bound radioactivity was then determined with charcoal assay B. As a control, the effect of the
various compounds on the binding capacity of the fresh preparation was also
tested. Of the compounds tested, only molybdate (10 mM) and citrate (10
mM) caused a slight reduction in the initial specific binding (82% and 86%,
respectively). After 3 days at 8"C, the control specific binding was reduced
to 41% of the initial level. The stability of the hormone binding was improved
by 25 mM DTE (74% of control initial binding), 10 mM citrate (6O%), 10% wl
v sucrose (%YO),and 10% vlv glycerol (60%). Molybdate (10 mM) and 5 mM
EDTA (29% and 23%, respectively) apparently hasten the decay of the
receptor.
The long-term effect of those compounds, which had proved beneficial in
the preliminary experiment, was then tested (Fig. 12). Sucrose (10% wlv)
improves the stability of the unoccupied receptor for 3 days, and then the
specific binding capacity declines dramatically. This is probably due to microbial contamination, since the addition of sodium azide extends the life of the
receptor (50% of the activity remaining after - 5 days). Addition of citrate
and DTE, as well as sucrose and sodium azide, improves the stability of the
receptor still further (50% of activity remaining after 8.5 days).
If the receptor preparation is frozen in the presence of sucrose (10% wlv),
citrate (10 mM), and DTE (25 mM) at -20°C or -79"C, the specific binding
capacity is totally stable for at least 8 weeks. The receptor shows the same S-
-
Ecdysteroid Receptors
i
311
i
STORAGE TIME AT 8%
[DAYS]
Fig. 12. Stability of the Bll ecdysteroid receptor at 8OC in the absence of hormone. Aliquots
(100 PI) of the cell extract were incubated at B0C with 50 pI of solutions containing the
required additives in 10 mM HEPES buffer (pH 8). At the required time points, 50 pl of 36 nM
[3H]pon A solution (+200-fold excess of unlabelled pon A) were added, and the tubes were
incubated for a further 2 h at 2 5 T . Specifically bound hormone was then determined with
+ sucrose
DCC assay B. Control ( + buffer), 0 ; + sucrose (final concentration, 10% w/v),
(10%) + NaN, (0.002%, w/v), A; + sucrose (10%) + NaN3 + citrate (10 mM) + DTE (25 mM),
*;
0.
value after being frozen for 4 weeks at -20°C as the receptor in freshly
prepared extract (data not shown).
Effect of NaCl on the Stability of the Hormone-Receptor Complex
The receptor preparation was prelabelled with 13H]pon A (9 nM 200fold excess of unlabelled pon A) for 2 h at 25°C. Aliquots were then brought
to the required temperature ( l ° C or 17°C)and a concentrated NaCl solution
was added to give a final NaCl concentration of 0.5 M. The specifically bound
label was determined at fixed time points with charcoal assay B. In the
presence of 0.5 M NaC1, the hormone-receptor complex is unstable in a
temperature-dependent manner. At 1°C the half-life of the H-R complex is
approximately 80 min, while at 17°C the half-life is approximately 11 min
(data not shown).
DISCUSSION
The data which are presently available concerning ecdysteroid receptors
in insects are derived from four systems: Drosophila salivary glands [3],
Drosophilu imaginal discs [2], Drosophilu embryos [MI, and Drosuphilu &-cells
[l], which, although of uncertain cell typel are believed to be imaginal disc
cells [16]. The characterisation of the receptor species from &-cells and
imaginal discs, although far from complete, shows many similarities in their
properties [2-61. This is perhaps not surprising if they really are of the same
cell type. It is therefore of great interest to extend these studies to other cell
types of Drosophilu in order to determine if the various target tissues have
identical ecdysteroid receptors,
312
Dinan
The tumorous blood cell line used in this study is an excellent model
system for answering this question and many others concerning the mode of
action of ecdysteroids. In addition to being of defined origin [S], the cell line
shows an interesting and unusual response to ecdysteroids. The phagocytotic response induced by ecdysteroids [17-191 is similar to the role of haemocytes during metamorphosis in Drosophila [20,21]. The histolysis of larval
tissues occurring at the end of dipteran larval life has stimulated the interest
of many research workers [22-241, and there is evidence that this process is
induced by ecdysteroids [23,25]. The BII and BIII cell lines offer simpler
systems for the study of the induction of phagocytosis in haemocytes. In the
mutants from which the cell lines are derived, the tumorous blood cells not
only phagocytose dying larval tissues, but also larval tissues and imaginal
discs, which should survive metamorphosis [S]. Consequently, these cell
lines are also important for the study of neoplastic growth.
The [3H]pon A used in this study deserves some comment. It was produced by the reductive tritiation of a probable mixture of 24- and 26-dehydro25-deoxy-20-hydroxyecdysone,yielding a product with a specific radioactivity of 180 Cilmmol. This is equivalent to -6 tritium atoms per molecule of
ponasterone A. Localisation studies show that effectively all the tritium is
located in the side-chain. Since this region of the molecule is structurally
important for biological activity [26], it is perhaps not surprising that the very
'hot' pon A is not recognised by the BII cells in the same way as unlabelled
pon A. This isotope effect causes several methodological problems, since
standard receptor studies are based on the assumption that labelled and
unlabelled ligands are identical with regard to receptor affinity and binding
[27l.
Radioactive steroids of high specific radioactivity can be expected to be
unstable. Hptlc analysis of the radiochemical purity of the [3H]pon A (Fig.
2A) erroneously indicates that there is no degradation, even after 2 years.
However, reverse-phase HPLC separation of the same preparation (Fig. 2B)
reveals that extensive degradation (-50%) has in fact occurred during this
period. Consequently, it is not sufficient to determine the radiochemcial
purity by tlc alone.
A half-life of -2 years was to be expected for the I3H]pon A since each
molecule contains an average of six tritium atoms (tI,* for tritium = 12.1
years). BII cells do not metabolise any of the three ecdysteroids tested and
cannot convert ecdysone to 20-hydroxyecdysone. This is in agreement with
studies on ecdysteroid metabolism, which have shown that insect haemolymph is not an important metabolic site for ecdysteroids.
The specific uptake of pon A by intact BII cells is saturable (Fig. 3).
Scatchard plots of this data indicated that the cells do not recognise the
labelled and unlabelled pon A equivalently (Fig. 3A). A Scatchard plot
obtained solely with [3H]p~nA shows this to be the case (Fig. 3C) and also
reveals the finer structure of the binding curve for [3H]pon A. One plausible
interpretation of this curve is that it reveals the presence of two specific
binding sites, type I being of very high affinity (KD for [3H]pon A = 0.3 nM)
with approximately 650 sites per cell, and type I1 being of high affinity (KD
for [3H]pon A = 2 nM) with approximately 1,200 sites per cell. However,
Ecdysteroid Receptors
313
proof of this interpretation requires further experimentation. Multiple sites
for a steroid hormone have previously been described for the oestrogen
receptors in rat uterus [28, and references cited therein].
The association of [3H]pon A with BII cells is second order. The association
rate constant for [3H]pon A was determined to be 2.6 x 1O7M-'rnin-' at
21°C and 9.6 x 105M-lmin-' at 1°C. Yund et a1 [2] have determined k, for
pon A with imaginal discs to be 1.2 x 107M -'min-' at 25°C and 1.4 x
106M-'min-' at 0°C to 4°C. Beckers et a1 [5] have determined the k, for K,
cells to be 3 X 107M-'min-' at 20°C. These values are in very good agreement with those determined in the present work.
The dissociation kinetics of [3H]pon A show two components (Fig. 5): a
rapid dissociation of nonspecifically bound [3H]pon A, which is complete by
25 min at 22"C, followed by a slower dissociation of the specifically bound
[3H]pon A with a half-life of 145 min. The dissociation rate constant for this
slower component is 4 x 10-3/min. This compares with values of 5.8 x lop3/
min for K, cells [4] and 3.6 x 10-2/min for imaginal discs [2]. The values for
imaginal discs and K, cells were obtained with a r3H]pon A (122 Cilmmol),
which apparently has the same biological activity as unlabelled pon A [2].
The lower kd obtained with BII cells might be due to the altered affinity of
the [3H]ponA, or it might reflect a difference between the previously studied
ecdysteroid receptors and those of BII cells.
A value of 0.15 nM for KD is obtained from the quotient of kd and k,,
which is in good agreement with the value obtained from equilibrium studies
(0.3 nM). From the competition studies (Fig. 6), it is possible to calculate the
approximate KD values 1291 for unlabelled pon A (2 nM), 20-hydroxyecdyand
,ecdysone (30 pM).
sone (200 a)
The KD for unlabelled pon A is similar to that determined for labelled and
unlabelled pon A in previous studies [1,2]. Therefore, it would appear that
the higher affinity of the BII receptor for the [3H]pon A used in this study, in
comparison with that obtained in previous studies, is not due to a difference
in the receptor per se, but to the altered stereostructure of the hormone
caused by the high degree of tritiation in the side-chain, which fortuitously
results in a closer fit of the hormone in the binding site. Therefore, the
uptake and binding of [3H]pon A by intact BII cells show three classical
attibutes which indicate the presence of ecdysteroid receptors in these cells:
specificity, high affinity, and saturability. As a prerequistite to the purification
of the ecdysteroid receptor, it is necessary to obtain a cell-free extract containing binding activity and to characterise this binding activity as fully as
possible in order to avoid artefactual results during the purification. Additionally, it is important to determine conditions under which the receptor is
as stable as possible, both in the presence and absence of hormone.
It proved difficult to lyse BII cells homogeneously in hypotonic buffer,
and, therefore, a cell-free extract was prepared by sonification. This extract
binds [3H]pon A specifically as shown by both DCC assay and chromatography on Sephadex G-25. A good yield of receptor was obtained from the cells
if the cells were sonified with one 15-s burst at 40 W. Approximately 60% of
the specific binding activity found with whole cells is recovered in the
supernatant after centrifugation at 20,OOOg. The optimum pH for extraction
314
Dinan
and binding to the receptor is 8.2. This compares with a broad optimum pH
of 7-9, which has been found for the ecdysteroid receptor in the crayfish
Astucus Zeptodactylus [30].
The presence of divalent metal ions results in reduced binding to the
receptor. Calcium and magnesium ions cause 85% inhibition of the control
binding at 50 mM. Magnesium is not so inhibitory (50% inhibition at 100
mM). Monovalent cations (Na+ and K+)show a reproducible stimulation of
specific binding at low concentrations ( <75 mM) if the preparation is prepared in TrislHCl buffer (75mM, pH 8). If, however, the cells are sonified in
HEPES buffer (10 mM, pH 8), no such stimulation is seen at low NaCl
concentrations; all concentrations of NaCl(25-250 mM) were inhibitory, with
50% inhibition at 200 mM (Dinan, unpublished observation). The reason for
this difference between the two buffer systems is unknown.
The H-R complex is also unstable in the presence of high salt concentrations. It decays rapidly in the presence of 0.5 M NaCl in a temperature
dependent fashion. Mammalian steroid hormone receptors have also been
found to be unstable in the presence of high salt concentrations [31,32], a
process which can be hindered by the addition of glycerol [32].
The sonification preparation shows a saturable specific binding of [3H]pon
A (Fig. 8A). If these results are expressed according to Scatchard [14](Fig.
8B), a curve is obtained, which is similar to that obtained with whole cells
(Fig. 3C). This curve can be resolved into two components: a very high
affinity specific component (KD = 10-loM; 10 fmollmg protein) and a high
affinity specific component (KD = 1nM; 30 fmollmg protein).
Specifically bound [3H]pon A is macromolecularly bound, as shown by
chromatography of the preincubated cell extract on Sephadex G-25 (Fig. 9).
The radioactivity eluting in the macromolecular fraction is totally compatible
with 100-fold excess of unlabelled pon A. The nature of the macromolecular
binding component was investigated using several digestive enzymes (Table
1).The most effective enzymes were the proteases, pronase and trypsin,
indicating that pon A binds to a protein. The heat instability of the binding
and its susceptibility to NEM also indicate this. The effect of NEM also
suggests that a sulphydryl group is important for binding activity. Both
DNase and RNase also slightly reduce the amount of [3H]pon A specifically
bound. In the case of the DNase, this can probably be attributed to the
presence of contaminating protease activity (detected by the "azocasein
method"), which is not inhibited by aprotinin (Dinan, unpublished observation). The RNase preparation does not show any protease activity in the
azocasein test, but azocasein may not be a suitable substrate and the test is
very insensitive.
Triton X-100 and Nonidet NP-40, when present at concentrations as low
as 0.1% vlv, totally prevented the adsorption of unbound [3H]pon A to
charcoal. Consequently, it was necessary to separate the free and bound
hormone on Sephadex G-25 in order to test the effect of these compounds
on binding. When added at the same time as the labelled pon A, both
detergents prevent all specific macromolecular binding, When the receptor
preparation was equilibrated with C3H]pon A and then NP-40 (0.8%) was
added just before application to the Sephadex G-25 column, the amount of
Ecdysteroid Receptors
315
[3H]pon A macromolecularly bound was identical with the control without
NP-40 (24% of added label). Therefore, these detergents cannot be used for
the extraction of naive ecdysteroid receptors, but could be used to lyse cells
which have already been incubated with hormone. Triton X-100 has been
shown to increase the dissociation of mammalian steroid hormones from
their receptors [33].
The ecdysteroid-receptor complex from BII cells sediments as one peak on
sucrose density gradients with an S-value of approximately 6S, which is
similar to the value obtained for the ecdysteroid receptors from Dvosophilu
embryos and imaginal discs under low salt conditions [34] and for that
obtained for the ecdysteroid receptor from freshly harvested K, cells [6]. A
similar value (6.6s) was obtained if an unlabelled BII cell extract was separated by sucrose gradient centrifugation, fractionated, and the specific binding determined in the individual fractions (data not shown).
In common with many other steroid hormone receptors, the ecdysteroid
receptor is more stable in crude preparations in the presence of ligand than
in its absence (Fig. 11).The half-life for the unloaded receptor at 8°C in the
presence of aprotinin (which was added as a protease inhibitor) is 3 days.
Aprotinin extends the life of the H-R complex at room temperature (Dinan,
unpublished observation). The H-R complex is stable for at least 3 days at
8°C but rapidly decays at 25°C. The stability of the unloaded receptor at 8°C
is improved by the presence of thiol reagents, sucrose, glycerol, or citrate.
The first three compounds have been shown to be beneficial for the stability
of many steroid receptors [35-381. Citrate has been shown to improve the
stability of the rat hepatic glucocorticoid receptor [39], although the mechanism is not clear.
Both molybdate and EDTA have been shown to stabilise certain receptors
[31,36,38,40,41]. However, this does not appear to be the case with the
ecdysteroid receptor from BII cells since they both accelerate the decay of
binding activity. A combination of the additives 10% sucrose, 0.02% NaN3,
10 mh4 citrate, and 25 mh4 DTE considerably improves the stability of the
ecdysteroid receptor at 8°C (Fig. 12). When frozen at -20°C in the presence
of these additives, the preparation is totally stable for a considerable length
of time. If the frozen receptor preparation (4weeks old) is thawed, incubated
with [3H]ponA (2 h at 25°C) and then separated by sucrose gradient centrifugation, a single peak at 6s is obtained (Dinan, unpublished observation).
Thus, it has been possible to partially characterise the high affinity binding
proteins for [3H]pon A in a cell-free extract of a tumorous blood cell line of
Drosophilu, which responds to ecdysteroid treatment. Many interesting questions remain to be investigated. Particularly important is the location of the
receptor within the cell since both imaginal discs [2] and K, cells [6] possess
unoccupied nuclear as well as cytosolic receptors.
LITERATURE CITED
1. Mar6y P, Dennis R, Beckers C, Sage BA, O’Connor JD: Demonstration of an ecdysteroid
receptor in a cultured cell line of Drosophilu melnnoguster. Proc Natl Acad Sci USA 75, 6035
(1978).
316
Dinan
2. Yund MA, King DS, Fristrom JW: Ecdysteroid receptors in imaginal discs of Drosophila
melnnoguster. Proc Natl Acad Sci USA 75, 6039 (1978).
3. Schaltman K, Pongs 0: Identification and characterization of the ecdysterone receptor in
Drosophilu melanoguster by photoaffinity labeling. Proc Natl Acad Sci USA 79, 6 (1982).
4. Beckers C, Mar6y P, Dennis R, O’Connor JD, Emmerich H: The uptake and release of
ponasterone A by the K, cell line of Drosophila melunoguster Mol Cell Endocrinol 17, 51,
(1980).
5. Beckers C, MarBy P, O’Connor JD, Emmerich H: Uptake and release of 3H-ponasterone
A by the K, cell line of Drosophila melanogaster. In: Developments in Endocrinology, Vol.
7. Progress in Ecdysone Research, Hoffmann JA, ed. ElsevierlNorth-Holland Biomedical
Press, Amsterdam, pp 335-347 (1980).
6. Sage BA, Tannis MA, O’Connor JD: Characterization of ecdysteroid receptors in cytosol
and naive nuclear preparations of Drosophilu K, cells. J Biol Chem 257, 6373 (1982).
7. Sang JH: Drosophilu cells and cell lines. Adv Cell Culture 2, 125 (1981).
8. Gateff E, Gissmann L, Shrestha R, Plus N, Pfister H, Schroder J, Zur Hausen H: Characterization of two tumorous blood cell lines of Drosophila melunoguster and the viruses they
contain. In: Invertebrate Systems in Vitro. Kurstack E, Maramorosch K, Diibendorfer A,
eds. ElsevierlNorth-Holland Biomedical Press, Amsterdam, pp 517-533 (1980).
9. Heinrich G: Isolierung, Struktur und Stoffwechselvon Insektenhautungshormonen.Ph.D.
dissertation, Math Nat Fakultat, Universitat Hamburg, FRG,pp 29-35, 65-67 (1970).
10. Dinan LN, Rees HH: The identification and titres of conjugated and free ecdysteroids in
developing ovaries and newly-laid eggs of Schistocercu greguriu. J Insect Physiol 27, 51
(1981).
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