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Ligand binding is without effect on complex formation of the ligand binding domain of the ecdysone receptor (EcR).

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Archives of Insect Biochemistry and Physiology 59:1–11 (2005)
Ligand Binding Is Without Effect on Complex
Formation of the Ligand Binding Domain of the
Ecdysone Receptor (EcR)
B. Greb-Markiewicz, T. Fauth, and M. Spindler-Barth*
The ligand-binding domain (LBD) encompassing the C-terminal parts of the D- and the complete E-domains of the ecdysteroid
receptor (EcR) fused to Gal4(AD) is present in two high molecular weight complexes (600 and 150 kDa) in yeast extracts
according to size exclusion chromatography (Superdex 200 HR 10/30). Hormone binding is mainly associated with 150-kDa
complexes. Complex formation is not influenced by hormone, but the ligand stabilizes the complexes at elevated salt concentrations. Mutational analysis of Gal4(AD)-EcR(LBD) revealed that formation of 600-kDa, but not 150-kDa, complexes depends
on dimerization mediated by the EcR(LBD). Deletion of helix 12 is without effect. Mutation of K497 in helix 4, known to be
essential for comodulator binding, abolishes 600-KDa complexes, but does not interfere with the formation of 150-kDa
complexes. In contrast, the DE-domains of USP fused to Gal4(DBD) elute as monomer after elimination of the dimerization capacity of the ligand-binding domains by mutation of P463 in helix 10. The data presented here reveal that the
complex formation of ligand-binding domains EcR and USP ligand is different. Arch. Insect Biochem. Physiol. 59:1–11,
2005. © 2005 Wiley-Liss, Inc.
KEYWORDS: Drosophila; Gal4 fusion protein; hormone; insect; nuclear receptor; steroid; Ultraspiracle
INTRODUCTION
Nuclear receptors (Kumar and Thompson,
1999; Spindler-Barth and Spindler, 2003; Whitfield
et al., 1999) are ligand-dependent transcription factors composed of different domains with well-defined functions. The individual receptor domains
influence each other. For example, the ligand-binding domain (LBD) encompassing the C-terminal
parts of the D- and the complete E-domains (Grebe
et al., 2003, 2004) is responsible for hormone
binding. In addition, ligand binding enhances
DNA binding mediated by the C-domain, and in-
fluences transactivation potency in a ligand-dependent manner, in addition to the hormone-independent transactivation function associated with the
A/B region. The LBD interacts also with other proteins like comodulators (Dressel et al., 1999;
Thormeyer et al., 1999), heat shock proteins, and
immunophilins (Song et al.,1997), which also affects receptor function.
Generally, nuclear receptors are present as
homo- or heterodimers and dimerization/oligomerization is an important prerequisite for receptor function, including hormone binding (Grebe
et al., 2003), nuclear transport (Prüfer et al., 2000),
Department of General Zoology and Endocrinology, University of Ulm, Ulm, Germany
B. Greb-Markiewicz’s present address is Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw University of Technology, Wybrzeze
Wyspianskiego 27,50-370 Wroclaw, Poland.
Contract grant sponsor: DFG; Contract grant number: Sp 350, 5-1.
*Correspondence to: M. Spindler-Barth, Department of General Zoology and Endocrinology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.
E-mail:margarethe.spindler-barth@biologie.uni-ulm.de
Received 27 July 2004; Accepted 28 July 2005
© 2005 Wiley-Liss, Inc.
DOI: 10.1002/arch.20054
Published online in Wiley InterScience (www.interscience.wiley.com)
2
Greb-Markiewicz et al.
and interaction with DNA (Khorasanizadeh and
Rastinejad, 2001; Przibilla et al., 2004; Lezzi et al.,
2002). Dimerization/oligomerization of nuclear
receptors is a rather complex process, which may
involve several receptor domains.
Dimerization mediated by the DNA-binding
domains of EcR and Ultraspiracle (USP), depends
on the presence of DNA, and is well characterized.
The 3D structure of this complex has been elucidated recently for USP (Devarakonda et al., 2003).
An additional dimerization interface is present in
the E-domain of all nuclear receptors and involves
the participation of helix 10 (Bourget et al., 1995).
As reported also from several vertebrate nuclear receptors (Savory et al. 2001; Tetel et al., 1997), an
additional dimerization site in the hinge region was
found in EcR of Choristoneura fumiferana (Perera
et al.,1999). Dimerization/oligomerization mediated by the N-terminal A/B domain of USP was
also described (Rymarczyk et al., 2003).
The functional ecdysteroid receptor is generally
considered as the heterodimer of EcR and USP (Yao
et al., 1993; Spindler-Barth and Spindler, 2003).
In all insects studied so far, high-affinity binding of
ecdysteroids is restricted to the complex of EcR/USP
(Grebe and Spindler-Barth, 2002; Hayward et al.,
2003; Swevers et al., 1996) with the exception of
Drosophila EcR, which binds hormone already in the
absence of USP (Grebe et al., 2003) although with
lower affinity (KD = 42 nM (Grebe et al., 2004).
The X-ray structure of the heterodimer of
EcR(LBD) and USP(LBD) stabilized by ponasterone A or a nonsteroidal hormone agonist has been
elucidated (Billas et al., 2003). In contrast to the
LBD of USP (Billas et al., 2001; Clayton et al.,
2001), the 3D structure of the EcR(LBD) alone is
not known so far. Therefore, only functional studies allow the identification of amino acids or amino
acid sequences involved in homodimerization of
EcR at present.
The functional role of amino acids involved in
hormone binding and ligand-dependent dimerization of EcR was characterized recently (Grebe et
al., 2003; Bergman et al., 2004). Wild type and
mutated ligand-binding domains of the ecdysteroid
receptor and its dimerization partner USP from
Drosophila melanogaster, fused to Gal4 domains were
examined by two hybrid assays (Lezzi et al., 2002),
ligand-binding and gel mobility shift assays (Grebe
et al., 2003, Grebe et al., 2004; Przbilla et al., 2004).
From these experiments, the minimal receptor fragment necessary for ligand binding of Drosophila EcR,
which consists of the C-terminal part of the D- and
the complete E-domain, was inferred. In addition
the participation of defined amino acids in ligand
binding and dimerization was studied.
The aim of this report was to identify the complex associated with ligand binding to the EcR(LBD)
from Drosophila melanogaster and to characterize
complex formation and dimerization/oligomerization of the ligand-binding domains of EcR and USP
in the absence of the heterodimerization partner.
We determined complex formation in a direct approach with size exclusion chromatography. The
study was performed with the same receptor fragments (EcR: aa375-652, Usp: aa172-508), which
were used previously for functional studies (Grebe
et al., 2004).
MATERIALS AND METHODS
Yeast Strain
Saccharomyces cerevisiae strain Y190 (Harper et
al., 1993) was cultured according to the instructions of the manufacturer (Clontech Laboratories,
Palo Alto, CA). Cells were transformed with
lithium acetate (Guthrie and Fink, 1991) and selected by auxotrophy for tryptophan (pAS2-1) and
leucine (pACT2), respectively.
Yeast Expression Plasmids
DNA encoding the C-terminal part of the Dand E-domains of the Drosophila ecdysone receptor EcR (aa 375-652) was cloned into the expression vector pACT2 (Li et al.,1994; Lezzi et al.,
2002), resulting in a Gal4(AD)-EcR(LBD) fusion.
For expression of Gal4(DBD)-USP(LBD), the corresponding domain of Ultraspiracle (aa 172-508)
was cloned either into the vector pGBKT7 (Louvet
et al., 1997) or into the vector pAS2-1 (Harper et
al., 1993; Lezzi et al., 2002). Mutated receptor doArchives of Insect Biochemistry and Physiology
Ligand Binding and Complex Formation of EcR/USP
mains (Grebe et al., 2003; Przibilla et al., 2004)
were kindly provided by V. Henrich (University of
North Carolina, Greensboro, NC).
Preparation of Yeast Extracts
Single colonies (not older than 4 days) of yeast
transformants carrying the expression plasmids
were picked and cultured at 30°C overnight in 5
ml selective medium containing 2% glucose with
vigorous shaking (225 rpm). They were then diluted in 50 ml YPD medium (20 g/l peptone, 10
g/l yeast extract, 2% glucose) and grown under the
same conditions until the OD600 reached 0.6. Cells
were harvested by centrifugation (1,500g, 5 min,
4°C) in prechilled tubes and washed with 50 ml
ice-cold buffer (20 mM HEPES, 20 mM NaCl, 20%
glycerol, 1 mM EDTA, 1 mM 2-mercaptoethanol,
pH 7.9). The pellets were frozen in liquid nitrogen and disrupted for 2 min at 2,000 rpm using a
Micro-dismembrator S (B. Braun Biotech International, Melsungen, Germany). After thawing,
homogenates were diluted with buffer and supplemented with a mixture of protease inhibitors
(aprotinin, leupeptin, pepstatin, benzamidine,
antipain, chymostatin; final concentration 2 µg/ml
each and 1 mM phenylmethyl-sulfonyl fluoride)
immediately before use. After short treatment with
ultrasonic power (microtip 2 × 2 s, 90 Watt,
Branson Sonifier, B-12, Branson, Danbury, CT) the
samples were centrifuged (100,000g, 1 h, 4°C) and
frozen in aliquots at –80°C until use. To eliminate
DNA-dependent dimerization of Gal4(DBD)USP(LBD), extracts were prepared in the presence
of 0.4 M NaCl. DNA was destroyed by incubation
with 20 µg/ml DNAse I (Serva, Heidelberg, Germany) for 45 min at 37°C. The reaction was
stopped by addition of EDTA (final concentration
20 mM). Extracts were desalted prior to chromatography with Ultrafree-0.5 filter units (Millipore,
Schwalbach, Germany).
Western Blot And Quantitative Determination Of
Fusion Proteins
Yeast extracts were diluted with sample buffer
(100 mM Tris, 3% SDS, 2% 2-mercaptoethanol,
May 2005
3
10% glycerol, 0.05% bromphenol blue, pH 8.8)
and boiled for 3 min (Laemmli, 1970). Ten micrograms of protein (Bradford, 1976) were applied
on each lane of an acrylamide gel and subjected
to electrophoresis using a Hoefer miniVe, (Amersham Biosciences, Freiburg, Germany) at 300 V and
15 mA. Gels were electroblotted on nitrocellulose
membranes (BA 85, 45 µm pore size, Schleicher
and Schuell, Dassel, Germany) according to KhyseAndersen (1984). The membranes were soaked in
blocking buffer (3% milk powder, 1% fat in 20
mM Tris-HCl, 137 mM NaCl, 0.1% Tween 20, pH
7.6, 0.02% Thimerosal). EcR(LBD) fusion protein
was probed with a Gal4(AD)- specific antibody
(no. 5398-1, Clontech Laboratories, Palo Alto, CA)
diluted in blocking buffer 1:5,000. USP fusion proteins were probed either with Gal4(DBD) specific
antibody (no. sc-577, Santa Cruz Biotechnology
Inc, Santa Cruz, CA) diluted 1:100 or with c-Myc
specific antibody (3800-1, Clontech Laboratories)
diluted 1:1,000. Specific Western signals were detected with peroxidase-conjugated secondary antibodies diluted 1:1,000 (anti-mouse IgG, Sigma
A-5906, Sigma-Aldrich, Taufkirchen, Germany) or
1:500 (anti- rabbit IgG, Sigma A-6667), in TBS-T
(20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween 20,
pH 7.6). Detection and quantification were done
as described in detail (Rauch et al., 1998). Specific
signals were imaged with Fluor-S MultiImager (BioRad Laboratories, Hercules, CA) and evaluated with
the software Multi-Analyst/PC (Version 1.1).
Chromatography
Two hundred microliters yeast extract (2 µg/µl),
incubated for 30 min at room temperature with
10–5 M 20-OH-ecdysone if indicated, was subjected
to size exclusion chromatography (Superdex 200
HR 10/30 , Amersham Pharmacia Biotech) using
an Äkta™ purifier(Amersham Pharmacia Biotech,
Uppsala, Sweden). After equilibration of the column with elution buffer (20 mM K-phosphate, pH
7.4, 50 mM KCl, 1 mM EDTA, 10% glycerol), the
sample was loaded on the column and separated
(flow rate of 0.25 ml/min, 4°C). Fractions were
4
Greb-Markiewicz et al.
collected (500 µl) and proteins precipitated with
7.5% TCA (final concentration). A molecular
weight marker kit (Sigma-Aldrich, Taufkirchen,
Germany) was used to calibrate the column. Fractions were subjected to Western blotting and specific bands quantified as described above.
Ligand Binding Assays
Yeast extracts were diluted with buffer and
supplemented with protease inhibitors as described
above immediately before use. Ligand binding was
determined with [3H]-ponasterone A (specific activity 2.5 TBq/mmol; kind gift of Dr. H. Kayser,
Syngenta, Basel, Switzerland) using a filter assay
as already described (Turberg and Spindler, 1992).
Radiolabeled ponasterone A was used, because the
affinity to EcR is higher compared to 20-OH-ecdysone. Fusion proteins were quantified by Western
blots as described above and normalized based on
wild type expression levels. Receptor proteins were
mixed with 4-5 nM [3H]-ponasterone A and incubated for 1 h at room temperature. Nonspecific
binding determined in the presence of 0.1 mM
non-labeled 20-OH-ecdysone was subtracted. Purity of [3H]-ponasterone A was checked routinely
by HPLC analysis. Ligand binding data of mutated
receptors were expressed as % of wild type hormone binding (= 100%).
RESULTS
The Ligand-Binding Domain of EcR Is Mainly
Associated With Complexes of Approximately 150 kDa
Gal4 fusions of receptor domains were used
to allow detection of receptor domains with Gal4specific antibodies after gel filtration, since no antibodies directed against LBDs are available.
Gal4(AD)-EcR(LBD) is present in yeast extracts
mainly as high molecular weight complexes of various sizes with two predominant peaks with an apparent Mτ of approximately 600 and 150 kDa (Fig.
1A). No monomers (Mτ = 49 kDa) were present at
low salt concentrations. Hormone binding is observed to a small degree in many fractions, but is
predominantly found in fractions corresponding
to an Mr of about 150 kDa (Fig. 1B). The complexes are highly sensitive to even moderate salt
concentrations (Fig. 2A), but complex formation
is restored by addition of ligand to 0.4 M NaCl
extracts (Fig. 2B). Western blots of the same fractions obtained by gel filtration (Fig. 2A) were
probed with a Gal4 (AD) specific antibody to show,
which complexes are associated with EcR (Fig. 2C).
The molecular weight of specific receptor bands
detected by Western blots is about 49 kDa, which
corresponds to the molecular weight of the fusion
protein monomer and demonstrates that no proteolytic degradation has occurred.
Helix 10 of EcR(LBD) Is Essential for Formation of 600
kDa-, But Not 150 kDa- Complexes
To elucidate complex formation in more detail,
ligand-binding domains of EcR with mutations in
helix 10, which is essential for heterodimerization
(Grebe et al., 2003, Bergman et al., 2004), were
examined (Table 1). Amino acids at position 612,
615, and 617 in helix 10 and EcRN626K adjacent to
helix 10 were selected. The amount of high molecular weight complexes is reduced or even abolished in mutated receptor fusion proteins (EcRA612V,
EcRL615A, and EcRI617A) and indicates that these complexes consist at least partially of dimers/oligomers
of the EcR fusion protein. Obviously, 150-kDa
complexes do not depend on the dimerization interface of the EcR(LBD) and are present even at a
higher degree compared to wild type.
Complex Formation Is Not Changed, If Helix 12
Is Truncated
Although it was assumed previously that Gal4
fusion proteins with nuclear receptor ligand binding domains do not interact with comodulators
present in yeast cells, Bergman et al. (2004) and
Tran et al. (2001) already showed that ligand-mediated activity of ecdysteroid receptor expressed in
yeast cells depends on coactivators. VomBaur et al.
(1998) also demonstrated that the yeast Ada complex consists of at least four proteins of different
sizes and acts as a comodulator in yeast cells by
Archives of Insect Biochemistry and Physiology
Ligand Binding and Complex Formation of EcR/USP
5
Fig. 1. Size exclusion chromatography of Gal4(AD)EcR(LBD) wild type. A: Fractions were subjected to Western blot and specific signals were quantified. Receptor
content (black bars) is expressed as % of total amount of
receptor applied to the column, which is set as 100%. B:
Hormone-binding capacity of the corresponding fractions
determined with 3H- ponasterone A.
Fig. 2. Size exclusion chromatography of Gal4(AD)EcR(LBD) wild type. Fractions were subjected to Western
blot and specific receptor bands quantified (black bars).
Total amount of receptor applied to the column was set
as 100%. A: 0.4 M NaCl was added to the extraction buffer.
B: 0.4 M NaCl extracts were incubated subsequently with
an excess of 20-OH-ecdysone (10–5 M) for 30 min at room
temperature. The same hormone concentration was present
in the elution buffer to prevent dissociation of the ligand
May 2005
during chromatography. C: Western blots of the fractions
shown in Figure 2A using a Gal4 (AD = specific antibody)
revealed specific receptor bands at 49 kDa (←) even at
higher retention volumes and demonstrate that that no
proteolytic degradation has occurred.
6
Greb-Markiewicz et al.
TABLE 1. Complex Formation of Wild Type and Mutated
Gal4(AD)-EcR(LBD) and Gal4(DBD)-USP(LBD)*
Receptor domain
[fused to Gal4 (AD)]a
EcR (LBD) wild type
EcR-K497E
EcR-A612V
EcR-L615A - hormone
EcR-L615A + hormone
EcR-1617A
EcR-N626K
EcR∆H12
600-kDa complexes
(% of total amount)
150-kDa complexes
(% of total amount)
28.8 ± 10.1
0
0
10.4 ± 5.1
15.5 ± 13.1
0
0
28 ± 3.5
33.8 ± 4.8
68.3 ± 0.4
82.3 ± 4.6
40.7 ± 5.0
49.2 ± 13.7
82.5 ± 4.3
90 ± 2.3
33 ± 1.8
*Total amount of receptor applied to the column is 100%. The sum of 600 and
150 kDa complexes is less than 100% because complexes of intermediate size
were not considered.
a
Low salt conditions (100 mmol Tris, pH = 7.4).
binding to the ligand-dependent transactivation
domain AF2 in helix 12. We, therefore, tested
EcR(LBD)s with truncated or mutated comodulator
binding sites.
Deletion of helix 12 does not change the elution pattern under low salt conditions (Table 1,
Fig. 3A), but salt sensitivity is increased. At 0.4 M
salt, all complexes disintegrate completely and
mainly monomers were observed (Fig. 3B). Stabilization by hormone is not possible in this case,
since ligand binding is abolished after deletion of
Helix 12 (Grebe et al., 2003).
Mutation of lys at position 497 to glu localized
within the signature motif for co-regulator recruitment (Kao et al., 2003) selectively destroys the capability to form high molecular weight complexes
(600 kDa), but formation of 150-kDa complexes
is not impaired.
Gal4(DBD)-USP(LBD) Dimer/Oligomer Formation (600
kDa) Requires Participation of Helix 10
In contrast to EcR(LBD), USP(LBD) fused to
Gal4(DBD) is exclusively present in high molecular weight complexes of about 600 kDa (Fig. 4A).
Is seems reasonable to assume that these complexes consist of dimers/oligomers of Gal4(DBD)USP(LBD), since mutation of P463—an amino
acid located in helix 10 and essential for dimerization (Bourguet et al., 1995)—to glu abolishes complex formation mediated by the ligand-binding
domain. Remaining complexes of about 150 kDa
Fig 3. Size exclusion chromatography of Gal4(AD)EcR(LBD) ∆H12. A: Low salt extraction (100 mM Tris, pH
= 7.4). B: Extract prepared with buffer containing 0.4 M
NaCl.
(Fig. 4B) disintegrate after DNAse treatment into
monomers (Fig. 4C), and demonstrates that these
complexes are due to DNA-dependent dimerization of the Gal4(DBD).
DISCUSSION
High molecular weight complexes of full-length
EcR/USP are present in cytosol and nuclear extracts
of insect cells (Spindler-Barth and Spindler, 1998),
but the composition of these complexes is not
known so far. Two types of complexes of EcR can
Archives of Insect Biochemistry and Physiology
Ligand Binding and Complex Formation of EcR/USP
7
be discriminated: a high molecular weight complex is dependent on dimerization mediated by the
E-domain, especially helix 10. A smaller complex
of about 150 kDa is resistant to mutation of amino
acids known to be involved in ligand-dependent
dimerization (Grebe et al., 2003; Bergman et al.,
2004).
The minimum sequence necessary for hormone
binding (Grebe et al., 2003) and hormone-dependent transactivation (Grebe et al., 2003; Lezzi et al.,
2002), was used in this study, which encompasses
the C-terminal part of the D-domain in addition to
the E-domain and underlines that the modular structure composed of domains A–E and functional
nuclear receptor domains are not equivalent (Xu et
al., 1996). As reported previously for the estrogen
receptor (Salomonsson et al., 1999), ligand binding is not required for formation of 150-kDa complexes of EcR(LBD). Therefore, the participation of
an additional ligand-independent dimerization site
in complex formation localized in the hinge region is likely, as reported previously for vertebrate
receptors (Tetel et al., 1997).
The D-domain was originally considered as an
inert link between DNA-binding and ligand-binding domains. Meanwhile, it became apparent that
the hinge region is involved in several receptor
functions and modifies the activity of both domains adjacent to the D-domain at the N-terminal and C-terminal end. The hinge region is
indispensable for hormone binding, modifies
ligand-binding specificity, for example, for diacylhydrazines (Spindler et al., 2001). A nuclear localization signal is present in the D-domain of
vertebrate nuclear receptors (Dingwall and Laskey,
1998). A similar sequence is also found in the Ddomain of EcR, although its function is not yet
elucidated (Nieva, personal communication). In
addition, the interaction of high-mobility group
box 1 proteins (Verrijdt et al., 2002), which enFig. 4. Size exclusion chromatography of Gal4(DBD)USP(LBD). Fractions were subjected to Western blot and
quantified (black bars). Total amount of receptor applied
to the column is 100%. A: Wild type, B: Gal4(DBD)USP(LBD) with P463 mutated to D. C: The same extract
May 2005
as in B was extracted with 0.4 M NaCl and incubated with
DNAse to destroy DNA-dependent dimerization mediated
by the Gal4(DBD) moiety.
8
Greb-Markiewicz et al.
hance DNA-binding, requires the participation of
the hinge region.
The essential role of the D-domain for the functionality of the ligand-binding domain was shown
previously by Perera et al. (1999) for Choristoneura
EcR, and was attributed to the enhanced stability of
the ligand-binding pocket due to interaction with
two additional helices present in the D-domain
(Spindler et al., 2001). Complex formation occurs
in the absence of the heterodimerization partner as
shown previously for both EcR (Spindler-Barth et
al., 2004) and USP (Przibilla et al., 2004).
Heterodimerization of DmEcR with DmUSP is
strongly preferred (Yao et al., 1993) and the participation of the ligand-binding domain in dimerization has been demonstrated (Grebe et al., 2003;
Przibilla et al., 2004).
Receptor dimerization is mediated by several
domains. Depending on the physiological conditions, different dimerization interfaces might be
predominantly involved, which may explain that
the results of X-ray studies and functional tests can
be different (Ribeiro et al., 2001). In the absence
of DNA and ligand, for example in the cytoplasm,
homodimerization/oligomerization seems to be
mediated mainly by the hinge region. The importance of the dimerization site in the ligand-binding
domain E is well established for heterodimerization
with USP especially in the presence of ligand and is
important for stimulation of nuclear transport by
hormones (Nieva et al., unpublished results). Later
on, DNA-dependent dimerization is required for
transactivation.
Different dimerization sites may participate in
homo- and heterodimerization as reported several
fold for vertebrate receptors, e.g., glucocorticoid receptor (Savory et al., 2001), retinoic X receptor
(Zhang et al., 1994), and thyroid receptor (Ribeiro
et al., 2001; Kitajima et al., 1995). Moreover, the
importance of each dimerization site seems to be
different for individual receptors. In USP, the participation of the hinge region of USP seems not to
be important, since only monomers of Gal4(DBD)USP(LBD) are obtained in the absence of DNA, if
the dimerization mediated by the ligand-binding
domain is interrupted.
Three different dimerization sites are identified
in EcR, which can be active selectively and independent from each other, although a mutual influence of receptor domains certainly modifies
their activity (Kumar et al., 1999). The impact of
different dimerization interfaces on complex formation and individual receptor functions of EcR
and USP and the interaction between dimerization
sites certainly deserves further investigation.
Complex formation does not require a specific
insect milieu, and also is observed in yeasts. It is
reasonable to assume that factors present in yeast
cells, can replace additional insect proteins. Several proteins like heat shock proteins (Arbeitman
and Hogness, 2000), imunophilins (Song et al.,
1997), and comodulators (VomBaur et al., 1998;
Tran et al., 2001) are described. Additional proteins might be involved in the formation the 600kDa complexes, since deletion of helix 12 and
mutation of K497, known to interact with comodulators, either destabilize the complex or reduce
the size of the high molecular weight ecdysteroid
receptor complexes. In contrast, 150-kDa complexes are not affected and are also independent
of the dimerization interface in the E-domain of EcR.
The analysis of these additional proteins is not possible in crude extracts, since yeast proteins dominate the protein pattern obtained after silverstaining
of SDS gels. Therefore, purification of a receptor in
a functional state is required. After purification, only
a minor fraction of about 2–35% (Arbeitmann and
Hogness, 2000; Grebe and Spindler-Barth, 2002)
of the total receptor content is still functional,
which is not sufficient to analyse association of
receptor proteins with additional proteins.
ACKNOWLEDGMENTS
Plasmids encoding wild type and mutated
ligand binding domains of EcR and USP were
kindly provided by V.C. Henrich (University of
North Carolina, Greensboro, NC). Radiolabeled
Pon A was a kind gift of Dr. H. Kayser (Syngenta,
Basel, Switzerland). We gratefully acknowledge the
careful technical assistance of N. Möbius). The
Archives of Insect Biochemistry and Physiology
Ligand Binding and Complex Formation of EcR/USP
work was supported by a DFG grant (Sp 350, 5-1)
to M.S.B.
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