close

Вход

Забыли?

вход по аккаунту

?

Transcriptional activity of ecdysone receptor isoforms is regulated by modulation of receptor stability and interaction with Ab- and C-domains of the heterodimerization partner ultraspiracle.

код для вставкиСкачать
A r t i c l e
TRANSCRIPTIONAL ACTIVITY OF
ECDYSONE RECEPTOR ISOFORMS
IS REGULATED BY MODULATION
OF RECEPTOR STABILITY AND
INTERACTION WITH AB- AND
C-DOMAINS OF THE
HETERODIMERIZATION PARTNER
ULTRASPIRACLE
Heike Ruff, Christian Tremmel, and
Margarethe Spindler-Barth
Institute of General Zoology and Endocrinology, University of Ulm,
Ulm, Germany
The stability of ecdysone receptor (EcR) expressed in a heterologous
system is regulated in an isoform-specific manner and modified by ligand
and heterodimerization partner. Transcriptional activities of various
receptor complexes with Usp and ligand as determined by reporter assays
are the result of two effects: change in receptor concentration and altered
transcriptional capability. Transcriptional activity of EcR-A is low when
compared to EcR-B1 independent of the absence or presence of
Ultraspiracle (Usp). Ligand increased the concentration of EcR-A, but
had no effect on the transcriptional capability, in contrast to EcR-B1,
which is not stabilized by hormone or Usp, but the transcriptional
capability is enhanced by heterodimerization and ligand. Exchange of the
AB-domain of Usp by the activation domain (AD) of Vp16 revealed that
the N-terminus of Usp inhibited transcriptional activity only with EcR-B
isoforms, whereas the hexapeptide in the AB-domain of wild type Usp
adjacent to the C-domain of Usp harbours an activating function.
Deletion of the C-domain of Usp did not affect the stability of the receptor
Grant sponsor: Deutsche Forschungsgemeinschaft (to M.S.B.); Grant numbers: Spi 350/5-1 and /5-2; Grant
sponsor: Cusanuswerk.
Correspondence to: Prof. Dr. Margarethe Spindler-Barth, Institute of General Zoology and Endocrinology,
University of Ulm, 89081 Ulm, Germany. E-mail: margarethe.spindler-barth@uni-ulm.de
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 72, No. 3, 154–171 (2009)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20309
Transcriptional Activity of Ecdysone Receptor
155
complex, but reduced the transcriptional capability of heterodimers with
all EcR-isoforms, indicating that the stability of the receptor, which is
important for termination of the hormone signal transduction, is
regulated in a cooperative manner by the AB-domains of EcR and Usp,
and ligand. We show the active role of Usp in modulation of the
transcriptional activity of the heterodimer in an isoform-specific manner
by the inhibitory N-terminus, the activating hexapeptide in the ABC 2009 Wiley Periodicals, Inc.
domain, and the C-domain of Usp. Keywords: insect; Drosophila; hormone; nuclear receptor; receptor protein
stability; hormonal regulation
INTRODUCTION
Transcriptional activity of nuclear receptors is modulated by many factors such as
dimerization partner, phosphorylation pattern, interaction with comodulators, and
other accessorial proteins. These interactions enable fine-tuning of the hormonal
response and adaption of receptor activity to the physiological requirements of the
target cells. In addition, qualitative and quantitative differences in the receptor activity
of receptor isoforms with different AB-domains allow variation of the hormone
response (Cheng, 2005; Duma et al., 2006a,b).
In Drosophila melanogaster, three ecdysone receptor isoforms (EcR-A, EcR-B1, and
EcR-B2) have been described with various lengths and sequences of the N-terminal
AB-domain (Talbot et al., 1993). Each isoform mediates a particular program of
ecdysone response during Drosophila development (Truman et al., 1994; Hiruma and
Riddiford, 2004). Phenotypic analysis of EcR isoform mutants showed that distinct
lethal phases and morphologies are associated with each isoform (Bender et al., 1997;
Schubiger et al., 2003). EcR-A is predominantly expressed in cells that proliferate and
differentiate during metamorphosis, while the EcR-B1 and EcR-B2 are present in
larval cells, which are eliminated by apoptosis during metamorphosis.
Robinow et al. (1993) showed that EcR-A is involved in remodelling of neurons
during metamorphosis. EcR-B1 mediates the ecdysteroid response in cells of the
salivary gland (Bender et al., 1997), whereas EcR-B2 most efficiently rescues larval
development in EcR mutants (Li and Bender, 2000). Usp, the invertebrate orthologue
of RXR, is the most important heterodimerization partner of EcR and is widely
expressed during development. Usp has thus far been present in all tissues analysed at
the onset of metamorphosis (Henrich et al., 1994), and is involved in activation of
ecdysone-dependent genes (Hall and Thummel, 1998). Usp’s absence during the late
third instar induces rather different phenotypic effects as compared to the absence of
EcR, indicating that not only the heterodimer is involved in regulation of gene
expression, but each nuclear receptor separately modulates expression of target genes.
The heterologous expression of insect nuclear receptors in vertebrate cells, which
are themselves devoid of the corresponding endogenous receptor proteins, provides
an opportunity to examine the functional properties of EcR isoforms separately and to
study the impact of Usp on a molecular level as pointed out in detail by Henrich et al.
(2009). The RXR content of the CHO-K1 subline used for our experiments is below
the detection limit and does not cause any measurable physiological effect (Nieva et al.,
2008; Azoitei and Spindler-Barth, 2009). Therefore, these cells were routinely used for
Archives of Insect Biochemistry and Physiology
156
Archives of Insect Biochemistry and Physiology, November 2009
the characterization of the molecular properties of EcR and Usp (Henrich et al., 2003,
2009; Beatty et al., 2006; Betanska et al., 2007, 2009; Dutko-Gwóźdź et al., 2008; Nieva
et al., 2007, 2008). At least some accessorial proteins like comodulators, which are
required for receptor action, seem to be sufficiently conserved to allow structurefunction studies (Tsai et al., 1999; Tran et al., 2001b; Henrich et al., 2009). Because the
presence and the combinations of different comodulators varies in different target
tissues, and their activities, which are regulated by posttranslational modification
(Hermanson et al., 2002; Lonard and O’Malley, 2007), depend on the physiological
state, selection of a typical insect-specific environment seems questionable. The
considerable excess of wild type Usp in insect cell lines especially prohibits study of the
influence of Usp variants necessary for functional studies of individual receptor
domains of Usp. This is because quantitative knock-out of wild type Usp is difficult to
achieve and it can not be excluded with certainty that traces of remaining wild type
Usp may influence the results of EcR isoforms without a heterodimerization partner or
with a less active Usp variant.
Two dimerization interfaces are described for ecdysteroid receptors (Perera et al.,
2005). The dimerization site, localized in the C-domain, is dependent on the presence
of DNA. A second dimerization interface in the E-domain is regulated by ligand
binding. As described by Ghbeish et al. (2001), mutations in the DNA binding domain
of Usp alter the physiological responses of the heterodimer. We subsequently also used
a Usp variant in which the DNA binding domain was deleted (Beatty et al., 2006) to
separately study the impact of each dimerization site on receptor function.
It was previously shown that ecdysone receptor isoforms expressed in vertebrate
cells show isoform-specific differences, which were further modulated by heterodimerization with Usp (Beatty et al., 2006; Gwozdz et al., 2007; Azoitei and SpindlerBarth, 2009). It is known from vertebrate nuclear receptors that the AB-domain is
involved in the regulation of receptor stability (Zhou et al., 1995), which is important
for termination of the hormonal signaling cascade, thus allowing isoform-specific
modulation of ecdysone action. EcR is further stabilized by heterodimerization with
Usp and by ligand binding (Nieva et al., 2008), which also alters basal and hormoneinduced transcriptional activity of the receptor complex (Beatty et al., 2006). In order
to investigate the regulatory potential of EcR isoforms in more detail, we determined
the influence of the AB-domains, the heterdimerization partner, and ligand on
receptor stability and transcriptional activity separately.
MATERIALS AND METHODS
Plasmids
The following plasmids were used for transfection studies: the three EcR isoforms A,
B1, and B2 were cloned in a pEYFP-C1 vector (BD Biosciences/Clontech, SaintGermain-en Laye, France) as described previously (Nieva et al., 2005), and were
provided by Dr. A. Ozyhar (Technical University of Wrozlaw, Wrozlaw, Poland). These
plasmids express full-length EcR isoforms with the fluorescent tag YFP at the Nterminus, which allows microscopical evaluation of transfection efficiencies and
facilitates detection and quantification of EcR isoforms on Western blots.
Wild type Usp cloned into pEYFP-N1 (BD Biosciences/Clontech) was also kindly
provided by Dr. A Ozyhar. The plasmid was further modified by Dr. S. Braun
Archives of Insect Biochemistry and Physiology
Transcriptional Activity of Ecdysone Receptor
157
(University of Ulm, Ulm, Germany) to allow expression of wild type Usp without the
YFP-tag. The plasmids pVP16-UspI (encoding the amino acids 98–507 of Usp, which
includes the last six amino acids of the N-terminal AB-domain of the original wild type
Usp), pVP16-UspII (amino acids 104–507 of wild type Usp encoding the CEdomains), and pVP16-UspIII (amino acids 170–507 of wild type Usp encoding the
DE-domains of wild type Usp) were kindly provided by Dr. Henrich (Greensboro,
NC). In these constructs (VP16-UspI, VP16-UspII, VP16-UspIII; Beatty et al., 2006),
the original AB-domain of Usp is replaced by the VP16 activation domain to overcome
the inhibitory effect of the AB-domain of wild type Usp on transactivation of reporter
genes in vertebrate cells (Henrich, 2005). The plasmid maps of the EcR and Usp
constructs are available upon request.
For determination of transcriptional activity, the plasmid pEcRE-tk-Luc was
cotransfected carrying the luciferase reporter gene attached to an ecdysone response
element of hsp27 (Riddihough and Pelham, 1987) and a constitutive thymidine kinase
promoter (Henrich et al., 2003).
Cell Culture and Transfection
CHO-K1 cells (Puck et al., 1958) were grown in DMEM/F12 medium (Gibco/
Invitrogen, Carlsbad, CA) supplemented with 5% of fetal bovine serum (Sigma,
Deisenhofen, Germany). Cells were seeded in six-well plates (Nunc, Wiesbaden,
Germany) with 4 105 cells per well. After 24 h, cells were transfected with
lipofectamine LTX (Invitrogen, Carlsbad, CA) according to the manufacturer’s
instructions. Each well received 3 mg of plasmid DNA (1 mg of the plasmid pEcRE-tkLuc and 1 mg of plasmids expressing EcR and Usp each; in case of experiments without
Usp, 2 mg of the plasmid expressing EcR). Cells were incubated with plasmid DNA in
medium without FCS. After 4 h, the medium was replaced by DMEM/F12
supplemented with 5% FCS. One hour later, muristerone A (Sigma, Deisenhofen,
Germany) was added (final concentration 1 mM). Cells were lysed 24 h after
transfection by shaking in 1 passive lysis buffer (PLB 5 , Promega, Madison, WI;
100 ml per well). To improve homogenization, the suspension was passed several times
through a thin syringe (0.4 20 mm, Terumo, Leuven, Belgium).
Reporter Gene Assay
Luciferase reporter activities were assayed with either the luciferase assasy system (no.
E1500, Promega) or the dual luciferase reporter assay system (no. E1910) according to
the manufacturer’s instructions.
Determination of Transfection Efficiency
Transfection efficiency (7575%) was controlled by fluorescence microscopy, which
allowed determination of the percentage of cells showing fluorescence due to the YFPtag and/or by cotransfection of pCIII-LacZ (0.5 mg/well) and subsequent determination
of galactosidase activity in the extracts as described by Beatty et al. (2006). Cell cultures
with lower transfection efficiencies as indicated above were discarded.
Western Blotting and Quantification of Receptor Content
Cell extracts (25 mg protein/lane) were separated on SDS gels (Laemmli, 1970). Gels
were electroblotted on nitrocellulose membranes (BA 85, 45-mm pore size, Schleicher
Archives of Insect Biochemistry and Physiology
158
Archives of Insect Biochemistry and Physiology, November 2009
and Schuell, Dassel, Germany). Membranes were stained with Ponceau to check
transblotting efficiency. Membranes were soaked in blocking buffer (20 mM Tris-HCl,
137 mM NaCl, 0.1% Tween 20, pH 7.6, 0.02% Thimerosal) containing 3% milk powder
(low fat, o1%) and 1% bovine serum albumine (Sigma, Deisenhofen, Germany) for 1 h
and then probed overnight with a monoclonal anti-GFP antibody (BD Biosciences/
Clontech, Saint-Germian-en Laye, France) diluted in the same buffer 1:500. For
detection of specific Western signals, peroxidise-conjugated secondary antibody (Antimouse IgG, no. A-5906, Sigma, Deisenhofen, Germany) diluted 1:500 in TBS (0.1%
Tween 20) was used. Signals were visualized with SuperSignal West Dura Extended
Duration Substrate (Pierce, Rockford, IL). Specific bands of the expected molecular
weight were visualized by the Chemi-Smart 5000 photodocumentation system (Vilber
Lourmat, Eberhardzell, Germany) and quantified using the ‘‘Rolling ball method’’
(User manual, Bio-1D advanced, Vilber Lourmat, Eberhardzell, Germany; Serra,
1984) in relation to a standard probe (5 100%) using the software Bio-1D (Vilber
Lourmat) as described. The linearity of the intensity of specific receptor bands was
verified by a calibration curve as described previously (Przibilla et al., 2004; Braun
et al., 2009).
RESULTS
The Concentration of Heterogenously Expressed EcR Is Affected by Its AB-Domain, the
Heterodimerization Partner, and Ligand Binding
If expressed separately, the concentration of all EcR isoforms was approximately equal
in the absence of hormone (Fig. 1A). This was expected, because the amounts of DNA
used for transfection and the transfection efficiencies were the same. Hormone
enhanced only the concentration of EcR-A, but not EcR-B1 and -B2 (Fig. 1A),
presumably due to stabilization of the receptor protein by the ligand.
Heterodimerization with wild type Usp differently modified the EcR isoform
concentrations. Addition of hormone selectively enhanced the concentrations of EcR-A
and -B2, whereas the concentration of EcR-B1 was neither improved by hormone nor
by the heterodimerization partner (Fig. 1B). Comparison of EcR concentrations in the
presence of Usp variants (UspI, II and III) revealed that the influence of Usp on EcR
concentrations was mainly due to the combined influence of the AB-domain of wild
type Usp and hormone and only a slight hormone-induced increase was observed for
receptor complexes with EcR-B2/UspII, if the AB-domain was replaced by the
AD-domain of Vp16 (Fig. 2A–C). Deletion of the C-domain of Usp as shown with Usp
III (5 Vp16AB-UspDE) did not reduce EcR concentrations (Fig. 2C). Taken together,
these results showed that hormone and the AB-domains of both EcR and Usp
modified EcR concentrations in a cooperative manner.
Transcriptional Activity of EcR Isoforms
When compared to empty vector, all EcR isoforms increased the activity of the
luciferase reporter, coupled to a promoter containing the ecdysone response element
1 hsp 27 approximately 8–11-fold (data not shown).
In the absence of Usp, basal transcriptional activity of EcR-A was very low
compared to EcR-B isoforms (Ao
oB1 5 B2) (Fig. 3A). This is remarkable, because the
Archives of Insect Biochemistry and Physiology
Transcriptional Activity of Ecdysone Receptor
A
159
rec. protein conc.
[ rel. units ]
6
4
2
0
EcR-A
EcR-B1
EcR-B2
122 kDa
91 kDa
B
rec. protein conc.
[ rel. units ]
8
6
2
0
EcR-A
EcR-B1
EcR-B2
122 kDa
91 kDa
Figure 1. Determination of EcR concentration by quantification of specific Western blot signals. White
bars 5 absence of hormone; black bars 5 with 1 mM muristerone A. A: In the absence of a heterodimerization
partner, the hormone effect of EcR-A (Po0.02) and the difference of the hormone-induced increase between
EcR-A and -B1 (Po0.04) and -B2 (Po0.02) are significant. B: In the presence of wild type Usp. The
hormone effects of EcR-A (Po0.004) and -B2 (Po70.03) are significant (M1SD, n 5 3–5).
concentration of EcR-A was comparable to those of EcR-B1 and -B2 (Fig. 1A). No
hormone-induced stimulation of transcriptional activity was observed for any of the
EcR isoforms (Fig. 3A). Comparison of the rather high EcR-A concentration in the
presence of hormone (Fig. 1A) and the very low transcriptional activity (Fig. 3A)
revealed that receptor concentration and transcriptional capability are regulated
independently.
Influence of the Heterodimerization Partner on Transcriptional Activity
Heterodimerization with wild type Usp considerably enhanced basal activity especially
of EcR-A and hormone-induced transcriptional activity of EcR-A (Po0.0005)
and EcR-B2 (Po0.005) of the receptor complex in comparison to unpaired EcR,
but had no effect on either basal or hormone-induced transcriptional activity of
EcR-B1 (Fig. 3B).
Exchange of the AB-domain of wild type Usp, which is routinely done to overcome
the inhibitory action of wild type Usp in vertebrate cells (Henrich, 2005), caused a
considerable increase in basal and hormone-induced transcriptional activity in all EcR
isoforms paired with UspI (Po0.03–0.05) that still contained the conserved
hexapeptide of the original AB domain of Usp adjacent to the DNA-binding domain.
Archives of Insect Biochemistry and Physiology
160
Archives of Insect Biochemistry and Physiology, November 2009
A
rec. protein conc.
[ rel. units ]
6
4
2
0
EcR-A
EcR-B1
EcR-B2
122 kDa
91 kDa
B
rec. protein conc.
[ rel. units ]
6
4
2
0
EcR-A
EcR-B1
EcR-B2
122 kDa
91 kDa
C
rec. protein conc.
[ rel. units ]
6
4
2
0
EcR-A
122 kDa
EcR-B1
EcR-B2
91 kDa
Figure 2. Determination of EcR concentration by quantification of specific Western blot signals
coexpressed wit Usp variants. White bars 5 absence of hormone; black bars 5 with 1 mM muristerone A.
A: UspI; the difference in the hormone effects of EcR-A and -B1 is significant (Po0.05). B: UspII. C: UspIII;
the hormone effect of EcR-B2 and the difference in the hormone-induced increase between EcR-B1 and
EcR-B2 are significant (Po0.05) (M7SD, n 5 3–5).
In contrast to EcR-B1/wild type Usp (Fig. 3B), a clear hormone effect was obtained
with all EcR isoforms including EcR-B1 paired with UspI (Fig. 4A). When the
hexapeptide was deleted (UspII), only basal but not hormone-induced transcriptional
activity was reduced (Fig. 4B). The influence of the N-terminus of the AB-domain of
wild type Usp can be summarized as follows: both the AB-domain of EcR isoforms and
the N-terminus of Usp are involved in the regulation of basal and hormone-induced
Archives of Insect Biochemistry and Physiology
Transcriptional Activity of Ecdysone Receptor
161
A
x-fold luciferase act.
40000
10000
5000
0
EcR-A
EcR-B1
EcR-B2
EcR-A
EcR-B1
EcR-B2
B
x-fold luciferase act.
40000
30000
20000
10000
0
Figure 3. Luciferase activity of EcR isoforms expressed in CHO-K1 cells. White bars 5 absence of
hormone; black bars 5 with 1 mM muristerone A. A: In the absence of a heterodimerization partner. The
difference between EcR-A and EcR-B1 and -B2 is significant (Po0.003). B: In the presence of wild type Usp.
The difference in basal activities between all EcR isoforms (Po0.04) and the hormone effects of EcR-A and
-B2 (Po0.005) is significant (M7SD, n 5 3–5). Y-axis: Luciferase activity of EcR-A in the absence of Usp 5 1.
transcriptional activity of the receptor complexes. The AB-domain of wild type Usp
contains two different regions: an inhibitory function is located at the N-terminus and
the conserved hexapeptide harbours an activating function that selectively increases
only basal transcriptional activity.
Influence of the C-Domain of Usp on Transcriptional Activity
The AB-domains of UspII and UspIII were identical; however, the C-domain was
deleted in UspIII, meaning the dimerization site in the DNA binding domain was
missing. In this case, heterodimerization was only mediated by the ligand-binding
domains. The weaker interaction between EcR and Usp reduced transcriptional
activities considerably (Fig. 4C) compared to heterodimers with UspII (Fig. 4B). Basal
activity of EcR-B1/UspIII is especially reduced, and demonstrates that the impact of
DNA binding and dimerization via the C-domain of Usp on transcriptional activity was
different for individual EcR isoforms.
Transcriptional Capability of the Various Receptor Complexes
We then investigated how these differences in reporter activities were achieved. As
described above, the concentration of receptor complexes varied despite comparable
transfection efficiencies (7575%). Consequently, changes in luciferase activities were
Archives of Insect Biochemistry and Physiology
162
Archives of Insect Biochemistry and Physiology, November 2009
A
x-fold luciferase act.
120000
+
*
40000
20000
x
0
EcR-A
EcR-B1
EcR-B2
B
x-fold luciferase act.
120000
40000
+
20000
*
0
EcR-A
EcR-B1
EcR-B2
x-fold luciferase act.
C
40000
30000
20000
10000
+
*
x
0
EcR-A
EcR-B1
EcR-B2
Figure 4. Luciferase activity of EcR isoforms expressed in CHO-K1 cells. White bars 5 absence of
hormone; black bars 5 with 1 mM muristerone A. A: UspI. B: UspII. C: UspIII; (M7SD, n 5 3–5). Significant
differences of basal activities of heterodimers with different Usp variants are indicated (x 5 Po0.04;
1 5 Po0.01, Po0.02). Y-axis: Luciferase activity of EcR-A in the absence of Usp and hormone 5 1.
the result of two variables: transcriptional capability of the receptor complexes and
receptor protein concentration. In order to eliminate the influence of variations in
receptor concentration and to determine the specific transcriptional potency of the
Archives of Insect Biochemistry and Physiology
Transcriptional Activity of Ecdysone Receptor
163
individual receptor complexes, we normalized the luciferase activities on receptor
concentration (Figs. 5 and 6).
No significant differences in transcriptional activity normalized on receptor
protein concentration were observed with any EcR isoform in the absence of Usp
(Figs. 3A and 5A) and the hormone-induced increase in luciferase activity in the case of
EcR-A (Fig. 3A) was only due to the hormone-induced stabilization of the receptor
protein.
Basal transcriptional capabilities of heterodimers with wild type Usp and EcR-A
and -B2 normalized on receptor concentration (Fig. 5B) were also rather low and no
hormone effect was observed. In contrast, basal and hormone-induced transcriptional
activity normalized on the receptor concentration of EcR-B1 paired with wild type Usp
was significantly higher (B14B24A). This was due exclusively to an increase in
transcriptional capability of EcR/Uspwt, because the concentration of the receptor
complex was quite low (Fig. 1B). The hormone-induced increase in luciferase activities
(Fig. 2B) of EcR-A and EcR-B2 heterodimers with wild type Usp compared to
unpaired EcR-A and -B2 was only due to the increase in receptor protein
concentration and was not caused by the improved transcriptional capabilities of the
heterodimers (Fig. 5B).
Exchange of wild type Usp by UspI or UspII (Fig. 6A, B) caused similar results
and confirmed that basal and hormone-induced transcriptional activities did not
change in parallel to the stabilization of the receptor protein. After elimination of the
x-fold luciferase act. /
rec. prot. conc. [rel. units]
A
250
50
0
EcR-A
EcR-B1
EcR-A
EcR-B1
EcR-B2
x-fold luciferase act. /
rec. prot. conc. [rel. units]
B
250
200
150
100
50
0
EcR-B2
Figure 5. Luciferase activity of EcR isoforms expressed in CHO-K1 cells normalized to receptor
concentration. White bars 5 absence of hormone; black bars 5 with 1 mM muristerone A. A: In the absence
of a heterodimerization partner. B: In the presence of wild type Usp. The hormone effect of EcR-B1 in the
presence of wild type Usp (Po0.02) and the differences in basal activities between EcR-A and -B- isoforms in
A and B are significant (Po0.008–0.003) (M7SD, n 5 3–5). Y-axis: Luciferase activity of EcR- A (normalized
to receptor concentration) in the absence of Usp and hormone 5 1.
Archives of Insect Biochemistry and Physiology
164
Archives of Insect Biochemistry and Physiology, November 2009
A
1000
x-fold luciferase act. /
rec. prot. conc. [rel. units]
750
500
250
200
150
100
50
0
EcR-A
EcR-B1
EcR-B2
EcR-A
EcR-B1
EcR-B2
EcR-A
EcR-B1
EcR-B2
x-fold luciferase act. /
rec. prot. conc. [rel. units]
B
250
200
150
100
50
0
x-fold luciferase act. /
rec. prot. conc. [rel. units]
C
250
200
150
100
50
0
Figure 6. Luciferase activity of EcR isoforms coexpressed with Usp variants in CHO-K1 cells normalized to
receptor concentration. White bars 5 absence of hormone; black bars 5 with 1 mM muristerone A. A: UspI.
B: UspII. C: UspIII. Brackets indicate significant difference between basal and hormone-induced activity
(Po0.02). (M7SD, n 5 3–5). Y-axis: Luciferase activity of EcR- A (normalized to receptor concentration) in
the absence of Usp and hormone 5 1.
influence of receptor concentration by normalization on EcR content, the activating
function of the conserved hexapeptide adjacent to the C-domain of Usp, which was
still present in UspI, in contrast to Usp II, became apparent in both the presence and
absence of hormone (Fig. 6A and B).
Archives of Insect Biochemistry and Physiology
Transcriptional Activity of Ecdysone Receptor
165
Basal transcriptional activity normalized on receptor concentration of EcR-A/
UspIII (Fig. 6C) was reduced, but was still about 12-fold higher compared to EcR-A
(Fig. 5A) in the absence of Usp. This demonstrated the importance of the dimerization
site in the ligand-binding domain, even in the absence of hormone. Dimerization was
less efficient in the absence of the C-domain of Usp (Azoitei et al., 2009). We, therefore,
verified whether additional Usp may improve transcriptional activity by increasing the
number of the heterodimeric complexes, but no increase was observed (Tremmel,
unpublished observations).
A pronounced hormone-induced increase in transcriptional capability was still
present in the case of EcR-B1/UspIII, but no hormone-induced stimulation of
transcriptional capability of EcR-B2/UspIII was observed any more after normalization
on receptor concentration. This confirms that hormone selectively increases
transcriptional activity of EcR-B1, whereas the influence of hormone on EcR-B2/
UspIII was restricted to stabilization of the receptor protein.
DISCUSSION
Selection of the Heterologous Expression System
As pointed out in detail by Henrich et al. (2009), vertebrate cells, especially CHO cells,
are suited for the study of the molecular properties of EcR and Usp. These cells do not
show any endogenous response to ecdysteroids and the results obtained from
vertebrate cells were basically confirmed ‘‘in vivo’’ (Henrich et al., 2009). In contrast to
insect cells, which contain a mixture of endogenously expressed EcR isoforms and
Usp, vertebrate cells allow the characterization of individual EcR isoforms even in the
absence of Usp.
Although some co-repressors seem to be rather conserved between vertebrates
and insects (Tsai et al., 1999; Tran et al., 2001b), the mixture of co-modulators
certainly differs between vertebrates and insects. Differences also exist between
different insect tissues and comodulator concentrations and activities vary depending
on the physiological state of the target cells. In our opinion, the disadvantages of insect
cells cultures as described above are not compensated by an insect milieu, which
contain an unknown combination of co-modulators, which also do not reflect the in
vivo situation in a certain animal tissue at a defined developmental stage. Our aim was
to study the differences in the functionality of the receptor protein molecules in a
constant environment. Comparative studies with CHO-, Cos-, and Hela-cells did not
show significant differences with the results obtained with CHO cells used in this study
(Tremmel, Betanska, and Nieva, unpublished observations).
Stability of the Receptor Protein Depends on the AB-Domain of EcR, the
Heterodimerization Partner, and/or the Ligand
Although the transfection efficiencies were comparable and the same plasmid was used
for constitutive expression of EcR isoforms, which should guarantee identical rates of
receptor protein synthesis, the concentration of EcR isoforms varied considerably. This
can only be explained by different rates of receptor protein degradation. According to
Alarid (2006), AF-1 and AF-2 regions are essential for recognition of the ubiquitinylation machinery that is responsible for processing of vertebrate nuclear receptors. The
Archives of Insect Biochemistry and Physiology
166
Archives of Insect Biochemistry and Physiology, November 2009
same mechanism may also result in isoform-specific differences in the stability of EcR.
The stability of the receptor protein is influenced by many factors, such as interaction
with comodulators and additional proteins like heat shock proteins (Cronauer,
unpublished observations), or cyclins (Betanska et al., 2009). Stabilisation, which is
enhanced additionally by heterodimerization and ligand binding, is clearly regulated
in a complex cooperative manner.
FUNCTIONAL IMPORTANCE OF RECEPTOR STABILITY ON MOLTING
HORMONE ACTION
Isoform-specific differences in receptor function contribute to the diversification of the
hormonal signal transduction in vivo and allow gene- and tissue-specific variations of
ecdysone action, which can be further modified according to the developmental stage
of the animal. Adaption to the changing physiological requirements of target cells
affords that the signalling cascade is turned off after appropriate hormonal
stimulation. This can be achieved by several mechanisms including target
cell-specific hormone metabolism, tissue-specific composition, and concentration of
co-modulators as well as their intracellular localization and their posttranslational
modifications like phosphorylation, which may contribute to the regulation of receptor
activity, thus modifying the efficiency of the hormonal signal transduction (Hermanson
et al., 2002; Lonard and O’Malley, 2007). Aside from interaction of co-modulators with
the E-domain of nuclear receptors (Tsai et al., 1999), binding to the N-terminus (Tian
et al., 2006; Hollenberg et al., 1995) is also described at least for vertebrate receptors
and allows isoform-specific regulation. Here we showed that receptor stability can
additionally contribute to the fine tuning of the hormone response and that receptor
stability is regulated in a cooperative manner dependent on the EcR isoform, presence
of hormone, and the AB- and C-domains of the heterodimerization partner, which
underline the active role of Usp in the modulation of EcR function.
Reporter Gene Activity Is the Result of Variations in Receptor Concentration and
Transcriptional Capability of the Receptor Complex
Changes in transcriptional activity of nuclear receptors are the result of two effects:
altered receptor concentration and modification of the transactivation capability of the
receptor complexes. If only one type of receptor is considered, protein concentration
correlates with the amount of transfected DNA; however, for comparison of different
receptor complexes, changes in stability, which alter receptor concentrations and
depend on the EcR isoform, ligand, and/or heterodimerization partner (Nieva et al.,
2008), afford determination of receptor concentrations, in order to compare the
transcriptional capabilities of the receptor complexes.
Influence of the AB-Domain of EcR on Transcriptional Activity
Basal transcriptional activity of EcR-A is very low compared to EcR-B isoforms,
although still higher compared to empty vector (Ruff, unpublished results). The
differences in basal transcriptional activity between EcR isoforms (EcR-A: EcR-B1:
EcR-B2 5 1:50:50) are most pronounced in the absence of Usp and hormone, but are
still present in heterodimers and after hormonal stimulation, although to a lesser
extent. The constitutive activation function AF-1 in the AB-domain of EcR mediates
Archives of Insect Biochemistry and Physiology
Transcriptional Activity of Ecdysone Receptor
167
transcriptional activity independent from the cellular context (Mouillet et al., 2001; Hu
et al., 2003). Reduced activity of EcR-A compared to EcR-B isoforms is always observed
and seems to be of functional importance also in flies (Cherbas et al., 2003; Schubiger
et al., 2003), even though the cellular context may modulate the relative transcriptional activities of the receptor isoforms.
The AB-Domain of Usp Harbours an N-Terminal Inhibitory Function and an Activating
Hexapeptide
Both the AB-domain of EcR and the AB-domain of Usp modulate the transcriptional
activity of the ecdysone receptor complex and underline the active role of the
heterodimerization partner. The influence of wild type Usp on basal transcriptional
capability varies according to EcR isoform (9-fold increase for EcR-A, 2-fold for
EcR-B1, slight decrease for EcR-B2; Fig. 5A and B). The influence of hormone is
restricted mainly to stabilization of the EcR. The relatively low transcriptional activity
of all EcR isoforms paired with wild type Usp gave rise to the hypothesis that the
AB-domain of wild type Usp may harbour an inhibitory function. The inhibitory
function located at the N-terminus of Usp (Henrich, 2005) becomes apparent by
exchanging the originally AB-domain with the activation domain of Vp16. The
increased transcriptional capabililty (normalized to receptor concentration) of
heterodimers with UspI, which still contain the highly conserved hexapeptide
adjacent to the C-domain of wild type Usp in comparison to UspII, reveals the
presence of an additional activating function in the AB-domain of wild type Usp:
however, the level of induction by hormone remains relatively constant and indicates
that the influence of the hexapeptide is restricted mainly to non-liganded receptor
complexes. The transcriptional activity of the receptor complex is clearly regulated by
the N-termini of both dimerization partners, which is in accordance with cross-talk
between both N-terminal domains as described for vertebrate receptors, e.g., ER/RAR
(Rousseau et al., 2003). Participation of the AB-domain of Usp in the regulation of
constitutive transcriptional activity was also reported for the ecdysone receptor of
Choristoneura (Tran et al., 2001a), but is in contrast to the conclusions of Hu et al.
(2003) based on experiments with mutated EcRs on a wild type Usp background.
Deletion of the C-Domain of Usp Impairs Basal Activity But Increases Hormone-Induced
Stimulation of Transcriptional Activity
Deletion of the C-domain of Usp reduces constitutive transcriptional activity to a
degree similar to that found in the absence of Usp. This is especially so for EcR-B1/
UspIII (5 Vp16-UspDE, whereas basal activity of UspIII complexes with EcR-A
remains still considerably higher, i.e., 12-fold). As shown by Niedziela-Maijka et al.
(2000), the C-domain of Usp plays an important role in DNA binding of the receptor
complex and directs EcR to the 30 end of the EcRE. The decrease in constitutive
transcriptional activity of heterodimers with UspIII may also be due to improper
folding of the N-terminus. The C-domain of nuclear receptors is essential for correct
folding of the AB-domain, which is a prerequisite for proper AF-1 function (Lavery
and McEwan, 2005).
The considerable ligand-induced stimulation of transcriptional activity of EcR-B1/
UspIII compared to heterodimers with full-length Usp is especially remarkable,
because DNA binding of EcR/UspIII complexes are drastically reduced as determined
by gel mobility shift assays (Beatty et al., 2006; Braun et al., 2009). The modulation of
Archives of Insect Biochemistry and Physiology
168
Archives of Insect Biochemistry and Physiology, November 2009
receptor activity with and without participation of DNA binding via Usp corresponds
to physiological differences described in vivo, given that some ecdysone responses
mediated by the heterodimeric complex require binding of Usp to DNA and others do
not (Ghbeish et al., 2003). It is interesting to note that a naturally occurring RAR
variant, which lacks the B- and C-domains, acts as a functional nuclear receptor after
dimerization with RXR, which mediates interaction of the heterodimeric complex with
DNA (Parrado et al., 2001).
Regulation of Transcriptional Activity of the Ecdysone Receptor Complex
Although ligand binding of receptor complexes with all EcR isoforms combined with
all Usp variants is identical (Azoitei and Spindler-Barth, 2009), hormone-induced
stimulation of transcriptional activity varies. This may be at least partially caused by
isoform-specific interaction with co-modulators and differences in DNA binding
(Braun, personal communication). Additional possibilities are isoform-specific interaction of AF-1 and AF-2, as was described for vertebrate nuclear receptors, e.g., RAR
and RXR (Nagpal et al., 1993; Gianni et al., 2003). This interaction may occur either
directly (Kumar and Thompson, 2003) or indirectly by bridging via a co-modulator
(Wang and Simons, 2005). Moreover, inhibitory as well as activating functions of wild
type Usp on basal and hormone-induced transcriptional capabilities indicate an
interaction of the AB-domain of Usp with the ligand-binding domain of EcRs.
ACKNOWLEDGMENTS
A plasmid coding for wild type Usp was kindly provided by Dr. S. Braun, who
modified a precursor plasmid obtained by Prof. A. Ozyhar (Technical University of
Wroclaw, Poland) thus allowing expression of wild type Usp. Expression plasmids for
EcR-A, EcR-B1, EcR-B2 and Usp I, II and III, were kindly provided by Dr. V.C.
Henrich (University of North Carolina, Greensboro, NC, USA). The technical
assistance of N. Möbius and E. Arnold, the help of A. Sarno, who corrected and
improved the English version of the manuscript, and M. Burret, who provided the
figures, is gratefully acknowledged. The work was supported by a grant from the DFG
to MSB (Spi 350/5-1 and /5-2) and a PhD-scholarship to Ch.T. (Cusanuswerk).
LITERATURE CITED
Alarid ET. 2006. Lives and times of nuclear receptors. Mol Endocrinol 20:1972–1981.
Azoitei A, Spindler-Barth M. 2009. DNA affects ligand binding of the ecdysone receptor of
Drosophila melanogaster. Mol Cell Endocrinol 303:91–99.
Azoitei A, Ruff H, Tremmel Ch, Braun S, Spindler-Barth M. 2009. Functional analysis of
ecdysteroid receptor from Drosophila melanogaster in vitro. In: Smagghe G, editor. Ecdysone,
structures and functions. Berlin: Springer. p 377–388.
Beatty J, Fauth T, Callender JL, Spindler-Barth M, Henrich VC. 2006. Analysis of transcriptional
activity mediated by Drosophila melanogaster ecdysone receptor isoforms in a heterologous
cell culture system. Insect Mol Biol 15:785–795.
Bender M, Imam FB, Talbot WS, Ganetzky B, Hogness DS. 1997. Drosophila ecdysone receptor
mutations reveal functional differences among receptor isoforms. Cell 91:777–788.
Betanska K, Nieva C, Spindler-Barth M, Spindler KD. 2007. Nucleocytoplasmic shuttling of the
ecdysteroid receptor (EcR) and of ultraspiracle (Usp) from Drosophila melanogaster in
Archives of Insect Biochemistry and Physiology
Transcriptional Activity of Ecdysone Receptor
169
mammalian cells: energy requirement and interaction with exportin. Arch Insect Biochem
Physiol 65:134–142.
Betanska K, Czogalla S, Spindler-Barth M, Spindler K-D. 2009. Influence of cell cycle on
ecdysteroid receptor in CHO-K1 cells. Arch Insect Biochem Physiol (in press).
Braun S, Azoitei A, Spindler-Barth M. 2009. DNA binding properties of Drosophila ecdysone
receptor. Arch Insect Biochem Phyisol (in press).
Cheng SY. 2005. Isoform-dependent actions of thyroid hormone nuclear receptors: lessons from
knockin, mutant mice. Steroids 70:450–454.
Cherbas L, Hu X, Zhimulev I, Belyaeva E, Cherbas P. 2003. EcR isoforms in Drosophila: testing
tissue-specific requirements by targeted blockade and rescue. Development 130:271–284.
Duma D, Jewell CM, Cidlowski JA. 2006a. Multiple glucocorticoid receptor isoforms and
mechanisms of post-translational modification. J Steroid Biochem Mol Biol 102:11–21.
Duma D, Jewell CM, Cidlowski JA. 2006b. Glucocorticoid receptor isoforms generate
transcription specificity. Trends Cell Biol 16:301–307.
Dutko-Gwóźdź J, Gwóźdź T, Or"owski M, Greb-Markiewicz B, Duś D, Dobrucki J, Ozyhar A.
2008. The variety of complexes formed by EcR and Usp nuclear receptors in the nuclei of
living cells. Mol Cell Endocrinol 294:45–51.
Ghbeish N, Tsai CC, Schubiger M, Zhou JY, Evans RM, McKeown M. 2001. The dual role of
ultraspiracle, the Drosophila retinoid X receptor, in the ecdysone response. Proc Natl Acad
Sci USA 98:3867–3872.
Giannı́ M, Tarrade A, Nigro EA, Garattini E, Rochette-Egly C. 2003. The AF-1 and AF-2
domains of RAR gamma 2 and RXR alpha cooperate for triggering the transactivation and
the degradation of RAR gamma 2/RXR alpha heterodimers. J Biol Chem 278:34458–34466.
Gwozdz T, Dutko-Gwozdz J, Nieva C, Betanska K, Orlowski M, Kowalska A, Dobrucki J,
Spindler-Barth M, Spindler K-D, Ozyhar A. 2007. EcR and Usp, components of the
ecdysteroid nuclear receptor complex, exhibit differential distribution of molecular
determinants directing subcellular trafficking. Cell Signal 19:490–503.
Hall Bl, Thummel CS. 1998. The RXR homolog ultraspiracle is an essential component of the
Drosophila ecdysone receptor. Development 125:4709–4717.
Henrich VC. 2005. The ecdysteroid receptor. In: Gilbert LJ, Iatrou K, editors. Comprehensive
molecular insect science, Vol. 3. Oxford: Elsevier, Pergamon Press. p 243–282.
Henrich VC, Szekely AA, Kim SJ, Brown NE, Antoniewski C, Hayden MA, Lepesant JA,
Gilbert LI. 1994. Expression and function of the ultraspiracle (usp) gene during
development of Drosophila melanogaster. Dev Biol 165:38–52.
Henrich VC, Burns E, Yelverton DP, Christensen E, Weinberger C. 2003. Juvenile hormone
potentiates ecdysone receptor-dependent transcription in a mammalian cell culture system.
Insect Biochem Mol Biol 33:1239–1244.
Henrich VC, Beatty J, Ruff H, Callender J, Grebe M, Spindler-Barth M. 2009. The multiple
partnership of EcR and Usp. In: Smagghe G, editor. Ecdysone, structures and functions.
Berlin: Springer. p 361–376.
Hermanson O, Glass CK, Rosenfeld MG. 2002. Nuclear receptor coregulators: multiple modes
of modification. Trends Endocrinol Metab 13:55–60.
Hiruma K, Riddiford LM. 2004. Differential control of MHR3 promoter activity by isoforms
of the ecdysone receptor and inhibitory effects of E75A and MHR3. Dev Biol 272:510–521.
Hollenberg AN, Monden T, Wondisford FE. 1995. Ligand-independent and -dependent
functions of thyroid hormone receptor isoforms depend upon their distinct amino termini.
J Biol Chem 270:14274–14280.
Hu X, Cherbas L, Cherbas P. 2003. Transcription activation by the ecdysone receptor
(EcR/USP): identification of activation functions. Mol Endocrinol 17:716–731.
Archives of Insect Biochemistry and Physiology
170
Archives of Insect Biochemistry and Physiology, November 2009
Kumar R, Thompson EB. 2003. Transactivation functions of the N-terminal domains of nuclear
hormone receptors: protein folding and coactivator interactions. Mol Endocrinol 17:1–10.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227:680–685.
Lavery DN, McEwan IJ. 2005. Structure and function of steroid receptor AF1 transactivation
domains: induction of active conformations. Biochem J 391:449–464.
Li T, Bender M. 2000. A conditional rescue system reveals essential functions for the ecdysone
receptor (EcR) gene during molting and metamorphosis in Drosophila. Development
127:2897–2905.
Lonard DM, O’Malley BW. 2007 Nuclear receptor coregulators: judges, juries, and executioners
of cellular regulation. Mol Cell 27:691–700.
Mouillet JF, Henrich VC, Lezzi M, Vögtli M. 2001. Differential control of gene activity by
isoforms A, B1 and B2 of the Drosophila ecdysone receptor. Eur J Biochem 268:1811–1819.
Nagpal S, Friant S, Nakshatri H, Chambon P. 1993. RARs and RXRs: evidence for two
autonomous transactivation functions (AF-1 and AF-2) and heterodimerization in vivo.
EMBO J 12:2349–2360.
Niedziela-Majka A, Kochman M, Ozyhar A. 2000. Polarity of the ecdysone receptor complex
interaction with the palindromic response element from the hsp27 genepromoter. Eur J
Biochem 267:507–519.
Nieva C, Gwóźdź T, Dutko-Gwóźdź J, Wiedenmann J, Spindler-Barth M, Wieczorek E,
Dobrucki J, Duś D, Henrich V, Ozyhar A, Spindler KD. 2005. Ultraspiracle promotes the
nuclear localization of ecdysteroid receptor in mammalian cells. Biol Chem 386:463–470.
Nieva C, Spindler-Barth M, Azoitei A, Spindler KD. 2007. Influence of hormone on intracellular
localization of the Drosophila melanogaster ecdysteroid receptor (EcR). Cell Signal 19:
2582–2587.
Nieva C, Spindler-Barth M, Spindler K-D. 2008. Impact of heterodimerization on intracellular
localization of the ecdysteroid receptor (EcR). Arch Insect Biochem Physiol 68:40–49.
Parrado A, Despouy G, Kraiba R, Le Pogam C, Dupas S, Choquette M, de Robledo M,
Larghero J, Bui H, Le Gall I, Rochette-Egly C, Chomienne C, Padua RA. 2001. Retinoic
acid receptor alpha1 variants, RARalpha1DeltaB and RARalpha1DeltaBC, define a new
class of nuclear receptor isoforms. Nucleic Acids Res 29:4901–4908.
Perera SC, Zheng S, Feng QL, Krell PJ, Retnakaran A, Palli SR. 2005. Heterodimerization of
ecdysone receptor and ultraspiracle on symmetric and asymmetric response elements. Arch
Insect Biochem Physiol 60:55–70.
Przibilla S, Hitchcock WW, Szécsi M, Grebe M, Beatty J, Henrich VC, Spindler-Barth M. 2004.
Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila
melanogaster. Biol Chem 385:21–30.
Puck TT, Cieciura SJ, Robinson A. 1958. Genetics of somatic mammalian cells. III. Long-term
cultivation of euploid cells from human and animal subjects. J Exp Med 108:945–956.
Riddihough G, Pelham HR. 1987. An ecdysone response element in the Drosophila hsp27
promoter. EMBO J 6:3729–3734.
Robinow S, Talbot WS, Hogness DS, Truman JW. 1993. Programmed cell death in the Drosophila
CNS is ecdysone-regulated and coupled with a specific ecdysone receptor isoform.
Development 119:1251–1259.
Rousseau C, Pettersson F, Couture MC, Paquin A, Galipeau J, Mader S, Miller Jr WH. 2003.
The N-terminal of the estrogen receptor (ERalpha) mediates transcriptional cross-talk
with the retinoic acid receptor in human breast cancer cells. J Steroid Biochem Mol Biol
86:1–14.
Schubiger M, Tomita S, Sung C, Robinow S, Truman JW. 2003. Isoform specific control of gene
activity in vivo by the Drosophila ecdysone receptor. Mech Dev 120:909–918.
Archives of Insect Biochemistry and Physiology
Transcriptional Activity of Ecdysone Receptor
171
Serra J. 1984. Image Analysis and mathematical morphology series: image analysis and
mathematical morphology, Vol. 1. New York: Academic Press. p 601.
Talbot WS, Swyryd EA, Hogness DS. 1993. Drosophila tissues with different metamorphic
responses to ecdysone express different ecdysone receptor isoforms. Cell 73:1323–1333.
Tian H, Mahajan MA, Wong CT, Habeos I, Samuels HH. 2006. The N-Terminal A/B domain of
the thyroid hormone receptor-beta2 isoform influences ligand-dependent recruitment of
coactivators to the ligand-binding domain. Mol Endocrinol 20:2036–2051.
Tran HT, Askari HB, Shaaban S, Price L, Palli SR, Dhadialla TS, Carlson GR, Butt TR. 2001a.
Reconstruction of ligand-dependent transactivation of Choristoneura fumiferana ecdysone
receptor in yeast. Mol Endocrinol 15:1140–1153.
Tran HAT, Shaaban S, Askari HB, Walfish PG, Raikhek AS, Butt TR. 2001b. Requirement of cofactors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast. J Mol
Endocrinol 27:191–209.
Truman JW, Talbot WS, Fairbach SE, Hogness DS. 1994. Ecdysone receptor expression in the
CNS correlates with stage-specific responses to ecdysteroids during Drosophila and Manduca
development. Development 120:219–234.
Tsai CC, Kao HY, Yao TP, McKeown M, Evans RM. 1999. SMRTER, a Drosophila nuclear
receptor coregulator, reveals that EcR-mediated repression is critical for development. Mol
Cell 4:175–186.
Wang D, Simons Jr SS. 2005. Corepressor binding to progesterone and glucocorticoid receptors
involves the activation function-1 domain and is inhibited by molybdate. Mol Endocrinol
19:1483–1500.
Zhou ZX, Lane MV, Kemppainen JA, French FS, Wilson EM. 1995. Specificity of liganddependent androgen receptor stabilization: receptor domain interactions influence ligand
dissociation and receptor stability. Mol Endocrinol 9:208–218.
Archives of Insect Biochemistry and Physiology
Документ
Категория
Без категории
Просмотров
1
Размер файла
163 Кб
Теги
interactions, domain, regulated, modulation, ultraspiracle, activity, transcription, isoforms, ecdysone, receptov, partner, heterodimerization, stability
1/--страниц
Пожаловаться на содержимое документа