G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane

Abstract

Animal steroid hormones are conventionally known to initiate signaling via a genomic pathway by binding to the nuclear receptors. The mechanism by which 20E initiates signaling via a nongenomic pathway is unclear.
We illustrate that 20E triggered the nongenomic pathway through a plasma membrane G-protein-coupled receptor (named ErGPCR) in the lepidopteran insect Helicoverpa armigera. The transcript of ErGPCR was increased at the larval molting stage and metamorphic molting stage by 20E regulation. Knockdown of ErGPCR via RNA interference in vivo blocked larval-pupal transition and suppressed 20E-induced gene expression. ErGPCR overexpression in the H. armigera epidermal cell line increased the 20E-induced gene expression. Through ErGPCR, 20E modulated Calponin nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca2+ levels. The inhibitors of T-type voltage-gated calcium channels and canonical transient receptor potential calcium channels repressed the 20E-induced Ca2+ increase. Truncation of the N-terminal extracellular region of ErGPCR inhibited its localization on the plasma membrane and 20E-induced gene expression. ErGPCR was not detected to bind with the steroid hormone analog [3H]Pon A.
These results suggest that ErGPCR participates in 20E signaling on the plasma membrane.

Full text

R E S E A R C H Open Access
G-protein-coupled receptor participates in
20-hydroxyecdysone signaling on the plasma
membrane
Mei-Juan Cai
†
, Du-Juan Dong
†
, Yu Wang, Peng-Cheng Liu, Wen Liu, Jin-Xing Wang and Xiao-Fan Zhao
*
Abstract
Background: Animal steroid hormones are conventionally known to initiate signaling via a genomic pathway by
binding to the nuclear receptors. The mechanism by which 20E initiates signaling via a nongenomic pathway is
unclear.
Results: We illustrate that 20E triggered the nongenomic pathway through a plasma membrane G-protein-coupled
receptor (named ErGPCR) in the lepidopteran insect Helicoverpa armigera. The transcript of ErGPCR was increased at the
larval molting stage and metamorphic molting stage by 20E regulation. Knockdown of ErGPCR via RNA interference
in vivo blocked larval–pupal transition and suppressed 20E-induced gene expression. ErGPCR overexpression in the H.
armigera epidermal cell line increased the 20E-induced gene expression. Through ErGPCR, 20E modulated Calponin
nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca
2+
levels. The inhibitors of
T-type voltage-gated calcium channels and canonical transient receptor potential calcium channels repressed the
20E-induced Ca
2+
increase. Truncation of the N-terminal extracellular region of ErGPCR inhibited its localization on
the plasma membrane and 20E-induced gene expression. ErGPCR was not detected to bind with the steroid hormone
analog [
3
H]Pon A.
Conclusion: These results suggest that ErGPCR participates in 20E signaling on the plasma membrane.
Keywords: Steroid hormones, G-protein-coupled receptors, Protein phosphorylation, Calcium influx, Signal transduction
Background
Animal steroid hormones are lipid-soluble molecules
conventionally known to initiate signaling via a genomic
pathway. Steroid hormones enter the nucleus by freely
diffusing through cell membranes to combine with intra-
cellular nuclear receptors for gene transactivation. Nuclear
receptors act by forming homodimers or heterodimers.
For example, the glucocorticoid receptor [1] and estrogen
receptor [2] form a homodimer, and insect ecdysone
receptor (EcR) forms a heterodimer with an ultraspiracle
protein (USP), the ortholog of retinoid X receptor in ver-
tebrates [3]. The heat shock proteins Hsp90 and Hsp70
interact with the nuclear receptors to facilitate their DNA
binding activity in fruit flies [4] and mammals [5]. Hsp90
[6] and Hsc70 [7] have been found to be involved in insect
steroid hormone signaling by differential interaction with
the nuclear receptors. However, plant steroid hormones,
such as brassinosteroids, employ a nongenomic pathway
to initiate signaling by combining with plasma membrane
receptor kinases for gene transactivation [8]. The bras-
sinosteroids exhibit structural similarity to the steroid
hormones of vertebrates and insects [9]. These studies
suggest the existence of similar pathways in plants and
animals, which should be further studied.
Previous studies indicated that animal steroid hormones
can trigger nongenomic actions through the cytoplasmic
membrane [10]. For example, estrogen activates phos-
phoinositide 3 kinase to recruit protein kinase B to the
membrane in mammals [11]. A G-protein-coupled seven
transmembrane receptor (GPR30) can act as the membrane
receptor for estrogen [12], and it has been renamed G-pro-
tein-coupled estrogen receptor 1 (GPER) [13]. However,
* Correspondence: xfzhao@sdu.edu.cn
†
Equal contributors
The Key Laboratory of Plant Cell Engineering and Germplasm Innovation,
Ministry of Education, Shandong Provincial Key Laboratory of Animal Cells
and Developmental Biology, School of Life Sciences, Shandong University,
Jinan 250100, Shandong, China
© 2014 Cai et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Cai et al. Cell Communication and Signaling 2014, 12:9
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GPR30 is located in the endoplasmic reticulum [14] and
may be translocated to the plasma membrane [15]. New
studies have suggested that GPER is ubiquitinated at the
cell surface, and constitutively internalized in an arrestin-
independent manner. Moreover, GPER does not recycle to
the plasma membrane [16]. GPER acts as a stand-alone
membrane receptor of pregenomic action independent
on the estrogen nuclear receptor [17]. GPER also func-
tions in the nervous system, and may be a pharmaceutical
target [18].
In insects, the ATP-binding cassette transporter E23
can act as a general negative regulator of ecdysteroid
signaling by transporting 20E outside of the cell [19]. 20E
promotes neuroblast proliferation during metamorphosis
partly by suppressing nitric oxide production in <15 min
without protein synthesis or transcription [20], and the cell
membrane receptor of 20E is assumed to be a leucine-rich
repeat receptor kinase [21]. The plasma membrane of the
anterior silk gland of Bombyx mori binds [
3
H] ponasterone
A([
3
H]Pon A), suggesting that the anterior silk gland may
express an unknown membrane 20E receptor [22]. 20E in-
duces intracellular Ca
2+
release into the cytoplasm via an
unknown G-protein-coupled receptor (GPCR) pathway in
the anterior silk gland of silkworms [23]. The Drosophila
dopamine receptor DmDopEcR binds [
3
H]Pon A, and is
considered as a 20E membrane receptor [24]. Ecdysteroids
trigger rapid Ca
2+
increase, including intracellular Ca
2+
release, and extracellular Ca
2+
influx through GPCR in
mouse skeletal muscle cells [25]. In our previous study,
we demonstrated that 20E regulates the rapid nuclear
translocation and phosphorylation of Calponin for gene
expression in Helicoverpa armigera [26]. These findings
suggest that 20E has membrane receptors and a nonge-
nomic signaling pathway.
In this study, we reported an ecdysone-responsible
GPCR (ErGPCR) participates in 20E signaling on the
plasma membrane. The knockdown of ErGPCR disrupted
several biological processes, including the larval–pupal
metamorphosis, expression of 20E-induced genes, subcel-
lular translocation and phosphorylation of Calponin, and
20E-induced cytosolic Ca
2+
increase.
Results
ErGPCR is involved in 20E-regulated gene expression
It has been known that 20E regulates the gene expression
of the nuclear receptor EcRB1 and transcription factors
Br,USP1,E75B,andHR3 [27]. Suramin disrupts GPCR
binding with the G protein by blocking the association of
Gproteinαand βγ subunits [28]. Suramin is widely used
to study GPCR- and G-protein-initiated cell signaling,
including the 20E-induced GPCR pathway in the anterior
silk gland of silkworms [23], cytosolic Ca
2+
increase, and
protein kinase C activation [29]. Thus, the involvement of
GPCRs in 20E-induced gene expression was analyzed
using the GPCR inhibitor suramin in a lepidopteran H.
armigera epidermal cell line (HaEpi cell line, established
in our laboratory) [30]. 20E significantly promoted the
expression of EcRB1,BrZ2,HHR3,andUSP1 compared
with the DMSO solvent control. However, the 20E-induced
transcript increase was repressed by the addition of
suramin (Figure 1). These results suggest that GPCRs
are probably involved in 20E-regulated mRNA levels.
We identified six GPCR candidates from the expressed
sequence tags (EST) of the cDNA library of the HaEpi
cell line using BLASTX assay (http://www.ncbi.nlm.nih.
gov/) (Additional file 1: Figure S1, Table S1). The mRNA
levels of six GPCR candidates were upregulated by 20E
induction, and two non-GPCR ESTs were unaffected.
Knockdown of No. 16666 and ErGPCR in the HaEpi
cells using RNA interference (RNAi) decreased EcRB1,
BrZ2,HHR3,andUSP1 transcript levels in 20E induction.
The knockdown of the other four GPCR candidates
affected one to three 20E-induced gene transcripts
(Additional file 1: Figure S2). These results suggest the
involvement of GPCRs in 20E-induced gene expression.
ErGPCR was further studied regarding its expression
profile during development. The deduced amino acid
sequence of ErGPCR contains a signal peptide at the
N-terminus and seven transmembrane domains (Additional
file 1: Figure S3). ErGPCR belongs to methuselah-like
proteins in the class B secretin GPCR family based on
NCBI Blast analysis (http://blast.ncbi.nlm.nih.gov/Blast.
cgi). ErGPCR has 57% identity with Spodoptera frugiperda
GPCR, 32% with Tribolium castaneum GPCR, and 30%
with Drosophila melanogaster GPCR (Additional file 1:
Figure 1 Involvement of GPCRs in the 20E pathway in HaEpi cells
as determined by quantitative real-time reverse transcription
polymerase chain reaction (qRT-PCR) analysis. DMSO treatment
was used as the solvent control for 20E. DMSO plus suramin 50 μM
treatment for 1 h was used to determine the toxic effects of suramin
on the cells. The HaEpi cells were pretreated with 50 μM suramin for
1 h and then exposed to 1 μM 20E for another 6 h. The results are
based on the ΔΔCT calculation by normalization of the β-actin
gene. Error bars represent the standard deviation of three
independent replicates. Asterisks indicate significant differences
(Student’sttest, *p<0.05).
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Figure S4). However, D. melanogaster DmDopEcR, Homo
sapiens GPR30, and H. sapiens beta-2 adrenergic receptor
(AR) are not found by BLASTX analysis. This finding sug-
gests that ErGPCR is less similar to DmDopEcR, GPR30,
and AR. Phylogenetic analysis indicated that ErGPCR does
not cluster with DmDopEcR, GPR30, and AR. These
results illustrate that these GPCRs belong to different
GPCR groups (Additional file 1: Figure S5).
The transcript level of ErGPCR was increased at the
larval molting stage (5 M) and metamorphic molting stage
(sixth-instar 72 h larvae to pupae) in the tissues (Figure 2).
Given that the 20E titer is higher during molting and
metamorphosis in lepidopteran insect Manduca sexta
[27], the hormone induction on the mRNA levels of
ErGPCR was examined. The ErGPCR transcript level was
upregulated in the midgut from 3 h to 24 h after 20E
injection into the sixth-instar larvae. JH III injection
into the sixth-instar larvae did not affect the ErGPCR
transcript levels, but repressed the 20E-induced upreg-
ulation of ErGPCR (Figure 3). These data suggest that
ErGPCR mRNA level is upregulated by 20E signaling.
To confirm that 20E upregulates ErGPCR, we knocked
down the nuclear receptor of 20E, EcRB1, and analyzed
the transcript of ErGPCR.WhenEcRB1 was knocked
down, the upregulation of ErGPCR induced by 20E was
blocked (Additional file 1: Figure S6). These results reveal
that 20E upregulates ErGPCR transcript via the nuclear
receptor EcRB1.
Figure 2 The increased transcripts of ErGPCR during molting
and metamorphosis. (A),(B) and (C) ErGPCR is highly expressed
during molting and metamorphosis in epidermis, midgut and fat
body detected by qRT-PCR. 5Fis the fifth instar 12 h larvae; 5Mis
the fifth instar molting larvae; 6–0to6–120 h are the 6th instar
larvae in hours; p 0 to p 8 are the pupae in days.
Figure 3 Hormonal induction of ErGPCR in the midgut by
injection of 20E (A) or JH III (B) or 20E +JH III (C) into the 6th
instar 6 h larvae (500 ng/larva) analyzed by qRT-PCR. DMSO is a
solvent control. β-actin gene was used as the quantitative control
for the mRNA. The asterisks indicate significant differences between
20E treatment and DMSO solvent control by student’sttest analysis
from three independent repeats (*p< 0.05, n = 3).
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ErGPCR is involved in the larval–pupal transition in vivo
by regulating gene expression
The function of ErGPCR in larval–pupal transition was
determined through RNAi by injecting dsErGPCR into the
larval hemocoel. The knockdown of ErGPCR blocked lar-
val–pupal transition (Figure 4A). In the dsRNA of green
fluorescent protein (dsGFP)-injected control, 90% of the
larvae pupated, whereas 10% died. However, in dsErGPCR
treatment, only 29% of the larvae pupated, 50% died, and
21% displayed larval–pupal chimeras (Figure 4B). Of the
29% that pupated after ErGPCR knockdown, the duration
of development was significantly delayed compared with
the dsGFP control: a 23 h delay from fifth instar to the
sixth instar, and a 52 h delay from the sixth instar to the
pupal stage (Figure 4C). RT–PCR showed that ErGPCR
was significantly knocked down by four consecutive
Figure 4 ErGPCR knockdown by dsRNA injection blocks larval-pupal transition. (A) Phenotypes after ErGPCR knockdown (dsErGPCR injection
into the 5th instar larval hemocoel). Scale bar = 1 cm. (B) Statistical analysis of the phenotype in A.(C) 5–0 h to 6–0 h: 0 h of the 5th instar to 0 h
of the 6th instar 0 h; 6–0 h to pupation: 0 h of the 6th instar to pupation. (D) The efficiency of ErGPCR knockdown, analyzed by semi-quantitative
RT-PCR. (E) The gene transcripts in the larval epidermis after ErGPCR knockdown were determined by qRT-PCR analysis. Error bars represent the
standard deviations of three replicates. Asterisks denote significant differences (*p< 0.05; **p< 0.01, via Student’sttest) based on 30 larval
samples with three replicates.
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dsErGPCR injections into the larvae (Figure 4D). The
transcript levels of the genes involved in the 20E pathway,
including EcRB1,USP1,HHR3,BrZ2,andE75B,werede-
creased in the larval epidermis after ErGPCR knockdown
(Figure 4E). These results suggest that ErGPCR is related
to larval–pupal transition and gene expression in vivo.
ErGPCR is located on the membrane and is necessary for
20E-induced gene expression
Immunocytochemical analysis using rabbit anti-ErGPCR
polyclonal antibodies showed that ErGPCR was located
on the plasma membrane of HaEpi cells. The green
fluorescence of ErGPCR overlapped with the red-stained
cell membrane to display an orange color by confocal laser
scanning microscopy (Figure 5A). Moreover, the overex-
pression of ErGPCR-GFP (C-terminal with GFP) was also
located on the cell plasma membrane (Figure 5B). ErGPCR
knockdown in the HaEpi cells decreased the 20E-induced
transcript levels of EcRB1,USP1,HHR3,BrZ2,andE75B
compared with the dsGFP-treated control (Figure 5C).
By contrast, ErGPCR overexpression led to an increase
in the transcript levels of EcRB1,BrZ2,HHR3,andUSP1
(Figure 5D). Moreover, 20E upregulated the mRNA level
of insulator body protein mod(mdg4)1a not through
EcRB1 but through ErGPCR (Figures 5E and F). These
results suggest that ErGPCR may serve as the initiation
step of 20E signal amplification on the plasma membrane
as nongenomic or pregenomic action before the hierarch-
ical control of 20E-induced genomic action.
20E regulates protein nuclear translocation and
phosphorylation via ErGPCR
The nongenomic pathway is characterized by rapid protein
translocation and phosphorylation [31]. The Calponin
protein has been demonstrated to undergo quick nuclear
translocation and phosphorylation by 20E induction [26].
Thus, ErGPCR was knocked down in the HaEpi cells to de-
termine its function in 20E-regulated rapid translocation
and phosphorylation of Calponin. Calponin was mainly lo-
calized in the cytoplasm in the DMSO-negative control
cells, but was translocated into the nucleus after 20E induc-
tion. However, after ErGPCR knockdown, 20E could not
induce the nuclear translocation of Calponin (Figure 6A).
Moreover, the 20E-mediated Calponin phosphorylation was
suppressed when ErGPCR was silenced (Figure 6B). The
protein synthesis inhibitor anisomycin did not inhibit
20E-induced Calponin phosphorylation (Figure 6C). These
results suggest that 20E regulates Calponin nuclear trans-
location and phosphorylation via ErGPCR.
20E regulates cellular Ca
2+
release and influx via ErGPCR
to regulate gene expression
The increase in cellular Ca
2+
is another characteristic of
the nongenomic pathway of steroid hormones [32]. Thus,
ErGPCR was knocked down in the HaEpi cells to deter-
mine the function of ErGPCR in the rapid 20E-regulated
Ca
2+
increase. When the cells were incubated in calcium-
free buffer (DPBS), cytosolic Ca
2+
level increased rapidly
by 20E treatment, and peaked at approximately 50 s, then
declined to a lower level at 120 s. Following the addition
of 1 mM calcium into DPBS at 120 s, the cytosolic Ca
2+
levels gradually increased and then remained constant.
However, suramin (50 μM) pretreatment for 1 h inhibited
the 20E-induced rapid increase in cytosolic Ca
2+
levels
(Figure 7A). When ErGPCR was knocked down by RNAi,
the 20E-induced Ca
2+
increase, including intracellular Ca
2+
release, and extracellular Ca
2+
influx, was also inhibited
compared with the control (Figure 7B). These findings
suggest that 20E induces rapid intracellular Ca
2+
release
and extracellular Ca
2+
influx via ErGPCR.
Various calcium channel blockers, including the T-type
voltage-gated calcium channel inhibitor flunarizine dihy-
drochloride (FL), L-type calcium channel inhibitor verap-
amil hydrochloride (Ve) [33], transient receptor potential
(TRP) calcium channel store-operated channel (SOC)
inhibitor 2-aminoethoxydiphenyl borate (2-APB) [34],
and receptor-operated (ROC) TRPC3 channel inhibitor
pyrazole (Pyr3) [35], were employed to determine the
involvement of calcium channels in 20E-induced extracellu-
lar Ca
2+
influx. The 20E-induced Ca
2+
influx was restrained
by 50 μM FL without affecting intracellular Ca
2+
release.
By contrast, 20E-induced Ca
2+
release and influx were un-
affected by 100 μM Ve (Figure 7C). The 2-APB inhibitor
(10 μMto100μM) had no effect on the 20E-induced Ca
2+
release and influx. However, 10 μM Pyr3 suppressed the
20E-induced Ca
2+
influx, but had no effect on intracellular
Ca
2+
release (Figure 7D). These results reveal that T-type
calcium channels and TRPC3 channels are involved in
20E-induced Ca
2+
flux.
To investigate the effect of the cellular Ca
2+
increase
on the 20E-induced gene expression and 20E-induced
Calponin phosphorylation, we performed qRT–PCR and
western blot. The 20E-induced upregulation of EcRB1,
BrZ2,HHR3,andUSP1 was suppressed by FL and Pyr3
(Figure 7E). Meanwhile, the 20E-induced phosphorylation
of Calponin was inhibited (Figure 7F). By contrast, Ve and
2-APB inhibitors had no effect on the 20E-induced gene
expression and 20E-induced Calponin phosphorylation.
These results show that the 20E-induced rapid intracellu-
lar Ca
2+
increase is required for 20E-regulated gene ex-
pression and protein phosphorylation.
To examine the mechanism by which 20E regulates
gene expression through ErGPCR and Ca
2+
signaling,
ChIP experiments were performed by anti-RFP antibody
in the EcRB1-RFP-overexpressing HaEpi cells. In M. sexta,
one ecdysone response element (EcRE1, GGGGTCAATG
AACCG) was identified in 20E reporter gene hormone
receptor 3 (HR3). 20E regulates EcRB1/USP1 heterodimer
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Figure 5 ErGPCR is located on the plasma membrane and is involved in 20E-induced gene expression in HaEpi cells. (A) Green fluorescence
indicated ErGPCR protein detected with anti-ErGPCR (antibody specificity is shown in Additional file 1: Figure S7) and ALEXA 488–labeled
goat anti-rabbit secondary antibody by confocal microscope. The nuclei were stained with 4′-6′-diamidino-2-phenylindole dihydrochloride
(DAPI). (B) Green fluorescence protein (GFP, green) alone was detected in the entire cell; the plasma membrane (red) was marked with
1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI); and the orange color are the overlapping of ErGPCR-GFP (green)
and membrane (red) by confocal microscope. Scale bar =20 μm. (C) and (D) The effects of ErGPCR knockdown and overexpression on
20E-induced gene expression, analyzed by qRT-PCR. (E) and (F) 20E upregulated mod(mdg4)1a expression not through EcRB1 but through
ErGPCR, by qRT-PCR analysis. β-actin was used as the quantitative control. * indicates significant differences (p< 0.05) among the treatments
via Student’sttest from three independent replicates.
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binding to EcRE to regulate gene transcription [36].
We cloned the 5' regulatory region of Helicoverpa HR3
(HHR3) that contains putative EcRE (GGGGTCAA
TGAACTG), which has one “T”different from EcRE1 in
MHR3. The EcRE from HHR3 is proven to be active by
GFP-plasmid examination. Fewer PCR product (EcRE)
Figure 6 Knockdown of ErGPCR by RNAi blocks 20E-induced nuclear translocation and phosphorylation of Calponin in the HaEpi cells.
(A) Green fluorescence represents Calponin detected with anti-Calponin antibody. Blue fluorescence indicates DAPI-stained nuclei. The image in the
white box shows the efficacy of ErGPCR knockdown (by dsRNA incubation for 24 h followed by 1 μM 20E induction for 1 h), determined by RT-PCR; N,
C, and dsG indicate ErGPCR transcripts in normal cells, dsGFP-treated cells, and dsErGPCR-treated cells, respectively. (B) 20E-induced phosphorylation of
Calponin was detected with anti-Calponin antibody by western blot analysis.Cal-P indicates phosphorylated Calponin. (C) The effect of anisomycin on
20E-induced phosphorylation of Calponin was analyzed using anti-His monoclonal antibody by western blot analysis. Cal-GFP (56 kDa) is the fused
expressed Calponin with GFP in the HaEpi cells; Cal-GFP-P indicates phosphorylated Calponin-GFP; GFP (35 kDa) was expressed alone in the cells as a
control. The cells were incubated with 10 μM anisomycin for 1 h and then treated with DMSO or 1 μM 20E for 1 h.
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was detected from the immunoprecipitates in the pIEx-4-
RFP-transfected control samples after various treatments
by anti-RFP antibody, because the RFP recognized by
anti-RFP antibody did not bind to DNA. By contrast, in
the pIEx-4-EcRB1-RFP-transfected cells, the PCR product
(EcRE) was detected from the immunoprecipitates in 20E
induction by anti-RFP antibody. However, after ErGPCR
knockdown, the PCR product (EcRE) was significantly
decreased compared with the dsGFP treatment control
(Figure 8A). These results suggest that 20E via ErGPCR
regulates EcRB1 binding to EcRE to regulate the 20E-
induced gene transcription.
Similarly, in the pIEx-4-RFP-transfected control samples,
fewerPCRproduct(EcRE)wasdetectedintheimmuno-
precipitate by anti-RFP antibody after various treatments.
By contrast, in the pIEx-4-EcRB1-RFP-transfected samples,
the PCR product containing EcRE was detected in 20E
induction by anti-RFP antibody, which recognized the
binding of EcRB1-RFP to EcRE. However, fewer DNA
product containing EcRE was detected after the addition
of inhibitors FL and Pyr3. Ve and 2-APB had no effect
on the PCR product (EcRE) (Figure 8B). These results
suggest that 20E via cellular Ca
2+
signaling modulates the
binding of EcRB1 to EcRE, and regulates the 20E-induced
gene transcription.
N-terminal extracellular region is essential to ErGPCR
function in the 20E signaling pathway
Truncated mutations of ErGPCR in the N-terminal region
(aa 20 to 197) (OVErGPCR
Δ20–197 aa
) or second inner loop
Figure 7 ErGPCR is involved in the 20E-induced increase in cytosolic Ca
2+
levels. (A) Suramin inhibited the 20E-induced Ca
2+
increase (1 μM
20E, 1 mM calcium chloride, 50 μM suramin). F represents the fluorescence of cells after treatment, whereas F
0
denotes the average fluorescence of
cells before treatment. Fluorescence was detected every 6 s for 360 s using a Laser Scan Confocal Microscope Carl Zeiss LSM 700 (Thornwood, NY,
USA) at 555 nm and then analyzed using the Image Pro-Plus software. (B) ErGPCR knockdown using dsRNA (5 μg/mL) incubation inhibited the
20E-induced Ca
2+
increase. (C) The T-type calcium channel blocker flunarizine dihydrochloride (FL, 50 μM) inhibited the 20E-indued Ca
2+
influx. The
L-type calcium channel blocker verapamil hydrochloride (Ve, 100 μM) did not affect the 20E-induced Ca
2+
influx. (D) The transient receptor potential
(TRP) channel blocker pyrazole (Pyr3, 10 μM) inhibited the 20E-induced Ca
2+
increase. 2-Aminoethoxydiphenyl borate (2-APB) (10 μMto100μM) did
not affect 20E-induced Ca
2+
influx. (E) qRT-PCR showing the involvement of Ca
2+
signal in 20E-induced gene expression. Cells were incubated in 1 μM
20E for 6 h after different inhibitors pretreatment for 1 h and the RNA was isolated for qRT-PCR. β-actin was used as the quantitative control. *indicates
significant differences (p< 0.05) among the treatments via Student’sttest from three independent replicates. (F) Western blot showing the
involvement of Ca
2+
signal in 20E-induced Calponin phosphorylation. pIEx-4-Cal-GFP was overexpressed in HaEpi cells for 48 h. The cells were
treated with 1 μM 20E for 1 h after different inhibitors pretreatment for 1 h. The GFP was overexpressed as controls.
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(aa 275 to 308) (OVErGPCR
Δ275–308 aa
) were overexpressed
by fusing with GFP to analyze the functional domain of
ErGPCR in the 20E signaling pathway (Figure 9A). Over-
expressed full-length ErGPCR (OVErGPCR) was located
ontheplasmamembrane.However,theN-terminal
region-truncated ErGPCR (OVErGPCR
Δ20–197 aa
)was
in the cytoplasm, whereas the second inner loop truncation
(OVErGPCR
Δ275–308 aa
) was located in both the plasma
membrane and cytoplasm (Figures 9B and C). Correlated
with its location, the truncation of the N-terminal extra-
cellular region of ErGPCR (OVErGPCR
Δ20–197 aa
)caused
a decrease in the 20E-induced transcript levels of EcRB1,
BrZ2,HHR3,andUSP1 compared with the full-length
OVErGPCR. By contrast, truncation of the second inner
loop of ErGPCR (OVErGPCR
Δ275–308 aa
)didnotaffect
the 20E-induced transcript levels of EcRB1,BrZ2,HHR3,
and USP1 (Figure 9D). These results indicate that the
complete structure of ErGPCR is essential to its plasma
membrane location, and the N-terminal extracellular
region is required for ErGPCR function in the 20E sig-
naling pathway.
ErGPCR is not detected to bind with the 20E analog
[
3
H]Pon A
ErGPCR and EcRB1 were overexpressed in HaEpi cells by
fusing with GFP at the C-terminus (Figure 10A) for the
binding experiments. No increase in [
3
H]Pon A (0.1 nM)
was detected with the increase in cell numbers (1 × 10
4
cells to 100 × 10
4
cells) in ErGPCR-GFP-transfected cells
compared with the normal cells, GFP-overexpressed cells,
or EcRB1-GFP-overexpressed cells (Figure 10B). The
increase in [
3
H]Pon A was not detected in the plasma
membrane fractions (5 μgto500μg) from HaEpi cells
that overexpressed ErGPCR-GFP compared with that
in the membrane fractions from the normal cells and
GFP-overexpressed cells (Figure 10C). These results sug-
gest that cells or cell membrane fractions could bind with
[
3
H]Pon A in a cell- or cell membrane fraction-dependent
manner. However, overexpression of ErGPCR does not
increase [
3
H]Pon A binding by this analysis.
Discussion
Although studies have shown that GPCRs are involved
in 20E signaling, definitive evidence of this involvement
is scarce. Our study reveals that ErGPCR regulates 20E
signaling on the plasma membrane. Through ErGPCR,
20E regulates gene expression, fast protein translocation
and phosphorylation, rapid intracellular Ca
2+
increase, and
larval–pupal transition.
20E regulates genomic action through the ErGPCR-mediated
nongenomic pathway
20E initiates the genomic pathway by binding with its
nuclear hormone receptor EcR to regulate gene expression
for metamorphosis [37]. 20E upregulated the mRNA levels
of EcRB1,USP1,HHR3,BrZ2,andE75B.Knockdownof
ErGPCR repressed the binding of EcRB1 to EcRE thus
blocked 20E-induced expression of EcRB1,USP1,HHR3,
BrZ2,andE75B in the cell line and larvae, which resulted
in blocking the 20E genomic pathway, thereby inhibiting
metamorphosis. These results indicate that 20E initiates a
nongenomic pathway to regulate a 20E-mediated genomic
pathway via ErGPCR. In positive feedback, ErGPCR tran-
script was upregulated by 20E via EcRB1. 20E does not act
through EcRB1 to upregulate the mRNA level of the insula-
tor body protein mod(mdg4)1a in HaEpi cells [38]. Instead,
mod(mdg4)1a is upregulated by 20E through ErGPCR.
These results suggest the existence of various pathways
in 20E signaling. The reason that knockdown of EcRB1
repressed ErGPCR but did not repress mod(mdg4)1a
Figure 8 ChIP analysis showing ErGPCR regulates 20E-induced
EcRB1 binding to EcRE through cellular Ca
2+
signaling. (A) ErGPCR
knockdown suppresses the EcRB1 binding to EcRE under 20E treatment.
Cells were transfected with pIEx-4-RFP or pIEx-4-EcRB1-RFP for 24 h. The
cells were treated by dsErGPCR or dsGFP for 12 h, then exposed in 1
μM 20E or DMSO for 6 h. (B) FL and Pyr3 repress EcRB1 binding to
EcRE in 20E treatment. Cells were transfected with pIEx-4-RFP or
pIEx-4-EcRB1-RFP for 48 h. The cells were subjected in 1 μM20Eor
DMSO for 6 h after different calcium channel inhibitors pretreatment
for 1 h. ChIP was carried out without antibody (negative control) or
with anti-RFP antibody. The DNA fragment (EcRE) was amplified by
qRT-PCR using HHR3F/R primers. Input is the positive control of
nonimmunoprecipitated chromatin.
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might because the time difference of mRNA transcription
and protein translation of ErGPCR.
Steroid hormones, such as mammalian estrogen and
insect ecdysone, are conventionally thought to exert
their actions through binding to intracellular receptors
because of their small molecules and lipid solubility.
However, growing evidence indicates that steroid hor-
mones also exert rapid cell surface-initiated actions by
binding to membrane receptors [39], such as the estro-
gen membrane receptor GPR30 [14]. Rapid protein
subcellular translocation and phosphorylation (within
minutes) are the outcomes of a nongenomic signaling
pathway [24]. 20E regulates the rapid nuclear trans-
location and phosphorylation of Calponin for gene trans-
activation in H. armigera [26]. We found that 20E
regulated Calponin nuclear translocation and subse-
quent phosphorylation through ErGPCR. This finding
suggests that 20E functions in the membrane via a
nongenomic pathway to regulate protein translocation
and phosphorylation, which may contribute to the activa-
tion of transcription factors and formation of transcription
complexes.
Figure 9 The N-terminus of ErGPCR is the key domain for its cell membrane localization and function. (A) mutation sites of ErGPCR,
△20-197 indicates the truncation of amino acids 20–197; △275-308 indicates the truncation of amino acids 275–308. (B) The localization of the
overexpressed GFP (OVGFP), full length of ErGPCR fused with GFP (OVErGPCR-GFP), truncation of N-terminal extracellular region of ErGPCR fused
with GFP (OVErGPCR
△20-197aa
-GFP), and truncation of the second inner loop of ErGPCR fused with GFP (OVErGPCR
△276-308aa
-GFP) in the HaEpi cells
by confocal microscope. (C) Subcellular localization of ErGPCR and two mutants were confirmed by western blot with His-tag antibody. (D) The
effects of the mutations of ErGPCR on the transcripts of EcRB1,BrZ2,HHR3 and USP1 induced by 20E were examined by qRT-PCR, with β-actin as
control. Bars indicate mean ± S.D. from three independent experiments. Asterisk indicates significant difference (p < 0.05).
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ErGPCR is involved in 20E-increased cytosolic Ca
2+
levels
20E increases the cytosolic Ca
2+
levels by promoting
the release of Ca
2+
from the intracellular endoplasmic
reticulum via an unknown GPCR in silkworm silk glands
[23]. 20E also regulates Ca
2+
influx from extracellular
sources via an unknown GPCR that activates calcium
channels in murine skeletal muscles [25]. Voltage-gated
calcium channels are essential in regulating extracellular
Ca
2+
influx in a wide variety of tissues [40]. T-type Ca
2+
channels are involved in 20E-induced nuclear and DNA
fragmentation in silkworm silk glands [23]. GPCRs
serve as chaperones and interact with voltage-gated cal-
cium channels to form complexes [41]. Our data show
that 20E regulates rapid intracellular Ca
2+
release and
extracellular Ca
2+
influx through ErGPCR, and T-type
voltage-gated Ca
2+
channels are involved in Ca
2+
influx.
In addition, we found that the 20E-induced Ca
2+
influx
was also inhibited by the TRP channel inhibitor Pyr3. This
result suggests that TRP channels are also involved in 20E-
induced Ca
2+
influx. TRP channels are non-voltage-gated
Ca
2+
channels involved in Ca
2+
entry [42]. TRP channels
are classified into six subfamilies according to their primary
structure and function, including ROC and SOC [43].
GPCRs directly or indirectly modulate several TRP chan-
nels [44,45]. TRP channels are associated with steroid hor-
mones in mammals [46]. Rapid calcium release or influx in
the cells is the outcome of nongenomic signaling. Calcium
is an important secondary messenger that regulates numer-
ous essential physiologic processes, including protein kinase
C activation, for further protein phosphorylation [47] and
gene transcription. In our study, when the cellular Ca
2+
was
blocked by inhibitors, 20E-induced gene expression and the
phosphorylation of Calponin were blocked. These findings
confirm the function of calcium on gene expression and
protein phosphorylation as the secondary messenger, and
reveal that 20E regulates the cellular calcium via ErGPCR
to regulate the genomic pathway.
ErGPCR does not bind with the steroid hormone
analog [
3
H]Pon A
In classical GPCR signaling pathways, ligands bind to
cell surface transmembrane receptors, such as the β2 ARs,
and cause conformational changes in their transmembrane
and intracellular domains [48]. Numerous studies have
reported the binding of several GPCRs with 20E, such
as the binding of DmDopEcR with [
3
H]Pon A [24], or an
unknown GPCR in the anterior silk gland of silkworms
binding with [
3
H]Pon A [22]. However, we did not detect
the binding of ErGPCR with [
3
H]Pon A using the whole
cells and cell membrane fractions by overexpressing
ErGPCR in HaEpi cells. Thus, ErGPCR is likely transiently
activated by 20E without any stable ligand binding.
Based on NCBI Blast analysis (http://blast.ncbi.nlm.nih.
gov/Blast.cgi), ErGPCR belongs to methuselah-like pro-
teins in the class B secretin GPCR family, but DmDopEcR
shows homology with vertebrate ARs [24]. Identification
and phylogenetic analysis using amino acid sequences
show that ErGPCR differs from GPR30, beta-2 AR, or
Drosophila DmDopEcR, which may explain the differences
in ligand binding activity. Another possibility is the
analytical method, which needs further study in next work.
GPR30 has shown negligible binding to estrogen (17β-
estradiol) in several studies [49], which may be due to
the different analytical methods [17]. A major challenge
in the study of steroid hormone nongenomic pathways
is the binding assay of GPCR with the steroid hormone
[50]. Although ErGPCR did not bind ecdysteroid in our
study, this result is of particular importance in many
cellular responses to 20E, including 20E-induced mRNA
levels, protein subcellular translocation and phosphoryl-
ation, and cellular Ca
2+
increase. Whether other GPCRs
can bind with 20E needs further exploration.
A total of 800 GPCRs have been discovered in mammals
[51], 1000 in Caenorhabditis elegans [52], and 200 in D.
melanogaster [53]. GPCRs involved in steroid membrane
Figure 10 Binding assay of [
3
H]Pon A to whole cells or membrane fractions. (A) Western blot analysis of the overexpressed GFP, ErGPCR-
GFP and EcRB1-GFP. (B) Binding of [
3
H]Pon A to the whole HaEpi cells that expressed GFP, ErGPCR-GFP or EcRB1-GFP, respectively. The cells (1–100 ×
10
4
) were incubated with 0.1 nM [
3
H]Pon A (5740 cpm) for 1 h at 27°C, respectively. (C) The membrane fractions (5, 50, 250, 500 μgmembranepro-
teins) isolated from above cells were incubated with 0.1 nM [
3
H]Pon A for 1 h at 27°C, respectively. cpm: counts per minute of [
3
H]Pon A. The data
marked with different small letters indicate a significant difference at 0.05 levels by the one-way ANOVA with Duncan′s test.
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signaling may differ among various organisms. GPCRs
form homodimers, heterodimers, oligomers, or complexes
in the signalsomes of the membrane [54]. The mechanism
underlying GPCRs in the 20E membrane pathway requires
further study.
Conclusions
ErGPCR participates in 20E-regulated gene expression,
rapid Calponin nuclear translocation and phosphorylation,
rapid intracellular Ca
2+
release, and extracellular Ca
2+
in-
flux via T-type calcium channels and TRP channels. The
N-terminal extracellular region is critical for the function
of ErGPCR in the 20E signaling pathway. ErGPCR is
necessary for the larval–pupal transition in H. armigera
development (Figure 11).
Methods and materials
Insect
Cotton boll worms, H. armigera, were reared in our
laboratory on an artificial diet of wheat, soybean, vitamins,
and inorganic salts under a 14 h light:10 h dark cycle at
27°C.
Quantitative real-time reverse-transcriptase PCR (qRT-PCR)
Approximately 5 μg of the total RNA from each sample
was reverse transcribed into first-strand cDNA for qRT-
PCR analysis (First Strand cDNA Synthesis Kit, Sangon,
China). qRT-PCR was performed using SsoFast™EvaGreen
Supermix (BIO-RAD, Shanghai, China). Thermocycling
(94°C for 20 s, 58°C for 20 s, and 72°C for 20 s) was
performed for 40 cycles using the CFX96™real-time sys-
tem (BIO-RAD). The experiment was repeated three
times using three independent RNA samples for statistical
analysis. β-actin was used as the cDNA quality and
quantity controls. The primers used for qRT-PCR are
listed in Additional file 1: Table S2.
Suramin inhibition
H. armigera epidermal cells (HaEpi) cells were cultured
until 80% confluence at 27°C in 25 cm
2
culture flasks
using Grace’s medium (Invitrogen, Carlsbad, CA, USA)
supplemented with 10% fetal bovine serum (FBS, Mdgenics,
St. Louis, MO, USA). The cells were incubated with 50 μM
suramin (sodium salt; Sigma Chemical, St. Louis, MO,
USA) for 1 h and then exposed to 1 μM20Eforanother
6 h. Gene expression was determined via qRT-PCR.
Screening of the target GPCR by qRT-PCR
dsRNA was produced using a MEGAscript™RNAi kit
(Ambion, Austin, TX, USA). Some GPCR ESTs obtained
through random sequencing of the HaEpi cells, a cell line
previously established in our laboratory, were individually
knocked down using RNA interference. Lipofectamine
2000 (Invitrogen, Carlsbad, CA, USA) was used for dsRNA
transfection. Briefly, 5 μgofdsRNAand8μL of Lipofecta-
mine 2000 were added to 125 μL of FBS-free Grace’s
medium incubated at room temperature for 30 min. The
reagents were mixed and incubated for another 20 min,
Figure 11 Diagram showing the ErGPCR-mediated nongenomic pathway response to 20E. 20E through ErGPCR nongenomic pathway
regulates the cytosolic calcium fast increase, including the release of the intracellular Ca
2+
, and the influx of the extracellular Ca
2+
via T-type
calcium channels and TRP channels. 20E also through ErGPCR regulates the rapid phosphorylation and nuclear translocation of Calponin, and
through ErGPCR regulates gene transcription in the genomic pathway for metamorphosis.
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and then directly added into HaEpi cells in a six-well plate
containing 0.8 mL of Grace’s medium per well. After incu-
bation at 27°C for 24 h, the cells were rinsed and then re-
fed with fresh medium containing 10% FBS. The cells
were cultured with 1 μM 20E for 6 h. Control cells were
treated with the same amount of dsGFP. Total RNA was
then extracted from the cells for qRT-PCR based on three
independent replicates.
Cloning of full-length cDNA of ErGPCR
We obtained an EST with the 3′end of ErGPCR by
random sequencing of the cDNA library of the insect
during metamorphosis. The 5′end of the gene was
amplified via PCR using the gene-specific reverse pri-
mer ErGPCRF1 and the 5′primer through the Genome
Walker method as described by Clontech Laboratories
Inc. (cat. no. 638904; Clontech, CA, USA).
Recombinant expression of ErGPCR in Escherichia coli and
antiserum preparations
The ErGPCR fragment was amplified using the primers
ErGPCRExpF and ErGPCRExpR. The PCR product was
cloned into pET30a plasmid, expressed in Escherichia
coli rosette cells, and then cultured in a Luria–Bertani
medium (1% tryptone, 0.5% yeast extract, 1% NaCl, and
25 μg/mL kanamycin). The target protein was purified
using His-bind resin to produce polyclonal rabbit anti-
serum.Thespecificityoftheantibodywasdetermined
via western blot analysis using horseradish peroxidase–
labeled goat anti-rabbit polyclonal secondary antibodies
(Zhongshan, Beijing).
Immunocytochemistry
The cells grown on cover slips were fixed with 4% parafor-
maldehyde in phosphate-buffered saline (PBS; 140 mM
NaCl, 2.7 mM KCl, 10 mM Na
2
HPO
4
, 1.8 mM KH
2
PO
4
,
pH 7.4) for 10 min. The fixed cells were incubated with
0.2% Triton-X 100 in PBS for 8 min, blocked with 2% bo-
vine serum albumin (BSA) in PBS for 30 min, and then in-
cubated with primary antibody against the target gene
(1:100 dilution in 2% BSA/PBS) overnight at 4°C. The cells
were washed and then incubated with the ALEXA 488–
labeled goat anti-rabbit secondary antibodies (diluted
1:1000 in 2% BSA/PBS) for 1 h at 37°C. Nuclei were
stained with DAPI (1 μg/mL in PBS) for 10 min. Fluores-
cence was detected using a Laser Scan Confocal Microscope
Carl Zeiss LSM 700 (Thornwood, NY, USA).
ErGPCR overexpression and truncated mutation of ErGPCR
PCR was used to prepare truncated mutations of ErGPCR.
ErGPCR fragments were amplified via PCR with various
primers (Additional file 1: Table S2) using proofreading
DNA polymerase. The mutated ErGPCR was amplified via
PCR using the ErGPCR fragments as templates. The open
reading frame of ErGPCR and different mutated ErGPCRs
were inserted into the pIEx-4 plasmid (Merck, Darmstadt,
Germany), fused with GFP. The plasmid was transfected
into HaEpi cells with Cellfectin following the protocol of
the supplier (Invitrogen, Carlsbad, CA, USA). Afterward,
20E was added to the cells at a final concentration of
1μM. An equal volume of DMSO was used as the
solvent control for 20E. DiI (1,1′-dioctadecyl-3,3,3′,3′-tetra-
methylindocarbocyanine perchlorate; Beyotime, Shanghai,
China) was used for plasma membrane staining.
Examination of Calponin translocation and phosphorylation
Subcellular Calponin translocation and phosphorylation
were detected by immunocytochemistry and immunoblot-
ting using rabbit polyclonal antibodies against Helicoverpa
Calponin. After ErGPCR knockdown, the cells were treated
with 1 μM 20E for 0.5 h to 3 h. Control cells were treated
via the same method using GFP dsRNA. Fluorescence was
detected using an Olympus BX51 fluorescence microscope.
The phosphorylation analysis was performed by western
blot.
Calcium ion detection
HaEpi cells were seeded and cultured for 72 h in a six-well
tissue culture plate with 10% FBS Grace’smediumat27°C.
The cells were incubated with dsRNA for 24 h as previously
described. The cells were incubated in a 3 μMAMester
Calcium Crimson™dye (Invitrogen, Carlsbad, CA, USA) in
Grace’s medium for 30 min at 27°C. The cells were then
washed with DPBS (2.7 mM KCl, 1.5 mM KH
2
PO
4
,and
8mMNa
2
HPO
4
)andexposedto1μM20EinDPBS
for 2 min for detection of intracellular calcium release.
Afterward, 1 mM calcium chloride was added to induce
extracellular calcium influx. Fluorescence was detected at
555 nm every 6 s for 360 s using a Laser Scan Confocal
Microscope Carl Zeiss LSM 700 (Thornwood, NY, USA).
Data were analyzed using the Image Pro-Plus software.
For the inhibition experiments, the cells were pretreated
with different inhibitors for 1 h prior to 20E treatment.
The GPCR inhibitor suramin, T-type voltage-gated calcium
channel inhibitor flunarizine dihydrochloride, L-type
calcium channel inhibitor verapamil hydrochloride, and
TRP channel inhibitors 2-APB and Pyr3 were purchased
from Sigma Chemical (St. Louis, MO, USA).
Chromatin immunoprecipitation (ChIP)
The HaEpi cells were seeded in a six-well plate. Cells
were transfected with pIEx-4-EcRB1-RFP at a density
of 2 × 10
6
. After 24 h, the cells were transfected with
dsErGPCR, and the controls were incubated with
dsGFP. After 24 h, the cells were subjected to either
DMSO or 1 μM 20E. After 6 h, the cells were cross-linked
with 0.5% formaldehyde at 37°C for 10 min, followed by
quenching at 0.125 M glycine at room temperature for
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10 min. The cells were then washed with ice-cold 1 × PBS
and harvested at 6,000 rpm for 5 min. Cells were re-
suspended with SDS lysis buffer (1% SDS, 10 mM EDTA,
50 mM Tris–HCl, pH 8.1) and sonicated to yield average
DNA fragments of 200 bp to 1000 bp. After centrifugation
to remove cell debris, the lysates were pre-cleared with
protein A resin at 4°C for 1 h, followed by incubation with
no antibody (negative control) or anti-RFP antibody over-
night. Immunoprecipitated protein-DNA complexes were
incubated with protein A for an additional 2 h at 4°C. The
complexes were washed with low-salt buffer [0.1% SDS,
1.0% Triton X-100, 2 mM EDTA, 200 mM Tris–HCl
(pH 8.0), 150 mM NaCl] once, high-salt wash buffer [0.1%
SDS, 1.0% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl
(pH 8.0), 500 mM NaCl] once, LiCl wash buffer [10 mM
Tris–HCl (pH 8.1), 0.25 M LiCl, 1 mM EDTA, 1% NP-40,
1% deoxycholate] once, and TE buffer [10 mM Tris–HCl
(pH 8.1), 1 mM EDTA] two times. The bound proteins
were eluted with elution buffer (1% SDS, 0.1 M NaHCO
3
).
DNA-protein crosslinks were reversed at 65°C overnight,
followed by RNase and proteinase K treatment. DNA was
purified with phenol/chloroform and ethanol precipita-
tion, and analyzed by qRT–PCR using HHR3F/R primers
(Additional file 1: Table S2). The negative control cells
were transfected with the same volume of pIEx-4-RFP,
and the cells received the same treatment as above.
RNAi in larvae
T7 promoter-containing PCR primers (GPCRRNA-iF,
GPCRRNA-iR, GFPRNAiF and GFPRNAiR in Additional
file 1: Table S2) were used to amplify the gene fragments.
The PCR product purified with phenol–chloroform was
used as a template to synthesize dsRNA using the MEGA-
script RNAi Kit, as previously described. The dsRNA was
diluted in nuclease-free water to 0.4 μg/μL. Afterward,
5μL was injected into the fifth instar larvae at 6 h and
at 30 h, as well as into the sixth instar at 6 h and 30 h. The
controls were injected with dsGFP. Three independent
experiments were performed using 30 larvae each.
[
3
H]Pon A binding assays
Cell membranes that express ErGPCR, EcRB1 and GFP
were prepared from HaEpi cells with the plasmid
ErGPCR-GFP-pIEx-4, EcRB1-GFP-pIEx-4, and GFP-pIEx-4.
The details are as follows: cells were collected by centrifu-
gation (1700×g, 10 min, 4°C) and then resuspended in
15 mL of HEPES buffer [20 mM HEPES, 6 mM MgCl
2
,
1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM
ethylene glycol bis (2-aminoethyl) tetraacetic acid (EGTA),
pH 7.4]. After sonication, the homogenate was centrifuged
at 1700×g for 10 min. The resulting supernatant was cen-
trifuged at 48000×g for 1 h at 4°C. The pellet was resus-
pended in HEPES buffer, and the protein concentration
was determined via the Bradford method. For the binding
assay, a range of membrane fractions were incubated with
1nM[
3
H]Pon A (Perkin Elmer, MA, USA) at 27°C for 1 h
in 200 μL of binding buffer (20 mM HEPES, 100 mM
NaCl, 6 mM MgCl
2
, 1 mM EDTA, 1 mM EGTA). For the
saturation experiments, reaction mixtures containing
50 μg of the membrane fraction were incubated at 27°C
for 1 h in the presence of the appropriate [
3
H]Pon A con-
centration in the binding buffer. Nonspecific binding was
determined in the presence of 1 μM 20E. After incubation,
particulate proteins were collected on glass fiber filters.
The filters were then added to 5 mL of scintillation fluid.
Radioactivity was determined using a SN-6930 liquid scin-
tillation counter (Shanghai Hesuo Rihuan Photoelectric
Instrument Co., Ltd., China). The whole cell binding ex-
periments used the same method but without sonication
and membrane preparation.
Additional file
Additional file 1: “The data sets supporting the results of this
article are included within the article”. Figure S1. Alignment of the
GPCR ESTs obtained by random sequencing the Helicoverpa epidermal
cell line. Figure S2. Screen of the target GPCR involved in 20E-induced
gene expression by qRT-PCR. Figure S3. Nucleotide and deduced amino
acid sequence of ErGPCR. Figure S4. Multiple alignments of ErGPCR with
other G-protein-coupled receptors from different insects or vertebrates.
Figure S5. Phylogenetic analysis of ErGPCR. Figure S6. 20E upregulates
ErGPCR through EcRB1.Figure S7. The recombinant expression of ErGPCR
fragments in E. coli.Table S1. Identification of the GPCRs. Table S2.
Primers used in dsRNA synthesis and qRT-PCR.
Abbreviations
20E: 20-hydroxyecdysone; JH: Juvenile hormone; GPCR: G protein-coupled re-
ceptor; EcRB1: Ecdysone nuclear receptor B1; USP1: Ultraspiracle protein 1;
HR3: Hormone receptor 3; BrZ2: Broad isoform Z2; DMSO: Dimethylsulfoxide;
dsRNA: Double-stranded RNA; RNAi: RNA interference; GFP: Green fluorescent
protein; RFP: Red fluorescent protein; HaEpi: An epidermal cell line from
Helicoverpa armigera; FBS: Fetal bovine serum; DAPI: 4′-6-diamidino-2-
phenylindole dihydrochloride; BSA: Bovine serum albumin; TRP: Transient
receptor potential; SOC: Store-operated channel; ROC: Receptor-operated
channel; DiI: 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
perchlorate; 2-APB: 2-aminoethoxydiphenyl borate; Cpm: Counts per minute
of [
3
H] Pon A; qRT-PCR: quantitative reverse transcription polymerase chain
reaction; ChIP: Chromatin immunoprecipitation; EcRE: Ecdysone response
element.
Competing interests
The authors declare no conflict of interests.
Authors’contributions
Mei-Juan Cai performed the overexpression of ErGPCR and Ca
2+
detection.
Du-Juan Dong constructed the pIEx-4-ErGPCR-GFP plasmid. Yu Wang cloned
the gene. Wen Liu overexpressed ErGPCR in cell line. Peng-Cheng Liu examined
the phosphorylation and translocation of Calponin. Jin-Xing Wang directed the
research. Xiao-Fan Zhao designed the studies and wrote the manuscript. All
authors read and approved the final manuscript.
Acknowledgments
This work was supported by the grants from the National Natural Science
Foundation of China (No. 31230067) and the National Basic Research
Program of China (973 Program, No. 2012CB114101) and Ph. D. Programs
Foundation of Ministry of Education of China (No. 20120131110025). The
L4440 and pIEx-4 plasmids and HT115 (DE3) are kindly provided by
Dr. Marek Jindra and Masako Asahina of the Biology Center, Czech Academy
Cai et al. Cell Communication and Signaling 2014, 12:9 Page 14 of 16
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of Sciences and Department of Molecular Biology, University of South Bohemia,
Czech Republic. We thank professor Lynn Moorhead Riddiford in Janelia Farm
Research Campus, Howard Hughes Medical Institute in United States giving
suggestions on preparing this manuscript.
GenBank number: JQ809653.
Received: 1 November 2013 Accepted: 3 February 2014
Published: 10 February 2014
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doi:10.1186/1478-811X-12-9
Cite this article as: Cai et al.:G-protein-coupled receptor participates
in 20-hydroxyecdysone signaling on the plasma membrane. Cell
Communication and Signaling 2014 12:9.
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