From the Departments of Entomology and
§ Cell Biology and Neuroscience, University of
California, Riverside, California 92521 and
Euroscreen s.a.,
Rue Adrienne Bolland 47, B-6041 Gosselies, Belgium
Received for publication, December 20, 2002, and in revised form, February 3, 2003
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ABSTRACT |
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Insect ecdysis is a hormonally programmed
physiological sequence that enables insects to escape their old cuticle
at the end of each developmental stage. The immediate events leading to
ecdysis, which are initiated upon release of ecdysis-triggering
hormones (ETH) into the bloodstream, include respiratory inflation and sequential stereotypic behaviors that facilitate shedding of the cuticle. Here we report that the Drosophila gene
CG5911 encodes two functionally distinct subtypes of G
protein-coupled receptors through alternative splicing (CG5911a and
CG5911b) that respond preferentially to ecdysis-triggering hormones of
flies and moths. These subtypes show differences in ligand sensitivity
and specificity, suggesting that they may play separate roles in ETH
signaling. At significantly higher concentrations (>100-fold), certain
insect and vertebrate peptides also activate these receptors, providing evidence that CG5911 is evolutionarily related to the
thyrotropin-releasing hormone and neuromedin U receptors. The
ETH signaling system in insects is a vital system that provides
opportunities for the construction of models for the molecular basis of
stereotypic animal behavior as well as a target for the design of more
sophisticated insect-selective pest control strategies.
A defining feature of growth and metamorphosis in insects is the
periodic shedding of cuticle known as ecdysis (1). This complex
process, by which the insect escapes its outer shell and sheds the
cuticular lining of the foregut, hindgut, and respiratory system, is
mediated by a peptide hormone-signaling cascade that results in a
sequence of precisely timed physiological and behavioral events.
Initiation of the ecdysis sequence coincides with the appearance of
ecdysis-triggering hormones
(ETHs)1 in the bloodstream,
which act directly on the central nervous system to elicit patterned
motor output characteristic of pre-ecdysis and ecdysis behaviors (2,
3). In the moth Manduca sexta, two peptides called MasPETH
and MasETH act in concert to trigger successive phases of the ecdysis
behavioral sequence. Remarkably, the temporal features of these motor
patterns recorded from the isolated nervous system closely reflect the
behavior observed in whole animals. Related peptides in
Drosophila melanogaster known as ETH1 and ETH2 trigger
ecdysis, although their respective roles in the recruitment of
different parts of the ecdysis sequence are less clear. As yet the
molecular and cellular targets of ETHs have not been described.
Additional signaling molecules operating downstream of ETH within the
central nervous system include the eclosion hormone and the crustacean
cardioactive peptide (2, 4-7).
Identification of ETH receptors would be of great help in defining the
cellular elements involved in ecdysis behaviors. With this objective,
we took advantage of the Drosophila genome project to
investigate G protein-coupled receptors (GPCRs) that are likely to
respond to ETH (8, 9). The search for ETH receptors was narrowed to a
small number of candidates on the basis of an assumption of
ligand-receptor co-evolution, in which receptors for peptides having a
C-terminal amino acid motif consisting of PRX amide would have high sequence homology. An exhaustive analysis of the
Drosophila genome yielded two groups of GPCRs, the
neuromedin U (NMU) group and the vasopressin group (10). Functional
analysis revealed that the NMU group likely arose through
ligand-receptor co-evolution, because it responds to several groups of
peptides with a C-terminal PRX amide motif characteristic of
the ETHs. In contrast, the vasopressin group responds to unrelated
peptides, and thus the PRX amide in this group appears to
have arisen through convergent evolution.
The monophyletic group of NMU receptors was found to be activated by
all three categories of insect PRX amide peptides (10): pyrokinins or pheromone biosynthesis-activating neuropeptide
(PBAN)-like peptides ending with the amino sequence motif
-FXPRXa (where final "a" means amide)
(11, 12), cardioactive CAP2b-like peptides with the -FPRXa
motif (13, 14), and ecdysis-triggering hormones having a
-PRXa motif (2, 5, 10, 15, 16). Surprisingly, some receptors
exhibited sensitivity to multiple peptides. In particular, CG8795 was
activated by DrmPK2, HUG The responsiveness of CG8795 to only relatively high concentrations of
ETH1 suggested that it is unlikely to be the physiological ETH
receptor. We therefore expanded the search profile for ETH receptors to
include CG5911, which encodes a receptor related to the NMU
group. As predicted (8), CG5911 gene products occur as two
alternative splice variants termed CG5911a and CG5911b. CG5911b
responds to ETH1 and the related peptides MasETH and MasPETH from moths
at subnanomolar concentrations while showing relative insensitivity to
other peptides sharing C-terminal PRX amide motifs. A
pattern of cross-reactivity in a second set of peptides provides further support for the hypothesis that CG5911 encodes the
authentic ETH receptor.
Cloning of CG5911--
cDNA was synthesized using
Superscript (Invitrogen) with mRNA isolated by Dynabeads (Dynal)
from whole flies (~50 individuals of Canton S) in both larval and
adult stages by priming at poly(A) sites. PCR reactions conducted in
20-µl volumes contained 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTP, 0.5 µM of each primer, 0.5 unit of
Taq polymerase (Invitrogen), and 0.5 unit of Pfu
polymerase (Stratagene). The reaction mixture was denatured initially
for 5 min at 94 °C and then subjected to 35 cycles of 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min and 30 s. The
RT-PCR products were cloned in pGEM-T-EZ vector (Promega) and
sequenced. Start and stop codons of the open reading frames (ORF) were
confirmed by the primers in the RT-PCR designed on the outside of the
in-frame stop codons of the ORF. The primers shown in Fig. 1 are Fa
(5'-AAGGACGATGGCGATGCTGA) paired with Ra (5'-GCTTACCCCTTACTGCCAGT) and Fb (5'-CCCGAGGATGTTGTCCCTGT) paired with
Rb (5'-GATGGGATTCAGATCTTGCT). The CG5911 ORFs were inserted into the
pXOON vector (17), which contains an enhanced green fluorescent
protein marker. Phylogenetic analysis was done in PAUP version
4.0b8a for generation of a distance tree with 1000 bootstrapping samplings.
Cell Line Expression--
Transient expression of
CG5911 gene products was accomplished in the CHO-WTA11 cell
line exhibiting stable expression of the luminescent reporter aequorin
(18) and the G protein G Functional Analysis--
Luminescence assays were performed in
opaque 96-well microplates (Corning) using a Beckman model LD400
Luminometer. After the addition of cells to a well, luminescence was
monitored using an 0.5-s sampling time for 20 s. Each 96-well
microplate contained multiple wells for positive controls (100 and 300 µM ETH1) and negative controls (buffer only).
Luminescence at each ligand concentration was integrated during the
20-s response interval and normalized to the largest positive control
response in each plate after the subtraction of background values
obtained from negative controls. Luminescence measured in multiple
replica wells (2-4 wells) for one concentration of ligand was averaged
for the analysis. Data collected from at least three replica plates
were used for analysis using the Origin analysis program (OriginLab
Corp., Northampton, MA). The sources of peptides were described
previously (10), and additional vertebrate peptides (NMU-8, TRH, and
ghrelin) were purchased from the American Peptide Company. MasETHacid
was synthesized at the University of California, Riverside, using
standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry
and automated solid state techniques. A list of peptides used in this
study is provided in Table I.
Molecular Cloning of CG5911a and CG5911b--
The
CG5911 gene has homologous mutually alternative exons 4a and
4b encoding the protein sequence from the middle of transmembrane segment 4 to the C terminus (Figs. 1 and
2). The clones used for the expression
contain the coding sequence from nucleotide position
Initial BLASTP searches and phylogenetic analyses identified the
translation of CG5911 (GenBankTM accession number AAF55872)
as a Drosophila GPCR closely related to the monophyletic
group of NMU receptors responding to PRX amide peptides
(10). Putative transcripts generating CG5911a and -b are grouped with
thyrotropin-releasing hormone (TRH) receptor in a distance tree
with a bootstrapping value of 83.8% (Fig.
3).
The prediction of gene structure revealed the presence of homologous
mutually alternative exons 4a and 4b from transmembrane segment 4 to
the C terminus encoding two splice variants of the receptor (Figs. 1
and 2). Stop codons occurring at C-terminal ORFs for CG5911a and
CG5911b were identified by RT-PCR using primers located after the stop
codon. RT-PCR with the primer covering a stop codon at the 5' end of
the ORF for CG5911b revealed a transcript supporting the ORF prediction.
CG5911a and CG5911b Are Preferentially Sensitive to
ETHs--
Ecdysis-triggering hormones of both Drosophila
and Manduca elicited robust luminescence responses in the
CHO-WTA11 cell line stably expressing G
Both CG5911a and CG5911b showed preferential sensitivity to ETHs, with
CG5911b showing a significantly higher apparent affinity for all
peptides tested (Fig. 4, C and D).
Drosophila ETH1 was the most active ligand for both
receptors. For CG5911b, responses were evident at 0.25 nM
and reached peak values at 10 nM with an EC50
value of 0.9 nM. ETH2 and Manduca PETH were only
slightly less active, with EC50 values of ~2
nM. Interestingly, Manduca ETH was approximately
5 times less active on CG5911b than PETH.
CG5911a was markedly less sensitive to all peptides tested compared
with CG5911b. The most active peptide again was ETH1, with an
EC50 of 414 nM, making it ~400-fold less
active on this receptor than on CG5911b. ETH2, MasPETH, and MasETH had
EC50 values greater than 2 µM.
Cross-reactivity of CG5911 to Other Peptide Ligands--
CG5911b
showed significant sensitivity to a range of ETH-related peptides
having the signature PRXa C-terminal amino acid motif (Fig.
5). These peptides include
SCPB, HUG Ecdysis-triggering hormones are vital regulatory signals that
govern the stereotypic physiological sequence leading to cuticle shedding in insects. In previous work we identified ETHs in two moth
species, Manduca sexta and Bombyx mori, and in
the fruit fly Drosophila melanogaster (2, 15, 16). Using
genetic tools in Drosophila to excise the eth
gene, lethal ecdysis deficits were demonstrated at the end of the first
larval instar (21). Lethality was reversed by the injection of ETH
peptides (21). The ETH signaling system therefore has been demonstrated
to be both necessary and sufficient for insects to survive the earliest ecdysis. To expand our understanding of the molecular basis for ETH
action, we conducted a search for its likely receptor in
Drosophila.
In this paper we report on the cloning and functional expression of the
Drosophila gene CG5911, predicted by Hewes and
Taghert (8) to encode splice variants called CG5911a and CG5911b. Our results show that both splice variants of CG5911 are highly
selective for ETH1 and related peptides from both fly and moth, with
CG5911b responding to subnanomolar concentrations of
Drosophila ETH1. For each receptor subtype the highest
sensitivity was observed to Drosophila ETH1 and ETH2, but
comparable sensitivity also was observed for MasPETH and MasETH. Other
insect and vertebrate peptides, particularly those having C-terminal
PRX amide motifs, also activated CG5911b, but only at
~100-1000-fold higher concentrations. The selectivity and high
sensitivity of CG5911 to ETHs alone strongly supports the hypothesis
that this gene encodes the authentic ETH receptor.
The gene structure of CG5911 with the results of RT-PCR
revealed two homologous exons presenting the mutually alternative splice variants CG5911a and CG5911b. Phylogenetic analysis suggests that divergent exons 4a and 4b are the product of duplication. The
portion of the receptor protein predicted by exon 4 extends from the
middle of transmembrane segment 4 (T4) to the C terminus and therefore
includes two extracellular loops (T4-T5 and T6-T7), one intracellular
loop (T5-T6), and the C-terminal intracellular tail. Clearly,
alternative splicing of exons 4a and 4b to create these receptor
isoforms could affect both their ligand and G protein-coupling specificities.
Functional differences in CG5911a and CG5911b are suggested by several
observations. CG5911b is activated by subnanomolar concentrations of
ETH1 (EC50 = 0.9 nM), whereas the sensitivity of CG5911a is more than 400-fold lower (EC50 = 410 nM). The lower sensitivity of CG5911a to all ligands may be
a consequence of poor coupling efficiency to the G In addition to these differences, the ligand sensitivity for the two
receptor subtypes is significantly different. The relative potency of
ligands on CG5911a (ETH1 > MasETH > ETH2 > MasPETH; see Fig. 4) reflects the same order of biological activity observed for
the induction of ecdysis behavior (16). In the case of CG5911b, we
observed a somewhat different order of potency (ETH1 > ETH2 > MasPETH > MasETH). The reversal in receptor sensitivity to
MasPETH and MasETH suggests that these receptor subtypes discriminate between MasPETH and MasETH and raises the intriguing possibility that
CG5911 orthologs in the Manduca system encode
receptors that are ligand-specific. Indeed, preliminary findings
involving the Manduca ortholog of CG5911b confirm the
preference for MasPETH.2 The
possible ligand specificity of the two receptor subtypes may account at
least in part for divergent activities of these two ligands in the
induction of natural pre-ecdysis behavior (2, 3). Further study of
Manduca orthologous isoforms of CG5911 is under way to test
this hypothesis.
Responses of CG5911b to a second tier of ligands that are active only
at >100-fold-higher concentrations reveal further insights into its
properties and the likely physiological role of CG5911 as the ETH
receptor. All ETHs are C-terminally amidated, and this post-translational modification is essential for biological activity in
the silkworm, B. mori (15). Although the injection of
synthetic Bombyx ETH elicits the entire ecdysis behavioral
sequence in silkworm larvae, the free acid form proved to be largely
inactive (15). In the present study, the free acid form of MasETH
(MasETHacid, Fig. 5) was found to be more than 100 times
less active than native MasETH. CG5911b therefore discriminates between
these subtly different forms of the peptide just as we observed
previously in behavioral assays (2).
We tested a variety of both invertebrate and vertebrate peptides on
CG5911b, and most were inactive. Nevertheless, some interesting patterns of activity are apparent among the few peptides that showed
some biological activity. Of the invertebrate peptides tested, the
peptide HUG The mammalian peptides neuromedin and TRH, the receptors of which are
related phylogenetically to CG5911, also activate CG5911b, albeit at
relatively high concentrations. The significant bioactivity of these
peptides, with the complete absence of responses to neurotensin and
ghrelin (assayed at 10 µM), strongly support our
phylogenetic analysis, which indicates that CG5911 evolved
from a GPCR ancestral to the NMU/TRH receptors.
Myomodulin (PMSMLRLamide), a neuropeptide first identified in molluscs,
also was found to activate CG5911b. We were unable to find
myomodulin-like peptides in the Drosophila genomic data base, but a similar sequence occurs in the Caenorhabditis
elegans data base
(Y45F10A.5).3 Myomodulin
could play an analogous role to ETH in nematodes, which with the
arthropods have been newly classified in the Ecdysozoa (23). Indeed,
antibodies raised against myomodulin cross-react with ETHs in endocrine
Inka cells (24), indicating structural similarities between these peptides.
During preparation of this manuscript, independent evidence appeared to
support the hypothesis that CG5911 functions as the ETH receptor (25).
These findings confirm that two functional subtypes of CG5911 are
produced by alternative splicing and that ETH1 is a more potent agonist
against both receptors. These authors (25) also demonstrated the
presence of an additional 5'-untranslated exon. However, our results
demonstrate a higher sensitivity of CG5911 to both ligands and a
considerably greater divergence in receptor sensitivity between the two
subtypes. These findings could indicate that CG5911a and CG5911b
interact with different G proteins and may activate different signal
transduction pathways. Our pharmacological and phylogenetic analyses
also indicate that ETH and the pyrokinin/CAP2b groups of insect
neuropeptides constitute a set of ligands that have evolved with CG5911
and the neuromedin U group of vertebrate receptors and their respective ligands.
In summary, we have demonstrated that alternative splice variants of
CG5911 encode two functional receptor subtypes of ETH receptor in Drosophila. Although further work is necessary
to confirm their physiological roles in natural ETH signaling, the pronounced ligand sensitivity and specificity of CG5911 gene
products along with the phylogenetic relationships of CG5911
to both Drosophila and mammalian receptors for
PRX amide peptides provide strong evidence for their roles
as the physiological ETH receptors.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and ETH1, with sensitivity to DrmPK2 and
HUG
being far greater than to ETH1 (DrmPK2 > HUG
> ETH1).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
16 (19). Cells were grown in
complete Ham's F12 medium at 37 °C in 5% CO2. Transfection with pXOON-CG5911a and pXOON-CG5911b was performed using
FuGene6 (Roche Molecular Biochemicals) according to the manufacturer's protocol at a DNA to FuGene6 ratio of 3:1. Transfected cells were observed under an epifluorescence microscope 1 day later to
determine transfection efficiency as measured by the number of cells
expressing enhanced green fluorescent protein. Before functional
assays, cell suspensions were incubated in coelanterazine h (Molecular
Probes) according to previously defined protocols (20).
Sequences for signaling peptides used in this study
RESULTS
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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24 upstream of
the Met site to +368 nucleotides after the 3' stop codon for CG5911a
and from
490 to +8 for CG5911b (see GenBankTM accession numbers
AY220741, AY220742, and Celera Genomics sequence
GenBankTM accession number AE003734) (Fig.
2). The ORF is supported by the in-frame
stop codon in the 5'-untranslated region (Fig. 1).
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Fig. 1.
Genomic structure of CG5911
encoding putative ETH receptors. The receptor has two
alternative transcripts with mutually alternative exons 4a and 4b.
Transcripts were experimentally confirmed by RT-PCR with primers
(arrows) located at the boundary of start and stop codons in
the predicted open reading frames. The 5' and 3' ends of the transcript
were not determined and are depicted as slanted ends of exon
1 (5') and exon 4 (3').
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Fig. 2.
Amino acid sequence alignment of G
protein-coupled receptors in the clade including the neuromedin U
receptor. Sequences are from the GH secretagogue receptor
(GHSR, NP_114464), neurotensin receptor
(NTR, JH0164), CG8784 (AF522189),
CG8795 (AF522190), CG9918 (AF522191),
CG14575 (AF522193), neuromedin U receptors 1 and 2 (NMUR1 and -2, AB038649 and NP_071611),
thyrotropin-releasing hormone receptors 1, 2, and 3 (TRHR1,
-2, and -3, CAD12658, CAD12657, and CAD12656,
respectively), and CG5911a and b in this study.
Inverted and shaded letters indicate identical
and similar sequences, respectively, in more than 50% of the taxa.
· · · and ~ ~ ~ indicate gaps introduced for sequence
alignment in the middle of the sequences and for the N-terminal end,
respectively. Transmembrane domains are indicated by prediction from
the NMUR1 sequence. The location of mutually alternative exons 4a and
4b in CG5911 is indicated in TM4.
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Fig. 3.
Phylogenetic tree for the G protein-coupled
receptors in the clade including the neuromedin U receptor. The
phylogenetic tree was generated in PAUP (version 4.0b8a) for the
neighbor-joining distance method, with numbers at the nodes
representing the bootstrapping values in 1000 replications (see Fig. 2
legend for a description of the taxa). The sequence for CG5911b was
used only for exon 4b (Fig. 1), which is a mutually exclusive
alternative exon.
16 and aequorin
transfected with pXOON-CG5911a and pXOON-CG5911b (Fig.
4). The time course of cellular responses was concentration-dependent, with delayed slow responses
beginning at 0.1 nM and rapid peak responses occurring at 1 µM for CG5911b (Fig. 4A). At the lowest
effective concentrations (0.1 nM), the luminescence
response was delayed more than 10 s. Integration of each response
shown in Fig. 4A during a 20-s sampling period is shown in
Fig. 4B.
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Fig. 4.
Concentration-response relationships for ETH1
and related peptides on the cell line CHO-WTA11
(G 16 with aequorin) expressing CG5911a and
CG5911b. A, response of CG5911b to increasing
concentrations of ETH1. Plotted are the time courses of luminescent
responses after the addition of CHO cells to a well containing
increasing concentrations of ligand. B, integrated
luminescence units from the experiment shown in A. C, concentration-response relationships for CG5911a to ETH1,
ETH2, MasETH, and MasPETH. Each point is a mean value + S.E. for
integrated luminescence responses shown in B as a function
of ligand concentration. D, concentration-response
relationships for CG5911b exposed to the same series of
Drosophila and Manduca ETH
peptides.
, CAP2b-1, CAP2b-2, PBAN, and MasETH acid. The
most active of these peptides were SCPB, MasETH acid,
HUG
, and CAP2b-1; the EC50 values of these peptides were
in the range of 300 nM to 3 µM. Furthermore,
the vertebrate peptides NMU and TRH caused low but significant receptor activation when applied at 10 µM as did the molluscan
peptide myomodulin (p < 0.001 in Student's
t test; Fig. 5B). No activity was observed for a
number of other peptide ligands, including some with the C-terminal
PRXa motif. These ligands include Drm-PK-2, CAP2b-3, and Hez
PMP (Fig. 5B).
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Fig. 5.
Activity of ETH-related ligands on CHO cells
expressing CG5911b. Note that this group of ligands has much lower
activity compared with the ETH group shown in Fig. 4D.
A, concentration-response relationships of ETH-related
ligands on cells expressing CG5911b. B, activity of an
additional set of ligands applied at a 10 µM
concentration on CHO cells expressing CG5911b. The responses shown in
the bar graph are all significant levels compared with buffer control
(p < 0.001). Other ligands having no activity when
applied at 10 µM are also indicated.
DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
16
used in our assay system. Indeed, the smaller size of peak luminescence
responses recorded for CG5911a as compared with CG5911b (data not
shown) supports this idea. Further investigation of CG5911a sensitivity
using different G proteins will be necessary to resolve this question. Nevertheless, differences in G protein coupling and downstream signal
transduction steps could provide a basis for functionally distinct
roles for these two receptor subtypes in ETH signaling.
, encoded by the ortholog of the moth PBAN
gene as well as PBAN itself, were active at several hundred-fold higher
concentrations than authentic ETHs. The low affinity response of
CG5911b to HUG
and its high affinity response to ETH1 and ETH2
complement our previous results, which showed that the related GPCR
encoded by CG8795 is highly sensitive to HUG
but considerably less
sensitive to ETHs (10). These results confirm our phylogenetic analyses, which indicate the close evolutionary relationship between CG8795 and CG5911. The question remains as to
whether the cross-reactivities of these two ligands (ETH and
HUG
) against CG8795 and CG5911b reflect genuine biological
phenomena. Evidence in support of this comes from Roos and colleagues
(22), who showed that ectopic expression of the hugin
gene produced lethal ecdysis defects.
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ACKNOWLEDGEMENTS |
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We thank Dr. Tiesheng Shi for the synthesis of MasETHacid and Beckmann Coulter, Inc., for the use of the LD400 luminometer.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AI-40555.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY220741 and AY220742.
¶ Present address: Dept. of Entomology, 123 West Waters Hall, Kansas State University, Manhattan, KS 66506. E-mail: ypark@oznet.ksu.edu.
** To whom correspondence should be addressed: Dept. of Cell Biology and Neuroscience, 5429 Boyce Hall, University of California, Riverside, CA 92521. Tel.: 909-787-4746; Fax: 909-787-3087; E-mail: adams@mail.ucr.edu.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M301119200
2 Y. J. Kim and M. E. Adams, unpublished data.
3 Y. Park and M. E. Adams, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ETH, ecdysis-triggering hormone; PETH, pre-ecdysis-triggering hormone; GPCR, G protein-coupled receptor; NMU, neuromedin U; ORF, open reading frame; RT-PCR, reverse transcriptase PCR; CHO, Chinese hamster ovary; TRH, thyrotropin-releasing hormone; PBAN, pheromone biosynthesis-activating neuropeptide.
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