Biochemical and Biophysical Research Communications 412 (2011) 578–583

Contents lists available at SciVerse ScienceDirect

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Identification of the Drosophila and Tribolium receptors for the recently

discovered insect RYamide neuropeptides q
Caitlin Collin, Frank Hauser, Peter Krogh-Meyer, Karina K. Hansen, Ernesto Gonzalez de Valdivia,
Michael Williamson, Cornelis J.P. Grimmelikhuijzen ⇑
Center for Functional and Comparative Insect Genomics, Department of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark

a r t i c l e i n f o a b s t r a c t

Article history: One year ago, we discovered a new family of insect RYamide neuropeptides, which has the C-terminal
Received 29 July 2011 consensus sequence FFXXXRYamide, and which is widely occurring in most insects, including the fruitfly
Available online 6 August 2011 Drosophila melanogaster and the red flour beetle Tribolium castaneum (F. Hauser et al., J. Proteome Res. 9
(2010) 5296–5310). Here, we identify a Drosophila G-protein-coupled receptor (GPCR) coded for by gene
Keywords: CG5811 and its Tribolium GPCR ortholog as insect RYamide receptors. The Drosophila RYamide receptor is
GPCR equally well activated (EC50, 1  109 M) by the two Drosophila RYamide neuropeptides: RYamide-1
Neuropeptide
(PVFFVASRYamide) and RYamide-2 (NEHFFLGSRYamide), both contained in a preprohormone coded
Neuropeptide Y
Neuropeptide F
for by gene CG40733. The Tribolium receptor shows a somewhat higher affinity to Tribolium RYamide-
Short neuropeptide F 2 (ADAFFLGPRYamide; EC50, 5  109 M) than to Tribolium RYamide-1 (VQNLATFKTMMRYamide; EC50,
RYamide 7  108 M), which might be due to the fact that the last peptide does not completely follow the RYamide
Feeding consensus sequence rule. There are other neuropeptides in insects that have similar C-terminal
Drosophila sequences (RWamide or RFamide), such as the FMRFamides, sulfakinins, myosuppressins, neuropeptides
Tribolium F, and the various short neuropeptides F. Amazingly, these neuropeptides show no cross-reactivity to the
Insects Tribolium RYamide receptor, while the Drosophila RYamide receptor is only very slightly activated by high
concentrations (>106 M) of neuropeptide F and short neuropeptide F-1, showing that the two RYamide
receptors are quite specific for activation by insect RYamides, and that the sequence FFXXXRYamide is
needed for effective insect RYamide receptor activation. Phylogenetic tree analyses and other amino acid
sequence comparisons show that the insect RYamide receptors are not closely related to any other known
insect or invertebrate/vertebrate receptors, including mammalian neuropeptide Y and insect neuropep-
tide F and short neuropeptide F receptors. Gene expression data published in Flybase (www.flybase.org)
show that the Drosophila CG5811 gene is significantly expressed in the hindgut of adult flies, suggesting a
role of insect RYamides in digestion or water reabsorption.
Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction well-established model organisms, such as the fruitfly Drosophila

More than 80% of all animal species are insects, making insects
extremely important players in the ecology of our planet. Insects
are also crucial for agriculture, because most flowering food plants
depend on insects for their pollination, and insects can be severe
agricultural pests, destroying about 30% of our potential annual
harvest. Furthermore, insects are also medically important as vec-
tors for serious diseases, such as malaria, sleeping sickness, ele-
phantiasis (lymphatic filariasis), Dengue fever, Yellow fever, West
Nile, Chagas disease, and many more. Finally, some insects are
q
The sequence data of this paper have been submitted to the GenBank database
under Accession Nos. HQ709383, JN200817, JN222358, and JN234381.
⇑ Corresponding author. Fax: +45 3532 1200.
E-mail address: cgrimmelikhuijzen@bio.ku.dk (C.J.P. Grimmelikhuijzen).
URL: http://www2.bio.ku.dk/insect_genomics/ (C.J.P. Grimmelikhuijzen).
melanogaster, and the red flour beetle Tribolium castaneum.
Because insects are so important, more than 50 insect genome
projects have been initiated, of which about 25 now have been
completed [1–5]. This large number of sequenced insect genomes
represents an invaluable resource for insect biologists and will help
to better understand insect evolution and physiology and, eventu-
ally, enable them to improve crop protection and prevent the
transmission of insect-borne diseases.
G-protein-coupled receptors (GPCRs) and their ligands (bio-
genic amines, neuropeptides, and protein hormones) represent
important groups of molecules, because they occupy a high hierar-
chic position in the physiology of insects and steer central pro-
cesses, such as feeding, behavior, reproduction, and development
[6–8]. One year ago, when studying the genome from the parasitic
wasp Nasonia vitripennis, we discovered a new class of neuropep-
tides, the insect RYamides, characterized by the C-terminal

0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2011.07.131
 C. Collin et al. / Biochemical and Biophysical Research Communications 412 (2011) 578–583 579

Fig. 1. Structures of some processed and non-processed insect and mammalian RYamide/RFamide/RWamide neuropeptides. (A) A partial representation of the Aedes aegypti
(Aa), D. melanogaster (Dm), and T. castaneum (Tc) preprohormones, showing the immature (non-processed) sequences of the RYamides-1 and -2 in these species. The
complete preprohormones have been published in [9]. Aa-RYamide-1 has been sequenced previously using MALDI–TOF/CID mass spectrometry [9], showing that processing
occurs close to proline residues. The neuropeptide sequences are highlighted in green, the mono- or dibasic cleavage sites are highlighted in red, and the resulting C-terminal
glycine that normally is converted into an amide is highlighted in yellow. (B) Structures of the sequenced and/or proposed processed RYamides-1 and -2 from A. aegypti, D.
melanogaster, and T. castaneum (a long list of other RYamides from other insect species is given in [9]), the pig (Sus scotra domesticus) neuropeptide Y (Ss-NPY), D. melanogaster
neuropeptide F and the various D. melanogaster short neuropeptides F. A complete list of D. melanogaster RFamide/RWamide neuropeptides is given in [8]. Amino acid
residues that are identical to Aa-RYamide-1 are highlighted in green. Conserved amino acid residues common to Aa-RYamide-1 are highlighted in orange. Note that porcine
neuropeptide Y (Ss-NPY) has two conserved and two identical amino acid residues in common with Aa-RYamide-1. This is not the case with Dm-NPF and short neuropeptide
F sequences.

sequence FFXXXRYamide [9]. These RYamides are structurally dif-
ferent from other insect neuropeptides containing the RFamide or
RWamide C termini, such as the FMRFamides, sulfakinins, myosup-
pressins, neuropeptides F, and short neuropeptides F [9]. Further-
more, we found that the insect RYamides occur in all insects
with a sequenced genome, with the only exception being four
ant species [5,9]. Finally, we also sequenced one such insect RYa-
mide (PFFVGSRYamide) from the terminal ganglion of the yellow
fever mosquito Aedes aegypti, using MALDI–TOF/CID mass spec-
trometry, showing that the insect RYamide genes are indeed ex-
pressed and that RYamides are genuine insect neuropeptides [9].
Insect RYamides also occur in the twelve Drosophila species
with a sequenced genome and in the sequenced agricultural pest,
the red flour beetle T. castaneum (Fig. 1) [9]. In the current paper,
we have identified the RYamide receptor from both the fruitfly D.
melanogaster and the red flour beetle T. castaneum. We expect that
these identifications will improve our understanding of insect
biology.
2. Materials and methods
The cDNA from the Drosophila GPCR gene CG5811 was cloned
by PCR (GenBank database Accession No. JN234381), using primers
based on the genomic sequence, and cDNA made from a mixture of
different developmental stages of the fly. The PCR product was
subsequently inserted into the expression vector pIRES2-EGFP
(Clontech, Mountain View, USA). The cDNA from the Tribolium
GPCR gene LOC664001 was cloned in a similar way (GenBank data-
base Accession No. HQ709383) and subsequently inserted into the
expression vector pIRES2-ZsGreen1 (Clontech, Mountain View,
USA).
Chinese hamster ovary (CHO) cells stably expressing the human
G-protein G16 (CHO/G16) were grown as described previously [11]
and transfected with the expression vectors using JetPEI™ (Poly-
plus, Illkirch, France). The bioluminescence assay was performed
as described earlier [10–12] with the only difference that JetPEI™
was used as transfection medium. We tested eight Drosophila neu-
ropeptides with an RFamide or RWamide C-terminus and the novel
neuropeptides Drosophila RYamide-1 and -2 and Tribolium RYa-
mide-1 and -2 (all synthesized by Genemed Synthesis, San Antonio,
USA).
Multiple sequence alignments and phylogenetic tree analyses
were performed with ClustalW (http://www.ebi.ac.uk/Tools/msa/
clustalw2/) and the Lasergene software package, using receptor se-
quences with the following GenBank database Accession Nos.: Dm
sNPF-R (AAF49074); Tc sNPF-R1 (XP_966794.1); Tc sNPF-R2
(EFA01301.1); Dm NPF-R (AAF51909.3); Tc NPF-R (EFA10681.1); Mm
NPY-Y2 (NP_032757.2); Mm NPY-Y1 (NP_035064.1); Mm NPY-Y6
(AAB18624.1); Mm NPY-Y4 (NP_032945.3); Mm NPY-Y5 (NP_
057917.2); Mm PGR15L (NP_001028533.1); Mm GPR83 (NP_
034417.1); Mm NPFF-R2 (NP_573455); Mm NPFF-R1 (AAK94198.1);
Mm QRFP-R (NP_937835.1); Dm RYa-R (JN234381); Tc RYa-R
(HQ709383); Mm NK1 (NP_033339); Mm NK3 (NP_067357); Mm
NK2 (NP_033340) and Dm FMRFamide receptor (AAL83921). In
Fig. 2, N- and C-terminally truncated sequences (yielding the core
receptor regions from transmembrane domains I–VII) were used.
Transmembrane helices were predicted using the TMHMM server
(http://www.cbs.dtu.dk/services/TMHMM/).
580 C. Collin et al. / Biochemical and Biophysical Research Communications 412 (2011) 578–583

Dm sNPF-R (CG7395)
100 Tc sNPF-R1
84
Tc sNPF-R2
Ce NPR-1
100 Dm NPF-R (CG1147)
Tc NPF-R
Mm NPY-Y2
91 Mm
mm NPY-Y1
Y1_tm.pro
84 Mm
mmNPY-Y6
Y6_tm.pro
58
Mm
mmNPY-Y4
Y4_tm.pro
Mm
mmNPY-Y5
Y5_tm.pro
100 Mm
mmPGR15L
GPR15-l_tm.pro
Mm
mmGPR83
GPR83_tm.pro
100 Mm
mmNPFF-R2
NPFF-R2_tm.pro
50 Mm
mmNPFF-R1
NPFFR1_tm.pro
Mm QRFP-R
99 Dm RYa-R (CG5811)
Tc RYa-R
96 Mm NK1-R
99 Mm NK3-R
Mm NK2-R
Dm FMRFa-R (CG2114)
CG2114_tm.pro
217.4

200 150 100 50 0

Fig. 2. Phylogenetic tree analysis of the evolutionary relationships between the insect RYamide receptors (RYa-R; highlighted in pink), neuropeptide F receptors (NPF-R;
highlighted in blue), short neuropeptide F receptors (sNPF-R; highlighted in green), and some related non-insect receptors. The length of each branch represents the
evolutionary distance (measured as amino acid substitutions) between each receptor and the common ancestor of that receptor and its neighbor. Bootstrap values above 50
are given at nodes. The tree is rooted by the Drosophila FMRFamide receptor. It can be seen that the insect short neuropeptide F receptors and neuropeptide F receptors are
related to the mammalian neuropeptide Y receptors, while the insect RYamide receptors form a separate, more distantly related group. In this analysis only the seven
transmembrane regions of the various GPCRs were taken into account. An analysis using complete receptor sequences (including the N- and C-terminal regions of the GPCR)
lead to similar results (Fig. S3). Also, when software programs different from Lasergene (e.g., ClustalW Phylip protdist and Phylodendron) were used, similar results were
obtained. Abbreviations: Dm, Drosophila melanogaster; Mm, Mus musculus; Tc, Tribolium castaneum; Ce, Caenorhabditis elegans; sNPF-R, short neuropeptide F receptor; NPR,
neuropeptide receptor; NPF-R, neuropeptide F receptor; NPY-Y, neuropeptide Y receptor; PGR15L, probable G protein-coupled receptor 15-like; GPR83, G-protein coupled
receptor 83; NPFF-R, neuropeptide FF receptor; QRFP-R, pyroglutamylated RFamide peptide receptor; NK, neurokinin receptor; FMRFa-R, FMRFamide receptor. For GenBank
Accession Nos. see Section 2.

3. Results
3.1. The Drosophila and Tribolium RYamide peptides
We have previously annotated and published the RYamide prep-
rohormone sequences from 13 insect species, among them those
from several Drosophila species [9]. Based on the presence of single-
and dibasic cleavage sites in the prohormones, we proposed likely
structures for the various processed biologically active insect RYa-
mides (Fig. 1) [9]. A help during this peptide predicting process
was the sequencing by mass spectrometry of an RYamide peptide
from the terminal ganglion of the yellow fever mosquito A. aegypti,
showing that the precursor was preferentially cleaved adjacent or
close to a proline residue (Fig. 1) [9]. Based on these considerations
and findings, we could predict two RYamide neuropeptides from D.
melanogaster, Drosophila RYamide-1 (PVFFVASRYamide) and Dro-
sophila RYamide-2 (NEHFFLGSRYamide) and two peptides from T.
castaneum (Tribolium RYamide-1, VQNLATFKTMMRYamide; and
Tribolium RYamide-2, ADAFFLGPRYamide) (Fig. 1).
We also cloned the preprohormone containing the two Dro-
sophila RYamides (GenBank database Accession No. JN222358).
This cDNA contained the whole coding region for the preprohor-
mone (identical to the one predicted earlier, see Ref. [9]), including
a start and stop codon, but the 30 -end, containing the polyadenylation
signal and polyA-tail could not, despite numerous efforts using
30 -RACE, be identified. This difficulty in obtaining the complete
30 -end of the cDNA could be due to the fact that the D. melanogaster
RYamide gene (CG40733) is located in the heterochromatin region of
chromosome 2 [13]. This location might imply a very low expression
of the gene and, therefore, a difficulty of obtaining enough
mRNA/cDNA material for our RACE experiments.
In addition, we cloned the preprohormone containing the two
Tribolium RYamide peptides (yielding a coding region identical to
the one, we predicted earlier, see Ref. [9]; GenBank database Acces-
sion No. JN200817). In contrast to the Drosophila sequence, this
preprohormone cDNA contained a polyadenylation signal and
polyA-tail.
3.2. Cloning of the Drosophila and Tribolium RYamide receptors
About 70% of all Drosophila neuropeptide GPCRs have currently
been deorphanized, among them the receptors for most of the Dro-
sophila neuropeptides with an RFamide or RWamide C terminus: the
various FMRFamides, sulfakinin, myosuppressin, neuropeptide F,
and the various short neuropeptides F [7,14]. From the remaining
30% of the Drosophila neuropeptide receptors, we selected the GPCR
coded for by gene CG5811 as a promising candidate for being the
Drosophila RYamide receptor, because it is distantly related to the
mammalian neuropeptide Y receptors (Fig. 2), and neuropeptide Y
has the C-terminal RYamide sequence in common with the insect
RYamides (Fig. 1). Also, 20 years ago, neuropeptide Y and neuropep-
tide YY were found to activate the CG5811 receptor, albeit at
relatively high concentrations [15], but these mammalian neuro-
peptides are not intrinsic to Drosophila or other insects and the
CG5811 receptor has, therefore, still to be regarded as an orphan
receptor [6,7]. Thus, we recloned the CG5811 cDNA based on the
 C. Collin et al. / Biochemical and Biophysical Research Communications 412 (2011) 578–583 581

Fig. 3. Alignment of the Drosophila and Tribolium RYamide receptor sequences from Figs. S1 and S2. The seven transmembrane sequences are indicated by TMI-VII. Identical
amino acid residues between the two receptors are highlighted. Intron positions in the corresponding genes are indicated by boxes (for their exact positions, see Figs. S1 and
S2). There are three common introns between the genes; a fourth intron has only shifted one codon and has conserved the intron phasing.

A B
8000 100
0-5 sec. Dm-RYa1
5-10 sec. 80 Dm-RYa2
6000
Luminescence

10-15 sec. Dm-NPF

L/L.Max

60 Perisulfakinin
4000 Dm-sNPF1
40 Dm-sNPF3
2000 Dm-FMRFa2
20
Dm-FMRFa3
0 0 Dm-FMRFa7
-13 -12 -11 -10 -9 -8 -7 -6 Dm-MS
Dm-RYa1 Dm-RYa2 PBS
Log M

C D
15000 100
0-5 sec. Tc-RYa1
5-10 sec. 80 Tc-RYa2
10-15 sec. Dm-NPF
Luminescence

10000
L/L.Max

60 Perisulfakinin
Dm-sNPF1
40
5000 Dm-sNPF3
Dm-FMRFa2
20
Dm-FMRFa3
0 Dm-FMRFa7
-13 -12 -11 -10 -9 -8 -7 -6 -5 Dm-MS
Tc-RYa1 Tc-RYa2 PBS
Log M

Fig. 4. Bioluminescence responses of cloned CHO/G-16 cell lines transfected with DNA coding for either the Drosophila melanogaster (A and B) or Tribolium castaneum (C and
D) RYamide receptor. The vertical bars represent SEM (n = 2 in A and C; n = 3 in B and D), which sometimes are smaller than the symbols (or lines) used. In these cases, only
the symbols (or lines) are given. (A) Bioluminescence response of CHO/G-16/Drosophila RYamide receptor cells after addition of Drosophila RYamide-1 (Dm-RYa1) (final
concentration, 5  106 M), Dm-RYa2 (final concentration, 5  106 M) or phosphate-buffered saline (PBS). (B) Dose–response curves of the effects of Dm-RYa1, Dm-RYa2,
Drosophila neuropeptide F (Dm-NPF), perisulfakinin, Drosophila short neuropeptide F-1 (Dm-sNPF1), Dm-sNPF3, Drosophila FMRFamide-2 (Dm-FMRFa2), Dm-FMRFa3, Dm-
FMRFa7, and Drosophila myosuppressin (Dm-MS) on the transfected cells from A. The transfected cells were equally well activated by both Drosophila RYamides (EC50,
1  109 M), but not by any of the other neuropeptides except for a slight activation at higher concentrations (>106 M) by neuropeptide F and short neuropeptide F-1. (C)
Bioluminescence response of CHO/G-16/Tribolium RYamide receptor cells after addition of Tribolium RYamide-1 (Tc-RYa1) (final concentration, 5  106 M), Tc-RYa2 (final
concentration, 5  106 M) or phosphate-buffered saline (PBS). (D) Dose–response curves of the effects of Tc-RYa1, Tc-RYa2, Drosophila neuropeptide F (Dm-NPF),
perisulfakinin, Drosophila short neuropeptide F-1 (Dm-sNPF1), Dm-sNPF3, Drosophila FMRFamide-2 (Dm-FMRFa2), Dm-FMRFa3, Dm-FMRFa7, and Drosophila myosuppressin
(Dm-MS) on the cells transfected in C. The transfected cells were activated by Tc-RYa2 (EC50, 5  109 M), Tc-RYa1 (EC50, 7  108 M), but not by any of the other
neuropeptides.

sequence given in [15], which codes for a protein with seven trans- We also cloned the Tribolium ortholog of the Drosophila gene
membrane regions and a DRY sequence after the third CG5811 [7] (Fig. S2; GenBank database Accession No.
transmembrane region, a feature typical for family A GPCRs HQ709383). The two insect receptors are closely related, having
(Fig. S1; GenBank database Accession No. JN234381). 52% amino acid sequence identity (Figs. 2 and 3, Figs. S3 and S4).
582 C. Collin et al. / Biochemical and Biophysical Research Communications 412 (2011) 578–583

Their genes contain three introns at identical positions, highlight- RWamide sequence such as neuropeptide F and short neuropep-
ing again their close evolutionary relationship (Fig. 3). tides F (Fig. 1). Both insect neuropeptide F and short neuropeptides
F are involved in feeding (reviewed in [16]). Also, the insect RYa-
mides have the C-terminal RYamide sequence and two other con-
3.3. Identifying the Drosophila and Tribolium receptors as the insect
served residues in common with mammalian neuropeptide Y
RYamide receptors
(LIXXXRYamide). Mammalian neuropeptide Y is the most potent
feeding stimulant, when injected into the hypothalamus [17,18].
We stably transfected Chinese hamster ovary (CHO) cells with
These structural similarities might perhaps suggest that also the
cDNAs coding for either the Drosophila or Tribolium receptor, and
insect RYamides would be involved in feeding and energy
selected clones that expressed the receptors effectively (as tested
homeostasis.
by bioassays, see below). These cells were also stably transfected
Mammals have at least five neuropeptide Y receptors (Y1,
with cDNA coding for the promiscuous G protein, G-16, and tran-
Y2, Y4, Y5, and Y6), which have 30–50% amino acid sequence
siently transfected with cDNA, coding for apoaequorin, [10–12].
homology among each other [18]. However, after a phylogenetic
Two hours before the bioassays, coelenterazine, a co-factor for apo-
tree analysis, we do not find that the insect RYamide receptors
aequorin, was added to the cell medium. An activation of the
are structurally closely related to any of these five mammalian
receptors would, under these circumstances, lead to an IP3/Ca2+
neuropeptide Y receptors (Fig. 2, Fig. S3). This conclusion is
cascade followed by a light (bioluminescence) response of the
confirmed by other ways of amino acid sequence comparison
transfected CHO cells. This bioluminescence response can be easily
(amino acid sequence alignments show 18–28% amino acid
measured [10–12].
sequence identities between the insect RYamide and mammalian
Fig. 4A and B show the activation of the Drosophila receptor by
neuropeptide Y receptors, see Fig. S4). These analyses, therefore,
the two Drosophila RYamide peptides. The activation of the recep-
indicate that the insect RYamide signaling systems are evolu-
tor occurs within 5 s after addition of the peptides, after which the
tionarily not significantly related to the mammalian neuropep-
receptor quickly desensitizes (Fig. 4A). Both Drosophila RYamide
tide Y system, despite the fact that, nearly 20 years ago,
peptides are about equally potent with an EC50 of 1  109 M
neuropeptide Y (and neuropeptide YY) was found to activate
(Fig. 4B). Other insect neuropeptides with an RFamide or RWamide
the Drosophila RYamide (CG5811) receptor [15]. We conclude
C terminus, such as the various Drosophila FMRFamides, Drosophila
that this activation was due to accidental structural similarities
short neuropeptide F-3, Drosophila myosuppressin, and the cock-
(pharmacology) between neuropeptide Y and the insect
roach neuropeptide perisulfakinin, did not activate the receptor
RYamides (Fig. 1), and that it does not imply close evolutionary
(Fig. 4B). However, Drosophila neuropeptide F and short neuropep-
or physiological relationships. Thus, although the Drosophila
tide F-1 also slightly activated the receptor, albeit at much higher
CG5811 receptor has been named Drosophila neuropeptide Y
(100,000) concentrations than the two Drosophila RYamides
receptor during nearly two decades [15], it does clearly not
(Fig. 4B).
appear to be particularly related to the mammalian neuropeptide
Fig. 4C and D show the effects of the two Tribolium RYamides on
Y receptor.
cells expressing the Tribolium receptor. The two peptides show a
Fig. 2 also shows that the insect neuropeptide F [19], and
similar kinetics (Fig. 4C), but Tribolium RYamide-2 (EC50, 5 
short neuropeptide F receptors [20] are the insect receptors that
109 M) is somewhat more potent than Tribolium RYamide-1
are most closely related to the neuropeptide Y receptors. A
(EC50, 8  108 M) for activation of the receptor (Fig. 4D). The
clear ortholog relationship between these insect receptors and
lower potency of Tribolium RYamide-1 might be due to the fact that
any of the neuropeptide Y subtypes, however, cannot be
it does not completely follow the FFXXXRYamide consensus
deduced.
sequence rule, because one phenylalanine residue of the consensus
Finally, Fig. 2 shows that there are other mammalian GPCRs that
sequence is replaced by a lysine residue (Fig. 1). In contrast to the
are moderately related to the insect RYamide receptors. However,
Drosophila receptor, the Tribolium RYamide receptor cannot be
the phylogenetic distances between these receptors are quite long
activated by other insect peptides with an RFamide or RWamide
and, therefore, are not supporting any close evolutionary
C terminus, such as the FMRFamides, perisulfakinin, myosuppressin,
relationships.
neuropeptide F, and the various short neuropeptides F (Fig. 4D).
Thus, so far, the structures of the insect RYamide receptors can-
These findings show that both the Drosophila and Tribolium
not give us any clear hint to the possible functions of the insect
RYamide receptors are quite selective for insect RYamides.
RYamide signaling system. Yet, our current identifications of the
insect RYamide receptors offer many new experimental possibili-
4. Discussion ties for elucidating these roles, for example by using RNAi, silenc-
ing receptors in Drosophila or Tribolium and observing the resulting
The insect RYamide peptides are a large group of neuropeptides phenotypes. Expression studies are another means to gain more in-
with the C-terminal consensus sequence FFXXXRYamide that we sight into the insect RYamide system. Flybase (www.flybase.org)
discovered last year, when annotating the genome from the para- contains a very large set of microarray expression data for nearly
sitic wasp N. vitripennis [9]. The RYamides occur in all insects with every Drosophila gene. Inspecting these data, we found that the
a sequenced genome [9], with the exception of four sequenced ant CG5811 gene is significantly expressed in the hindgut of adult male
genomes [5]. Furthermore, we know that insect RYamides occur in and female flies, while it is not, or hardly, expressed in any of the
the terminal ganglion from the yellow fever mosquito A. aegypti, other investigated tissues (Fig. S5). Although these microarray data
from which we isolated and sequenced it using MALDI–TOF/CID have to be taken with some precaution, this might suggest that the
mass spectrometry, establishing that insect RYamides are genuine insect RYamides are possibly involved in digestion, or water
neuropeptides [9]. So far, nothing is known about the physiological reabsorption.
roles of insect RYamides, but the current identification of the insect
RYamide receptors further confirms that insect RYamides are gen- Acknowledgments
uine signaling molecules, activating classical family A GPCRs
(Fig. 4, Figs. S1 and S2). We thank Anders Bo Rønnegaard Hansen for typing the manu-
The insect RYamides have some structural features in common script and the Danish Research Agency, and Novo Nordisk Founda-
with other insect neuropeptides bearing the C-terminal RFamide or tion for financial support.
 C. Collin et al. / Biochemical and Biophysical Research Communications 412 (2011) 578–583
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2011.07.131.
References
[1] C.J.P. Grimmelikhuijzen, G. Cazzamali, M. Williamson, et al., Perspective. The
promise of insect genomics, Pest Manage. Sci. 63 (2007) 413–416.
[2] J.H. Werren, S. Richards, C.A. Desjardins, et al., Functional and evolutionary
insights from the genomes of three parasitoid Nasonia species, Science 327
(2010) 343–348.
[3] S. Richards, R.A. Gibbs, N.M. Gerardo, et al., Genome sequence of the pea aphid
Acyrthosiphon pisum, PLoS Biol. 8 (2010) e1000313.
[4] P. Arensburger, K. Megy, R.M. Waterhouse, et al., Sequencing of Culex
quinquefasciatus establishes a platform for mosquito comparative genomics,
Science 330 (2010) 86–88.
[5] S. Nygaard, G. Zhang, M. Schiøtt, et al., The genome of the leaf-cutting ant
Acromyrmex echinatior suggests key adaptations to advanced social life and
fungus farming, Genome Res. 21 (2011) 1339–1348.
[6] F. Hauser, G. Cazzamali, M. Williamson, et al., A review of neurohormone
GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis
mellifera, Prog. Neurobiol. 80 (2006) 1–19.
[7] F. Hauser, G. Cazzamali, M. Williamson, et al., A genome-wide inventory of
neurohormone GPCRs in the red flour beetle Tribolium castaneum, Front.
Neuroendocrinol. 29 (2008) 142–165.
[8] D.R. Nässel, A.M. Winther, Drosophila neuropeptides in regulation of
physiology and behavior, Prog. Neurobiol. 92 (2010) 42–104.
[9] F. Hauser, S. Neupert, M. Willamson, et al., Genomics and peptidomics of
neuropeptides and protein hormones present in the parasitic wasp Nasonia
vitripennis, J. Proteome Res. 9 (2010) 5296–5310.
583
[10] J. Stables, A. Green, F. Marshall, et al., A bioluminescent assay for agonist
activity at potentially any G-protein-coupled receptor, Anal. Biochem. 252
(1997) 115–126.
[11] T. Secher, C. Lenz, G. Cazzamali, et al., Molecular cloning of a functional
allatostatin gut/brain receptor and an allatostatin preprohormone from the
silkworm Bombyx mori, J. Biol. Chem. 276 (2001) 47052–47060.
[12] F. Staubli, T.J.D. Jørgensen, G. Cazzamali, et al., Molecular identification of the
insect adipokinetic hormone receptors, Proc. Natl. Acad. Sci. USA 99 (2002)
3446–3451.
[13] P. Dimitri, R. Caizzi, E. Giordano, et al., Constitutive heterochromatin: a
surprising variety of expressed sequences, Chromosoma 118 (2009) 419–
435.
[14] K.K. Hansen, F. Hauser, M. Williamson, et al., The Drosophila genes CG14593
and CG30106 code for G-protein-coupled receptors specifically activated by
the neuropeptides CCHamide-1 and CCHamide-2, Biochem. Biophys. Res.
Commun. 404 (2011) 184–189.
[15] X.-J. Li, Y.-N. Wu, R.A. North, M. Forte, Cloning, functional expression and
developmental regulation of a neuropeptide Y receptor from Drosophila
melanogaster, J. Biol. Chem. 267 (1992) 9–12.
[16] D.R. Nässel, C. Wegener, A comparative review of short and long neuropeptide
F signaling in invertebrates: any similarities to vertebrate neuropeptide Y
signaling? Peptides 32 (2011) 1335–1355.
[17] M.J. Chee, W.F. Colmers, Y eat? Nutrition 24 (2008) 869–877.
[18] M.M. Kamiji, A. Inui, Neuropeptide Y receptor selective ligands in the
treatment of obesity, Endocr. Rev. 28 (2007) 664–684.
[19] S.F. Garczynski, M.R. Brown, P. Shen, et al., Characterization of a functional
neuropeptide F receptor from Drosophila melanogaster, Peptides 23 (2002)
773–780.
[20] I. Mertens, T. Meeusen, R. Huybrechts, et al., Characterization of the short
neuropeptide F receptor from Drosophila melanogaster, Biochem. Biophys. Res.
Commun. 297 (2002) 1140–1148.