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Briefings in Functional Genomics and Proteomics 2006 4(4):321-330; doi:10.1093/bfgp/eli003
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions please email: journals.permissions@oxfordjournals.org

Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data

Frank Hauser, Michael Williamson, Giuseppe Cazzamali and Cornelis J. P. Grimmelikhuijzen

Corresponding author. Frank Hauser, Department of Cell Biology and Comparative Zoology, Universitetsparken 15, DK-2100 Copenhagen, Denmark. Tel: +45 3532 1206; Fax: +45 3532 1200; E-mail: fhauser{at}bi.ku.dk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 DISCUSSION
 NOTE
 References
 
Most neuropeptide and protein hormone receptors belong to the large superfamily of G-protein-coupled receptors (GPCRs). These cell membrane proteins steer many important processes such as development, reproduction, homeostasis and behaviour when activated by their corresponding ligands. The first insect genome, that of the fruitfly Drosophila melanogaster, was sequenced in 2000, and about 200 GPCRs have been annnotated in this model insect. About 50 of these receptors were predicted to have neuropeptides or protein hormones as their ligands. Since 2000, the cDNAs of most of these candidate receptors have been cloned and for many receptors the endogenous ligand has been identified. In this review, we will give an update about the current knowledge of all Drosophila neuropeptide and protein hormone receptors, and discuss their phylogenetic relationships.

Keywords: receptor, GPCR, neuropeptide, neurohormone, insects, Drosophila


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
DISCUSSION
 NOTE
 References
 
Insects form the largest animal group in the world. They are economically and ecologically important, because most plants depend on insects for their pollination. But insects can also be agricultural pests and vectors for major diseases such as malaria, sleeping sickness, yellow fever and elephantiasis. Despite their importance, our knowledge of their biology is still incomplete. The fruitfly Drosophila melanogaster has long been used as the model organism to study insect biology. In 2000, the complete euchromatic portion of the genome from the fruitfly was published [1]. This was the first insect genome sequence that became publicly available and, therefore, represented a milestone in insect research. It has been followed in 2002 by the publication of the genome from the malaria mosquito Anopheles gambiae [2]. Recently, the genomes of two other insect species (the honeybee Apis mellifera and the silkworm Bombyx mori) have been released and several other insect genome projects are underway.

All the genomic sequences from insects are goldmines to identify important insect proteins. This identification is first done by computer-assisted gene annotation using prediction programs like Genie or Genscan [1]. From these hypothetical gene structures, predictions of all transcript and protein sequences and the function for each protein can be made. Thereafter, these annotations have to be verified or corrected by experimental data.

Our research group focuses on the identification and characterization of insect neurohormone receptors and their ligands (neuropeptides, protein hormones, biogenic amines), because these proteins steer many important processes, such as development, reproduction, feeding and behaviour. Most neurohormone receptors belong to the large family of G-protein-coupled receptors (GPCRs) that are activated by extracellular ligands and thereafter initiate an intracellular second messenger cascade. All GPCRs have a uniform topology with a transmembrane domain consisting of seven hydrophobic {alpha}-helices and several well conserved sequence motifs [3].

Using this transmembrane and conserved motif information, 163 GPCRs have been identified within the about 13 600 annotated genes from Drosophila [1]. A more detailed analysis by Brody and Cravchik [4] identified about 200 genes coding for Drosophila GPCRs. These are classified into four families: rhodopsin-like (family A), secretin receptor-like (family B), metabotropic glutamate receptor-like (family C) and atypical receptors (family D). Hewes and Taghert [5] categorized 21 of these Drosophila GPCRs as classical neurotransmitter (biogenic amine or a related compound) receptors and 44 as candidate peptide receptors. Since these early analyses that appeared shortly after the publication of the Drosophila genome, nearly all of the annotated candidate receptors have been cloned (and their sequences had often to be corrected) and about half of the originally ‘orphan’ receptors have now been ‘deorphanized’ (which means that their endogenous ligands have been found), using the orphan receptor or reverse-pharmacology strategies [6]. This information has given new insights into the neurobiology of Drosophila, and also into the evolution and co-evolution of receptor/ligand couples. Today, Drosophila knock-out mutants of individual neuropeptide receptor genes have become available from gene disruption projects, where P-element insertions are systematically introduced into each Drosophila gene [7, 8]. Together with other tools in functional insect genomics, e.g. the piggyBac system to disrupt individual Drosophila genes [9], we will get a much better understanding of general insect biology in the near future.

In this review, we will give an update about the knowledge of all the Drosophila neuropeptide and protein hormone GPCRs and we will discuss their phylogenetic relationships. These receptors belong all to either family A or B. Both families have the same seven transmembrane topology, but different amino acid residues at several characteristic positions. For example, family A receptors have a conserved Asp-Arg-Tyr (DRY) sequence motif just after the third transmembrane helix, whereas this motif is absent in family B receptors.

Rhodopsin-like (family A) neuropeptide and protein hormone receptors in Drosophila
Figure 1 shows a phylogenetic tree analysis of all Drosophila neuropeptide and protein hormone receptors belonging to the rhodopsin-like family A. The tree is rooted using the Drosophila metabotropic glutamate receptor CG11144 as an outgroup. Below follows a short description of these Drosophila receptors, according to their phylogenetic relationships.


Figure 1
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  		Figure 1: Phylogenetic tree of family A-type neuropeptide and protein hormone receptors from Drosophila melanogaster. Sequence alignments and phylogenetic tree calculations were done with the MegAlign program of the DNASTAR program package, using the TM1–TM7 region of each receptor. The metabotropic glutamate receptor CG11144 was used as an outgroup. The length of each branch represents the evolutionary distance between the receptor and the ancestor, which it has in common with its nearest neighbour, while the units at the bottom of the figure indicate the number of amino acid residue substitutions. The identified endogenous Drosophila ligands are given in parentheses. Orphan receptors are indicated by stars. The receptor subgroups are numbered according to the chapters in the text. ETH, eclosion triggering hormone; NPF, neuropeptide F; AKH, adipokinetic hormone; CCAP, crustacean cardioactive peptide; DLGR, Drosophila leucine-rich repeat containing G-protein-coupled receptor; GPA2, glycoprotein hormone-alpha2; GPB5, glycoprotein hormone beta5; mGluR, metabotropic glutamate receptor.

 
Myosuppressin, FMRFamide and proctolin receptors
The upper two receptors of Figure 1 (CG13803 and CG8985) have been deorphanized recently by us as the first specific insect myosuppressin receptors [10]. Insect myosuppressins are decapeptides with the general structure X1DVX2HX3FLRFamide (X1 is pQ,P,T; X2 is D,G,V; X3 is V,S) that inhibit contraction of a variety of visceral muscles [11]. The two receptors appear to be paralogues; they are closely related to CG13229, a receptor that was cloned by us as a third myosuppressin receptor candidate, but which could not be activated by myosuppressin or any other ligand from our neuropeptide library in our assay system. It is therefore still an orphan.

A fourth receptor distantly related to myosuppressin receptors is CG2114. This receptor was recently identified by us [12] and later by others [13] as the first invertebrate FMRFamide receptor. In invertebrates, FMRFamide peptides are involved in various physiological processes like feeding, reproduction and behaviour.

CG6986 was identified by us and others as the first insect proctolin receptor [14, 15]. Proctolin was the first invertebrate neuropeptide to be isolated and fully sequenced back in 1975 [16]. The pentapeptide structure (RYLPT) is conserved in a wide range of insects and crustaceans and proctolin shows not only myostimulatory, but also neurostimulatory effects in insects [11]. There are two other receptors (CG16726 and CG30340) that are distantly related to myosuppressin, FMRFamide and proctolin receptors. They are still uncharacterized orphan receptors.

Pyrokinin, capa and ETH receptors
The neuropeptides pyrokinin, capa and ETH (ecdysis triggering hormone) have their C-terminal sequence (PRL/V/Iamide) in common. Interestingly, their receptors (CG8784, CG8795, CG9918, CG14575 and CG5911, see Figure 1) are also structurally and evolutionary related. This points to a receptor/ligand co-evolution in insects [17].

CG8784 and CG8795 are two receptor paralogues that can be activated by nanomolar concentrations of Drosophila pyrokinin-2 (SVPFKPRLamide) [17, 18], a neuropeptide encoded by the hugin gene [19]. Pyrokinins (generally characterized by the FXPRLamide C-terminal sequence) have diverse myotropic effects, but they also initiate sex pheromone biosynthesis and embryonic diapause in certain insects [20].

There exists a second pyrokinin peptide in Drosophila, pyrokinin-1 (TGPSASSGLWFGPRL amide), which is encoded by the capability gene [21]. Pyrokinin-1 acts only weakly on the receptors CG8784 and CG8795. However, pyrokinin-1 can activate the closely related receptor CG9918. This was first demonstrated by expressing this receptor in Xenopus oocytes [17], where it responded to a rather high pyrokinin-1 concentration (10 µM). However, when expressed in Chinese hamster ovary (CHO) cells together with the promiscuous G protein, G-16, the potency of pyrokinin-1 to activate CG9918 increased 100-fold [22]. Therefore, CG9918 can be regarded as a true pyrokinin-1 receptor.

Recently, CG14575 was cloned and identified as a capa receptor [17, 23]. The Drosophila neuropeptides capa-1 and capa-2, encoded together with pyrokinin-1 by the capability gene, are small neuropeptides with the C-terminal sequence FPRVamide that stimulate fluid secretion in Malpighian (renal) tubules [21].

The gene CG5911 gives rise to two alternatively spliced receptors, CG5911-A and CG5911-B. They have identical amino acid sequences from the start methionine to the end of transmembrane domain 4. The use of two homologous mutually alternative exons each encoding a sequence from the end of transmembrane domain 4 to the C terminus produces the two splice variants. Both variants can be activated by low concentrations of the Drosophila ecdysis triggering hormones ETH-1 (DDSSPGFFLKITKNVPRLamide) and ETH-2 (GENFAIKNLKTIPRIamide) [24, 25]. These two hormones initiate and regulate ecdysis (cuticle shedding), a behavioral program needed for insect growth and metamorphosis [26].

Allatostatin A and B receptors
The two related receptor paralogues, CG2872 and CG10001, have been shown to be activated by four different Drosophila allatostatin A peptides encoded by the allatostatin gene [27–30]. This group of insect peptides has a common C-terminal motif Y/FXFGLamide [11, 27]. The precise physiological role of type A allatostatins in Drosophila is still unclear. In other insects, they inhibit the corpora allata, block muscle contraction in several parts of the insect gut [11], and stimulate the activity of various digestive enzymes [31].

CG14003 is somewhat more distantly related to the allatostatin A receptors (Figure 1). However, we were unable to activate this receptor by any available ligand in our assay system, so CG14003 is still an orphan receptor (Arler et al., unpublished).

Using a GPCR-ß-arrestin2 interaction assay, visualizing a green fluorescent protein-tagged ß-arrestin molecule in living cells, Johnson et al. have recently shown that CG30106 specifically responds to allatostatin B [32]. Allatostatin B neuropeptides are characterized by the C-terminal sequence W(X)6Wamide resembling the mammalian galanin peptides [33]. In Drosophila, no physiological function has been associated with allatostatin B so far, but in crickets B-type allatostatins inhibit juvenile hormone production of the corpora allata [33]. The paralogous receptor CG14593 might be a second allatostatin B receptor, but this has not been demonstrated, so far.

Drosokinin, neuropeptide F and sulfakinin receptors
CG10626 was identified by Radford et al. as a leucokinin receptor [34]. Leucokinins are 6–15 amino acids long neuropeptides that are characterized by the C-terminal pentapeptide FXXWGamide [35]. The Drosophila leucokinin (drosokinin) stimulates fluid secretion in the Drosophila Malpighian tubules by raising the chloride shunt conductance [36].

In 1992, it was postulated that CG5811 is a neuropeptide Y-type receptor, because it could be activated by mammalian NPY at micromolar concentrations [37]. However, no real neuropeptide Y exists in Drosophila and the true endogenous ligand for CG5811 is still elusive.

CG7395 was characterized by Mertens et al. as the short neuropeptide F receptor [38]. Drosophila short neuropeptide Fs are four shorter peptides (6–11 amino acids in length) contained within a single preprohormone and having the common C-terminal sequences RLRFamide or RLRWamide [39].

Another related receptor, CG1147, was characterized as a functional neuropeptide F receptor [40]. Drosophila neuropeptide F (dNPF) is a 36 amino acid residue long peptide with the C-terminal sequence RVRFamide. It is structurally related to the vertebrate neuropeptide Y family and is expressed in Drosophila midgut endocrine cells and in the brain [41]. Members of the vertebrate neuropeptide Y family regulate feeding behaviour and digestion, and also dNPF was shown to control food intake and social behaviour in Drosophila [42].

CG10823 is distantly related to neuropeptide F receptors, but this receptor is not deorphanized yet.

CG6857 and CG6881 are two closely related receptor paralogs that can be activated by the Drosophila sulfakinin DSK-1 (FDDY(SO3H) GHMRFamide) [43] (Kobberup et al., unpublished). The Drosophila sulfakinin preprohormone encodes three different putative drosulfakinins strongly resembling mammalian gastrin and CCK. Sulfakinins are, like their mammalian counterparts, involved in feeding processes [44].

Allatostatin C and tachykinin receptors
The physiological ligand for the receptor paralogues CG7285 and CG13702 has been discovered recently as Drosophila allatostatin C (pEVRYRQCYFNPIS CF) [45]. Allatostatin C is a cyclic neuropeptide originally isolated from the moth Manduca sexta on the basis of its ability to inhibit juvenile hormone production in the moth corpora allata, two endocrine glands located near the insect brain [46]. The action of allatostatin C in Drosophila is unknown.

CG13995 is distantly related to allatostatin C receptors, but its ligand is not identified yet. Two other paralogous receptors belonging to this subgroup, CG6515 and CG7887, have been deorphanized by Johnson et al. [33]. These receptors responded specifically to the tachykinin DTK-1, one out of five different endogenous Drosophila tachykinin peptides with the common C-terminal sequence FXGXRamide. Drosophila tachykinin peptides potently stimulate contractions of the insect gut [47].

CG13575 is distantly related to allatostatin C and tachykinin receptors, but it is still an orphan receptor.

Corazonin, adipokinetic hormone (AKH) and crustacean cardioactive peptide (CCAP) receptors
CG11325 shows structural and evolutionary relationship to vertebrate gonadotropin-releasing hormone (GnRH) receptors [48]. Insects do not have GnRH, but one might expect that CG11325 and its ligand have a central function in reproduction. Therefore, it was a little surprising when we identified Drosophila adipokinetic hormone (pELTFSPDWamide) as the endogenous ligand for this receptor [49]. Insect AKHs are neuropeptides controlling lipid and sugar mobilization from the fat body during energy-consuming activities such as flight and locomotion [50].

CG10698 is closely related to the AKH receptor and was cloned and characterized by us and others as the Drosophila corazonin receptor [17, 51]. The neuropeptide corazonin (pETFQYSRGWTN amide) was originally isolated from cockroaches based on its cardio-excitatory actions on the isolated cockroach heart [52]. In locusts, corazonin induces a strong darkening of the exoskeleton in association with swarm formation and migration [53]. Not only do the AKH and corazonin receptors seem to have a common evolutionary origin (Figure 1), but also their ligands are structurally related. This is another example indicating a co-evolution of receptors and their ligands [17, 51].

CG6111 is a third member of this receptor subfamily and it was shown to be a receptor for crustacean cardioactive peptide (CCAP) [17, 54], a highly conserved cyclic neuropeptide (PFCNAFTGCamide) originally isolated from the shore crab Carcinus maenas but present in identical form also in insects, including Drosophila, [11, 55]. It is a multifunctional peptide that shows cardioexcitatory and AKH-releasing activity, but that also controls the motor behaviour program associated with ecdysis [11, 25].

Drosophila leucine-rich repeat containing GPCRs (DLGRs)
Drosophila has four different LGRs which we named DLGR1, –2, –3 and –4 [56, 57] (Bohn et. al., unpublished). These receptors are closely related to the mammalian glycoprotein hormone (LH/CG, FSH and TSH) receptors and are characterized by having a large, horseshoe-shaped extracellular N-terminal domain containing 9–18 leucine-rich repeats.

Recently, CG8930, DLGR2 or ‘rickets’ was shown by us and others to be the receptor for the Drosophila moulting hormone bursicon [58, 59], a neurohormone responsible for tanning and hardening of the cuticle, for wing expansion after adult eclosion and for apoptosis of the wing epithelial cells [60, 61]. A DLGR-2 defective mutant is associated with the ‘rickets’ phenotype, which shows lack of sclerotization and melanization of the cuticle, bent legs and abnormal wing expansion after adult eclosion [62]. Like the mammalian glycoprotein hormones, bursicon is a heterodimer composed of two larger N-glycosylated cystine-knot polypeptides.

Very recently, also the ligand for DLGR1 or CG7665 was identified. Hsueh's group showed that a heterodimer, composed of the two Drosophila homologues of the human glycoprotein subunits GPA2 and GPB5, can activate DLGR1 [63].

The cDNAs coding for DLGR3 (CG5042 or CG5046 or CG31096) and DLGR4 (CG4187) were cloned by us (Bohn et al., unpublished) and showed quite some differences to the earlier, incomplete annotations. Both receptors are still orphans.

Uncharacterized receptors
There are six additional genes coding for putative neuropeptide receptors belonging to family A that are still orphans. These genes are CG4322, CG3171, CG4313, CG12610, CG12290 and CG5936, and their phylogenetic positions are shown in Figure 1.

Family B-type neuropeptide receptors in Drosophila
Figure 2 shows a phylogenetic tree analysis of the five Drosophila neuropeptide receptors belonging to family B-type receptors. The tree is rooted using the Drosophila FMRFamide receptor CG2114 as an outgroup.


Figure 2
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  		Figure 2: Phylogenetic tree of all family B-type neuropeptide receptors from Drosophila melanogaster. Sequence alignments and phylogenetic tree calculations were done as in Figure 1. The FMRFamide receptor CG2114 was used as an outgroup. The identified endogenous Drosophila ligands are given in parentheses. Orphan receptors are indicated by stars.

 
Diuretic hormone receptors
The receptor encoded by CG8422 has been shown to be a functional diuretic hormone (DH44) receptor [64]. CG8422 is structurally related to mammalian corticotropin releasing factor (CRF) receptors, and also diuretic hormone resembles mammalian CRF peptides [65]. Its paralogue, CG12370, is still an orphan receptor.

A second family B-type receptor, encoded by the gene CG32843 (or CG17415), responded to the Drosophila neuropeptide diuretic hormone 31 (DH31) when co-expressed with the Drosophila accessory protein RCP (receptor component protein) required for proper function [66]. DH31 is related to calcitonin/calcitonin gene-related peptide (CGRP) [67]. Also its receptor, CG32843, resembles members of the calcitonin receptor-like receptor family. CG8422 and CG32843 are co-expressed in principal cells of Drosophila Malpighian tubules, which fits with the biological function of diuretic hormones in controlling water and salt homeostasis [66].


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 DISCUSSION
NOTE
 References
 
Gene finding programs, manual annotations and cloning experiments have identified 40 neuropeptide and four protein hormone GPCRs, belonging to family A (Figure 1) and five neuropeptide receptors, belonging to family B (Figure 2). Of these 49 GPCRs, 29 (60%) have been deorphanized, so far. The remaining 20 receptors (marked with stars in Figures 1 and 2) might be somewhat more difficult to deorphanize. This might be partly because they do not use IP3/Ca2+ in their second messenger pathways (most bioassay systems use Ca2+ signalling), and partly because auxiliary proteins might be needed to functionally insert these receptors into the cell membrane of cells in cell culture (heterodimeric receptors might be necessary for this functional expression, or the presence of helper proteins, such as Drosophila orthologues of mammalian RCP and RAMPs [68, 69]). In addition, their ligands might be completely new or have rather complex structures (e.g. the molecular nature of the moulting hormone bursicon was first resolved recently [58, 59], making a reverse pharmacology approach difficult or impossible. In these cases, ligand purification from tissue extracts will still be necessary. However, one could assume that despite these technical challanges we will have deorphanized a large portion of the Drosophila neuropeptide and protein hormone receptors within the coming years. Thereby, remaining ligands such as eclosion hormone (EH), prothoracicotropic hormone (PTTH), pigment-dispersing factor (PDF), SIFamide, ion transport peptides or the brain peptides encoded by the amnesiac gene will find their cognate receptors.

What could we do with such an (nearly complete) inventory of these neurohormone GPCRs in Drosophila? First, one could carry out receptor localization at the cellular level (using in situ hybridization or immunocytochemistry). This line of research will certainly give us surprising results and question many existing paradigms about the actions of insect neurohormones. For example, the insect neuropeptide myosuppressin has always been thought to act on insect muscles. Using Northern blot analyses, however, we have now found that these receptors are nearly exclusively located in the brain of Drosophila [10]. These results point to a completely new role of myosuppressins in insects. Receptor localization, therefore, is one on the first steps leading to a ‘New Insect Endocrinology’.

The Drosophila gene disruption project has already been able to disrupt more than 5600 genes (40% of all genes) from Drosophila and this number will certainly increase in the coming years [8]. Among the disrupted genes are several genes coding for neuropeptide and protein hormone receptors. Analyses of the phenotypes of the Drosophila receptor mutants, will also challenge existing paradigms on the actions of the various neurohormone receptor/ligand couples in insects and, thus, contribute to a new view on insect endocrinology.

Most of these scientific discoveries will first be made in Drosophila and can later be applied to other insects. The first insect groups to profit from these discoveries will, of course, be those species for which a genome project exists, i.e. the malaria mosquito Anopheles gambiae, the yellow fever mosquito Aedes aegypti, the honey bee Apis mellifera, the silkworm Bombyx mori and the red flour beetle Tribolium castaneum [70–73]. The annotation and identification of the Drosophila neuropeptide/protein hormone receptors are extremely helpful for the annotation and interpretation of the corresponding (orthologue) receptors in these insect species. The same is true for all the other experimental findings on the Drosophila receptors. Thus, Drosophila GPCR research will have a strong impact on insect molecular endocrinology.


   NOTE
TOP
 ABSTRACT
 INTRODUCTION
 DISCUSSION
 NOTE
References
 
After this article was accepted, two orphan receptors have been deorphanized: CG13575 (Figure 1) has been identified as the SIFamide receptor [74] and CG4395 (Figure 2) as the PDF receptor gene [75–77].


Key Points

  • Most insect neurohormone GPCRs are deorphanized in Drosophila.
  • This knowledge can be used to annotate and characterize orthologue GPCRs in other insects with sequenced genomes.
  • Drosophila GPCR research will lead to a new view on insect endocrinology.

 


   FOOTNOTES
 
Frank Hauser and Cornelis J. P. Grimmelikhuijzen are biochemists, Michael Williamson is a research technician, and Giuseppe Cazzamali is a postdoctoral fellow (biologist) at the University of Copenhagen. Their research interests are comparative molecular endocrinology and molecular neurobiology.


   References
TOP
 ABSTRACT
 INTRODUCTION
 DISCUSSION
 NOTE
 References
 

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