Elsevier

Progress in Neurobiology

Volume 80, Issue 1, September 2006, Pages 1-19
Progress in Neurobiology

A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera

https://doi.org/10.1016/j.pneurobio.2006.07.005Get rights and content

Abstract

G protein-coupled receptor (GPCR) genes are large gene families in every animal, sometimes making up to 1–2% of the animal's genome. Of all insect GPCRs, the neurohormone (neuropeptide, protein hormone, biogenic amine) GPCRs are especially important, because they, together with their ligands, occupy a high hierarchic position in the physiology of insects and steer crucial processes such as development, reproduction, and behavior. In this paper, we give a review of our current knowledge on Drosophila melanogaster GPCRs and use this information to annotate the neurohormone GPCR genes present in the recently sequenced genome from the honey bee Apis mellifera. We found 35 neuropeptide receptor genes in the honey bee (44 in Drosophila) and two genes, coding for leucine-rich repeats-containing protein hormone GPCRs (4 in Drosophila). In addition, the honey bee has 19 biogenic amine receptor genes (21 in Drosophila). The larger numbers of neurohormone receptors in Drosophila are probably due to gene duplications that occurred during recent evolution of the fly. Our analyses also yielded the likely ligands for 40 of the 56 honey bee neurohormone GPCRs identified in this study. In addition, we made some interesting observations on neurohormone GPCR evolution and the evolution and co-evolution of their ligands. For neuropeptide and protein hormone GPCRs, there appears to be a general co-evolution between receptors and their ligands. This is in contrast to biogenic amine GPCRs, where evolutionarily unrelated GPCRs often bind to the same biogenic amine, suggesting frequent ligand exchanges (“ligand hops”) during GPCR evolution.

Introduction

Insects are the largest animal group in the world (75% of all species are insects) and are economically and ecologically extremely important, because most flowering plants depend on insects for their pollination. The honey bees alone, for example, pollinate 20 billion dollars worth of crop yearly in the United States. But insects can also be severe agricultural pests, destroying 30% of our potential annual harvest, and can be vectors (intermediate pathogen carriers) for major diseases such as malaria, sleeping sickness, dengue fever, yellow fever, and elephantiasis.

There are, at present, highly exciting developments occurring within the field of insect research, because the genomes from the fruitfly Drosophila melanogaster and the malaria mosquito Anopheles gambiae (both belonging to the insect order Diptera, or flies) and that from the silkworm Bombyx mori (belonging to the order Lepidoptera, or moths and butterflies) have recently been sequenced (Adams et al., 2000, Holt et al., 2002, Xia et al., 2004). The latest published addition to insect genomics is the completion of the Honey Bee Genome Project (The Honey Bee Genome Sequencing Consortium, 2006).

Insects can be subdivided into two evolutionary lineages, the Holometabola (insects with a complete metamorphosis during development) and Hemimetabola (insects with an incomplete metamorphosis). The genomic sequences from the honey bee are especially exciting, because the honey bee belongs to an insect order (the Hymenoptera), which has very recently been shown to occupy the most basal position in the Holometabola lineage (Savard et al., 2006). In contrast, the Diptera are the most advanced holometabolous insects. A comparison between the two insect orders, as we do in the current review, therefore, will give us invaluable evolutionary information on holometabolous insects, which comprise 80–85% of all insect species. For the study of the evolution of insects it would, of course, be even more interesting to compare the genomes from holometabolous with that from hemimetabolous insects. Unfortunately, large-scale genomic information on hemimetabolous insects is not available yet.

The honey bee is extremely important for agriculture as a major pollinator and as a producer of honey. Furthermore, the honey bee is a well-studied social insect, which has fascinating social instincts and behavioral traits that involve learning, communication, and navigation. The honey bees even have their own language known as the honey bee dance (the only known non-primate symbolic language) and research in this area has been awarded with a Nobel Prize to Karl von Frisch in 1973 (von Frisch, 1994). It is expected that the sequencing of the honey bee genome will advance our knowledge of this insect enormously, which will have a strong impact on both the applied part of honey bee research (related to agriculture) and the more basic research on social behavior and learning of the bee. Moreover, all new results obtained in the honey bee will be highly relevant for our studies of other social insects, such as ants, wasps, and termites.

G protein-coupled receptor (GPCR) genes are large gene families in all animals, sometimes making up 1–2% of the animals’ genome. Also in the honey bee genome, a large number (about 240) of GPCR genes have been identified, which is about 1.5% of the total number of genes present in the bee (The Honey Bee Sequencing Consortium, 2006). GPCRs are transmembrane proteins with a characteristic topology, consisting of an extracellular N terminus, seven hydrophobic transmembrane α helices and an intracellular C terminus. GPCRs are activated by extracellular signals, after which they initiate a second messenger cascade in the interior of the cell, thus transducing the signals from the outside to the inside of the cell. GPCRs can be receptors for light (the rhodopsins), odorants (olfactory receptors), or neurotransmitters/neurohormones. They can be classified into four families: rhodopsin-like (or family A), secretin receptor-like (family B), metabotropic glutamate receptor-like (family C), and atypical receptors (family D). All families have the same seven transmembrane topology, but differ by amino acid residues at certain characteristic positions. For example, family A receptors have a conserved Asp-Arg-Tyr (DRY) sequence motif just after the third transmembrane α helix, whereas this motif is lacking in members of the other GPCR families (Gether, 2000).

Our research group is especially interested in neurohormone GPCRs from insects and their corresponding ligands (the neuropeptides, protein hormones, and biogenic amines), because these molecules play a central role in the physiology of these animals, i.e., they occupy a high “hierarchic” position in the steering or coordination of important processes, such as reproduction, development, growth, feeding, homeostasis, and behavior. Because the Drosophila Genome Project was the first insect genome project to be completed (Adams et al., 2000), most information on insect neurohormone GPCRs is available from Drosophila. The website of the Drosophila Genome Project contains a list of 41 genes predicted (“annotated”) to code for neuropeptide GPCRs, three for protein hormone GPCRs, and 21 for biogenic amine GPCRs (Hewes and Taghert, 2001) (www.flybase.org). We found one additional protein hormone GPCR and three neuropeptide GPCRs that were not annotated by flybase, thus the total number of neurohormone GPCRs in Drosophila is probably 69 (Hauser et al., 2006). We have further found, after cDNA cloning, that in many cases the flybase annotations were incorrect, because the predicted intron/exon organizations were wrong, or because exons from other annotated neighboring genes also were part of the correct receptor gene. Furthermore, the ligands for many of the annotated receptor genes are unknown, i.e., they are orphan receptors. Therefore, proper cDNA cloning of the annotated receptor genes and subsequent ligand identification are still necessary processes.

There are several ways to identify a ligand for an orphan receptor. They all imply the heterologous expression of the receptor cDNA in cells, which, for example, can be frog oocytes, or mammalian cells in cell culture. The activation of the expressed GPCRs by a tissue extract, containing the ligand, or by a synthetic ligand from a chemical library can be measured by the second messenger responses (for example, by changes in cytoplasmic cAMP or Ca2+ concentrations in mammalian cells, or cAMP- or Ca2+-induced ion currents across the cell membranes of frog oocytes). When a second messenger response occurs, the cells can be used as a bioassay and the ligand can be purified and identified (in the case of an extract) or directly be determined if the ligand comes from a library (Civelli et al., 2001, Civelli, 2005). We and others have been especially successful with a system, where we have stably transfected Chinese hamster ovary (CHO) cells in cell culture with a cDNA for an insect GPCR. These cells were also stably transfected with DNA, coding for the promiscuous G protein, G-16, and transiently transfected with DNA, coding for apoaequorin. Three hours before the assay, we added the co-factor of apoaequorin, coelenterazine, to the culture medium. An activation of the expressed receptor in such pretreated cells would initiate an IP3/Ca2+ cascade, leading to a strong bioluminescence response (Stables et al., 1997, Lenz et al., 2001, Secher et al., 2001, Staubli et al., 2002, Cazzamali and Grimmelikhuijzen, 2002, Meeusen et al., 2002, Mertens et al., 2002). A schematic drawing of this bioassay is given in Fig. 1. The system can be improved by selecting cell clones that express the GPCRs most effectively. This can be done by using a transfection vector that, in addition to DNA coding for the insect GPCR, also contains DNA coding for green fluorescent protein (GFP). In this case, the cell clones with strongest fluorescence can be selected and used in our assay system. An example of such an assay in cloned cell lines is given in Fig. 2, where the insect neuropeptide proctolin (RYLPT) induces a bioluminescence response in CHO cells expressing the proctolin receptor, which is 400× over background. These are robust responses that give clear answers as to the identity of the ligands for the insect orphan GPCRs.

In the last few years, our group and others have successfully cloned and identified (“deorphanized”) about 40 Drosophila neurohormone GPCRs, or more than half of all neurohormone GPCRs that are believed to be present in the fruitfly. This work has already given us impressive insights not only into the neuroendocrinology of Drosophila but also into the evolution of neurohormone receptors and co-evolution of the receptors and their ligands (Park et al., 2002, Rosenkilde et al., 2003, Mendive et al., 2005). All this knowledge gained in Drosophila can be used to understand the molecular endocrinology of other insects, and the first insect groups where this knowledge can be applied will, of course, be those groups for which a genome project exists.

Neurohormone receptors and their ligands are expected to play a central role in the learning and behavior of the honey bee. The releases of genomic sequencing data (December 2003–Spring 2006) prior to the current publication of the honey bee genome (The Honey Bee Sequencing Consortium, 2006), therefore, immediately raised the question about the presence of these molecules in the bee. For these reasons, we started an annotation project for neurohormone receptors right after the first genomic sequences from the honey bee were released (December 2003). In the present paper, we will describe the results of this annotation effort, where we discovered 51 novel honey bee neurohormone GPCRs in addition to five biogenic amine GPCRs characterized earlier. We will also review our current knowledge on Drosophila neurohormone GPCRs and describe the evolution of insect GPCRs based on our analyses of the two insect species.

Section snippets

Neurohormone receptor genes present in the fruitfly and honey bee genomes

We used the amino acid sequences of both the annotated and the identified (cloned and “deorphanized”) Drosophila neurohormone receptors (www.flybase.org; Hewes and Taghert, 2001, Hauser et al., 2006) to screen (BLAST search) the genomic sequences released by the Honey Bee Genome Project (www.hgsc.bcm.edu/projects/honeybee/). We started our manual annotations right after the first release of the honey bee genomic sequences in December 2003 (Amel_1.0), where we already could assign most of the

General discussion

In this paper, we have identified 56 neurohormone GPCRs in the honey bee. Of these, 35 are neuropeptide, two are protein hormone, and 19 are biogenic amine GPCRs (Fig. 3, Fig. 4, Fig. 5). Of the 19 biogenic amine receptors, five have already been cloned and deorphanized (Am 5–9 in Fig. 3). Of the remaining 14 biogenic amine receptor genes, 10 have close Drosophila orthologues that have been deorphanized, so we know what the likely ligands are for the honey bee receptors. Of the two protein

Methods

D. melanogaster neurohormone G protein-coupled receptor proteins were used as probes to search for similar proteins in Apis mellifera. tBLASTn searches were performed on the genomic sequences from various releases of the Honey Bee Genome Project (Amel_1.0 to Amel_4.0; http://www.hgsc.bcm.tmc.edu/projects/honeybee/). The full sequences of the candidate Apis receptor proteins were identified using a combination of Invitrogen Vector NTI Advance 9.0 package (InforMax), the Lasergene DNA Software

Note added in proof

After this review went to press, a paper was published (Schlenstedt et al., 2006), where gene Am 12 (Fig. 3) was experimentally identified (deorphanized) as a honey bee serotonin receptor gene, thus confirming our annotation.

Acknowledgements

We thank Sarah Preisler for typing the manuscript, Kristoffer Egerod for supplying Fig. 2, and the Danish Research Agency (Research Council for Nature and Universe), Lundbeck Foundation, Carlsberg Foundation, and Novo Nordisk Foundation (Fabrikant Vilhelm Pedersen og Hustrus Mindelegat) for financial support.

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    Nucleotide sequence data reported here are available in the CoreNucleotide or third party annotation section of the DDBJ/EMBL/GenBank databases under the accession numbers: AF498306; AJ245824; AJ547798; AY921573; AY_961388–AY_961391; AY_961393–AY_961396; BK005219; BK005220; BK005238–BK005242; BK005257–BK005259; BK005261–BK005269; BK005271; BK005273; BK005274; BK005684; BK005712; BK005714–BK005719; BK005754; DQ151547; DQ201783; XP_394102; XP_394798; XP_395101; XP_395760; XP_396348; XP_396445; XP_396491; XP_397077; Y13429.

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