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Mini-review: The evolution of neuropeptide signaling
Regulatory Peptides 177 (2012) S6–S9
Contents lists available at SciVerse ScienceDirect
Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Review
Mini-review: The evolution of neuropeptide signaling Cornelis J.P. Grimmelikhuijzen ⁎, Frank Hauser 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
Keywords: Adipokinetic hormone Corazonin GnRH GPCR Neuropeptide Evolution
a b s t r a c t Neuropeptides and their G protein-coupled receptors (GPCRs) have an early evolutionary origin and are already abundant in basal animals with primitive nervous systems such as cnidarians (Hydra, jellyfishes, corals, and sea anemones). Most animals emerging after the Cnidaria belong to two evolutionary lineages, the Protostomia (to which the majority of invertebrates belong) and Deuterostomia (to which some minor groups of invertebrates, and all vertebrates belong). These two lineages split about 700 million years (Myr) ago. Many mammalian neuropeptide GPCRs have orthologues in the Protostomia and this is also true for some of the mammalian neuropeptides. Examples are oxytocin/vasopressin, GnRH, gastrin/CCK, and neuropeptide Y and their GPCRs. These results implicate that protostomes (for example insects and nematodes) can be used as models to study the biology of neuropeptide signaling. © 2012 Elsevier B.V. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The evolution of the nervous system and neuropeptide signaling . . . . 3. How does neuropeptide signaling in Proto- and Deutorostomia compare? 4. What can we learn from these evolutionary insights? . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Most animals belong to two lineages of animal evolution, the Proto- and Deuterostomia that split about 700 million years (Myr) ago [1] (Fig. 1). The majority of invertebrates belongs to the Protostomia, while a few invertebrate groups and all vertebrates, such as mammals, belong to the Deuterostomia (Fig. 1). In addition, there are some very basal animal groups, such as cnidarians (sea anemones, corals, jellyfishes) and sponges that evolved long before the split of Proto- and Deuterostomia. The Protostomia contain many more animal species than the Deuterostomia. The arthropods (insects, crustaceans, spiders, and ticks), for example, constitute at least 1.2 million species, which is more than 85% of all animal species living on Earth. In contrast, mammals only comprise 0.3%.
⁎ Corresponding author. E-mail address: [email protected] (C.JP. Grimmelikhuijzen). 0167-0115/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2012.05.001
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An overview using a pie chart representation of the number of animal species (Fig. 2) visualizes how little mammals fill compared to the other animals. Yet, most research, also at this conference, is focused on mammals. Is this strong focus on ourselves and our own animal group (mammals) really justified? Some will say “yes”, because this research on mammals is medically relevant and will help us to cure diseases. Also, this type of research is economically important, because of its close link to the pharmaceutical industry. We agree with all these arguments. Yet, we feel that a focus only on mammals is dangerous and will block both researchers and clinicians from seeing “the whole picture”. Therefore, we would like to invite the reader of this Abstract Book to also look at the other animals.
2. The evolution of the nervous system and neuropeptide signaling Nervous systems probably evolved in the common ancestor of cnidarians, because sponges (Porifera, Fig. 1) do not show any physiological or anatomical signs of a nervous system. The nervous systems in cnidarians are strongly peptidergic. We and others have isolated and
C.J.P. Grimmelikhuijzen, F. Hauser / Regulatory Peptides 177 (2012) S6–S9
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Fig. 1. A simplified phylogenetic tree of animal evolution. The number of species is highlighted in green behind each phylum. There are two larger basal phyla, the Porifera (sponges) and Cnidaria (sea anemones, corals, Hydra, jellyfishes) that originated more than 700 Myr ago. Nervous systems probably evolved within the Cnidaria or their close ancestors. About 700 Myr ago newly emerging animal groups split into two evolutionary lineages, the Protostomia (highlighted in blue) and Deuterostomia (highlighted in red).
sequenced a large number of neuropeptides, cloned their preprohormones and located these molecules, using immunocytochemistry and in situ hybridization, in neurons and dense neuronal plexuses in cnidarians [2,3]. A remarkable feature of cnidarian preprohormones is the very high neuropeptide copy numbers contained within the precursors, which can be up to 37 neuropeptide copies [4]. Attempts to demonstrate fast transmitters in cnidarians (Glu, Gly, GABA, acetylcholine, ATP), catecholamines, other biogenic amines, or gasotransmitters (NO, CO, H2S) have failed or have not been convincingly positive with the exception of serotonin, which appears to be produced by some neurons in the sea pansy Renilla köllikeri [5], but not in other cnidarians investigated so far. These data, therefore, show that neuropeptides are the dominant transmitters in cnidarians and also suggest that they played a key role in the first nervous systems that emerged in evolution.
Neuropeptides exert their actions by binding to their specific G protein-coupled receptors (GPCRs), a process that starts a second messenger cascade. Because of the many steps involved, neuropeptide actions are relatively slow. Cnidarians are interesting in this respect, because, although most neuropeptides bind to GPCRs, some neuropeptides in these animals activate ligand-gated ion channels, thus giving fast responses. We have cloned three genes from Hydra magnapapillata that code for degenerin (DEG)/epithelial Na + channel (ENaC) gene family members. When these three genes are coexpressed in frog oocytes, they form a Na + ion channel that is gated by the Hydra neuropeptides Hydra-RFamides I and II. These ion channels have pore properties and amiloride sensitivities that are very similar to the other known DEG/ENaC channels [6]. Neuropeptide-gated ion channels, therefore, may supply cnidarians with fast excitatory neurotransmission in a situation where other fast transmitters and their receptors are not available (i.e. have not been “invented” yet). Amiloride-sensitive FMRFamide-gated Na + channels have also been cloned and characterized from various molluscs [7,8]. Evolutionarily, cnidarians and molluscs are widely separated (Fig. 1), which suggests that all the animals in between (e.g. plathyhelminths, annelids) may also have peptide-gated ion channels. Even in the deuterostome lineage neuropeptide-gated ion channels may be discovered. 3. How does neuropeptide signaling in Proto- and Deutorostomia compare?
Fig. 2. A pie chart representation of present-day animal groups according to their number of species. Arthropods constitute about 85% of all animal species, while for mammals this number is 0.3%.
Because neuropeptides are closely associated with the emergence of the first nervous systems, it is no surprise that neuropeptide signaling still plays a key role in both Proto- and Deuterostomia (Fig. 1). An interesting question is whether there are structural similarities between the neuropeptides/GPCRs in these two animal groups. If yes, these peptide signaling systems should have originated before the split of Proto- and Deuterostomia and be evolutionarily very old (>700 Myr). Also, if yes, mammalian endocrinologists might learn from the protostomes: many of these animals are excellent laboratory models (e.g. the fruitfly Drosophila melanogaster and the nematode Caenorabditis
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C.J.P. Grimmelikhuijzen, F. Hauser / Regulatory Peptides 177 (2012) S6–S9
Neuropeptide name
Amino acid sequence
AKH ACP (AKH/corazonin-related peptide) Corazonin GnRH-1
Fig. 3. Comparison of the amino acid sequences of mosquito (Anopheles gambiae) AKH (adipokinetic hormone), mosquito ACP (AKH/corazonin-related peptide), mosquito corazonin, and mammalian GnRH-1. Residues that are identical between at least three peptides are highlighted in red. Amino acid residues that are identical between two peptides are highlighted in green. Conserved (but not identical) residues are highlighted in blue. Adapted from [9].
elegans). They are simple, have short generation times (12 days or shorter) and are amenable for genetic manipulations (gene disruption mutants, RNAi). Furthermore, the genomes from many protostomes have been sequenced, which also turns them into potential new models. Within the arthropods alone, for example, more than 50 genomes have recently been sequenced. The large numbers of sequenced genomes from both proto- and deuterostomes strongly facilitate answering the above question. When we look at neuropeptide genes it is often difficult to see whether a mammalian neuropeptide has an orthologue in protostomes, because the neuropeptide part in the preprohormone is often very small and difficult to “hit” when TBLASTN screening of the genome sequences is used as a tool. Also, there has been longterm neuropeptide/GPCR evolution and co-evolution in these
A
B
receptors
Drosophila, Apis, Daphnia, pea aphid
CRZR
mosquitoes, Bombyx, Nasonia, Rhodnius
CRZR
Tribolium
animals, changing the peptide structures dramatically and masking their evolutionary relationships [9]. However, there are a few exceptions. Oxytocin/vasopressin is one such example, where a nearly intact oxytocin/vasopressin-like peptide, and its associated neurophysin part on the preprohormone can be found in many protostomes, including arthropods. These findings are supported by the presence of functional oxytocin/vasopressin receptors [10–13]. Other examples are neuropeptide Y (called neuropeptide F in arthropods) and its receptor [14,15], a sulfated gastrin/CCK-like peptide (called sulfakinin in arthropods) and its receptor [16,17], and glycoprotein hormones related to FSH, LH, and TSH and their leucine-rich repeats containing GPCRs (LGRs). In fact, glycoprotein hormones and their LGRs are ubiquitous in proto- and deuterostomes and are even occurring in cnidarians showing that they are essential signaling systems already associated with the earliest nervous systems [18–24]. Although it is often difficult to find the protostome orthologue of a mammalian neuropeptide, it is much easier to do so for the neuropeptide GPCRs, which is mainly due to their much larger size, increasing the chances to find a “hit” using TBLASTN. When we started our insect work 15 years ago, we were very much interested in insect reproduction and in the question, whether it was related to mammalian reproduction. Finding a GnRH peptide gene orthologue in insects was impossible. However, we could readily find and clone a Drosophila orthologue of the GnRH receptor [25]. When we subsequently expressed the receptor in Chinese hamster ovary cells and isolated the ligand from Drosophila extracts, using a cell-based bioassay, we were very much surprised that this was not a GnRH-related peptide, but adipokinetic hormone (AKH), a well-known insect neuropeptide that mobilizes carbohydrates and lipids from the insect fat body during flight [26]. AKH has about the same size as GnRH, but it has hardly any amino acid residues in common with GnRH (Fig. 3). What does this example of the GnRH receptor show us? (i) It shows that the GnRH receptor already originated before the split
AKHR
CRZ
ACPR
AKHR
CRZ
ACPR
AKHR
ligands AKH
ACP
AKH
ACP
AKH
Fig. 4. Scenario for receptor/ligand co-evolution, leading to insect receptors that are specific for either AKH, ACP, or corazonin. A. Receptors for corazonin (CRZR), ACP (ACPR), and AKH (AKHR). These receptors are structurally related (indicated by the white parts not involved in ligand binding), but also have specific differences (indicated by the colored parts that bind their specific ligands). It is assumed that the three receptors have a common ancestor (bottom), which also is the ancestor of the GnRH receptor. The ancestor gene duplicated and, after mutations, these duplications gave rise to two receptors that are the ancestors of the corazonin and ACP/AKH receptors. This last receptor gene duplicated once again. B. The receptor ligands corazonin (CRZ), ACP, and AKH. These ligands have some amino acid residues in common (indicated by the white parts) but also some specific differences (indicated by the colored parts). It is assumed that these ligands have a common ancestor, which also might be the ancestor for GnRH. The gene duplication scenario is the same as in A. It is unknown whether the ligands or receptors duplicated first, but these duplications must have had time windows that overlap (= coevolution between A and B). This figure has been originally published in The Journal of Biological Chemistry ([9], © the American Society for Biochemistry and Molecular Biology).
C.J.P. Grimmelikhuijzen, F. Hauser / Regulatory Peptides 177 (2012) S6–S9
of Proto- and Deuterostomia, hence that it is more than 700 Myr old; (ii) it also shows that the ancestral ligand, which probably had a mixture of the AKH and GnRH amino acid sequences, has changed considerably during proto- and deuterostome evolution; (iii) the ancestral GnRH signaling system might have controlled carbohydrate and lipid release, reproduction, or both. Of course we do not know (and we will never know), but our intuition tells us that it might be both. During deuterostome evolution, the “release part” might have been lost, while during protostome evolution, the “reproduction part” might have been abandoned. The situation in insects is a little bit more complex than what was described in the preceding paragraph, because there are two additional GPCRs that are closely related to the AKH receptor (Fig. 4). These are the receptors for corazonin, a neuropeptide that stimulates heart beat, and AKH/corazonin-related peptide (ACP), whose function is unknown [9,27–29]. Not only are the structures of the three receptors related, but also the peptide structures of their ligands (Fig. 3). Although related, none of the peptide ligands crossreacts with the receptors for the other ligands [9,28]. Thus, this is a clear example of receptor/ligand co-evolution, where an ancestral receptor gene and its ligand gene have been duplicated twice during evolution, yielding three independent neuropeptide/GPCR signaling systems (Fig. 4). 4. What can we learn from these evolutionary insights? From the above-mentioned examples we can see that there can be rapid neuropeptide/receptor co-evolution. This rapid evolution can wash-out clear structural relationships between evolutionarily related ligands (for example GnRH and AKH). For other ligands there are constraints, probably ligand/receptor binding constraints, that prevent such rapid changes (for example, mammalian and insect oxytocin/vasopressin). It is important to understand neuropeptide signaling in protostomes and to understand neuropeptide/GPCR evolution, because this can give us an idea of the way the ancestral receptors and their ligands, existing before the split of Proto- and Deuterostomia, were functioning. Knowing more about the ancestral GnRH (or oxytocin/ vasopressin) receptor, for example, might contribute to a better understanding of the current mammalian receptor. Perhaps we overlook some biological actions for the mammalian neuropeptide/ GPCR couple that are evident in the protostome counterparts. Revealing the biological actions of the protostome neuropeptide/ GPCR counterparts can be done in many ways, but one straightforward approach is the use of RNAi and gene interruption mutants. We expect that the results of such comparative neuropeptide research might be rewarding for biologists and clinicians alike. Acknowledgments We thank Anders Bo Rønnegaard Hansen for typing the manuscript and the Danish Research Agency, Novo Nordisk Foundation, and Carlsberg Foundation for financial support. References [1] Douzery EJ, Snell EA, Bapteste E, Delsuc F, Philippe H. The timing of eukaryotic evolution: does a relaxed molecular clock reconcile proteins and fossils? Proc Natl Acad Sci U S A 2004;101:15386–91. [2] Grimmelikhuijzen CJP, Williamson M, Hansen GN. Neuropeptides in cnidarians. Can J Zool 2002;80:1690–702. [3] Grimmelikhuijzen CJP, Leviev I, Carstensen K. Peptides in the nervous systems of cnidarians: structure, function and biosynthesis. Int Rev Cytol 1996;167:37–89. [4] Leviev I, Grimmelikhuijzen CJP. Molecular cloning of a preprohormone from sea anemones containing numerous copies of a metamorphosis inducing neuropeptide: a likely role for dipeptidyl aminopeptidase in neuropeptide precursor processing. Proc Natl Acad Sci U S A 1995;92:11647–51.
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[5] Umbriaco D, Anctil M, Descarries L. Serotonin-immunoreactive neurons in the cnidarian Renilla koellikeri. J Comp Neurol 1990;291:167–78. [6] Dürrnagel S, Kuhn A, Tsiairis CD, Williamson M, Kalbacher H, Grimmelikhuijzen CJP, Holstein TW, Gründer S. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra. J Biol Chem 2010;285:11958–65. [7] Cottrell GA, Green KA, Davies NW. The neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) can activate a ligand-gated ion channel in Helix neurones. Pflugers Arch 1990;416:612–4. [8] Lingueglia E, Champigny G, Lazdunski M, Barbry P. Cloning of the amiloridesensitive FMRFamide peptide-gated sodium channel. Nature 1995;378:730–3. [9] Hansen KK, Stafflinger E, Schneider M, Hauser F, Cazzamali G, Williamson M, Grimmelikhuijzen CJP. Discovery of a novel insect neuropeptide signaling system closely related to the insect adipokinetic hormone and corazonin hormonal systems. J Biol Chem 2010;285:10736–47. [10] Van Kesteren RE, Smit AB, De Lange RP, Kits KS, van Golen FA, van der Schors RS, De With ND, Burke JF, Geraerts WP. Structural and functional evolution of the vasopressin/oxytocin superfamily: vasopressin-related conopressin is the only member present in Lymnaea, and is involved in the control of sexual behavior. J Neurosci 1995;15:5989–98. [11] Kanda A, Takuwa-Kuroda K, Iwakoshi-Ukena E, Furukawa Y, Matsushima O, Minakata H. Cloning of Octopus cephalotocin receptor, a member of the oxytocin/vasopressin superfamily. J Endocrinol 2003;179:281–91. [12] Levoye A, Mouillac B, Rivière G, Vieau D, Salzet M, Breton C. Cloning, expression and pharmacological characterization of a vasopressin-related receptor in an annelid, the leech Theromyzon tessulatum. J Endocrinol 2005;184:277–89. [13] Stafflinger E, Hansen KK, Hauser F, Schneider M, Cazzamali G, Williamson M, Grimmelikhuijzen CJP. Cloning and identification of an oxytocin/vasopressinlike receptor and its ligand from insects. Proc Natl Acad Sci U S A 2008;105: 3262–7. [14] Brown MR, Crim JW, Arata RC, Cai HN, Chun C, Shen P. Identification of a Drosophila brain-gut peptide related to the neuropeptide Y family. Peptides 1999;20:1035–42. [15] Garczynski SF, Brown MR, Shen P, Murray TF, Crim JW. Characterization of a functional neuropeptide F receptor from Drosophila melanogaster. Peptides 2002;23: 773–80. [16] Nachmann RJ, Holman GM, Haddon WF, Ling N. Leucosulfakinin, a sulfated insect neuropeptide with homology to gastrin and cholecystokinin. Science 1986;234: 71–3. [17] Kubiak TM, Larsen MJ, Burton KJ, Bannow CA, Martin RA, Zantello MR, Lowery DE. Cloning and functional expression of the first Drosophila melanogaster sulfakinin receptor DSK-R1. Biochem Biophys Res Commun 2002;291:313–20. [18] Nothacker H-P, Grimmelikhuijzen CJP. Molecular cloning of a novel, putative G protein-coupled receptor from sea anemones structurally related to members of the FSH, TSH, LH/CG receptor family from mammals. Biochem Biophys Res Commun 1993;197:1062–9. [19] Hauser F, Nothacker H-P, Grimmelikhuijzen CJP. Molecular cloning, genomic organization and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals. J Biol Chem 1997;272:1002–10. [20] Eriksen KK, Hauser F, Schiøtt M, Pedersen K-M, Søndergaard L, Grimmelikhuijzen CJP. Molecular cloning, genomic organization, developmental regulation, and a knock out mutant of a novel Leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster. Genome Res 2000;10:924–38. [21] Hsu SY, Nakabayashi K, Bhalla A. Evolution of glycoprotein hormone subunit genes in bilateral metazoa: identification of two novel human glycoprotein hormone subunit family genes, GPA2 and GPB5. Mol Endocrinol 2002;16:1538–51. [22] Mendive FM, Van Loy T, Claeysen S, Poels J, Williamson M, Hauser F, Grimmelikhuijzen CJP, Vassart G, Vanden Broeck J. Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2. FEBS Lett 2005;579:2171–6. [23] Luo CW, Dewey EM, Sudo S, Ewer J, Hsu SY, Honegger HW, Hsueh AJ. Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2. Proc Natl Acad Sci U S A 2005;102: 2820–5. [24] Van Loy T, Vandersmissen HP, Van Hiel MB, Poels J, Verlinden H, Badisco L, Vassart G, vanden Broeck J. Comparative genomics of leucine-rich repeats containing G protein-coupled receptors and their ligands. Gen Comp Endocrinol 2008;155: 14–21. [25] Hauser F, Søndergaard L, Grimmelikhuijzen CJP. Molecular cloning, genomic organization and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to gonadotropin-releasing hormone receptors from vertebrates. Biochem Biophys Res Commun 1998;249:822–8. [26] Staubli F, Jørgensen TJD, Cazzamali G, Williamson M, Lenz C, Søndergaard L, Roepstorff P, Grimmelikhuijzen CJP. Molecular identification of the insect adipokinetic hormone receptors. Proc Natl Acad Sci U S A 2002;99:3446–51. [27] Roch GJ, Busby ER, Sherwood NM. Evolution of GnRH: diving deeper. Gen Comp Endocrinol 2011;171:1–16. [28] Belmont M, Cazzamali G, Williamson M, Hauser F, Grimmelikhuijzen CJP. Identification of four evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae. Biochem Biophys Res Commun 2006;344: 160–5. [29] Veenstra JA. Isolation and structure of corazonin, a cardioactive peptide from the American cockroach. FEBS Lett 1989;250:231–4.
Contents lists available at SciVerse ScienceDirect
Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Review
Mini-review: The evolution of neuropeptide signaling Cornelis J.P. Grimmelikhuijzen ⁎, Frank Hauser 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
Keywords: Adipokinetic hormone Corazonin GnRH GPCR Neuropeptide Evolution
a b s t r a c t Neuropeptides and their G protein-coupled receptors (GPCRs) have an early evolutionary origin and are already abundant in basal animals with primitive nervous systems such as cnidarians (Hydra, jellyfishes, corals, and sea anemones). Most animals emerging after the Cnidaria belong to two evolutionary lineages, the Protostomia (to which the majority of invertebrates belong) and Deuterostomia (to which some minor groups of invertebrates, and all vertebrates belong). These two lineages split about 700 million years (Myr) ago. Many mammalian neuropeptide GPCRs have orthologues in the Protostomia and this is also true for some of the mammalian neuropeptides. Examples are oxytocin/vasopressin, GnRH, gastrin/CCK, and neuropeptide Y and their GPCRs. These results implicate that protostomes (for example insects and nematodes) can be used as models to study the biology of neuropeptide signaling. © 2012 Elsevier B.V. All rights reserved.
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The evolution of the nervous system and neuropeptide signaling . . . . 3. How does neuropeptide signaling in Proto- and Deutorostomia compare? 4. What can we learn from these evolutionary insights? . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Most animals belong to two lineages of animal evolution, the Proto- and Deuterostomia that split about 700 million years (Myr) ago [1] (Fig. 1). The majority of invertebrates belongs to the Protostomia, while a few invertebrate groups and all vertebrates, such as mammals, belong to the Deuterostomia (Fig. 1). In addition, there are some very basal animal groups, such as cnidarians (sea anemones, corals, jellyfishes) and sponges that evolved long before the split of Proto- and Deuterostomia. The Protostomia contain many more animal species than the Deuterostomia. The arthropods (insects, crustaceans, spiders, and ticks), for example, constitute at least 1.2 million species, which is more than 85% of all animal species living on Earth. In contrast, mammals only comprise 0.3%.
⁎ Corresponding author. E-mail address: [email protected] (C.JP. Grimmelikhuijzen). 0167-0115/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2012.05.001
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An overview using a pie chart representation of the number of animal species (Fig. 2) visualizes how little mammals fill compared to the other animals. Yet, most research, also at this conference, is focused on mammals. Is this strong focus on ourselves and our own animal group (mammals) really justified? Some will say “yes”, because this research on mammals is medically relevant and will help us to cure diseases. Also, this type of research is economically important, because of its close link to the pharmaceutical industry. We agree with all these arguments. Yet, we feel that a focus only on mammals is dangerous and will block both researchers and clinicians from seeing “the whole picture”. Therefore, we would like to invite the reader of this Abstract Book to also look at the other animals.
2. The evolution of the nervous system and neuropeptide signaling Nervous systems probably evolved in the common ancestor of cnidarians, because sponges (Porifera, Fig. 1) do not show any physiological or anatomical signs of a nervous system. The nervous systems in cnidarians are strongly peptidergic. We and others have isolated and
C.J.P. Grimmelikhuijzen, F. Hauser / Regulatory Peptides 177 (2012) S6–S9
S7
Fig. 1. A simplified phylogenetic tree of animal evolution. The number of species is highlighted in green behind each phylum. There are two larger basal phyla, the Porifera (sponges) and Cnidaria (sea anemones, corals, Hydra, jellyfishes) that originated more than 700 Myr ago. Nervous systems probably evolved within the Cnidaria or their close ancestors. About 700 Myr ago newly emerging animal groups split into two evolutionary lineages, the Protostomia (highlighted in blue) and Deuterostomia (highlighted in red).
sequenced a large number of neuropeptides, cloned their preprohormones and located these molecules, using immunocytochemistry and in situ hybridization, in neurons and dense neuronal plexuses in cnidarians [2,3]. A remarkable feature of cnidarian preprohormones is the very high neuropeptide copy numbers contained within the precursors, which can be up to 37 neuropeptide copies [4]. Attempts to demonstrate fast transmitters in cnidarians (Glu, Gly, GABA, acetylcholine, ATP), catecholamines, other biogenic amines, or gasotransmitters (NO, CO, H2S) have failed or have not been convincingly positive with the exception of serotonin, which appears to be produced by some neurons in the sea pansy Renilla köllikeri [5], but not in other cnidarians investigated so far. These data, therefore, show that neuropeptides are the dominant transmitters in cnidarians and also suggest that they played a key role in the first nervous systems that emerged in evolution.
Neuropeptides exert their actions by binding to their specific G protein-coupled receptors (GPCRs), a process that starts a second messenger cascade. Because of the many steps involved, neuropeptide actions are relatively slow. Cnidarians are interesting in this respect, because, although most neuropeptides bind to GPCRs, some neuropeptides in these animals activate ligand-gated ion channels, thus giving fast responses. We have cloned three genes from Hydra magnapapillata that code for degenerin (DEG)/epithelial Na + channel (ENaC) gene family members. When these three genes are coexpressed in frog oocytes, they form a Na + ion channel that is gated by the Hydra neuropeptides Hydra-RFamides I and II. These ion channels have pore properties and amiloride sensitivities that are very similar to the other known DEG/ENaC channels [6]. Neuropeptide-gated ion channels, therefore, may supply cnidarians with fast excitatory neurotransmission in a situation where other fast transmitters and their receptors are not available (i.e. have not been “invented” yet). Amiloride-sensitive FMRFamide-gated Na + channels have also been cloned and characterized from various molluscs [7,8]. Evolutionarily, cnidarians and molluscs are widely separated (Fig. 1), which suggests that all the animals in between (e.g. plathyhelminths, annelids) may also have peptide-gated ion channels. Even in the deuterostome lineage neuropeptide-gated ion channels may be discovered. 3. How does neuropeptide signaling in Proto- and Deutorostomia compare?
Fig. 2. A pie chart representation of present-day animal groups according to their number of species. Arthropods constitute about 85% of all animal species, while for mammals this number is 0.3%.
Because neuropeptides are closely associated with the emergence of the first nervous systems, it is no surprise that neuropeptide signaling still plays a key role in both Proto- and Deuterostomia (Fig. 1). An interesting question is whether there are structural similarities between the neuropeptides/GPCRs in these two animal groups. If yes, these peptide signaling systems should have originated before the split of Proto- and Deuterostomia and be evolutionarily very old (>700 Myr). Also, if yes, mammalian endocrinologists might learn from the protostomes: many of these animals are excellent laboratory models (e.g. the fruitfly Drosophila melanogaster and the nematode Caenorabditis
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Neuropeptide name
Amino acid sequence
AKH ACP (AKH/corazonin-related peptide) Corazonin GnRH-1
Fig. 3. Comparison of the amino acid sequences of mosquito (Anopheles gambiae) AKH (adipokinetic hormone), mosquito ACP (AKH/corazonin-related peptide), mosquito corazonin, and mammalian GnRH-1. Residues that are identical between at least three peptides are highlighted in red. Amino acid residues that are identical between two peptides are highlighted in green. Conserved (but not identical) residues are highlighted in blue. Adapted from [9].
elegans). They are simple, have short generation times (12 days or shorter) and are amenable for genetic manipulations (gene disruption mutants, RNAi). Furthermore, the genomes from many protostomes have been sequenced, which also turns them into potential new models. Within the arthropods alone, for example, more than 50 genomes have recently been sequenced. The large numbers of sequenced genomes from both proto- and deuterostomes strongly facilitate answering the above question. When we look at neuropeptide genes it is often difficult to see whether a mammalian neuropeptide has an orthologue in protostomes, because the neuropeptide part in the preprohormone is often very small and difficult to “hit” when TBLASTN screening of the genome sequences is used as a tool. Also, there has been longterm neuropeptide/GPCR evolution and co-evolution in these
A
B
receptors
Drosophila, Apis, Daphnia, pea aphid
CRZR
mosquitoes, Bombyx, Nasonia, Rhodnius
CRZR
Tribolium
animals, changing the peptide structures dramatically and masking their evolutionary relationships [9]. However, there are a few exceptions. Oxytocin/vasopressin is one such example, where a nearly intact oxytocin/vasopressin-like peptide, and its associated neurophysin part on the preprohormone can be found in many protostomes, including arthropods. These findings are supported by the presence of functional oxytocin/vasopressin receptors [10–13]. Other examples are neuropeptide Y (called neuropeptide F in arthropods) and its receptor [14,15], a sulfated gastrin/CCK-like peptide (called sulfakinin in arthropods) and its receptor [16,17], and glycoprotein hormones related to FSH, LH, and TSH and their leucine-rich repeats containing GPCRs (LGRs). In fact, glycoprotein hormones and their LGRs are ubiquitous in proto- and deuterostomes and are even occurring in cnidarians showing that they are essential signaling systems already associated with the earliest nervous systems [18–24]. Although it is often difficult to find the protostome orthologue of a mammalian neuropeptide, it is much easier to do so for the neuropeptide GPCRs, which is mainly due to their much larger size, increasing the chances to find a “hit” using TBLASTN. When we started our insect work 15 years ago, we were very much interested in insect reproduction and in the question, whether it was related to mammalian reproduction. Finding a GnRH peptide gene orthologue in insects was impossible. However, we could readily find and clone a Drosophila orthologue of the GnRH receptor [25]. When we subsequently expressed the receptor in Chinese hamster ovary cells and isolated the ligand from Drosophila extracts, using a cell-based bioassay, we were very much surprised that this was not a GnRH-related peptide, but adipokinetic hormone (AKH), a well-known insect neuropeptide that mobilizes carbohydrates and lipids from the insect fat body during flight [26]. AKH has about the same size as GnRH, but it has hardly any amino acid residues in common with GnRH (Fig. 3). What does this example of the GnRH receptor show us? (i) It shows that the GnRH receptor already originated before the split
AKHR
CRZ
ACPR
AKHR
CRZ
ACPR
AKHR
ligands AKH
ACP
AKH
ACP
AKH
Fig. 4. Scenario for receptor/ligand co-evolution, leading to insect receptors that are specific for either AKH, ACP, or corazonin. A. Receptors for corazonin (CRZR), ACP (ACPR), and AKH (AKHR). These receptors are structurally related (indicated by the white parts not involved in ligand binding), but also have specific differences (indicated by the colored parts that bind their specific ligands). It is assumed that the three receptors have a common ancestor (bottom), which also is the ancestor of the GnRH receptor. The ancestor gene duplicated and, after mutations, these duplications gave rise to two receptors that are the ancestors of the corazonin and ACP/AKH receptors. This last receptor gene duplicated once again. B. The receptor ligands corazonin (CRZ), ACP, and AKH. These ligands have some amino acid residues in common (indicated by the white parts) but also some specific differences (indicated by the colored parts). It is assumed that these ligands have a common ancestor, which also might be the ancestor for GnRH. The gene duplication scenario is the same as in A. It is unknown whether the ligands or receptors duplicated first, but these duplications must have had time windows that overlap (= coevolution between A and B). This figure has been originally published in The Journal of Biological Chemistry ([9], © the American Society for Biochemistry and Molecular Biology).
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of Proto- and Deuterostomia, hence that it is more than 700 Myr old; (ii) it also shows that the ancestral ligand, which probably had a mixture of the AKH and GnRH amino acid sequences, has changed considerably during proto- and deuterostome evolution; (iii) the ancestral GnRH signaling system might have controlled carbohydrate and lipid release, reproduction, or both. Of course we do not know (and we will never know), but our intuition tells us that it might be both. During deuterostome evolution, the “release part” might have been lost, while during protostome evolution, the “reproduction part” might have been abandoned. The situation in insects is a little bit more complex than what was described in the preceding paragraph, because there are two additional GPCRs that are closely related to the AKH receptor (Fig. 4). These are the receptors for corazonin, a neuropeptide that stimulates heart beat, and AKH/corazonin-related peptide (ACP), whose function is unknown [9,27–29]. Not only are the structures of the three receptors related, but also the peptide structures of their ligands (Fig. 3). Although related, none of the peptide ligands crossreacts with the receptors for the other ligands [9,28]. Thus, this is a clear example of receptor/ligand co-evolution, where an ancestral receptor gene and its ligand gene have been duplicated twice during evolution, yielding three independent neuropeptide/GPCR signaling systems (Fig. 4). 4. What can we learn from these evolutionary insights? From the above-mentioned examples we can see that there can be rapid neuropeptide/receptor co-evolution. This rapid evolution can wash-out clear structural relationships between evolutionarily related ligands (for example GnRH and AKH). For other ligands there are constraints, probably ligand/receptor binding constraints, that prevent such rapid changes (for example, mammalian and insect oxytocin/vasopressin). It is important to understand neuropeptide signaling in protostomes and to understand neuropeptide/GPCR evolution, because this can give us an idea of the way the ancestral receptors and their ligands, existing before the split of Proto- and Deuterostomia, were functioning. Knowing more about the ancestral GnRH (or oxytocin/ vasopressin) receptor, for example, might contribute to a better understanding of the current mammalian receptor. Perhaps we overlook some biological actions for the mammalian neuropeptide/ GPCR couple that are evident in the protostome counterparts. Revealing the biological actions of the protostome neuropeptide/ GPCR counterparts can be done in many ways, but one straightforward approach is the use of RNAi and gene interruption mutants. We expect that the results of such comparative neuropeptide research might be rewarding for biologists and clinicians alike. Acknowledgments We thank Anders Bo Rønnegaard Hansen for typing the manuscript and the Danish Research Agency, Novo Nordisk Foundation, and Carlsberg Foundation for financial support. References [1] Douzery EJ, Snell EA, Bapteste E, Delsuc F, Philippe H. The timing of eukaryotic evolution: does a relaxed molecular clock reconcile proteins and fossils? Proc Natl Acad Sci U S A 2004;101:15386–91. [2] Grimmelikhuijzen CJP, Williamson M, Hansen GN. Neuropeptides in cnidarians. Can J Zool 2002;80:1690–702. [3] Grimmelikhuijzen CJP, Leviev I, Carstensen K. Peptides in the nervous systems of cnidarians: structure, function and biosynthesis. Int Rev Cytol 1996;167:37–89. [4] Leviev I, Grimmelikhuijzen CJP. Molecular cloning of a preprohormone from sea anemones containing numerous copies of a metamorphosis inducing neuropeptide: a likely role for dipeptidyl aminopeptidase in neuropeptide precursor processing. Proc Natl Acad Sci U S A 1995;92:11647–51.
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