neuropeptide S system

neuropeptide S system

General and Comparative Endocrinology xxx (2014) xxx–xxx Contents lists available at ScienceDirect General and Comparative Endocrinology journal hom...

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General and Comparative Endocrinology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen

Evolutionary history of the neuropeptide S receptor/neuropeptide S system Ravisankar Valsalan, Narayanan Manoj ⇑ Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India

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Article history: Available online xxxx Keywords: Neuropeptide S Neuropeptide S receptor Evolution Gene duplication Coevolution

a b s t r a c t The neuropeptide S receptor (NPSR) belongs to the G protein-coupled receptor (GPCR) superfamily and is activated by the neuropeptide S (NPS). Although recently discovered, the vertebrate NPSR–NPS system has been established as an important signaling system in the central nervous system and is involved in physiological processes such as locomotor activity, wakefulness, asthma pathogenesis, anxiety and food intake. The availability of a large number of genome sequences from multiple bilaterian lineages has provided an opportunity to establish the evolutionary history of the system. This review describes the origin and the molecular evolution of the NPSR–NPS system using data derived primarily from comparative genomic analyses. These analyses indicate that the NPSR–NPS system and the vasopressin-like receptor–vasopressin/oxytocin peptide (VPR–VP/OT) system originated from a single system in an ancestral bilaterian. Multiple duplications of this ancestral system gave rise to the bilaterian VPR–VP/ OT system and to the protostomian cardioacceleratory peptide receptor–cardioacceleratory peptide (CCAPR–CCAP) system and to the NPSR–NPS system in the deuterostomes. Gene structure features of the receptors were consistent with the orthology annotations derived from phylogenetic analyses. The orthology of the peptide precursors closely paralleled that of the receptors suggesting an ancient coevolution of the receptor–peptide pair. An important challenge for the coevolution hypothesis will be to establish the molecular and structural basis of the divergence between orthologous receptor–ligand pairs in this system. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Neuropeptides are a group of diverse peptides that modulate and control neuronal signaling and activity in the eumetazoans. Most neuropeptides elicit response by binding to the transmembrane spanning G protein-coupled receptors (GPCRs). The neuropeptide S receptor (NPSR) is the cognate receptor for the endogenous 20 amino acid neuropeptide S (NPS) (Sato et al., 2002). The highly conserved peptide was named so because of a conserved N-terminal serine residue (human NPS sequenceSFRNGVGTGMKKTSFQRAKS) is encoded as a prepropeptide and is expressed in isolated cells of the amygdala and the dorsomedial hypothalamic nucleus and particularly confined to specific regions of the brainstem including the peri-locus coeruleus (LC) area, the principal sensory trigeminal nucleus and the lateral parabrachial nucleus (Liu et al., 2011; Xu et al., 2004, 2007). NPSR mRNA is widely expressed throughout the central nervous system (CNS), ⇑ Corresponding author. Fax: +91 44 22574102. E-mail addresses: [email protected] (R. Valsalan), [email protected] (N. Manoj).

particularly in regions in the brain that are associated with regulation stress response, memory, the olfactory system and regulation of arousal (Leonard and Ring, 2011; Reinscheid et al., 2005a; Xu et al., 2007). The NPSR (formerly GPR154) was first identified as an asthma susceptibility gene (Gloriam et al., 2005; Laitinen et al., 2001, 2004). Subsequently, several other physiological functions for the NPSR–NPS system were discovered, although the biochemical and physiological roles of the system are still not well understood (Xu et al., 2004). Functionally, dispensation of NPS produces strong anxiolytic-like behavior while increasing wakefulness at the same time (Cannella et al., 2009; Jungling et al., 2008; Kallupi et al., 2010; Meis et al., 2008; Paneda et al., 2009). Furthermore, the system modulates locomotor activity and enhances spatial memory and produces anti-nociceptive effects to thermal stimuli in mice (Beck et al., 2005; Duangdao et al., 2009; Han et al., 2009; Li et al., 2009; Pape et al., 2010; Peng et al., 2010; Rizzi et al., 2008; Xu et al., 2004). Studies have also demonstrated an anorectic effect of the NPS on food intake in rats and mice. This effect was influenced by NPSR antagonists confirming the role of the NPS– NPSR system (Cifani et al., 2011; Peng et al., 2010). Among the multiple isoforms of human NPSR products resulting from alternative

http://dx.doi.org/10.1016/j.ygcen.2014.05.011 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: Valsalan, R., Manoj, N. Evolutionary history of the neuropeptide S receptor/neuropeptide S system. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.05.011

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splicing, the seven transmembrane topology is encoded by three isoforms, two of which constitute functional receptors that are trafficked to the cell membrane (Laitinen et al., 2004; Vendelin et al., 2005). Furthermore, several single nucleotide polymorphisms (SNPs) and isoforms in the human NPSR gene are associated with risks of asthma, bronchial hyper-responsiveness, immunological disorders such as rhinoconjunctivitis, respiratory distress syndrome and irritable bowel syndrome (Bernier et al., 2006; D’Amato et al., 2007; Kormann et al., 2005; Melen et al., 2005; Nepomuceno et al., 2010; Pulkkinen et al., 2006; Reinscheid et al., 2005b). Potent NPSR antagonists thus have potential for multiple clinical applications for the treatment of obesity, hypersomia and anxiety disorders (Camarda et al., 2009; Li et al., 2009; Okamura et al., 2008; Ruzza et al., 2010). Recent studies reporting large scale comparative genomic and phylogenetic analyses of peptide and corresponding receptor genes strongly suggest conservation and common evolutionary origin of a large fraction of peptidergic systems in the earliest urbilaterian ancestor which had a nervous system capable of integrating complex sensory information. While the relationships between several receptor groups are unambiguous, the homology of the corresponding peptides is less clear since it is likely that the peptides may have arisen independently on multiple occasions. However, in the general case, the peptide and receptor genes appear to have coevolved in the different lineages leading to present day metazoans (Hoyle, 2011; Janssen et al., 2008, 2010; Jekely, 2013; Kim et al., 2010; Lindemans et al., 2009, 2011; Liu et al., 2008; Mirabeau and Joly, 2013; Park et al., 2002). The NPS and its cognate receptor is a recently deorphanized system and the origin and evolution of the NPSR–NPS system has been unclear. The system, until recently was thought to have first evolved in the tetrapods (Reinscheid, 2007). Furthermore, earlier comparisons of the human NPSR with other vertebrate GPCRs showed that the vertebrate vasopressin-like receptors are phylogenetically the closest group and hence the receptor was first named vasopressin receptorrelated receptor (VRR1). Following this, Gupte et al. (2004) showed typical GPCR signaling by a V1AR/NPSR chimeric receptor, which contained the human V1A receptor with all three intracellular loops (ICLs) and C terminus replaced by those of NPSR. The chimera based on receptor homology was functionally responsive to vasopressin and showed dual signaling by coupling to both Gq and Gs pathways, consistent with the activation of NPSR by NPS (Gupte et al., 2004). Furthermore, chemogenomic analyses of human non-olfactory GPCRs also indicated that the NPSR and the vasopressin-like receptors are clustered in a strongly supported clade, distinct from the other peptide receptors (Gloriam et al., 2009; Surgand et al., 2006). Subsequent comparative genomic analyses of many different species have now allowed for the resolution of the origin and divergence of the NPSR–NPS system in the eumetazoans (Elphick, 2012; Jekely, 2013; Mirabeau and Joly, 2013; Pitti and Manoj, 2012). This review aims to focus on the molecular evolution of the NPSR–NPS system with emphasis on the origin, divergence and evolutionary history of this receptor/ligand pair.

2. The phylogeny of neuropeptide S receptor orthologs Sequences for 40 tetrapod NPSRs have been identified from genomes of mammals, birds, reptilian and amphibian species. NPSR orthologs were not detected in available fish genomes including the lobe-finned fish coelacanth (actinistia), the ray-finned fishes (actinoptergyii), the elephant shark (Callorhinchus milii), a representative of the earliest jawed vertebrates and the sea lamprey (Petromyzon marinus), a jawless fish that represents the basal vertebrates. Furthermore, the NPSR was absent in the urochordates and in the basal eumetazoans including cnidaria, porifera and

placozoa. Genome searches also failed to identify the NPSR in the protostomes, including mollusks, nematodes, annelids and arthropods. Of particular interest is the identification of NPSR orthologs in the cephalochordate (Branchiostoma floridae) and in the ambulacrarian species including the hemichordate (Saccoglossus kowalevskii) and the echinoderm (Strongylocentrotus purpuratus) (Pitti and Manoj, 2012). The transmembrane regions of the lancelet (B. floridae), acorn worm (S. kowalevskii) and sea urchin (S. purpuratus) NPSR-like sequences share about 56%, 46% and 36% identity, respectively, to the equivalent region in human NPSR (Table S1, Data S1). To the best of our knowledge, only a single subtype of the NPSR/NPSR-like exists. The NPSR sequences showed strongest homology to the crustacean cardioacceleratory peptide receptors (CCAPR), followed by the vasopressin-like receptors (sequence identity between the human NPSR and the fruit fly CCAPR 44%). The CCAPR homologs are cognate receptors of the CCAP nonapeptide and are exclusive to the protostomians. Among the lophotrochozoans, homologs are present in the mollusk and annelid phyla and is absent in the platyhelminthes. In the ecdysozoa, the CCAPR is present only in the arthropods and is lost in the lineage leading to the nematodes (Table S1). Unlike the NPSR-like receptors, CCAPRs are well characterized, particularly in insects. The CCAPR is expressed in all developmental stages, with the highest expression in adult heads. Additionally, the role of the CCAPR/CCAP system in cardiac control and interruption of ecdysis behavior have been reported (Arakane et al., 2008; Belmont et al., 2006; Cazzamali et al., 2003; Estevez-Lao et al., 2013; Lee et al., 2013; Li et al., 2011). Fig. S1 presents a multiple sequence alignment of representative NPSR/NPSR-like/CCAPR sequences. A detailed analysis of the structure of the receptor and comparisons of conserved amino acid residues and sequence motifs that may play important roles in the function of the NPSR/NPSR-like/CCAPR proteins has been reported (Pitti and Manoj, 2012). For instance, structure– activity studies indicate that Asp1052.61 in human NPSR could play a direct role in agonist binding by ionic interactions with the conserved Arg residue at position 3 in the NPS peptide (Clark et al., 2010; Okamura et al., 2008). A negatively charged residue, at this position is completely conserved in all homologs except in frog NPSR where it is substituted by Asn. Substitution of residue N1072.63 to Ile is associated with asthma susceptibility in humans. Studies have shown that the N107I mutation led to an increase in potency and maximal efficacy of NPS due to higher levels of cell surface receptor expression of the mutant compared with wild type receptor (Reinscheid et al., 2005b). Interestingly, the sequence alignment indicates that the human NPSR is an exception among all tetrapods known to date with an Asn at this position whereas it is Ile in all other orthologs (Fig. S1). Phylogenetic reconstruction employing the Bayesian and Maximum likelihood methods were used to investigate the orthology of the predicted NPSR homologs across multiple lineages. In light of early studies which indicated strong homology of the tetrapod NPSR to the vertebrate vasopressin-like receptor, a large dataset of diverse peptide receptor sequences from both deuterostomian and protostomian species were used for the phylogenetic analyses. These included the family of vasopressin-like receptor subtypes (i.e., vertebrate vasopressin 1A (V1AR), vasopressin 1B (V1BR), vasopressin 2 (V2R), the oxytocin (OTR) receptors and their orthologs), the CCAPR, the Gonadotropin-releasing hormone receptor (GnRHR) orthologs and several related neuropeptide receptors (Pitti and Manoj, 2012). The trees constructed using both methods revealed good agreement in the topology of the well-supported clades containing mixed sets of protostomian and deuterostomian sequences. Tests for statistical robustness included nonparametric bootstrapping and Bayesian posterior probabilities. The trees had two major clades with the vasopressin-like receptor, CCAPR and NPSR clustered into one group (100% confidence value) and all other peptide receptors

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forming the second cluster, serving as an outgroup (Figs. 1, S2 and S3). This phylogenetic grouping has been consistently observed in subsequent large scale comparative analyses of the metazoan peptidergic receptor families (Jekely, 2013; Mirabeau and Joly, 2013). These results established the unambiguous orthology of the vertebrate NPSR and invertebrate NPSR-like and the protostomian CCAPR and indicated that the ancestral gene to this group is in fact of bilaterian origin. Functional ligand cross reactivity studies are needed to provide additional support for the assumption of orthology of NPSR/NPSR-like/CCAPR proteins. Furthermore, the strong association of the vasopressin-like receptors with the NPSR/ NPSR-like/CCAPR group (100% confidence value) revealed the tight evolutionary relationship between these two groups of receptors

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suggesting that these two groups most likely evolved from a common bilaterian receptor gene (Jekely, 2013; Mirabeau and Joly, 2013). Based on this assumption, a detailed analysis using position-specific rate shift variation had identified several putative amino acid residues that likely contributed to the functional divergence between the vertebrate NPSRs and the vasopressin-like receptors (Gu, 1999; Pitti and Manoj, 2012). 3. Gene structure analysis of the receptors Conservation of gene structure features have been used to support phylogenetic analyses of orthologous genes and in families of paralogous genes and protein superfamilies (Carmel et al., 2007;

Fig. 1. Phylogenetic relationship of the NPSR, CCAPR, GnRHR and vasopressin-like receptors from multiple bilaterian phyla. Bayesian tree of amino acid sequences from tetrapod NPSR (red), invertebrate NPSR-like receptor (purple), protostomian CCAPR (orange), bilaterian vasopressin-like receptors (V1AR/V1BR/OTR/V2R/VPR in green), bilaterian Gonadotropin releasing hormone receptor GnRHR (blue) sequences. The tree was generated using the Bayesian approach in MrBayes 3.1.2 using JTT+F+I+G model (Ronquist and Huelsenbeck, 2003). Bayesian posterior probabilities used as confidence values of tree branches are marked as a percentage value. Nodes were compressed to represent the metazoan lineages. Scale bar represents the number of estimated changes per site for a unit of branch length. The maximum likelihood tree of the same set of receptors showed identical topology and similar support for clusters using a bootstrap measure and is shown in Fig. S3. (Stamatakis, 2006). The receptor abbreviations, names, common and binomial names of the species and accession numbers of the sequences are listed in Table S1. In this figure and in Fig. 2, the sequence names marked with ⁄ and + symbols represent manually corrected sequences at the N terminus and C terminus, respectively. Sequence names marked with à symbol represent fragmented sequences.

Please cite this article in press as: Valsalan, R., Manoj, N. Evolutionary history of the neuropeptide S receptor/neuropeptide S system. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.05.011

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Sanchez et al., 2003). A comparative analysis of the genomic structures of the NPSR, NPSR-like and CCAPR genes strongly supported the receptor orthology hypothesis (Mirabeau and Joly, 2013; Pitti and Manoj, 2012,). Fig. 2 illustrates the distribution, position and phase of introns within representative amino acid sequences (Fig. S1). In general, the NPSR genes are composed of 9 coding exons. The introns are named as I to VIII according to the inserted positions in human NPSR to enable comparisons. The invertebrate NPSR-like sequences are composed of 7 or 8 exons. The CCAPRs contain a mostly conserved set of 6 to 9 exons (Cazzamali et al., 2003). Intron V appears to have been inserted in the common ancestor of vertebrates and lancelet, after branching off from the lineage leading to the acorn worm. The intron corresponding to VIII is mostly conserved across CCAPR, NPSR-like and NPSR genes with deletion occurring in the lancelet and the marine worm. However, this intron has undergone phase change in acorn worm and water flea. The intron corresponding to position VI is conserved in the vertebrates and appears to have evolved independently in the lancelet and acorn worm. Similarly, the insertion of introns V and VI in the sea urchin appear to be independent events. Other variations between the NPSR-like and NPSR genes include phase change in introns I and VI in lancelet. The few intron loss or gain events that occur appear to be lineage-specific events. The remarkable conservation of intron positions and phases is in line with the orthology of the NPSR/NPSR-like/CCAPR group derived from the phylogenetic analyses. Further, it follows that this receptor group shares an ancestral gene whose origin dates back to a bilaterian ancestor, concomitant with the emergence of the vasopressin-like receptors. The vasopressin-like receptors are mostly composed of 2 to 3 exons. The position of the single intron in human V1AR is the most conserved among this group of receptors (Gimpl and Fahrenholz, 2001; Kanda et al., 2006; Rozen et al., 1995; Zingg, 1996). This position is about 25 residues away from intron VII of

the human NPSR. Thus, conserved intron gain relative to the vasopressin-like receptors appears to be a common feature of the NPSR/ NPSR-like/CCAPR group of receptors. Alternatively, the vasopressin-like receptor lineage may have originated as a consequence of retrotranscription of an mRNA encoding a NPSR/NPSR-like/ CCAPR ancestor and subsequently gaining its single intron. The relatively large number of introns in this group suggests their involvement in exon shuffling, alternative splicing events and transcriptional regulatory events (Majewski and Ott, 2002; Qiu et al., 2004; Sverdlov et al., 2005). The biological functions of alternatively spliced variants of GPCRs are unclear (Schwarz et al., 2000; Vanetti et al., 1993). For instance, truncated variants of GnRHR isoforms have been shown to inhibit the signaling and transport of the wild-type receptor (Wang et al., 2001). In the human NPSR, two alternatively spliced forms show different pharmacological profiles (Vendelin et al., 2005). An analysis of the gene order of the chromosomal region containing the NPSR gene revealed the expected conserved synteny of the NPSR gene loci across the vertebrates. However, the NPSRlike and CCAPR genes did not display conserved synteny to the NPSR (Pitti and Manoj, 2012). 4. Orthology of the peptides Next, we examine the orthology of the peptide ligands using the receptor orthology as a guide. Direct orthology hypothesis for homologous peptides is often limited by the poor statistical support for analyses using short peptide sequences. The human NPS precursor is 89 amino acids long and contains features typical of a neuropeptide precursor, including a hydrophobic signal peptide followed by a linker and the mature peptide (20 amino acids) preceded by a pair of basic amino acids (Lys, Arg) that serve as a protease cleavage site (Data S2). NPS precursor sequences were first

Fig. 2. Conservation of intron positions and phases in the NPSR orthologs. Schematic of the intron positions and phases mapped onto multiple alignment of amino acid sequences from representative NPSR, NPSR-like, CCAPR and human vasopressin-like receptor subtypes is shown. The 0, 1 and 2 phase introns are marked with blue, red and orange lines, respectively. Introns corresponding to human NPSR are named I to VIII according to their mapped positions in the amino acid sequence. The representatives include tetrapod NPSRs, invertebrate deuterostome NPSR-like sequences from sea urchin (echinoderm), acorn worm (hemichordate), lancelet (cephalochordate), the protostomian CCAPRs from (water flea, mosquito and fruit fly – arthropods), limpet (mollusk) and marine worm (annelid) and the human vasopressin-like receptors. Intron positions were deemed conserved across the alignment for small changes in intron positions (±10 amino acid residues) (Betts et al., 2001). Receptor length in amino acid residues is shown for each sequence.

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reported to be present in all vertebrates with the exception of the fish genomes (Reinscheid, 2007). The NPS peptide coding sequence is highly conserved with few variations in the sequence. Structure–activity relationship studies using Ala and D-amino acid scans have demonstrated that the Nterminal region of the human peptide is crucial for biological activity. In particular, studies performed on Phe2, Arg3, and Asn4 revealed the chemical requirements of these positions for NPSR binding and activation. Furthermore, the sequence Gly5-Val6-Gly7 seems to be important for shaping the bioactive conformation of the peptide (Camarda et al., 2008a,b; Reinscheid et al., 2005a,b; Roth et al., 2006; Tancredi et al., 2007). Furthermore, in vivo studies have confirmed the antagonist action of NPS derived peptides in mouse models (Guerrini et al., 2009). The NPS were found only in tetrapods, including mammals, birds, reptiles, marsupials and amphibian species (Table S2). Homologs of NPS precursor proteins were not detected in several deuterostomian lineages. These include the available genomes of fish and lower chordates and invertebrates, including urochordate (Ciona intestinalis), cephalochordate (B. floridae), hemichordate (S. kowalevskii) and echinoderm (S. purpuratus). The peptide is also absent in the protostomian genomes that include the mollusks, arthropods, annelids and nematodes and in the eumetazoan cnidarian (Nematostella vectensis) genome. Thus it appeared that the NPS gene first evolved in tetrapods, concurrent with the appearance of the terrestrial vertebrates. Interestingly, the discovery of a novel family of neurophysinassociated neuropeptide (NG peptide) precursors in deuterostomian invertebrates provided the missing evolutionary link between the vertebrate NPS and their putative invertebrate homologs (Elphick, 2010). The NG peptide so called because of a conserved sequence motif NG, shares strong sequence similarity to the functionally critical N-terminal region of the NPS peptide (Fig. 3A). Genes encoding the NG peptide precursors comprise a signal peptide, one or more copies of NGFFFamide-like peptide followed by a C-terminal neurophysin (Np) domain. The NG peptides were identified in the echinoderm S. purpuratus (sea urchin NGFFFamide), in the hemichordate S. kowalevskii (acorn worm NGFYNamide) and in the cephalochordate B. floridae (amphioxus SFRNGVamide), but were absent in the vertebrates, urochordates, protostomes or the cnidarians (Table S2, Data S2, Fig. S4). Furthermore, no genome contained both the NPS and NG peptides. Taken together, the data suggested that the NG peptide may in fact be the invertebrate ortholog of the vertebrate NPS in the deuterostomes. This scenario also requires invoking the loss of genes encoding the NPS/NG peptide precursor in multiple lineages including the urochordates and in the fish genomes (i.e., agnathans, chondrichthyes, teleosts and actinistia). The Np domain was until recently thought to be uniquely associated with the vasopressin/oxytocin-type peptides and is required for biosynthesis of these neuropeptides (De Bree and Burbach, 1998; Elphick, 2010). It is noteworthy that the acorn worm, amphioxus and sea urchin NG peptide precursors and the vasopressin/ oxytocin-like peptide precursors share a common domain organization that comprises the neuropeptide domain followed by a C-terminal Np domain although the NG peptide is structurally unrelated to the cyclic, amidated vasopressin/oxytocin-like peptide (Fig. 4). The origin of the neurophysin-containing vasopressin/oxytocin-like peptide precursors and their receptor systems can be dated back to a common ancestor of bilaterian animals, 640–760 million years ago (Aikins et al., 2008; Gruber and Muttenthaler, 2012; Kawada et al., 2008; Stafflinger et al., 2008; Van Kesteren et al., 1992). In contrast, the occurrence of the neurophysin containing NG peptide precursors indicates the presence of the gene at least as far as the common ancestor of the deuterostomes. Thus, the association of neurophysin with vasopressin/

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oxytocin-like peptides appears to be evolutionarily more ancient than that in the NG peptide precursor. One interpretation for the origin of the NG peptide precursor gene involved the fusion of the neurophysin-encoding exon from a vasopressin/oxytocin-like gene with a gene containing a signal peptide-encoding exon and a NG peptide-encoding exon in a deuterostomian ancestor (Elphick, 2010; Elphick and Rowe, 2009; Rowe and Elphick, 2012; Semmens et al., 2013). However, Mirabeau and Joly (2013) propose that this domain association pattern indicates a case where the NG peptide precursors and the vasopressin/oxytocin-like precursors probably shared a common ancestral precursor protein in an ancestral bilaterian animal. Their interpretation is founded on the hypothesis of a common origin of the bilaterian NPSR/NPSR-like/ CCAPR group and the vasopressin-like receptors based on receptor phylogeny. Additional support for this hypothesis comes from the fact that genes encoding the NG peptide precursor (Joint Genome Institute (JGI) ID: 84803) and the vasopressin/oxytocin-like precursor (JGI ID: 84802) are located in tandem in the B. floridae genome indicating that they arose through a local duplication event (Mirabeau and Joly, 2013; Semmens et al., 2013). This pattern has not been observed in other available genomes. However, unlike the NG peptide precursor, the NPS precursor lacks the C-terminal neurophysin domain and appears to have lost the domain. This observation supports an evolutionary model wherein the ancestral deuterostomian gene encoding the NPS/NG peptide precursor comprised a NPS/NG peptide domain and a Cterminal neurophysin domain but the neurophysin encoding exon was lost in the lineage that gave rise to the NPS in tetrapods. More recently, the discovery of a sea cucumber (echinoderm) NG peptide precursor that lacks the neurophysin domain suggests that the neurophysin domain in the NG peptide precursor is dispensable (Elphick, 2012) (Fig. 4). The functional significance of the neurophysin domain in the NG peptide biosynthesis requires further examination. Comparative studies, including phylogenetic analyses have now clearly established the orthology of the deuterostomian NPS/NG peptide and the protostomian CCAP peptide implying a common ancestor of bilaterian origin (Jekely, 2013; Mirabeau and Joly, 2013). The CCAP is a cyclic nonapeptide and is involved in increasing heart rate, regulating ecdysis behavior and modulating oviduct contractions in arthropods (Donini et al., 2001; Park et al., 2003; Tublitz, 1989). Among the several protostomian genomes available to date, CCAP is present in the lophotrochozoan mollusk and annelid phyla but is missing in the platyhelminthes. Furthermore, in the ecdysozoans the peptide is retained in the arthropods and is absent in the nematodes. Interestingly, analysis of three ant (A. cephalotes, C. floridanus and H. saltator) whole genome shotgun (WGS) sequences suggests that the CCAP is lost in ants compared to other insects (Gruber and Muttenthaler, 2012). Although the similarity of the primary sequence of the two peptides within the deuterostomian and protostomian lineages is strong, the sequence similarity across the lineages is limited. However, shared motifs can be identified (Fig. 3A). For example, the shared K-R-x-F-x-N motif between the NPS and CCAP sequences, the shared R-N-G-x-(1-3)-G-(1)-K(KR) motif between NPS and NG sequences. There is no discernible homology at the level of primary sequence of the vasopressin/oxytocin peptides to the NPS/NG peptide/CCAP group although the CCAP and vasopressin/oxytocin peptides share some common molecular features. The mature peptides are both C-terminally amidated, intramolecular disulfide bridged nonapeptides. However, the positions of the Cys residues and the sequences are different (CCAP – PFCNAFTGC, vasopressin – CYFQNCPRG). The vasopressin/oxytocin peptides all contain an N-terminal cyclic 6residue structure stabilized by the disulfide bond and a flexible C-terminal tail whereas the CCAP contains a C-terminal cyclic 7residue structure and an N-terminal tail. An intriguing feature is

Please cite this article in press as: Valsalan, R., Manoj, N. Evolutionary history of the neuropeptide S receptor/neuropeptide S system. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.05.011

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Fig. 3. (A) Multiple alignment of representative NPS, NG and CCAP peptide sequences. Comparison of the conserved region of the amino acid sequences highlights specific sequence motifs. The shared K-R-x-F-x-N motif between the NPS and CCAP sequences and the shared R-N-G-x-(1–3)-G-(1)-K-(KR) motif between NPS and NG sequences. Common names, scientific names and the taxonomic class of the species included in the alignment are: human (Homo sapiens, mammalia), orangutan (Pongo abelii, mammalia), chimpanzee (Pan troglodytes, mammalia), mouse (Mus musculus, mammalia), duck (Anas platyrhynchos, aves), chicken (Gallus gallus, aves), lancelet (Branchiostoma floridae, cephalochordata), acorn worm (Saccoglossus kowalevskii, hemichordata), sea urchin (Strongylocentrotus purpuratus, echinodermata), star fish (Asterias rubens, echinodermata), sea cucumber (Apostichopus japonicus, echinodermata), fruit fly (Drosophila melanogaster, arthropoda), cockroach (Periplaneta americana, arthropoda), hornworm (Manduca sexta, arthropoda), capitella (Capitella teleta, annelida) and sea snail (Conus villepinii, mollusca). B) Multiple alignment of the mature CCAP and vasopressin peptide sequences. The vasopressin sequence is shown in reverse orientation of the peptide chain. Comparison of amino acid sequences highlights specific conserved residues.

the inverse sequence similarity of the CCAP peptide to the vasopressin peptide wherein four of the six conserved residues in vasopressin are identical to that in CCAP (Stangier et al., 1987) (Fig. 3B). It remains to be seen if this similarity (but in retro-direction) between the two sequences is indicative of evolutionary or functional relationships. The gene structure of the NPS precursor is conserved in all species. The gene is encoded by three exons with the first two exons coding for the signal peptide and the third exon coding for the remaining peptide. A phylogenetic analysis of the proteins displayed the expected clustering wherein sequences from the closest related species cluster together. The gene structure of the three NG peptide precursors is conserved with all three genes interrupted by two conserved introns positions (Elphick, 2010). 5. Evolutionary history of the NPSR–NPS system A remarkable feature of the evolution of the NPSR–NPS system is an indication of the deep bilaterian ancestry of the NPSR/NPSRlike/CCAPR/vasopressin-like receptors. While this pattern for the receptors is directly revealed by phylogenetic and cluster analyses, the more diverse peptide ligand sequences did not show obvious homologous relationships at the level of primary structure. However, existence of conserved domains outside the peptide region helped deduce the homology (Jekely, 2013; Mirabeau and Joly, 2013; Pitti and Manoj, 2012). The combined analysis of the receptor/peptide system provides an unambiguous picture of the origin, evolutionary history and divergence of the system (Fig. 5).

In this model, firstly the ancestral receptor–peptide system that contained the receptor VPR/OTR+NPSR/NPSR-like/CCAPR gene and the (VP/OT+NPS/NG/CCAP)-Np precursor gene first appeared in an ancestral bilaterian and duplicated to the bilaterian VPR/OTR–(VP/ OT)-Np system and the NPSR/NPSR-like/CCAPR-(NPS/NG/CCAP)Np system genes. The names VPR/OTR and VP/OT are used to denominate the vasopressin-like receptors and vasopressin/oxytocin peptides, respectively and all peptide denominations described in this section represent the primary structure of the precursor gene. The VPR/OTR–(VP/OT)-Np system independently duplicated in the protostomes and deuterostomes giving rise to a large family of diverse receptor/peptide systems. The evolution of this system in the vertebrates involves a complex pattern of multiple duplications and reciprocal losses in different vertebrate classes (Ocampo Daza et al., 2012; Lagman et al., 2013; Yamaguchi et al., 2012). For instance, the vertebrate receptor family consists of six ancestral members among which four receptors have been retained in mammals. The system has also been identified in several classes of invertebrates, such as mollusks, annelids, nematodes and arthropods. However, it is evident that not all invertebrate species contain the system. Lineage specific loss has been reported in some homometabolous insect species including the honey bee, fruit fly, mosquito and silkworm (Gruber and Muttenthaler, 2012; Gruber, 2014). Secondly, a duplication of the bilaterian NPSR/NPSR-like/ CCAPR-(NPS/NG/CCAP)-Np system must have occurred, giving rise to the CCAPR/CCAP system in the protostomes and the NPSR/NPSRlike-(NPS/NG)-Np system in the deuterostomes. Lineage specific loss of the system is observed in the platyhelminthes and nematodes in the protostomian lineage. A third duplication occurred in the NPSR/NPSR-like-(NPS/NG)-Np system in an ancestor of extant deuterostomes resulting in the emergence of the NPSRlike/NG-Np system in the invertebrate deuterostomes and the NPSR/NPS system in the tetrapods. Lineage specific loss of the system has occurred in the urochordates and in the basal vertebrates including the agnatha, chondrichthyes, teleosts and actinistia. Interestingly, the neurophysin domain is lost independently in the protostomian CCAP peptide and in the tetrapod NPS peptide. Additionally, a species specific loss of the Np domain in the sea cucumber NG peptide precursor is also seen, indicating that the function of the Np-domain in the NG peptide precursor may be dispensable unlike that in the VP/OT-Np peptides (De Bree and Burbach, 1998; Elphick, 2012) (Fig. 4). Multiple lines of evidence including the cluster studies of Jekely, 2013 and the phylogenetic analyses of Mirabeau and Joly (2013) and Pitti and Manoj (2012) point to the fact that the evolutionary history of the NPSR–NPS system represents a clear case of coevolution of receptors and peptides. Additional evidence in support of this hypothesis comes from observation of the concomitant lineage specific loss of the peptide and receptor genes in the nematodes, platyhelminthes and urochordates and in basal vertebrates including agnathans, chondrichthyes, teleosts and the actinistia. Furthermore, no lineage appears to have more than one ortholog of the receptor/peptide system suggesting the one-ligand-one receptor model of functional regulation of this neuropeptide signaling system in the metazoans. In particular, answers to several questions are of interest. Firstly, is the NPSR-like sequence the cognate receptor of the NG peptide? Secondly, what is the functional role of the neurophysin domain in the NG peptide precursor? Thirdly, is there definitive demonstration of functional cross reactivity of the receptors and peptide orthologs across the lineages to support the receptor– ligand coevolution hypothesis in this system? If so, what are the shared and divergent structural features across the ligand and the receptor sequences that contribute to the cross-reactivity? Several established examples of GPCR/peptide coevolution in the ver-

Please cite this article in press as: Valsalan, R., Manoj, N. Evolutionary history of the neuropeptide S receptor/neuropeptide S system. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.05.011

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Fig. 4. Domain organization of peptide precursors. Representatives of peptide precursors include tetrapod NPS, deuterostomian invertebrate NG, protostomian CCAP and bilaterian VP/OT. N-terminal signal peptides are shown in yellow, NG peptides are shown in cyan, NPS peptides are shown in red, CCAP peptides are shown in purple, VP peptides are shown in orange and neurophysin domains (Np) are shown in green. The X symbol indicates loss of the Np domain in the sequence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. A proposed scheme for the evolutionary history of the neuropeptide S receptor/neuropeptide system in the bilateria. The scheme is presented as a cladogram. The VPR/ OTR+NPSR/NPSR-like/CCAPR and the (VP/OT+(NPS/NG/CCAP))-Np precursor genes first appeared in an ancestral bilaterian and duplicated to the bilaterian VPR/OTR–(VP/OT)Np and the NPSR/NPSR-like/CCAPR–(NPS/NG/CCAP)-Np system genes. The names VPR/OTR and VP/OT are used to denominate the vasopressin/oxytocin-like receptor and vasopressin/oxytocin peptides, respectively and all peptide denominations described in this section represent the primary structure of the precursor gene. Independent duplications of the VPR/OTR–(VP/OT)-Np system in the protostomes and deuterostomes gave rise to a large family of homologous vasopressin-like receptor/peptide systems. A second duplication of a bilaterian NPSR/NPSR-like/CCAPR–(NPS/NG/CCAP)-Np system gave rise to the CCAPR/CCAP system in the protostomes and the NPSR/NPSR-like– (NPS/NG)-Np system in the deuterostomes. A third duplication occurred in the NPSR/NPSR-like–(NPS/NG)-Np system in an ancestor of extant deuterostomes resulting in the emergence of the invertebrate NPSR-like/NG-Np system and the NPSR/NPS system in the tetrapods. Lineage specific loss of orthologous systems have occurred in multiple lineages and is marked by an X. Independent loss of the neurophysin domain has occurred in the protostomian CCAP peptide and in the tetrapod NPS peptide. ⁄The VPR/OTR– (VP/OT)-Np system in arthropods has been lost in several insect species including honey bee, fruit fly, mosquito and silkworm. #The CCAP peptide is lost in the ant species.

Please cite this article in press as: Valsalan, R., Manoj, N. Evolutionary history of the neuropeptide S receptor/neuropeptide S system. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.05.011

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tebrates and in the insects is known (Dreborg et al., 2008; Hansen et al., 2010; Hoyle, 1999; Park et al., 2005; Roch et al., 2011; Sundstrom et al., 2010). Support for the coevolution hypothesis of the NPSR–NPS system across the bilaterian lineages requires evidence from complete characterization of the system involving comparisons with additional basal deuterostome genomes, structure–activity studies, relevant activation assays, as well as in vivo studies. 6. Conclusion In the last several years, remarkable progress has been achieved in the characterization of receptor–neuropeptide systems using reverse pharmacology methods. These studies have paralleled the increasing number of genome sequencing projects. Combination of data from both these sources has enhanced our knowledge of the orthology of multiple receptor–peptide systems, both within lineages and across diverse lineages. An evolutionary history of the NPSR–NPS system has been constructed using cluster and phylogenetic analyses. Data suggests common ancestry of the NPSR/ NPS system and the VPR/OTR–VP/OT system in an ancestral bilaterian. The molecular evolution of this ancestral system has been mainly shaped by duplication and divergence giving rise concurrently to the diverse bilaterian VPR/OTR–VP/OT system and to the NPSR/NPSR-like–NPS/NG peptide systems in the deuterostomes and the CCAPR–CCAP system in the protostomes. Other events include lineage specific gene loss of the system and lineage specific loss of the Np domain in the peptide precursors. Acknowledgments We acknowledge infrastructural support from the Bioinformatics Infrastructure Facility, supported by the Department of Biotechnology (DBT), Government of India. This work was supported by a grant (BT/PR11748/BID/07/285/2008) from the DBT, Government of India. We thank Thejkiran Pitti and Infant Sagayaraj for their assistance with the manuscript preparation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ygcen.2014. 05.011. References Aikins, M.J., Schooley, D.A., Begum, K., Detheux, M., Beeman, R.W., Park, Y., 2008. Vasopressin-like peptide and its receptor function in an indirect diuretic signaling pathway in the red flour beetle. Insect Biochem. Mol. Biol. 38, 740– 748. Arakane, Y., Li, B., Muthukrishnan, S., Beeman, R.W., Kramer, K.J., Park, Y., 2008. Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum. Mech. Dev. 125, 984–995. Beck, B., Fernette, B., Stricker-Krongrad, A., 2005. Peptide S is a novel potent inhibitor of voluntary and fast-induced food intake in rats. Biochem. Biophys. Res. Commun. 332, 859–865. Belmont, M., Cazzamali, G., Williamson, M., Hauser, F., Grimmelikhuijzen, C.J., 2006. Identification of four evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae. Biochem. Biophys. Res. Commun. 344, 160–165. Bernier, V., Stocco, R., Bogusky, M.J., Joyce, J.G., Parachoniak, C., Grenier, K., et al., 2006. Structure–function relationships in the neuropeptide S receptor: molecular consequences of the asthma-associated mutation N107I. J. Biol. Chem. 281, 24704–24712. Betts, M.J., Guigo, R., Agarwal, P., Russell, R.B., 2001. Exon structure conservation despite low sequence similarity: a relic of dramatic events in evolution? EMBO J. 20, 5354–5360. Camarda, V., Rizzi, A., Ruzza, C., Zucchini, S., Marzola, G., Marzola, E., et al., 2009. In vitro and in vivo pharmacological characterization of the neuropeptide S

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Please cite this article in press as: Valsalan, R., Manoj, N. Evolutionary history of the neuropeptide S receptor/neuropeptide S system. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.05.011