The role of G protein-coupled receptors in the early evolution of neurotransmission and the nervous system

G protein-coupled receptors (GPCRs) constitute the largest superfamily among membrane bound proteins that control key physiological functions, such as, neurotransmission, hormone releases, and immune responses among others (Katritch et al., 2013; Lagerström and Schiöth, 2008; Rosenbaum et al., 2009). As such, the large range of integral transmembrane proteins in this family respond to subsequent, diverse array of ligands: including neurotransmitters, hormones, lipids, and odorants (Civelli et al., 2013; Shoichet and Kobilka, 2012). GPCRs in mammals are classified into the five main families according to the GRAFS classification: glutamate, rhodopsin, adhesion, frizzled and secretin (Fredriksson et al., 2003). These five GPCR families are associated with various physiological functions, but the roles of the glutamate and the rhodopsin families are particularly well documented for neurotransmission and regulation of the nervous system (Niswender and Conn, 2010; Schoepp, 2001). Some functions include synaptic transmission, synapse formation, axon guidance and development of neuronal circuits (Collingridge et al., 2004; Hnasko and Edwards, 2012). Of the five GPCR families, the rhodopsin family is the largest, serving as molecular targets for the neurotransmitters serotonin, dopamine, acetylcholine, histamine, adrenaline and norepinephrine. GPCRs in this family also react to several neuropeptides, such as somatostatin, melanocortins, neuropeptide Y and neuropeptide FF. Similarly, glutamate GPCRs are molecular targets for the principal excitatory neurotransmitter of the central nervous system – glutamate. Upon activation, glutamate GPCRs crucially modulate synaptic transmission and neuronal excitability throughout the central nervous system (Cartmell and Schoepp, 2000; Ferraguti and Shigemoto, 2006; Niswender and Conn, 2010; Pinheiro and Mulle, 2008). Adhesion GPCRs are a growing research field, and several of these receptors have shown an important role in the CNS (Araç et al., 2012a; Langenhan et al., 2013; Strokes and Piao, 2010). For example, the brain-specific angiogenesis inhibitor (BAI) subfamily is involved in the control of synaptogenesis (Duman et al., 2013; Stephenson et al., 2014), whereas the latrophilins have been implicated in the control of synaptic transmission (O'Sullivan et al., 2012; O'Sullivan et al., 2014) and GPR56 is associated with brain development (Jeong et al., 2012; Luo et al., 2011; Singer et al., 2013).

GPCRs are well represented in several primitive metazoans, as well as in some pre-metazoan species (de Mendoza et al., 2014; Krishnan et al., 2012; Nordström et al., 2011). Interestingly, several of the pre-bilaterian metazoans, which lack most of the cell types that are commonly found in the bilaterians, still constitute a rich repertoire of GPCRs (Patel, 2012; Technau and Steele, 2011). In this work, we focus on four model organisms that are important for understanding the evolution of the nervous system, which also encode a large number of GPCRs: Amphimedon queenslandica, Trichoplax adhaerens, Nematostella vectensis and the ctenophore Mnemiopsis leidyi. A. queenslandica is a demosponge belonging to an ancient group of animals that diverged from other metazoans over 600 million years ago. T. adhaerens is a placozoan, which represents one of the simplest, free-living animals, whereas N. vectensis, a primitive animal that, along with corals, jellyfish and hydras, is contained in the oldest eumetazoan phylum, the cnidaria. The most common feature of these ancient metazoans is their lack of most the cell types found in bilaterians. For example, A. queenslandica are simple pore-bearing animals that lack a gut, nervous system and muscles (Degnan et al., 2008; Fahey and Degnan, 2010; Leys and Hill, 2012; Srivastava et al., 2010). T. adhaerens is a flat animal that lacks nerves, sensory cells and muscle cells (Ender and Schierwater, 2003; Miller and Ball, 2005; Schierwater, 2005; Srivastava et al., 2008). However, N. vectensis is regarded as one of the earliest organisms in animal evolution possessing a nervous system (Putnam et al., 2007; Technau and Steele, 2011). N. vectensis constitutes an ectodermal and endodermal nerve net making it a simple and diffuse nervous system that runs throughout the animal's body (Marlow et al., 2009; Nakanishi et al., 2012). Also, recently, the ctenophore (M. leidyi) genome has been sequenced and the organism also possesses a peripheral subepithelial nerve net, neurons and nerves associated with the tentacles (Jager et al., 2011; Richter and King, 2013; Ryan et al., 2013). Here, we analyse the large expansions of GPCRs, and present the evidence accumulated so far for the presence of neurotransmitters and their roles in early metazoans.

Understanding the evolutionary origins of the nervous system and neuronal gene families requires an accurate estimation of basal metazoan evolution and precise positioning of pre-bilaterian species (Moroz et al., 2014). The most current phylogenetic analysis based on molecular data places the cnidarians just basal to the bilateral animals (Putnam et al., 2007; Technau and Steele, 2011) (Fig. 1). Likewise, the placozoans are placed basal to cnidarians, whereas the sponges are considered as a sister lineage to all extant animals (Srivastava et al., 2008; Srivastava et al., 2010). Until recently, it was widely accepted that sponges form the earliest diverging and evolving metazoan lineage (a sister group to the rest of Metazoa) and this was supported by their morphological simplicity lacking most cell types. However, the positioning of sponges as basal to all animals is currently being challenged by a recent hypothesis using transcriptomic and genomic data from ctenophore M. leidyi (Ryan et al., 2013) (Fig. 1). This is surprising because there is a general understanding that morphological complexity evolved over time and that the sponges are morphologically simpler in comparison with the ctenophore, which possess nerve and muscle cells. Nevertheless, there is additional support from the genomic analysis of another ctenophore Pleurobrachia bachei, which places ctenophore as the most basal animal lineage using molecular phylogenetic analysis (Moroz et al., 2014). This topology is also supported by 10 additional transcriptomes from other ctenophores (Moroz et al., 2014). These results provide new concepts for understanding the origins of neural systems and also provide support for theories purporting independent evolution of nerve nets in both the common ancestor of ctenophores and in cnidarian/bilaterian ancestors (Moroz et al., 2014). This scenario is an alternative to another hypothesis, which argues that the first nerve cell originated close the origin of the cnidarians (Holland et al., 2013). Lastly, another hypothesis proposes a single origin of the neural system at the early metazoan ancestor with major losses and/or gains of sophisticated cell types, including nerve and muscle cells, during the course of the metazoan evolution. Additional genomes and a coherent phylogenetic approach using several molecular markers are likely to provide more data to test the robustness of these hypotheses (Nosenko et al., 2013).

Recent studies based on large-scale genomic analyses have shown that these primitive metazoans possess complex GPCR-mediated signalling systems (Fig. 1) (de Mendoza et al., 2014; Nordström et al., 2011). One of the most notable events is a vast expansion of the rhodopsin family at the origin of metazoans. The ctenophore M. leidyi contains around 600 genes coding for rhodopsin-like GPCRs, whereas the sponges and placozoans have ~150 and 400 genes, respectively (Fig. 1). The cnidarians, which are placed basal to the bilaterians and are known to have a nervous system, have the largest repertoire of GPCRs in pre-bilaterians, with a remarkable expansion of over 1000 genes coding for rhodopsin-like GPCRs. In addition to the rhodopsins, adhesion GPCRs expanded with highly diverse N-terminal functional regions, ranging from short N-termini with no domains, to very large N-termini containing multiple cadherin, calx-β, scavenger receptor cysteine-rich (SRCR) domain, hormone receptor (HRM) domain and immunoglobulin domain repeats. Furthermore, these early metazoans contain expansions of the glutamate family of GPCRs, which possess conserved N-terminal functional domains, including a ligand-binding domain (the so called Venus flytrap domain) and a cysteine-rich domain (CRD or NCD3G).

Genome-wide analysis and similarity-search approaches suggest that M. leidyi contains rhodopsin family receptors that have their top five closest hits as serotonin, dopamine and adrenergic receptors from bilaterians (Fig. 2). However, immunohistochemical and pharmacological data suggest that ctenophores do not use serotonin, acetylcholine, dopamine, noradrenaline, adrenaline, octopamine, histamine or glycine as neurotransmitters. Indeed, ctenophores lack any of the enzymes necessary to synthesise these conventional low molecular mass neurotransmitters (Moroz et al., 2014; Ryan et al., 2013). This is surprising because sponges have neurotransmitter-synthesising enzymes, although they lack the nerve-net-like environment found in ctenophores. A recent study, which attempted an extensive analysis on eight transcriptomes from all sponge classes, found that dopamine-β-hydroxylase, tryptophan hydroxylase, DOPA decarboxylase and phenylalanine hydroxylase are all present in sponges, and may be found across all sponge transcriptomes (Riesgo et al., 2014) (Fig. 2). Interestingly, Tethya wilhelma, a demosponge, uses chemical messengers, including serotonin, to induce rhythmic body contractions (Ellwanger and Nickel, 2006). Another study also reported serotonin-derived alkaloids in sponges (Salmoun et al., 2002). Furthermore, sponges have non-motile cilia that line the inner epithelium of the osculum (an excretory structure that regulates the water flow) and these cilia function as a sensory organ to coordinate whole-animal responses (Ludeman et al., 2014). Although sponges may harbour the molecular machinery required for synthesis of neurotransmitters, it is rather complicated to provide evidence for neurotransmitter signalling in sponges, because they lack a nervous system, nerve net or nerve cells. This may explain the differential roles for some of the conventional neurotransmitters in a nerve-cell-free environment.

A closer inspection of T. adhaerens revealed that the primitive metazoan also contains a few neurotransmitter-synthesising enzymes (Fig. 2). Components of T. adhaerens neurotransmitter biosynthesis include DOPA decarboxylase and dopamine-β-hydroxylase (Srivastava et al., 2008). Also, enzymes for proneuropeptide (pNP) processing, maturation and secretion have been identified in T. adhaerens (Jékely, 2013). A similarity search approach suggests that T. adhaerens contains receptors that have their top hits as bilaterian serotonin, dopamine, histamine, adrenergic, neuropeptide Y and neuropeptide FF receptors (Fig. 2). This is consistent with the whole genome report, which suggested the presence of putative neurotransmitter- and neuropeptide-binding receptors (Srivastava et al., 2008). Furthermore, it was recently discovered that T. adhaerens has four insulin genes, five neuropeptide precursors and most of the enzymes required for peptide processing, including the prohormone convertase, carboxypeptidase and amidating monooxygenase (Nikitin, 2014). Additionally, two leucine-rich repeat-containing GPCRs (LGRs) were found in T. adhaerens. However, glycoprotein hormones, the known ligands for LGRs, were absent (Nikitin, 2014). Although T. adhaerens contains several GPCRs, including the peptide and amine binding receptors, attempts to build phylogenetic trees and resolve their relationships with cnidarian and bilaterian GPCRs suggest that the T. adhaerens rhodopsins have diverged considerably and seem to have undergone lineage-specific diversification (Nikitin, 2014).

There is ample evidence that acetylcholine, glutamate, γ-aminobutyric acid (GABA), glycine, epinephrine, norepinephrine, dopamine and serotonin transmitters, as well as neuropeptides, are involved in cnidarian neurotransmission (Kass-Simon and Pierobon, 2007; Watanabe et al., 2009) (Fig. 2). Among these, the neuropeptide RFamide family of GPCRs is the most studied among the cnidarians. RFamide-positive neurons were found in N. vectensis, Hydra, Hydractinia and jellyfish, and they are found in all classes of cnidarians (Watanabe et al., 2009). Moreover, the components for neurotransmitter biosynthesis in cnidarians are more complete when compared with the other pre-bilaterians. Choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase, tryptophan hydroxylase, DOPA decarboxylase, dopamine-β-hydroxylase are all present in N. vectensis and other cnidarians (Anctil, 2009) (Fig. 2). Apart from the experimental verification for some of the neurotransmitters and neuropeptides, bioinformatic analyses found neuropeptide receptors similar to NPY, NPFF, galanin, tachykinin, GnRH/vasopressin-like, melanocortin, insulin-like and glycoprotein hormone-like receptors (Anctil, 2009). Furthermore, chordate-like olfactory receptors (ORs), a family of chemosensory receptors belonging to the large rhodopsin-like GPCRs, were discovered in N. vectensis. The presence of epithelial sensory cells supports the idea that the N. vectensis OR-like receptors may play a crucial role in sensing the environment (Churcher and Taylor, 2011; Marlow et al., 2009; Nakanishi et al., 2012; Niimura, 2009). These findings provide insights that the pre-bilaterians exhibit a GPCR-mediated sensory system and suggested that the genetic complexity commonly assumed to have arisen much later in animal evolution is actually an ancestral feature.

Functional roles of some of these neurotransmitters and neuropeptides are also demonstrated. Neuropeptides Hym-53, Hym-355 and CGLW amide trigger the oocyte maturation and spawning in hydrozoan jellyfish (Takeda et al., 2013). The GLW amide was shown earlier to have a role in muscle contraction and metamorphosis in Hydra (Takahashi et al., 2003). Similarly, another neuropeptide, Hym-176, induces ectodermal muscle contraction in Hydra (Yum et al., 1998). Also, FRamides in Hydra magnipapillata induce elongation as well as contraction of the body (Hayakawa et al., 2007). Thus, these neuropeptides are probably involved in regulating muscle contraction in cnidarians (Fujisawa, 2008; Grimmelikhuijzen et al., 1996; Takahashi, 2013). Furthermore, the roles of non-peptidergic neurotransmitters have been characterised in cnidarians. Serotonin is involved in metamorphosis of the hydrozoans Phialidium gregarium and Eudendrium racemosum (McCauley, 1997; Zega et al., 2007). And, the neurotransmitter glycine affects the endodermal pacemakers and thereby regulates elongation and contraction of the hydra body column (Ruggieri et al., 2004). Similarly, electrophysiological experiments show that glutamate and GABA and their receptors are involved in pacemaker activity in Hydra (Kass-Simon et al., 2003). Lastly, the amino acid transmitters, GABA, glycine and glutamate are implicated in the coordination of feeding response in Hydra.

Although studies suggest roles for neurotransmitters in cnidarians, the molecular functions of the pre-bilaterian rhodopsin GPCRs are still obscure. A current challenge is to understand the functional implications of the expansion of rhodopsin GPCRs in sponges and placozoans. Studying this topic is, however, difficult because sponges and placozoans are not readily suitable for laboratory purposes. Similarly, the functions of the most ancient rhodopsin GPCRs in ctenophores have not yet been characterised. Systematic cross-genome comparative analysis and robust phylogenetic analysis can serve as an initial step to explore the relationships between cnidarians and other bilaterian GPCRs. Based on the current evolutionary hypothesis, which argues for independent origins of the nerve cells in ctenophores and in a cnidarian/bilaterian ancestor, it could be possible that the rhodopsin family expanded independently in these pre-bilaterians and may have diverse functions based on the morphological characteristics of the organism. Also, considering the fact that rhodopsin shares a common ancestor before the divergence of metazoans, it is possible that these large expansions may have evolved to perform neuronal functions in ctenophores and cnidarians, and that this ability is secondarily lost in sponges and placozoans during the course of metazoan evolution. Further comparative genomics, as well as developmental or neurobiological studies would lead to a better understanding and reconcile the conjectures over the origins and evolution of metazoan neurotransmission. For this purpose, expansion of the rhodopsin family GPCRs could serve as one of the essential datasets to further understand and study the origins of the nervous system and neurotransmission from a GPCR perspective.

The functions of adhesion GPCRs in pre-bilaterian organisms are mostly unknown. Therefore, we approached the question of the roles of pre-bilaterian adhesion GPCRs by considering adhesion GPCRs in vertebrate model organisms and searching for putative adhesion orthologues or adhesion genes with similar domain compositions in the genomes of pre-bilaterian metazoans. This is a conventional approach, and has been used in practice to provide insights into the possible roles of genes in distant organisms. For example, the composition of the ursynapse and protosynapse (Emes and Grant, 2012; Emes et al., 2008; Ryan and Grant, 2009), as well as genetic components at the early origins of multicellularity (Brooke and Holland, 2003; Ruiz-Trillo et al., 2007), were previously inferred by comparing the gene repertoires of the distant metazoans with vertebrates and invertebrates (Brooke and Holland, 2003; Ruiz-Trillo et al., 2007). We primarily address the emerging roles of the adhesion GPCRs in vertebrate model organisms and compare the pre-bilaterian adhesion genes with a focus on the characteristics of N-terminal domains.

Cell–cell adhesion plays an integral role in synapse formation and development, synaptogenesis and synaptic plasticity (Bukalo and Dityatev, 2012; Dalva et al., 2007; Li and Sheng, 2003; Washbourne et al., 2004; Yamagata et al., 2003). The most extensively described cell adhesion molecules in synaptic transmission and formation are neurexins, neuroligins, cadherins and members of the immunoglobulin (Ig) superfamily (Craig and Kang, 2007; Dean and Dresbach, 2006; Lisé and El-Husseini, 2006; Scott and Palmer, 1993; Shapiro et al., 2007; Takeichi, 2007). Unlike the classical cell–cell adhesion molecules, the adhesion GPCRs are not characterised by their roles in regulation and/or development of the nervous system. However, in the past ten years, progress has been made in exploring the neurobiological roles of the adhesion GPCRs in mammals and other vertebrates. For example, among the nine adhesion GPCR subfamilies (see Fig. 2), subfamily I latrophilins are well known for their roles in synapse development and triggering neurotransmitter release. The presynaptic latrophilin 1 (LPHN1) interacts with the postsynaptic teneurin-2 (also known as Lasso, an endogenous ligand of latrophilin 1), forming a trans-synaptic complex and thus affecting synaptic development (Boucard et al., 2014; Silva et al., 2011). And, latrophilins interact with the postsynaptic fibronectin leucine-rich transmembrane (FLRT) family members. The ectodomains of the latrophilin 3 (LPHN3) and FLRT3 interact with high affinity in a ‘trans’ configuration and regulate development of excitatory synapses (O'Sullivan et al., 2012). In addition, latrophilin-1interacts with the postsynaptic neurexin I-α and -β, forming a trans-synaptic complex and thus modulating synaptic functions (Boucard et al., 2012; O'Sullivan et al., 2014; Südhof, 2001).

The brain-specific angiogenesis inhibitor family (BAIs; subfamily VII adhesion GPCRs) members are well characterised for their roles in synaptogenesis, involving regulation of synapses and synaptic density (for a review, see Stephenson et al., 2014). Recently, Duman and colleagues provided evidence that BAI1 can regulate synaptogenesis by coupling with Rac1, mediated by a specific interaction and recruitment of the Par3/Tiam1 polarity proteins to postsynaptic sites (Duman et al., 2013). Also, the C-terminal region of BAI1 interacts with PDZ-domain-containing proteins, including MAGI-2 and MAGI-3 (Stephenson et al., 2013). PDZ domains are protein-interaction domains that are concentrated in the post-synaptic density and regulate synaptic transmission (Kim and Sheng, 2004). Furthermore, the N-termini of BAIs encodes thrombospondin type 1 repeats (TSRs), which regulate synaptogenesis and neural development (Adams and Tucker, 2000; Risher and Eroglu, 2012). These TRS repeats act as major interacting regions for BAI interaction partners, including the C1q-like proteins (Bolliger et al., 2011), which are highly expressed in the CNS and aid in synapse formation (Yuzaki, 2010). Gene knockout studies also suggest a role in synaptogenesis for other BAI family members (Jeong et al., 2006; Lanoue et al., 2013; Okajima et al., 2011).

Cadherin EGF LAG seven-pass G-type receptors (CELSRs), or alternatively, 7TM-cadherins/flamingo, constitute the subfamily III adhesion GPCRs and comprise large N-termini (~2500–3500 amino acids) containing cadherin, EGF and laminin G (LAG) domain repeats. CELSRs play a crucial role in nervous system development and planar cell polarity (Boutin et al., 2012; Feng et al., 2012; Tissir and Goffinet, 2006), and are also involved in axonal development and dendritic patterning (Berger-Müller and Suzuki, 2011; Tissir et al., 2005). The CELSR homologue in Drosophila was demonstrated to play a crucial role in synaptogenesis, as well as for the survival of axons and synapses (Bao et al., 2007). In humans, mutations in CELSR1 lead to one of the most severe neural tube defects called craniorachischisis (Tissir and Goffinet, 2006). Moreover, knockout of CELSR2 and CELSR3 in mice leads to hydrocephalus (a lethal condition that accumulates excessive cerebrospinal fluid in the brain), suggesting that CELSR2 and CELSR3 are required for the development and function of ependymal cilia (Tissir et al., 2010). In addition, GPR56, which belongs to subfamily VIII is another adhesion GPCR that is involved in brain development (Singer et al., 2013). In humans, a mutation in GPR56 causes bilateral frontoparietal polymicrogyria (BFPP) syndrome, a condition where the cerebral cortex is malformed (Piao et al., 2004). Also, very large G-protein-coupled receptor 1 (VLGR1, subfamily IX) is highly expressed during embryonal neurogenesis in mouse (McMillan et al., 2002).

In comparison with their closest unicellular metazoan relatives, such as choanoflagellates and filastereans, the adhesion GPCRs expanded significantly in early metazoan lineages. Among all the pre-bilaterians, sponges, with a remarkable number of 72 genes, encode the largest repertoire of adhesion GPCRs. Similarly, N. vectensis and T. adhaerens harbour about 60 and 67 genes, respectively, whereas the ctenophore M. leidyi contains 26 genes coding for adhesion GPCRs (Fig. 1). It seems that adhesion GPCRs expanded significantly after the origin of metazoans (de Mendoza et al., 2014). One possible explanation is that expansion of adhesion GPCRs soon after the origin of metazoans was driven by the evolution of multicellularity, because cell–cell adhesion is one of the major factors involved in driving multicellularity (Abedin and King, 2010; Brooke and Holland, 2003; Müller, 2003; Ruiz-Trillo et al., 2007).

A BLAST search against the Swiss-Prot database using pre-bilaterian adhesion GPCRs as queries revealed that the ancient adhesion GPCRs are more similar to subfamilies I and VIII (Fig. 2.), which are associated with neurobiological roles in vertebrates (see above). Moreover, the VLGR1-like receptor (subfamily IX) is present in N. vectensis (Nordström et al., 2009) and a few receptors from T. adhaerens had their top hits as CELSRs (subfamily III) in the BLAST search. In an unpublished study, we attempted to classify these ancient adhesion GPCRs by performing a comparative phylogenetic analysis with the classified adhesion GPCRs from vertebrates. The unrooted topology obtained, based on the alignment of conserved seven transmembrane (7TM) regions, suggested that the pre-bilaterian adhesion GPCRs lack one-to-one orthologous relationships with known vertebrate homologues. However, we observed that a few sponge adhesion GPCRs are placed basal to human (used as a vertebrate representative) subfamily VIII adhesion GPCRs, and that a few adhesion GPCRs from T. adhaerens, N. vectensis and A. queenslandica are placed on the same node that clustered human subfamilies I and II. Although this phylogenetic clustering does not necessarily imply that these adhesion GPCRs have roles analogous to the mammalian homologues, it could be interesting to explore whether the ancestral Group I-, II- and VIII-like adhesion GPCRs in the pre-bilaterians are associated with neurobiological functions.

One of the most notable features of the adhesion GPCRs is that each subfamily has a unique pattern of N-terminal domain architecture (Langenhan et al., 2013). This molecular signature is often used as a marker to distinguish between the subfamilies as well as to hypothesise the potential functional roles of the receptors (Schiöth et al., 2010). For instance, N. vectensis encodes an adhesion GPCR containing cadherin domains, EGF and laminin G (LAG) domain repeats similar to the bilaterian CELSRs (Fig. 3). However, T. adhaerens, which lacks nerve or sensory cells, contains a CELSR-like homologue, with its signature cadherin and LAG domain repeats, implying that these ancient CELSR-like adhesions possess multiple roles, depending upon the physiology and behaviour of the organism. N. vectensis also has a VLGR1-like homologue, with multiple calx-β repeats, EPTP and laminin_G_3 repeats in their N-termini, sharing high sequence similarity with the vertebrate VLGR1 receptors. Interestingly, among the pre-bilaterians analysed in this study, the sponge adhesion GPCRs encode the most varied domain architecture: at least 11 of these contain a hormone receptor domain (HRM), although no hormones have been found in sponges (Fig. 3). Furthermore, seven sponge adhesion GPCRs have immunoglobulin domain repeats, and another two contain multiple repeats of the SRCR domain (Fig. 3). To the best of our knowledge, SRCR repeats are not found in other GPCRs, whereas SRCR repeats are often found in proteins associated with immune system functions (Martínez et al., 2011; Sarrias et al., 2004). In contrast to the sponges and other pre-bilaterians, M. leidyi adhesion GPCRs are relatively limited in the number of N-terminal domains and the majority of receptors contain only the 7TM domain and the GPS site. However, domains such as pentaxin, epimerase and GCC2 are present, to name a few.

Another important characteristic of adhesion GPCRs is the presence of a GPCR autoproteolysis-inducing (GAIN) domain located proximal to the first transmembrane helix (Fig. 3). The GAIN domain encompasses about 320 amino acids and contains the GPS (GPCR-proteolytic site) motif, a stretch of about 40 residues embedded within the large domain (Araç et al., 2012b). The GAIN domain mediates autoproteolytic events that trigger cleavage-based bipartite segmentation of adhesion GPCRs, which leads to the dissociation of the N-terminal fragment from the C-terminal fragment (Araç et al., 2012b). This intra-molecular processing at the GPS site in the GAIN domain is attributed to several factors, including signalling and membrane trafficking, as well as for the formation of heterodimeric GPCR complexes. The GAIN domain is evolutionarily ancient and is found in the amoebozoa Dictyostelium discoideum, the ciliate Tetrahymena thermophila and the choanoflagellate Monosiga brevicollis (Araç et al., 2012b). In pre-bilaterians, the GAIN domain seems to be present in about 50% of adhesion GPCRs, whereas the others lack a GAIN domain region, including the long N-terminus.

Recent studies suggest that vertebrate family I and II adhesion GPCRs interact with post-synaptic single-pass transmembrane proteins. Therefore, we wanted to explore whether pre-bilaterians comprise homologues of single transmembrane helix post-synaptic proteins. Interestingly, a recent evolutionary analysis on teneurins, an endogenous ligand of latrophilins (see above), suggested that these single-pass transmembrane proteins, which are involved in neurogenesis, were absent in the genomes of sponges, cnidarians, placozoans and ctenophores (Tucker et al., 2012). This implies that the pre-bilaterian adhesion GPCRs that are similar to family I do not have ligands or interactions partners analogous to those in vertebrates. Surprisingly, the pre-synaptic neurexins that are well renowned for their role in synaptogenesis, are found in pre-bilaterians, including the cnidarians (Putnam et al., 2007; Riesgo et al., 2014), placozoans (Srivastava et al., 2008), sponges (Riesgo et al., 2014) and ctenophores (Moroz et al., 2014; Ryan et al., 2013). Furthermore, to investigate whether putative homologues of fibronectin leucine-rich transmenbrane (FLRT) family are present in early metazoans, we performed a comprehensive search in the genomes of eumetazoans, placozoans, sponges and ctenophores. We identified several proteins with LRR repeats and a single transmembrane helix at the C-terminal end, but they are divergent from FLRT family members, indicating that the FLRT family is likely to be specific to bilaterians. Given that these early metazoans are rich in single-pass membrane proteins that constitute a substantial proportion of their membrane proteome, it would be interesting to explore whether these proteins interact with the adhesion GPCRs to aid cell-cell adhesion and communication affecting synapse formation and development.

In vertebrates, the glutamate family of receptors predominantly includes the metabotropic glutamate receptors (GRMs) and GABABRs, which are best characterised for their roles in modulation of synaptic transmission and neuronal excitability throughout the central nervous system (Niswender and Conn, 2010). Homologues of these neuromodulatory receptors are found in all metazoans, several pre-metazoan lineages, and also in the last eukaryotic common ancestor (LECA), suggesting that glutamate GPCRs have roles beyond their functions in a nervous system (de Mendoza et al., 2014; Krishnan et al., 2012). Although glutamate is largely known to be present in neurons and acts as the principal excitatory neurotransmitter, several lines of evidence suggest that glutamate and its receptors (including the GRMs) are present in a variety of non-excitable cells (for a review, see Nedergaard et al., 2002). For example, glutamate and GABA are both found in sponges that are often known to lack epithelial or sensory cells, or general tissue-level organisation. Early evidence for the presence of glutamate receptors in sponges was provided a decade ago when a metabotropic glutamate/GABA-like receptor was cloned from Geodia cydonium and was demonstrated to affect the concentrations of intracellular calcium upon activation by L-glutamate (Perovic et al., 1999). Recently, Elliott and Leys showed that glutamate triggers the contraction of the sponge canal system, whereas GABA inhibits glutamate-induced contraction, forming the synchronised behaviour of the incurrent and excurrent canal system of the demosponge Ephydatia muelleri (Elliott and Leys, 2010). Similarly, Tethya wilhelma, another demosponge, displayed contractions of its body column when exposed to GABA and L-glutamate (Ellwanger et al., 2007; Ellwanger and Nickel, 2006; Nickel, 2004). Sequencing of the T. adhaerens genome showed that this primitive metazoan encodes about 85 genes (76 were found in the latest Ensembl release) coding for glutamate family GPCRs, including a large expansion of GRMs (Srivastava et al., 2008), but the functions of these putative glutamate family receptors are mostly unknown. Several key enzymes, including glutamate dehydrogenase and glutaminase, which are involved in the synthesis of glutamate, as well as the glutamate decarboxylase, which synthesises GABA, are present in T. adhaerens. Thus it is conceivable that GRM-like receptors and GABAB-like receptors could be functioning as glutamate and GABA receptors in T. adhaerens, modulating the physiology and behaviour of the organism.

Several studies have provided biochemical evidence for the presence of glutamate and GABA, and their receptors in cnidarians (for a review, see Kass-Simon and Pierobon, 2007). More recently, immunohistochemistry studies have demonstrated that glutamate and GABA exist in the nerve plexus within the tentacles of a sea anemone Phymactis papillosa, suggesting that glutamate and GABA function as neuronal signalling molecules in the primitive nervous system of cnidarians (Delgado et al., 2010). Furthermore, GABA receptors modulate the discharge of nematocysts in hydra (Scappaticci et al., 2010; Scappaticci and Kass-Simon, 2008). Nematocysts, which are also called cnidae, are sophisticated stinging cells found in all cnidarians and are required for prey capture and defence mechanisms (Beckmann and Özbek, 2012). This suggests that the nematocyst discharge, induced by GABA-mediated neurotransmission in the hydra nervous system, could be required for prey capture and that this mechanism could also exist in other cnidarians. In contrast to the cnidarians, the role of glutamate GPCRs in ctenophores remains obscure. However, recent whole-genome analysis of the ctenophore P. bachei provided evidence that the comb jelly has eight genes coding for glutaminases and that L-glutamate acts as a neurotransmitter to induce muscle contractions (Moroz et al., 2014). It must be mentioned that L-glutamate has a role in inducing contractions of the E. muelleri canal system, and that it also affects the concentrations of intracellular calcium in G. cydonium (as discussed above). This suggests that glutamate and its receptors in pre-bilaterians are capable of inducing similar effects in both neuron-rich and neuron-free environments. Also noteworthy is that receptors such as kainate receptors (kainateGluR), AMPA receptors (AMPAGluR) and NMDA receptors, which belong to ionotropic glutamate family receptors (iGluRs), are present in early metazoans (Burkhardt et al., 2014). Specifically, kainate and AMPA receptors both have early metazoan origins and are present in T. adhaerens and N. vectensis, whereas NMDA receptors probably emerged in an early eumetazoan ancestor (Burkhardt et al., 2014). However, ctenophores contain a diversified set of iGluRs, because they seem to have undergone lineage-specific adaptations with unique exon and intron organisation (Moroz et al., 2014). Taken together, it seems evident that glutamate acts as a signalling molecule in early metazoans, and that it may induce diverse intracellular responses through a variety of receptors, depending on the morphological characteristics and environmental adaptations of the organism.

In this review, we highlight the large expansion of GPCR gene families before the divergence of bilateria. Comparative genomics reveals that the molecular components required for GPCR-mediated neurotransmission exist in organisms with a primitive nervous system, as well as in organisms that lack nerve cells or a nervous system. This includes some of the important enzymes required for the synthesis of neurotransmitters and neuropeptides, although these species do not comprise all the genes required for complete pathways as they appear in mammals. Genomic comparisons also demonstrate that adhesion GPCRs first expanded at the early origins of metazoans. Until recently, the roles of adhesion GPCRs were unclear; however, an emerging view suggests that they play a role as cell–cell adhesion molecules in the developing nervous system, which guide synaptic formation, neuronal patterning, and plasticity in vertebrates. Thus, an interesting question is whether pre-bilaterian adhesion GPCRs mediate cell–cell adhesion and have a possible role in the origins of synapses and synapse formation. Taken together, it is conceivable that some of the basic molecular components required for GPCR-mediated neurotransmission are an ancestral feature and during the course of metazoan evolution these genetic components have facilitated the evolution of a nervous system along with several other neuronal gene families. However, because the most recent hypothesis supports multiple origins of the neural systems, it is important to understand the functions of GPCRs and other neuronal gene families in T. adhaerens and A. queenslandica, which lack a nervous system. Progress on this front will require expanded comparisons of genomes with robust phylogenetic studies to analyse these GPCR families, as well as several other crucial elements, including all presynaptic and post-synaptic proteins. Important questions remain. For example, do GPCRs in sponges and placozoans share orthologous relationships with the GPCRs found in cnidarians and ctenophores? Are GPCRs in sponges and placozoans capable of inducing intracellular responses in non-excitable cells similar to those in excitable cells? What are the functional implications of the expansions of GPCRs in sponges, placozoans, cnidarians and ctenophores? Overall, the genomic machinery required for neuronal functioning in pre-bilaterians provides a great platform for further comparative genomics and functional studies, which aim to delineate the key evolutionary events that shaped the first nervous system in early metazoans.

This work was presented at the ‘Evolution of the First Nervous Systems II’ meeting, which was supported by the National Science Foundation (NSF). We thank Dr Lyle Wiemerslage, Dr Michael Williams and Dr Robert Fredriksson, Department of Neuroscience, Functional Pharmacology, Uppsala University for valuable comments and thorough proof reading of the manuscript.