The superfamily of G protein-coupled receptors (GPCRs) is one of the largest families of proteins in mammals 1. GPCRs participate in a tremendous diversity of physiological functions by playing a key role in the mediation of extracellular signals into cells while approximately 50% of all marketed drugs act on GPCRs 2. The superfamily can be divided according to the GRAFS system into five main families; Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin 3. These main families arose prior the split of nematodes from the chordate lineage. The Rhodopsin family has been very successful in evolutionary terms, representing about 60% of the GPCRs repertoire of eight bilateria species. However, the Rhodopsin family seems not to be present in Dictyostelium discoideum 456. The Rhodopsin family can be divided in four main groups (α-, β- γ- and δ) with 13 main branches and previous studies have shown that members within each of the four main groups seem to be found in most bilaterial species, while the representation of each of the main branches is highly variable 4678.

Amphioxus (Branchiostoma floridae) is a small cephalochordate that spends much of its time buried in the sand. It is one of the closest now living relatives to vertebrates. Amphioxus shares several features with vertebrates like a dorsal, hollow nerve cord, notochord, segmental muscles and pharyngeal gill slits. On the other hand, they are missing the pronounced head region of vertebrates as well as not having neural crest cells functioning similar to those in vertebrates, paraxial skeletal tissue and some visceral organs 9. Lately it has been argued on the basis of molecular phylogenetic data that tunicates, such as Ciona intestinalis, could be more closely related to vertebrates than cephalochordates are 10. GPCRs have been mined from the C. intestinalis genome while very little has been known about GPCRs in the most basal Cephalochordates such as amphioxus. However, this genome has just recently been sequenced and the first genome paper will soon be published. Version 1.0 is released in the form of scaffolds on the Joint Genome Institute homepage 11. It has been predicted that this genome will be very important for the understanding of the evolution of many vertebrate gene families.

We have performed a detailed mining and phylogenetic analysis of the gene repertoire of GPCRs in amphioxus. We found that this genome has a highly diversified set of genes coding for GPCRs which provides insight into evolutionary aspects of these.

We identified at least 664 unique GPCRs using our highly diversified seeding datasets. Most of these are found in the main families 3; Glutamate (18), Rhodopsin (570), Adhesion (37), Frizzled (6) and Secretin (16) (see Fig. 1). We did, however, not find any bitter taste (Taste 2) or vomeronasal (VR1) receptor. These types of receptors are abundant in rodents and most likely all other non-primate mammals but are rare in fish and chicken 8. Neither did we find evidence for any members of the many pre-vertebrate lineage-specific expansions such as the nematode chemosensory receptors, the gustatory receptors from insects, the odorant receptors from Drosophila melanogaster, MLO receptors in plants or fungal pheromone (STE2 and STE3) from yeast 412. We found, however, two sequences that show some similarity with the cAMP-binding receptors from slime moulds but the identity is fairly low (25.6%) and further work needs to determine if they are true cAMP receptors.

The Adhesion family of GPCRs has vague similarity with the Secretin receptors in the transmembrane (TM) regions but their structural and functional properties are very different 3. There are 33 known Adhesion receptors in human and these have characteristic long N-termini containing multiple functional domains 1314, known to be present in other protein families. Interestingly, we found a very rich repertoire of Adhesion GPCRs, in total 37, in amphioxus(see Fig. 2 and Additional file 1, figure S1). Some of the mammalian groups 813 are missing, such as the groups I (lectomedin receptors) and II (epidermal growth factor module-containing receptors and cell differentiating antigen receptor) that is likely to have evolved late in the vertebrate lineage 15. Three of the Adhesion groups have both mammalian and amphioxus members. These are group III (orphans, expressed in CNS), V (orphans) and VIII (orphans with highly variable N-terminal length). One orthologous receptor to the very long G protein-coupled receptor 1 (VLGR1) is also present. The remaining missing groups are group IV (CELSR with multiple cadherin domains), VI (GPR110, GPR111, GPR113, GPR115 and GPR116) and VII (brain-specific angiogenesis inhibitor receptors). Many of the amphioxus Adhesion GPCRs have the GPS domain, which are characteristic for the Adhesion family and not found in any other GPCR family including the Secretin receptors. Moreover, many of the amphioxus Adhesion GPCRs have multiple domains in the N-termini, which is another important feature of the mammalian Adhesion GPCRs. These N-termini are likely to mediate cell-to-cell interaction which for instance could allow participation in different types of cell guidance 16. Surprisingly, we found several novel domains in the N-termini of the amphioxus Adhesion GPCRs like Somatomedin B, Kringle, Lectin C-type and SRCR (for more details see Fig. 2) which to our knowledge are unique for amphioxus among the GPCRs. Also the domains LDLa, Immunoglobulin I-set, CUB and TNFR were found and they can not be found in mammal Adhesion GPCRs 13. Although; especially interesting is the Kringle and Somatomedin B domains which are found in sequences from a previously uncharacterized Adhesion expansion of ten genes (Fig 2). The Kringle domain (see alignment in Additional file 2, figure S2) is a protein-binding domain 17 present in urokinase-type plasminogen activator (uPA) while the Somatomedin B domain (Additional file 2, figure S1) can be found in vitronectin 18. These two proteins interact and the Somatomedin B domain and helps in the localization of uPA to focal adhesions in microvessel endothelial cells. Interestingly, all of these domains not previously identified in Adhesion GPCRs, have a large number of conserved cysteines which is a feature consistent with many other common domains found in this family. This similarity could suggest that these domains are inserted through domain shuffling of similar stretches of DNA. Interestingly, many of these new domains have recognised cell adhesion properties and participate in cell guidance. The genes in the other cluster in this family share a resemblance to invertebrate genes according to the top five hits in BLAST searches against the NCBIs (National Center for Biotechnology Information) non-redundant (nr) database. These invertebrate genes are primarily from either Strongylocentrotus purpuratus which is, according to Delsuc et al., closely related to amphioxus 10 or, more interestingly, from the more distantly related cnidarian Nematostella vectensis. In average 4.4 of these first five hits are from invertebrate species (see Additional file 3). These findings suggest that the Adhesion family, with its unique structural and functional characteristics, has a very long evolutionary history and that these are likely to be present in most vertebrates. Moreover, these properties seem to have undergone further diversification within the lineage leading to amphioxus, and are likely to have gained additional roles in cell guidance.

The Glutamate family of GPCRs, also known as family C, have previously been divided into eight groups with the largest, the metabotropic glutamate receptors (mGluRs) containing eight members in humans. In addition, the Glutamate family contains GABA (gamma amino butyric acid) B receptor, the calcium sensing receptor (CASR) and the related amino acid binding GPRC6A, taste receptors for sweet and umami (TAS1Rs), the orphan GPR158 as well as GPRC5 receptors, a group of four related orphan receptors. Moreover, the Glutamate family in mammals contain the large family of pheromone receptors designated vomeronasal receptors type 2 (V2Rs) which in the mouse genome contains over 50 functional genes while all V2Rs in the human genome are pseudogenes 19. In amphioxus, we identified 17 receptor sequences from the Glutamate family (Additional file 1, figure S4) while the human genome contains 22 sequences 19. All the amphioxus sequences except three are most similar to vertebrate sequences according to a BLAST search against NCBIs nr dataset (see Additional file 3). Two of the invertebrate-like sequences hit insect related genes while the third hits genes from the invertebrates Tribolium castaneum, S. purpuratus, Apis mellifera, N. vectensis and the vertebrate Tetraodon nigroviridis. Of the eight mammalian groups, four are represented in amphioxus while the branches of TAS1Rs, CASR, GPRC6A and V2Rs are missing. TAS1R, GPRC6A, and V2Rs were previously found to be present in the teleost Danio rerio and missing in C. intestinalis 19 and our findings here support that these receptor groups are specific for vertebrate species. Curiously, we did not find the gene for CASR in amphioxus which has previously been observed in Caenorhabditis elegans, indicating that this receptor was lost specifically in the lineage leading to amphioxus.

Amphioxus has also a characteristic Secretin family of GPCRs with distinctive features of a hormone binding domain in the N-termini. In line with all known vertebrate species, amphioxus does not have any of the methuselah receptors 20, which are related to the Secretin receptors and found in arthropods. The presence of typical Secretin GPCRs in amphioxus provides further evidence that Secretin and Adhesion are separate evolutionary branches in vertebrates. The Secretin family can be divided into five main branches a) Corticotrophin Releasing Factor (CRF); b) Secretin (SCT), Vasoactive Intestinal Peptide (VIP), Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Growth Hormone Releasing Hormone (GHRH); c) Glucagon (GCG), Glucagon-Like Peptide (GLP), Glucose Insulinotropic Peptide (GIP); d) Parathyroid Hormone (PTH) and e) Calcitonin (CAL) and Calcitonin Gene-Related Peptide (CGRP) 2122 of which groups a, d and e are found in amphioxus (see Additional file 1, figure S5). Recent studies on the Secretin family shows that the receptors in group a and e are ancient and found in both deuterostoma and protostoma 23, but the sequences we identified in amphioxus all show a closer relationship with vertebrates (see Additional file 3). These branches, together with group d, are also present in amphioxus while we do not find any receptors from group b and c in amphioxus. The missing groups contain the Secretin GPCRs binding gut peptides in mammals, suggesting that these functions are missing in amphioxus (see Additional file 4, table S2). On the other hand, several of the Secretin receptors (group a and e) that mediate hypothalamus/pituitary signalling in mammals are present (see Additional file 4, table S1). This may suggest that parts of the gut functions found in vertebrates are missing or are regulated differently in amphioxus compared to vertebrates. However, there are good evidence that amphioxus has GPCRs that are likely to participate in endocrine functions that may resemble that of hypothalamus and pituitary in mammals 24, such as corticotropin releasing hormone receptor, somatostatin receptors and vasopressin/oxytocin receptors (Additional file 4, table S1). It is also notable that several GPCRs known to participate in pancreatic functions are missing, such as cholecystokinin receptor A and B, gastric-inhibitory peptide receptor and the secretin receptor (Additional file 4, table S2). However, insulin like peptides 25 and receptors 26 have been shown to be present suggesting presence of some of the most fundamental pancreatic functions.

The Frizzled family contains ten frizzled and the smoothened receptor in most mammals and are the most well conserved of all GPCR families both regarding family members and primary amino acid sequence 8. Frizzled receptors are found in all bilateral species and are crucial for early embryonic development while being the receptor for Wnt and Sonic hedgehog. Frizzled-like receptors have also been found in the unicellular slime mold D. discodieum which was hypothesized to carry 25 frizzled genes in its genome 5, although these clearly have a borderline similarity to mammalian Frizzled GPCRs 6. We found five Frizzled receptors as well as an ortholog to smoothened in amphioxus (Additional file 1, figure S2). These are all vertebrate like according to their five best hit against NCBIs nr dataset. This is in line with our previous study indicating that vertebrates, including teleost fish, have a repertoire similar to that of humans, while invertebrates have fewer; between 5 (C. elegans) and 7 (D. melanogaster, Anopheles gambiae and C. intestinalis) Frizzled GPCRs 4. The increased number of Frizzled GPCRs probably reflects a requirement for higher signal diversity for formation of the more complex body plan and, in particular, the more complex nervous system of vertebrates.

The Rhodopsin family of GPCRs constitutes the largest family of GPCRs in vertebrates (Fig. 3). The largest group of Rhodopsin GPCRs in mammals are the mammalian type of olfactory receptors, comprising 388 members in humans. This type of olfactory receptors are also found in chicken 8 while there are markedly fewer in fish such as D. rerio and Takifugu rubripes 4 but we can not identify these in amphioxus. The Rhodopsin GPCRs have been divided into four main groups, termed α-,β-, γ- and δ-group 3, which in turn could be further subdivided into thirteen subgroups. Like most vertebrates, the Rhodopsin family in amphioxus contains the largest number of GPCRs (Fig 1). These GPCRs are found in all four main groups of Rhodopsin GPCRs. However, only eight of the thirteen subgroups are present in amphioxus (See Additional file 1, figure S6-S16); missing are the mammalian type of olfactory receptors, the chemokine, the melanin concentrating hormone (MCH), the MAS-related and the purin receptor subgroup. This is in line with our previous study indicating that MAS-related receptors are missing in teleost fish and that C. intestinalis is missing four of these five groups 4. The fact that C. intestinalis has a chemokine receptor cluster strengthens the argumentation forwarded by Delsuc et al. that tunicates and not cephalochordates are the closest living relatives to the vertebrates 10.

One dopamine receptor, AmphiD1/β, has been identified in amphioxus 2728. Recently, 11 receptors related to the large cluster of biogenic amine receptors were identified in amphioxus from unassembled genomic sequence reads and shown to have tissue specific expression 29. Our searches from the assembled genome identified additional 33 receptors belonging to the amine receptor cluster. Of the previously known receptors AmphiAmR3 and R7 cluster with histamine receptor 4 in our phylogenetic tree, AmphiAmR1 clusters with dopamine receptors D1 and D5, AmphiAmR4, R5, R6, R9 and R11 is found in a non-resolved cluster with only amphioxus sequences while we placed AmphiD1/β basal to the amine subgroup as it could not find a stable location in the tree. AmphiAmR2, R8 and R10 placed basal in the Rhodopsin family. Of the new amphioxus receptors we identified, 18 fall in groups containing only amphioxus sequences in the phylogenetic tree (Additional file 1, figure S6), indicating that they are not clearly orthologous to any of the mammalian receptors. However, we also identified receptors that fall into specific groups of human receptors, suggesting the possibility that they are sharing ligands with the orthologous human receptors. We identified receptors that place in the D2/D3 dopamine cluster, the muscarinic receptor cluster and the adenosine receptor cluster. Moreover, we found orthologs to three serotonin receptors, namely 1, 5 and 6 as well as six receptors that place close to histamine receptor 4 (Additional file 1, figure S6). In addition to the rich set of amine receptors, amphioxus has large set of peptide binding receptors and predicted peptide ligands, including several receptors (neuropeptide Y, neuropeptide FF, uterinbombesin, neuromedin and growth hormone secretagogues receptors) with ligands involved in appetite control (see further details in Additional file 4, table S1-S3).

The Rhodopsin family in amphioxus contains several larger expansions of GPCRs (see Additional file 1, figure S6-S16). The largest of these includes the Neuropeptide FF receptor in the β-group (39 sequences), somatostatin, opioid, galanin cluster (29 sequences), two expansions in the melanocortin, endothelin cannabinoid clusters with 21 and 24 sequences each and one in the melatonin receptor cluster (19). The β-group, which has several peptide receptors, also has a large expansion of twenty predicted genes that are similar to the prokineticin receptor (PK) 1 and 2 which are important for contraction of gastrointestinal smooth muscles 30. These are, on average, about 42 percent identical in the TM regions with PK1 and PK2 receptors. This expansion is surprising, however as prokineticin is a very potent muscle contractor, it is possible that these receptors are involved in these early specific pre-vertebrate muscles that create effective undulations of the amphioxus body.

163 amphioxus sequences had more resemblance to invertebrate sequences than to vertebrate transcripts according to the BLAST searches. Beside the expansion in the Adhesion family, most of these are categorized to the Rhodopsin family (111) or these are sequences we could not categorize (28). Still, none of the larger Rhodopsin clusters mentioned above show similarity to many invertebrate like sequences with exception of the melatonin cluster which shows similarity to several S. purpuratus genes. There is also a cluster in the β-group of the Rhodopsin family holding seven transcripts that all are insect like and have all hits related to the ecdysis triggering hormone receptor 31.

We found several examples of support for Ohno's 2R hypothesis that postulates that two whole genome duplication occurred in early vertebrate evolution 3233. The phylogenetic trees show that there are several branches with multiple human members where amphioxus sequences place basal (closer to the root of the tree) on the branch. For example, the tachykinin receptors 1–3 and frizzled receptor 1,2 and 7 both cluster with a single amphioxus sequence with high bootstrap values. These belong to paralogon groups (PG) 9 (Meta HOX) 3435 and 10 (HOX paralogon), respectively. Moreover, the muscarine receptors 1,3,5 in PG4 have basal amphioxustranscripts as well as adenosin binding receptors 1, 2A, 2B in PG11, brain specific angiogenesis-inhibitory receptors 1–3 in PG14 and somatostatin receptors 2,3,5 in PG12.

Overall, the amphioxus genome is in general terms surprisingly rich in GPCRs, with extensive representation of receptors from all of the main mammalian GPCRs families (See Fig 1). The repertoire resembles that of vertebrates but lacks the branches previously known to be vertebrate-specific. The groups that are missing include the chemokine receptors which are important for the immune system and receptors for purines which serve as important co-transmitters within the nervous systems. Amphioxus lacks many of the classical mammalian sensory receptors such as the pheromone receptors that are particularly important for non-primate mammals and also the mammalian olfactory receptors. Moreover, amphioxus does not have the bitter taste receptors (TAS2) or the sweet and umami (TAS1) receptors which suggest that amphioxus have a unique way of detecting these taste modalities. The amphioxus genome does not have any other classical GPCR group that recognizes exogenous stimuli, such as mammalian type olfactory receptors or pheromone receptors. On the other hand, amphioxus has opsin receptors, showing conservation in the visual system. It should however be mentioned that sensory GPCRs evolve rapidly and there are many species specific expansion of such receptors. It can thus not be excluded that there are sensory receptors that are not found in this type of searches. Amphioxus has fewer receptors that clearly can be recognised as monoamine receptors (only 1 muscarinic, 2 dopamine, 3 serotonin and one histamine) which are involved in neurotransmission in higher animals. Amphioxus does, however, have a large number of other receptors with high similarity to amine and peptide receptors that suggest early development of these systems that are very important for CNS functions.

KN carried out the study, participated in the design and drafted the manuscript. RF and HS conceived the study, participated in its design and in the writing of the manuscript. All authors read and approved the final manuscript.