The amphioxus (Branchiostoma floridae) genome contains a highly diversified set of G protein-coupled receptors
© Nordström et al; licensee BioMed Central Ltd. 2008
Received: 15 August 2007
Accepted: 16 January 2008
Published: 16 January 2008
G protein-coupled receptors (GPCRs) are one of the largest families of genes in mammals. Branchiostoma floridae (amphioxus) is one of the species most closely related species to vertebrates.
Mining and phylogenetic analysis of the amphioxus genome showed the presence of at least 664 distinct GPCRs distributed among all the main families of GPCRs; Glutamate (18), Rhodopsin (570), Adhesion (37), Frizzled (6) and Secretin (16). Surprisingly, the Adhesion GPCR repertoire in amphioxus includes receptors with many new domains not previously observed in this family. We found many Rhodopsin GPCRs from all main groups including many amine and peptide binding receptors and several previously uncharacterized expansions were also identified. This genome has however no genes coding for bitter taste receptors (TAS2), the sweet and umami (TAS1), pheromone (VR1 or VR2) or mammalian olfactory receptors.
The amphioxus genome is remarkably rich in various GPCR subtypes while the main GPCR groups known to sense exogenous substances (such as Taste 2, mammalian olfactory, nematode chemosensory, gustatory, vomeronasal and odorant receptors) in other bilateral species are absent.
The superfamily of G protein-coupled receptors (GPCRs) is one of the largest families of proteins in mammals . 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 . The superfamily can be divided according to the GRAFS system into five main families; Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin . 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 [4–6]. 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 [4, 6–8].
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 . 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 . 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 . 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.
Results & Discussion
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 . In amphioxus, we identified 17 receptor sequences from the Glutamate family (Additional file 1, figure S4) while the human genome contains 22 sequences . 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  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 , 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) [21, 22] 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 , 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 , 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  and receptors  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 . 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 , although these clearly have a borderline similarity to mammalian Frizzled GPCRs . 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 . 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.
One dopamine receptor, AmphiD1/β, has been identified in amphioxus [27, 28]. 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 . 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 . 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 .
We found several examples of support for Ohno's 2R hypothesis that postulates that two whole genome duplication occurred in early vertebrate evolution [32, 33]. 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) [34, 35] 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.
Search & confirmation
The amphioxus genome and protein sequences were downloaded from US Department of Energy Joint Genome Institute . Protein transcripts shorter than 250 amino acids were removed as they are too short to hold a 7TM-region. The remaining amphioxus proteins were searched with GPCR hmm models adapted from  using hmmer , which is available from . All hits with an E-value below 0.01 were aligned with BLAST  against a database consisting of the human RefSeq  together with nematode chemoreceptors, the gustatory receptors from insects, the odorant receptors from D. melanogaster, MLO receptors in plants , fungal pheromone receptors (STE2 and STE3) from yeast and cAMP receptors from D. discodieum. For each putative amphioxus GPCR, the top five hits were inspected and if a minimum of four of these were GPCRs, the transcript was accepted as a GPCR. Alternative splice variants were removed by aligning the amphioxus proteins to their genome with BLAT . As the amphioxus genome has not yet been assembled and only consists of scaffolds this method does not catch all splice-variants and duplicates.
In order to categorize the amphioxus GPCRs, human and other GPCRs were tagged with their family name. The human receptors were divided into the GRAFS families , Glutamate (GLU), Rhodopsin (Rhod), Adhesion (ADH), Frizzled (FZD) and Secretin (SEC). The Taste2 receptors were separated from Frizzled receptors. In addition, the Rhodopsin family was split in accordance to . The other GPCR families used in this step were nematode chemosensory receptors, the gustatory receptors from insects, the odorant receptors from D. melanogaster, MLO receptors from plants, fungal pheromone receptors (STE2 and STE3) from yeast and cAMP receptors from D. discodieum. The putative amphioxus GPCRs were aligned against a database consisting of these tagged GPCRs and had to have at least four of the five best hits from the same family to be assigned to this family. A multiple alignment was made for each family and the alignments were viewed in Jalview , and the amphioxus sequences were cut with the reference transcripts as a model to gain only the 7TM regions. This process was repeated three times in order to only remove sections in inverse proportion to how well they aligned, balanced against the alignment quality. First, the N and C terminals were removed, except for about twenty amino acids in each end. Second, internal loops were removed and in the last round N and C terminals were trimmed to exactly match the models. In order to remove duplicate sequences, all the cut amphioxus sequences were aligned against each other, using BLAT, and all pairs with identical sequences were removed. Maximum-likelihood trees were also created with PHYLIP and viewed in MEGA . All sequences in a family were aligned to each other and an identity distance matrix was constructed using Megalign from the DNASTAR package (DNASTAR, Madison, Wisconsin, United States). Amphioxus sequences with identities below ten percent towards their reference were discarded as random hits. If the identity was between ten and twenty, they were considered basal to the family. We calculated a neighbor-joining tree, bootstrapped five hundred times, using MEGA with default parameters based on an alignment made in MEGA using the ClustalW algorithm and batch ClustalW default parameters for the sequences with identities above twenty percent to at least one of the reference sequences. Sequences disrupting the tree were moved to the basal group on the basis of low bootstrap values and poor alignments through manual inspection. Additional identity checks were made with the EMBOSS  implementation of the Smith-Waterman local alignment algorithm. Domain search was performed with the PFAM domain dataset and the HMMER package.
The amphioxus sequence data was produced by the US Department of Energy Joint Genome Institute . The studies were supported by the Swedish Research Council. RF was supported by the Swedish Brain Foundation. We thank Chris Pickering for valuable comments to the manuscript.
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