The venus kinase receptor (VKR) family: structure and evolution
© Vanderstraete et al.; licensee BioMed Central Ltd. 2013
Received: 23 November 2012
Accepted: 24 May 2013
Published: 30 May 2013
Receptor tyrosine kinases (RTK) form a family of transmembrane proteins widely conserved in Metazoa, with key functions in cell-to-cell communication and control of multiple cellular processes. A new family of RTK named Venus Kinase Receptor (VKR) has been described in invertebrates. The VKR receptor possesses a Venus Fly Trap (VFT) extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity. VKR was shown to be highly expressed in the larval stages and gonads of several invertebrates, suggesting that it could have functions in development and/or reproduction.
Analysis of recent genomic data has allowed us to extend the presence of VKR to five bilaterian phyla (Platyhelminthes, Arthropoda, Annelida, Mollusca, Echinodermata) as well as to the Cnidaria phylum. The presence of NveVKR in the early-branching metazoan Nematostella vectensis suggested that VKR arose before the bilaterian radiation. Phylogenetic and gene structure analyses showed that the 40 receptors identified in 36 animal species grouped monophyletically, and likely evolved from a common ancestor. Multiple alignments of tyrosine kinase (TK) and VFT domains indicated their important level of conservation in all VKRs identified up to date. We showed that VKRs had inducible activity upon binding of extracellular amino-acids and molecular modeling of the VFT domain confirmed the structure of the conserved amino-acid binding site.
This study highlights the presence of VKR in a large number of invertebrates, including primitive metazoans like cnidarians, but also its absence from nematodes and chordates. This little-known RTK family deserves to be further explored in order to determine its evolutionary origin, its possible interest for the emergence and specialization of Metazoa, and to understand its function in invertebrate development and/or reproductive biology.
Receptor Tyrosine Kinases (RTKs) are transmembrane proteins that are involved in many fundamental intra- and inter-cellular processes. RTKs have essential multicellular-specific functions, including cell-to-cell communications, control of cell proliferation and differentiation . They have been found in all metazoan genomes, from the marine sponge Geodia cydonium to humans [2, 3]. Moreover, RTKs were also shown to be present in choanoflagellates [4, 5] and Filasterea , which are the sister groups of Metazoa. A large number of RTKs are conserved throughout evolution, but unique and organism-specific RTKs have been identified, such as Sweet tooth in Hydra vulgaris or kin15/kin16 in Caenorhabditis elegans[8, 9]. RTKs have been classified into distinct families, depending on the modular composition of their extracellular domains and their ability to bind different types of ligands, as well as by their kinase domain sequences. The human genome encodes 58 RTKs, and these receptors are classified into 20 families . According to the data regrouped in http://kinase.com, the invertebrate model organisms Drosophila melanogaster and C. elegans possess 16 and 29 RTKs and share 11 and 10 families with human RTKs, respectively .
Venus Kinase receptors (VKRs) constitute an RTK family, originally found in the parasite platyhelminth Schistosoma mansoni, then in several other invertebrates (insects and echinoderms). However, no VKR could be found in any chordate genome and, more strikingly, this receptor was not present in C. elegans and D. melanogaster. VKR proteins possess an atypical structure [11, 12], containing an intracellular tyrosine kinase (TK) domain similar to that of insulin receptors, and an extracellular Venus Flytrap (VFT) domain. VFTs were first identified as bacterial periplasmic-binding proteins involved in the transport of small molecules, such as amino acids, sugars or ions and they constitute the binding pocket of different receptor types such as class C G-protein coupled receptors . The VFT domain of VKR is related to that of the ANF receptor in protein family databases (pfam 01094) . VKR kinases can be activated following the binding of amino-acids to the extracellular VFT domain [12, 14], and this opens interesting perspectives on a novel mechanism for RTK activation as well as on possible specific and new functions of these receptors in cellular signalling.
In this paper, we present an up-dated version of the VKR family, resulting from an exhaustive research of VKR orthologs in the published genomes of a variety of organisms belonging to major phyla in Metazoa. We show that vkr genes are present at least in five major phyla in Bilateria (Platyhelminthes, Arthropoda, Annelida, Mollusca, Echinodermata), and also, more strikingly, in the cnidarian Nematostella vectensis. Phylogenetic analyses indicated that all the putative protein sequences grouped monophyletically, and a new version of the VKR phylogeny was built. In silico analyses and multiple alignments of the VFT and TK functional domains of VKRs allowed us to reinforce the structural model of the receptor and to get a better prediction of potential ligands and kinase activity of VKRs.
Results and discussion
A large distribution of vkrgenes in eumetazoan genomes
Previous studies have shown that VKRs constitute a distinct RTK family. These receptors were described in 15 different protostomes, including insects and the platyhelminth S. mansoni. A vkr gene was also found in the deuterostome Strongylocentrotus purpuratus. The aim of this study was to extend the VKR family to a large number of phyla in order to evaluate the place of these receptors in the animal kingdom and to contribute to a better understanding of their evolution.
Complete list of the 40 vkr genes found in genomic databases
Pediculus humanus corporis
Diversification of vkrgenes
Characteristics and structural organization of vkr genes
Coding exons for :
Vkr genes found in lophotrochozoan organisms (annelids, molluscs, platyhelminths), are overall more complex (15 to 18 exons) than the insect ones, except in the cestode H. microstoma (11 exons). Lophotrochozoan vkr genes are according to this more similar to that one detected in the phylogenetically basal animal N. vectensis. Indeed, Nvevkr is also intron-rich (15 exons), respecting therefore the known high complexity of the genes present in early animal genomes . Spvkr found in the echinoderm S. purpuratus remains the most complex vkr gene found with a size of 60kb and a total of 21 exons. In trematodes, the organisation of Smvkr1 and Smvkr2 genes was shown to be quite identical (see Table 2, and ), arguing for a duplication event in S. mansoni.
We also noticed that 12 out of 14 intron positions of Nvevkr were found in at least one phylum and that most of these conserved exon-intron boundaries were located in regions that encode VFT and TK domains. Most of these positions were shown to be conserved in the annelid (Ctvkr), mollusc (Lgvkr) and echinoderm (Spvkr) genes. Inversely, only a very limited number of intron positions seem to be conserved in arthropods as well as in platyhelminthes, indicating a profound reorganization of vkr genes along evolution. Finally, specific or non-conserved intron positions were also found in various vkr genes (Figure 3). Taken together, these studies demonstrate that vkr genes are highly variable in size and in complexity but that, in spite of their heterogeneity, all of them possess common features, which are conserved from Cnidaria to the other phyla.
Conservation of VKR tyrosine kinase domains
Using the multiple alignment ClustalW algorithm, we have compared the TK domain sequences of the 40 VKRs and generated an identity matrix. As it could be expected for catalytic structures, the sequences are relatively well conserved across all species, with the best scores of identity observed between the species belonging to a same order. For example, in Hymenoptera the TK domains of Bombus and Apis species are more than 92% identical, in Diptera, those of Drosophila species are more than 75% identical and those of the mosquitoes A. gambiae and A. aegypti are 93%. For platyhelminth VKRs, identities between TK domains scored between 57 to 96%, with the best score registered between the two cestode parasites E. multilocularis and E. granulosus. In Lepidoptera, the TK domains of Bombyx and Danaus VKRs were less conserved, except for BmVKR2 and DplVKR2 that share 81% of identity.
Conservation and divergence of VKR ligand-binding domains
This survey based on the analysis of newly released genomic data has allowed us to show that vkr genes actually represent a novel RTK family, widespread in the bilaterian branch of Eumetazoa. From this study we can extend the presence of VKR to a large variety of protostomes (Ecdysozoa and Lophotrochozoa). However, after the analysis of a large number of deuterostome genomes (Chordata and Hemichordata), it seems that deuterostomes would not contain vkr genes, with the exception of echinoderms. In these studies, an important information concerns the detection of NveVKR in the basal metazoan N. vectensis. From the presence of a vkr gene in Cnidaria, we can suggest that the origin of VKR would be anterior to the radiation of Bilateria, and possibly close in time to that of the setting-up of animal multicellularity. This would agree with the general acceptance that emergence of a series of cell surface receptors (including RTKs) necessary for cell adhesion, differentiation and cell-cell communications has driven evolution towards multicellularity . In this context, we have recently found putative RTKs exhibiting an architecture close to that of VKR proteins in the genomes of the choanoflagellates M. brevicollis and S. rosetta, which are free-living unicellular and colonial flagellates considered to be the closest living relatives of the animals. These findings (unpublished) encourage us to postulate that VKR could represent an ancient RTK present early in protists, that might have contributed to the establishment of multicellularity and to animal development.
Another important question concerns the distribution and stability of VKR throughout evolution. Vkr genes were found preferentially in the genomes of protostomes and particularly in insect genomes, but the large number of insect VKR sequences could likely result from the relative abundance of insect sequences in genomic databases. However, it was very interesting to observe that the finding of one vkr in all species of a given genus is not a general rule. For example, in the Drosophila genus, D. melanogaster and some others exceptionally do not possess a vkr gene . Also, it is surprising that no vkr exists in other ecdysozoa like nematodes, and in C. elegans particularly.
In this work, we have analysed the exon/intron structure of all vkr genes and shown that their organization is widely heterogeneous across the different phyla. However, all vkr genes share intron positions common to the ancestral gene Nvevkr, and this suggests that they might have been derived from this common ancestor, then subjected to more or less marked reorganization along evolution. Finally, the question of the existence of two vkr genes in Trematoda and Lepidoptera is still open, together with the problem of the “keep or loss” of vkr in some species. Further investigations about the functions of VKR in the biology and physiology of organisms should be required to answer these questions.
We have previously shown that vkr genes were preferentially expressed in larval stages and in gonads of several organisms, including sea urchin, mosquito and the trematode S. mansoni, thus suggesting a role of the receptor in embryogenesis and gonad development [11, 12, 14]. About their functional activity, we have been able to demonstrate the tyrosine kinase activity of several VKRs [12, 14] and sequence information here obtained for the TK domains of all VKRs confirms that most VKRs should be active kinases as well. VKR receptor activation was shown to be dependent on the binding of L-amino-acids, and specifically of L-arginine, to the extracellular VFT domain, in which conserved residue positions (for example S66 and R107) are essential for ligand-receptor interaction and probably involved in specificity and affinity of L-arginine. However, in some VKRs, these major conserved residues are not present, suggesting that they could bind other ligands and perhaps have different functional activities. We have shown that SmVKR1 and SmVKR2 of S. mansoni differ both in the primary sequence of their VFT domain and in their localization in the parasite. The observation that these receptors are activated respectively by L-arginine and Calcium  really illustrates this possible functional divergence between VKR.
Currently, diverse strategies are developed to analyse the consequences of VKR knock-down in organisms. Preliminary results of VKR targeting by RNA interference in S. mansoni have confirmed the importance of SmVKR1 and SmVKR2 for growth and differentiation of reproductive organs and parasite fertility (to be published). The use of other organisms as candidate models for studying the function of VKR is under investigation.
In conclusion, the VKR family is a little-known RTK family that deserves to be further explored in order to determine more precisely its evolutionary origin, its possible importance for the emergence and specialization of Metazoa, and to understand how its maintenance or its loss in various phyla or species could be in relation with development and physiological activities (like reproduction).
Genome database searches
Putative vkr sequences were searched using tBLASTn on genomic sequences available in the following databases: GenBank (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), FlyBase (http://flybase.org/), VectorBase (http://www.vectorbase.org), Wellcome Trust Sanger institute databases (http://www.sanger.ac.uk/resources/databases/) and the JGI genome portal (http://genome.jgi-psf.org/). Additionally, we also searched for vkr sequences in Hydra magnipapillata (hydrazome), Schmidtea mediterranea (SmedDB) and Schistosoma japonicum(schistoDB) genome databases.
Selected genomic sequences were analysed by GenScan (http://genes.mit.edu/GENSCAN.html) and Augustus (http://bioinf.uni-greifswald.de/augustus/) gene prediction servers, and putative VKR proteins were then determined. The presence of VFT, TM and TK domains was verified, and their delimitation defined using the SMART software (http://smart.embl-heidelberg.de/). Finally, TM regions were confirmed with the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM).
Protein sequences (listed in Additional file 1) were aligned using ClustalW algorithm in the BioEdit v7.1 software, and manually corrected. Maximum likelihood trees were built using MEGA5  under the JTT+I+G model, with 100 bootstrap repetitions.
Comparative modeling was performed on the VFT sequence domain of VKRs using the comparative modeling web-server Modweb (https://modbase.compbio.ucsf.edu/scgi/modweb.cgi) of the ModBase databases. Calculation and evaluation of models were performed with ModPipe software using sequence-sequence, sequence-profile and profile-sequence methods for fold assignment and target-template alignment.
This research was supported by the Institut de la Sante et de la Recherche Medicale and Université Lille Nord de France. MV, NG and MM fellowships were from the Ministere de l’Education Nationale et de la Recherche, France. The authors wish to thank Dr R.J. Pierce for his helpful advices.
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