In this study, we reported the discovery of a new family of tyrosine kinase receptors, called VKR, essentially present in invertebrates, and particularly in insects. Using a combination of bioinformatic, molecular biology and biochemical experiments, we demonstrated that the members of this family are single transmembrane-spanning molecules composed of an extracellular VFTM and an intracellular TK domain related to that of IR. At least for Apis mellifera, the VKR proteins are functional and their kinase activity can be induced by small ligands such as L-Arg.
The VKR proteins are largely represented in the class of insects, although the first two examples were discovered outside this class, in the trematode S. mansoni
(the parasite responsible for schistosomiasis, the second most important tropical disease after malaria 
, and in the sea urchin S. purpuratus
, and named SmVKR and SpVKR respectively. In 2006, during the inventory of the genes encoding RTK in the S. purpuratus
genome, two genes Sp-INSR and Sp-ILGFR related to IR family were identified 
, and we could recognize that the second one Sp-ILGFR was an RTK homologous to SmVKR. We could notice that gene structure was overall highly conserved in insects with an important restriction of size and of exon numbers when compared to other organisms. Owing to the substantial number of dipteran vkr
sequences identified, we could demonstrate that gene structures were homogenous in culicidae (mosquitoes) and drosophilidae (flies). Such a gene conservation in dipterans could argue for a potential importance of VKR receptors in insect biology.
gene could be detected in the lepidopteran genome of B. mori
and in the the genome of most of the drosophilidae belonging to the melanogaster
group, despite an extensive and complete analysis of the genome of these flies, suggesting the absence of the vkr
gene in these insects. In this group, only the D. ananasse
species was demonstrated to contain a putative vkr
. Moreover Davkr
has a genomic structure different from all of the other drosophilidae vkr
genes with an additional exon in the TK domain that could create an incorrect ATP-binding sequence in the protein and generate an inactive kinase. Such an observation is probably related to the disappearance of vkr
genes along the evolution of flies inside of the melanogaster
group. Moreover, for this interpretation, we have to consider that the currently defined Drosophila
genus has been shown to be paraphyletic, including more than 2 000 species, some but not all of them descending from a common ancestor 
. Gene mapping and analysis of vkr
gene environment in the various insects would be helpful for studying evolution of VKR as well as importance in insect biology.
This homogeneity of the VKR proteins in the phylogenetic analyses of VFT and TK domains suggests the existence of a common ancestral gene and the conservation of functional properties in all of these molecules. Although that no information can be given about the origin of vkr
, we can postulate that, according to the hypothesis already proposed by Yarden and Ullrich about the evolution of RTK 
, an ancestral vkr
gene would have resulted from the genetic combination of a pre-existing VFTM with a TK domain of a cytoplasmic protein. Until now, vkr
has never been detected in any vertebrate genome and therefore it can be supposed to be invertebrate specific. However, vkr
genes have not been found in every invertebrate species and for example are absent from the genome of the nematode C. elegans
. In platyhelminths, VKR has been characterized only in Schistosoma
species, but in-depth researches in turbellarian genome data bases have allowed the detection in the planarian Schmidtea mediterranea
of a genomic sequence (GenBank: AAWT01078636.1) encoding a putative TK domain of VKR proteins linked to a truncated VFT module. Further genomic and functional studies are still required to understand both the origin of vkr
genes and their selective existence/persistence in given invertebrate organisms.
The VFT domain being close to that of class-C GPCRs in particular GABAB
receptors and the TK one being close to that of IR, it suggests an original mode of functioning for a RTK. First, we showed that the recombinant AmVKR forms homo- or oligomers, as expected for membrane receptor containing a VFTM such as class-C GPCRs and ANFR that are well-known to function as homo- or hetero-dimers as well as the RTK 
. All motifs essential for TK activity were perfectly conserved in the TK domain of VKR suggesting that dimerized AmVKR receptors could autophosphorylate and exert kinase activity. Surprisingly, the basal level of auto-phosphorylation is quite low compared to that we could expect for a recombinant IR in the same conditions 
. In our study, we need to use constitutively active AmVKR mutants to obtain high levels of kinase activity strongly suggesting that VKR activation in cell signalling was dependent on the binding of a ligand in the extracellular domain of the receptor.
Although all RTKs are activated by dimerization, different ligands employ different strategies for inducing the active dimeric state 
. VFTMs of membrane receptors are functioning at least as dimers, and they contain the binding site for natural small ligands such as peptides 
, small sugar 
, amino-acids or derivatives 
and cations 
. Although the ligand binding site can be at the VFTM dimeric interface 
, usually ligands bind into the VFTM binding pocket and in Class-C GPCRs binding of the natural ligands induces closure of the VFT responsible for receptor activation 
. In VFTM VKR binding pocket, we observed a relative conservation of the residue responsible for binding of the alpha-amino acid functions of the glutamate in mGlu1, suggesting ligands of VKRs could be amino-acids or derivatives.
Accordingly, among the various molecules known to bind VFTM of class C GPCRs (glutamate, GABA, L-α- amino acids), we could show that L-arginine was able to increase in vitro
the basal kinase activity of AmVKR, suggesting that this aminoacid could be a preferential ligand for VKR receptors, similarly to the mammalian GPRC6A 
and the fish OR5.24 
receptors, two class C GPCRs for which L-arginine is also a potent agonist. Moreover, it is important to mention that in insects, VFTM-containing mX receptors are modulated by arginine and derivatives 
Future investigations will concern the biological role of VKR in development and reproduction, particularly in insects. The presence of high levels of vkr
transcripts in larval forms and in female gonads of different organisms already supposed the importance of VKR proteins in larval growth and differentiation as well as in reproduction. RNA interference experiments are currently performed in honey bee and mosquitoes in order to elucidate the function of VKR in these processes. Since ovary development in mosquitoes has been shown to be regulated by TOR (Target of Rapamycin)- mediated amino acid signaling 
, we will also investigate the importance of VKR signalling in anautogeny, ie activation of egg development after a blood meal, a unique feature in the life of mosquitoes.
In conclusion, VKR constitute a novel family of RTK specific for invertebrates, mainly expressed in reproductive organs and activated by aminoacids. The biological function of these receptors is yet unknown but if VKR are effectively implied in reproduction of parasites (like schistosomes) or in disease-transmitting insects (like Anopheles vector of malaria or Aedes vector of filaria and viruses), these receptors already represent interesting drug targets for novel strategies to combat parasitic and infectious diseases.