The tick Ixodes scapularis has five different GPCRs specifically activated by ACP (adipokinetic hormone/corazonin-related peptide).

Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none.

Cloning and deorphanization of three inotocin (insect oxytocin/vasopressin-like) receptors and their ligand from the tick Ixodes scapularis.

Many insects produce the cyclic neuropeptide inotocin (CLITNCPRGamide), which is the insect orthologue of the mammalian neuropeptides oxytocin and vasopressin. These insects also have one inotocin G protein-coupled receptor (GPCR), which is the orthologue of the mammalian oxytocin and vasopressin receptors. The tick Ixodes scapularis belongs to the subphylum Chelicerata, an arthropod taxon different from insects, to which also spiders, scorpions, and mites belong. I. scapularis is an ectoparasite and a health risk for humans, because it transfers pathogenic microorganisms to its human host during a blood meal, thereby causing serious neurological diseases, among them Lyme disease and tick-borne encephalitis (TBE). By annotating the genomic sequence of I. scapularis, we previously found one presumed tick inotocin preprohormone gene and, in contrast to insects, three genes coding for presumed inotocin GPCRs. We now find that these GPCR genes cluster on one genomic contig, suggesting that they originated by recent gene duplications. Closely located on the same contig are also four adipokinetic hormone/corazonin-related peptide (ACP) GPCR genes, and one crustacean cardioactive peptide (CCAP) GPCR gene, suggesting evolutionary relationships. These evolutionary relationships are confirmed by phylogenetic tree analyses of their gene products. We also cloned the tick inotocin preprohormone, which has a structural organization closely resembling mammalian oxytocin and vasopressin preprohormones, including the presence of a conserved neurophysin sequence, having seven cystine bridges. This neurophysin sequence has two cystine-knot domains, but in contrast to mammalian neurophysins, the tick neurophysin contains a canonical prohormone convertase cleavage signal and a peptide C-terminal amidation sequence (GKR), suggesting cleavage into two biologically active cystine-knot peptides. This cleavage/amidation sequence occurs in neurophysins from most hard tick species, but not in other chelicerates. Mature tick inotocin is different from insect inotocin and has the sequence CFITNCPPGamide. Finally, we cloned and stably expressed the three tick inotocin receptors in Chinese Hamster Ovary cells and found that each of them was activated by nanomolar concentrations of tick inotocin (EC50 for ITR1 = 1.6 × 10-8 M; EC50 for ITR2 = 5.8 × 10-9 M; EC50 for ITR3 = 9.3 × 10-9 M), thereby establishing that they are genuine tick inotocin receptors.