Abstract
Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides. In this study, we originally identified authentic TKs and their receptor from a protochordate, Ciona intestinalis. The Ciona TK (Ci-TK) precursor, like mammalian gamma-preprotachykinin A (gamma-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH(2) vertebrate TK consensus. Mass spectrometry of the neural extract revealed the production of both Ci-TKs. Ci-TK-I contains several Substance P (SP)-typical amino acids, whereas a Thr is exceptionally located at position 4 from the C terminus of Ci-TK-II. The Ci-TK gene encodes both Ci-TKs in the same exon, indicating no alternative generation of Ci-TKs, unlike the PPTA gene. These results suggested that the alternative splicing of the PPTA gene was established during evolution of vertebrates. The only Ci-TK receptor, Ci-TK-R, was equivalently activated by Ci-TK-I, SP, and neurokinin A at physiological concentrations, whereas Ci-TK-II showed 100-fold less potent activity, indicating that the ligand selectivity of Ci-TK-R is distinct from those of vertebrate TK receptors. Ci-TK-I, like SP, also elicited the typical contraction on the guinea pig ileum. The Ci-TK gene was expressed in neurons of the brain ganglion, small cells in the intestine, and the zone 7 in the endostyle, which corresponds to the vertebrate thyroid gland. Furthermore, the Ci-TK-R mRNA was distributed in these three tissues plus the gonad. These results showed that Ci-TKs play major roles in sexual behavior and feeding in protochordates as brain/gut peptides and endocrine/paracrine molecules. Taken together, our data revealed the biochemical and structural origins of vertebrate TKs and their receptors.
Highlights
Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides
TK-related peptides (TKRPs), but not TKs, have been found in invertebrates, which complicates the evolutionary aspect of the TK family [7, 20]
We showed the structure, activity, and localization of a TK peptide, Ciona TK (Ci-TK), and its receptor, Ci-TK-R, of an ascidian, C. intestinalis
Summary
Animals—Adults of C. intestinalis were cultivated and collected at the Maizuru Fisheries Research Station of Kyoto University, and maintained in sea water at 18 °C. The partial Ci-TK cDNA was obtained by PCR using the primers identical to nucleotides (nt) 46 – 66 and complementary to nt 499 –518. Preparation of digoxigenin (DIG)labeled cDNA probes, hybridization, and detection were performed in accordance with the DIG system protocol (Roche Applied Science). In Situ Hybridization—The Ci-TK cDNA fragment (nt 47–558) was inserted into the PST 18 vector (Roche Applied Science), and the linearized plasmid was supplied to the synthesis of DIG-labeled Ci-TK. RNA probe using a DIG RNA labeling kit (Roche Applied Science) Whole-mount in situ hybridization of the juvenile and adult neural complex and endostyle were performed as previously described [32, 33]. No positive signals were observed when sense probes were used, confirming the specificity of hybridization
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