Tachykinin and Tachykinin Receptor of an Ascidian, Ciona intestinalis

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 γ-preprotachykinin A (γ-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH2 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. 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 γ-preprotachykinin A (γ-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH2 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. The large diversity of neuropeptides is correlated with the evolution and divergence of the nervous systems as well as their biological roles in organisms. Tachykinins (TKs) 1The abbreviations used are: TK, tachykinin; Ci-TK, Ciona tachykinin; Ci-TK-R, Ci-TK receptor; GPCR, G-protein-coupled receptor; MS, mass spectrometry; NKA, neurokinin A; NKB, neurokinin B; PPTA, -B, and -C, preprotachykinin A, B, and C, respectively; RACE, rapid amplification of cDNA ends; SP, substance P; TKRP, TK-related peptide; nt, nucleotides; DIG, digoxigenin.1The abbreviations used are: TK, tachykinin; Ci-TK, Ciona tachykinin; Ci-TK-R, Ci-TK receptor; GPCR, G-protein-coupled receptor; MS, mass spectrometry; NKA, neurokinin A; NKB, neurokinin B; PPTA, -B, and -C, preprotachykinin A, B, and C, respectively; RACE, rapid amplification of cDNA ends; SP, substance P; TKRP, TK-related peptide; nt, nucleotides; DIG, digoxigenin. are vertebrate multifunctional brain/gut peptides involved in various central and peripheral functions including smooth muscle contraction, vasodilation, inflammation, and the processing of sensory information in a neuropeptidergic or endocrine/paracrine fashion (1Otsuka M. 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Neurol. 1992; 325: 572-580Crossref PubMed Scopus (25) Google Scholar). Since investigation of TKs or TKRPs in protochordates is expected to provide crucial findings concerning not only the biological roles of TKs or TKRPs in protochordates but also the evolutionary origins of the structures and functions of the TK family, we explored TK peptides and its receptor in an ascidian, Ciona intestinalis, which belongs to protochordates as a basal chordate, namely an emerging deuterostome model animal (29Moss C. Beesley P.W. Thorndyke M.C. Bollner T. Tissue Cell. 1998; 30: 517-524Crossref PubMed Scopus (11) Google Scholar, 30Corbo J.C. Di Gregorio A. Levine M. Cell. 2001; 106: 535-538Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 31Satoh N. Satou Y. Davidson B. Levine M. Trends Genet. 2003; 19: 376-381Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In this work, we present the structure, localization, and reactivity of Ciona TK, Ci-TK, and its receptor, Ci-TK-R, suggesting biological roles of the TK family in protochordates and the features of Ci-TK and Ci-TK-R as prototypes of vertebrate TK peptides and receptors.EXPERIMENTAL PROCEDURESAnimals—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.PCR Primers—All sequences of PCR primers are summarized in Supplemental Table I.Identification of the Ci-TK and Ci-TK-R cDNAs—Total RNA (0.5 μg) from the neural complex was reverse-transcribed to the template cDNA at 55 °C for 60 min using the oligo(dT) anchor primer and the avian myeloblastosis virus reverse transcriptase supplied in the 5′/3′-rapid amplification of the cDNA ends (RACE) kit (Roche Applied Science). The partial Ci-TK cDNA was obtained by PCR using the primers identical to nucleotides (nt) 46–66 and complementary to nt 499–518. RACE was performed using the gene-specific primers complementary to nt 268–288 (for 5′-RACE) and nucleotides identical with nt 268–288 (for 3′-RACE), respectively. Similarly, the Ci-TK-R cDNA was obtained by RT-PCR using the primers identical to nt 20–40 and complementary to nt 1464–1485 followed by nested PCR with primers identical to nt 45–68 and complementary to nt 1391–1410. 5′-RACE with primers complementary to 350–371 and 379–398, and 3′-RACE with primers identical to nt 1261–1280 and nt 1310–1329 were subsequently performed. Subcloned inserts were sequenced on an ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems) using a Big-Dye sequencing kit (Applied Biosystems) and universal primers (SP6 and T7 primers).Mass Spectrometry (MS)—Ten Ciona neural complexes were pulverized by grinding under liquid nitrogen and extracted in 20 ml of boiled water. The resultant extract was eluted using a Sep-pak plus C-18 cartridge (Waters; Tokyo, Japan), and the eluate was evaporated and lyophilized. To acquire MS/MS spectra of Ci-TKs, the crude peptide was dissolved in 50% (v/v) methanol containing 0.1% formic acid, followed by observation of the spectra for Ci-TK-I and -II with a Q-TOF tandem mass spectrometer equipped with a Z-spray nanoelectrospray interface (Micromass, Manchester, UK). The needle voltage was optimized at 1000 V; the cone voltage was set at 50 V. Argon was used as the collision gas, and the collision gas energy was set at 28 V.Functional Analysis of Ci-TK-R Expressed in Xenopus Oocytes—The open reading frame region of Ci-TK-R cDNA was amplified and inserted into the Xenopus expression vector pSPUTK (Stratagene). The cRNA was prepared from the plasmid linearized with HpaI using SP6 RNA polymerase (Ambion, Austin, TX). 50 nl of the cRNA solution (0.05 μg/μl) were injected into oocytes. The oocytes were incubated for 2–4 days at 17 °C and transferred to ND96 buffer (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES (pH 7.6)). The oocytes were voltage-clamped at -80 mV. The dose-response data and the EC50 values of the experiment were analyzed using Origin 6.1 software (Microcal Software, Tokyo). For the bioassay, the guinea pig ileum was a gift from Dr. Toshiaki Fujii. The contractile effects of Ci-TKs and other TKs were observed as described previously (19Kanda A. Iwakoshi-Ukena E. Takuwa-Kuroda K. Minakata H. Peptides. 2003; 24: 35-43Crossref PubMed Scopus (48) Google Scholar).Southern Hybridization of RT-PCR Products—The primer sets used for Ci-TK cDNA were identical with nt 20–40 in the Ci-TK cDNA and complementary to nt 582–602; the primer sets used for Ci-TK-R cDNA were identical with nt 45–68 in the Ci-TK-R cDNA and complementary to nt 752–773, and the primer sets used for Ciona β-actin cDNA were identical with nt 254–274 in the Ciona β-actin cDNA and complementary to nt 845–864. PCR was performed for 25 cycles for amplification of the Ci-TK cDNA and for 35 cycles for the Ci-TK-R cDNA consisting of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 72 °C. PCR products were resolved on a 1.5% agarose gel followed by transfer to a Hybond N+ membrane (Amersham Biosciences). 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 (32Ogasawara M. Satoh N. Biol. Bull. 1998; 195: 60-69Crossref PubMed Scopus (27) Google Scholar, 33Ogasawara M. Sasaki A. Metoki H. Shin-i T. Kohara Y. Satoh N. Satou Y. Dev. Genes. Evol. 2002; 212: 173-185Crossref PubMed Scopus (88) Google Scholar). The Ciona digestive tracts were dissected and fixed in Bouin's fluid at 4 °C overnight. Preparation of 5-μm serial sections, hybridization, washing, and detection were carried out as previously reported (34Satake H. Takuwa K. Minakata H. Matsushima O. J. Biol. Chem. 1999; 274: 5605-5611Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). No positive signals were observed when sense probes were used, confirming the specificity of hybridization.RESULTSIdentification of the Ci-TK Peptides, cDNA, and Genomic Structure—In an attempt to detect a Ciona TK cDNA, we performed BLAST searches on the draft genome data base of C. intestinalis (available on the World Wide Web at ghost.zool.kyoto-u.ac.jp/indexr1.html) (35Dehal P. Satou Y. Campbell R.K. Chapman J. Degnan B. De Tomaso A. Davidson B. Di Gregorio A. Gelpke M. Goodstein D.M. Harafuji N. Hastings K.E.M. Ho I. Hotta K. Huang W. Kawashima T. Lemaire P. Martinez D. Meinertzhagen I.A. Necula S. Nonaka M. Puntnam N. Rash S. H. Satake M. A. L. Wang S. K. J. M. S. R. S. P. S. H. K. K. S. K. M. C. G. M. Y. S. M. S. Nakayama A. S. Nomoto H. F. K. I. M. Sasaki A. Y. E. T. A. D. O. N. M. K. F. S. C. D. F. C. N. T. T. P. S. Kohara Y. Levine M. Satoh N. Science. 2002; PubMed Scopus Google Scholar). of only γ-PPTA to the data base searches in the detection of several on the Ciona genome data These were to encode an identical open reading frame including two TK sequences with the vertebrate TK consensus was of the structural organization of γ-PPTA (7Severini C. Improta G. Falconieri-Erspamer G. Salvadori S. Erspamer V. Pharmacol. Rev. 2002; 54: 285-322Crossref PubMed Scopus (508) Google Scholar, 12Nawa H. Hirose T. Takashima H. Inayama S. Nakanishi S. Nature. 1983; 312: 32-36Crossref Scopus (590) Google Scholar, 13Nawa H. Kotani H. Nakanishi S. Nature. 1984; 312: 729-734Crossref PubMed Scopus (448) Google Scholar). The cDNA was from the neural complex by RT-PCR followed by and in the amino encoded two TK sequences by a C-terminal signal at their C and typical at both suggesting that two TK peptides, and are from the we these two peptides as Ci-TK-I and respectively. The in analyses of the Ci-TK using the Ciona genome data base (available on the World Wide Web at that the Ci-TK gene of both Ci-TK-I and -II sequences were encoded in the to SP and NKA, which are encoded in the and of the vertebrate PPTA gene, respectively (7Severini C. Improta G. Falconieri-Erspamer G. Salvadori S. Erspamer V. Pharmacol. Rev. 2002; 54: 285-322Crossref PubMed Scopus (508) Google Scholar, 12Nawa H. Hirose T. Takashima H. Inayama S. Nakanishi S. Nature. 1983; 312: 32-36Crossref Scopus (590) Google Scholar, 13Nawa H. Kotani H. Nakanishi S. Nature. 1984; 312: 729-734Crossref PubMed Scopus (448) Google the of Ci-TK peptides as we performed Q-TOF MS/MS of the peptide of the ascidian neural 2 the of which was with and respectively. In addition, no mass values for of Ci-TK-I or were In these data that both Ci-TK-I and are from the we that TKs are conserved in protochordates as well as MS/MS spectra of Ci-TK-I and Ci-TK-II and several fragment are of Ci-TKs with was to a and an at 4 and 7 from the C respectively which is in with the that TKs with an amino and a or amino at of these the selectivity to NK1, the TK receptor (1Otsuka M. Yoshioka K. Physiol. Rev. 1993; 73: 229-308Crossref PubMed Scopus (1039) Google Scholar, 7Severini C. Improta G. Falconieri-Erspamer G. Salvadori S. Erspamer V. Pharmacol. Rev. 2002; 54: 285-322Crossref PubMed Scopus (508) Google Scholar). the other Ci-TK-I which is at position and/or from the C terminus of SP and several TKs (1Otsuka M. Yoshioka K. Physiol. Rev. 1993; 73: 229-308Crossref PubMed Scopus (1039) Google Scholar, 7Severini C. Improta G. Falconieri-Erspamer G. Salvadori S. Erspamer V. Pharmacol. Rev. 2002; 54: 285-322Crossref PubMed Scopus (508) Google Scholar). In addition, the region of Ci-TK-I no to any other TKs. Ci-TK-II a with NKA and several and TKs H. J. N. K. H. Biochem. Biophys. Res. Commun. PubMed Scopus (27) Google Scholar, Y. T. S. N. H. E. Conlon J.M. Biochem. J. 1992; PubMed Scopus (25) Google Scholar, D. S. C. Conlon J.M. Peptides. PubMed Scopus (25) Google Scholar, R.E. Peptides. 1997; PubMed Scopus Google at position from the C terminus as in Table I. However, the most of Ci-TK-II is that this ascidian TK is of only 7 amino acids, whereas other TKs amino (1Otsuka M. Yoshioka K. Physiol. Rev. 1993; 73: 229-308Crossref PubMed Scopus (1039) Google Scholar, 7Severini C. Improta G. Falconieri-Erspamer G. Salvadori S. Erspamer V. Pharmacol. Rev. 2002; 54: 285-322Crossref PubMed Scopus (508) Google Scholar). the Thr at position 4 from the C terminus of Ci-TK-II is as a amino that SP and its TKs contain an amino acid, whereas an amino is located at this position in NKA, NKB, and their counterparts (1Otsuka M. Yoshioka K. Physiol. Rev. 1993; 73: 229-308Crossref PubMed Scopus (1039) Google Scholar, 7Severini C. Improta G. Falconieri-Erspamer G. Salvadori S. Erspamer V. Pharmacol. Rev. 2002; 54: 285-322Crossref PubMed Scopus (508) Google Scholar). these the distinct of of Ci-TK-I and with several vertebrate M. Yoshioka K. Physiol. Rev. 1993; 73: 229-308Crossref PubMed Scopus (1039) Google Scholar, 7Severini C. Improta G. Falconieri-Erspamer G. Salvadori S. Erspamer V. Pharmacol. Rev. 2002; 54: 285-322Crossref PubMed Scopus (508) Google M. Yoshioka K. Physiol. Rev. 1993; 73: 229-308Crossref PubMed Scopus (1039) Google Scholar, 7Severini C. Improta G. Falconieri-Erspamer G. Salvadori S. Erspamer V. Pharmacol. Rev. 2002; 54: 285-322Crossref PubMed Scopus (508) Google M. Yoshioka K. Physiol. Rev. 1993; 73: 229-308Crossref PubMed Scopus (1039) Google Scholar, 7Severini C. Improta G. Falconieri-Erspamer G. Salvadori S. Erspamer V. Pharmacol. Rev. 2002; 54: 285-322Crossref PubMed Scopus (508) Google H. J. N. K. H. Biochem. Biophys. Res. Commun. PubMed Scopus (27) Google Y. T. S. N. H. E. Conlon J.M. Biochem. J. 1992; PubMed Scopus (25) Google R.E. Peptides. 1997; PubMed Scopus Google D. S. C. Conlon J.M. Peptides. PubMed Scopus (25) Google in a of Ciona data base with mammalian TK and TKRP provided a which the to mammalian The of the Ciona was by of and The amino was to the and several that are conserved in TK A that the of the Ciona amino to those of mammalian TK and TKRP Taken together, these results to that the Ciona is a for a Ci-TK receptor, of the amino sequences of TK and TKRP receptors. of Ci-TK-R, and three TKRP are are by The the in a and are with and selectivity of TK to their were by functional expression using Xenopus oocytes (1Otsuka M. Yoshioka K. Physiol. Rev. 1993; 73: 229-308Crossref PubMed Scopus