A novel tachykinin-related peptide receptor Sequence, genomic organization, and functional analysis Tsuyoshi Kawada 1 , Yasuo Furukawa 2 , Yoriko Shimizu 2 , Hiroyuki Minakata 1 , Kyosuke Nomoto 3 and Honoo Satake 1 1 Suntory Institute for Bioorganic Research, Osaka, Japan; 2 Department of Biological Science, Faculty of Science, Hiroshima University, Japan; 3 Faculty of Life Sciences, Toyo University, Gunma, Japan Structurally tachykinin-related peptides have been isolated from various invertebrate species and shown to exhibit their biological activities through a G-protein-coupled receptor (GPCR) for a tachykinin-related peptide. In this paper, we report the identification of a novel tachykinin-related pep- tide receptor, the urechistachykinin receptor (UTKR) from the echiuroid worm, Urechis unitinctus. The deduced UTKR precursor includes seven transmembrane domains and typ- ical sites for mammalian tachykinin receptors and inver- tebrate tachykinin-related peptide receptors. A functional analysis of the UTKR expressed in Xenopus oocytes dem- onstrated that UTKR, like tachykinin receptors and tachykinin-related peptide receptors, activates calcium- dependent signal transduction upon binding to its endo- genous ligands, urechistachykinins (Uru-TKs) IÀV and VII, which were isolated as Urechis tachykinin-related peptides from the nervous tissue of the Urechis unitinctus in our previous study. UTKR responded to all Uru-TKs equival- ently, showing that UTKR possesses no selective affinity with Uru-TKs. In contrast, UTKR was not activated by substance P or an Uru-TK analog containing a C-terminal Met-NH 2 instead of Arg-NH 2 . Furthermore, the genomic analysis revealed that the UTKR gene, like mammalian tachykinin receptor genes, consists of five exons interrupted by four introns, and all the intron-inserted positions are completely compatible with those of mammalian tachykinin receptor genes. These results suggest that mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors were evolved from a common ancestral GPCR gene. This is the first identification of an invertebrate tachykinin-related peptide receptor from other species than insects and also of the genomic structure of a tachykinin- related peptide receptor gene. Keywords: tachykinin-related peptide; Uru-TK; UTKR; Urechis unicinctus; G-protein-coupled receptor. Tachykinins are vertebrate multifunctional brain/gut pep- tides that play crucial roles not only in the various peripheral activities but also in the functions of the central nervous system including the processing of sensory information [1À5]. The major mammalian tachykinin family peptides are substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). Three mammalian tachykinin receptors, namely, NK1, NK2, and NK3 receptors, have also been well characterized. They belong to a G-protein-coupled receptor (GPCR) superfamily, and their interaction with their agonists causes the activation of phospholipase C (PLC) inducing the production of inositol 1,4,5-triphosphate (InsP 3 ) and an increase of intracellular calcium as second messengers [6]. Numerous structurally tachykinin-related peptides have been characterized from various invertebrates since locustatachykinins (Lom-TKs) I and II were purified [7]. Previously, we also identified urechistachykinins (Uru-TKs) I and II from the ventral nervous cord of the echiuroid worm Urechis unicinctus [8]. Furthermore, we cloned the Uru-TKs cDNA as the first example of cDNA encoding an invertebrate tachykinin-related peptide, showing that the Uru-TK precursor polypeptide encodes five more Uru-TK sequences (Uru-TKs IIIÀVII) as well as Uru-TKs I and II, and that six of seven Uru-TKs (Uru-TKs IÀV and VII, Table 1) are produced from this precursor [9,10]. Of particular importance in tachykinin-related peptides is that most tachykinin-related peptides share the C-terminal common sequence Phe-X-Gly-Y-Arg-NH 2 , which is ana- logous to the mammalian tachykinin consensus sequence Phe-X-Gly-Leu-Met-NH 2 . In addition, no tachykinin-rela- ted peptides containing the Phe-X-Gly-Y-Arg-NH 2 sequence have ever been isolated from vertebrates. Some biochemical activities of tachykinin-related pep- tides such as the contraction of cockroach hindgut and oviduct as well as depolarization or hyperpolarization of identified interneurons of locusts have been documented [7]. These bioactivities of tachykinin-related peptides are expec- ted to be exerted upon interaction with their receptors. To date, DTKR, NKD, and STKR have been cloned as tachykinin-related peptide receptors or receptor candidates Correspondence to H. Satake, Wakayamadai 1-1-1, Shimamoto-cho, Mishima-gun, Osaka 618À8503, Japan. Fax: + 81 75 962 2115, Tel.: + 81 75 962 3743, E-mail: Hono_Satake@suntory.co.jp Abbreviations: GPCR, G-protein coupled receptor; InsP 3 , inositol 1,4,5-triphosphate; NKA, neurokinin A; NKB, neurokinin B; PLC, phospholipase C; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; SP, substance P; Uru-TK, urechistachykinin; UTKR, Uru-TK receptor. Note: cDNA and genomic DNA sequence data are available in the DDBJ/EMBL/GenBank databases under accession numbers AB050456 and AB081457, respectively. (Received 26 April 2002, revised 8 July 2002, accepted 11 July 2002) Eur. J. Biochem. 269, 4238À4246 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03106.x [11]. More recently, a partial sequence of another putative tachykinin-related peptide receptor, LTKR was also iden- tified from the cockroach Leucophaea maderae [12]. These receptors or putative receptors show high amino-acid sequence similarity to mammalian tachykinin receptors [11À14],andNKDandSTKR,whichwereclonedfromthe fruitfly Drosophila melanogaster and the stable fly Stomoxys calcitrans, respectively, were found to interact with some tachykinin-related peptides [13,14]. Furthermore, recent studies revealed that STKR, like mammalian tachykinin receptors, activates the PLC-InsP 3 -calcium signal transduc- tion cascade [15,16]. These findings imply that tachykinin- related peptides are the invertebrate functional counterparts, at least partially, for vertebrate tachykinin family peptides. However, only a few tachykinin-related peptide receptors have been characterized from several insects as mentioned above. Furthermore, tachykinin-related peptides and their receptors from different species have so far been employed for studies of tachykinin-related peptide activity on insect tachykinin-related peptide receptors. Therefore, the bio- chemical characteristics of tachykinin-related peptides and their receptors such as the binding selectivity still need to be fully elucidated, and the interphyletic relationships and molecular evolution of tachykinin-related peptide receptors have not been investigated. To further study the biological functions and evolutionary and phylogenetic relationship of tachykinin-related peptide receptors and tachykinin recep- tors, we identified a novel tachykinin-related peptide recep- tor, UTKR from the echiuroid worm Urechis unicinctus. In this paper, we present a UTKR sequence, an exon/intron structure of the UTKR gene, and the response of the UTKR to Uru-TKs. To the best of our knowledge, this is the first characterization of a noninsect tachykinin-related peptide receptor and the structural organization of the tachykinin- related peptide receptor gene. MATERIALS AND METHODS Preparation of RNA from echiuroid worms Echiuroid worms were purchased from a fishing-bait shop. Total RNA was prepared from ventral nervous tissues using TRIzol reagent (Gibco, Gaithersburg, MD, USA), and mRNA was purified using Oligotex TM -dT 30 (Daiichikagaku, Tokyo, Japan) according to the manufac- turer’s instructions. Oligonucleotide primers All oligonucleotide primers were ordered from Kiko- Technology (Osaka, Japan). The oligo-dT anchor primer and the anchor primer were supplied in a 5¢/3¢ RACE kit (Roche Diagnostics, Basel, Switzerland). Identification of the partial fragment of UTKR cDNA All reverse transcription polymer chain reactions (RT-PCRs) and rapid amplifications of cDNA ends were performed using Taq Ex polymerase (Takara, Kyoto, Japan) or rTaq DNA polymerase (Toyobo, Osaka, Japan) and a thermal cycler (model GeneAmp PCR system 9600; PE-Biosystems, Foster City, CA, USA). The mRNA (0.5 lg) was reverse-transcribed to cDNA at 55 °C for 60 min using the oligo-dT anchor primer and the AMV reverse transcriptase supplied in the 5¢/3¢ RACE kit (Roche). The first-strand cDNA was amplified using the degenerate primers 5¢-AI(A/C)GIATG(A/C)GIACIGTIA CIAA(T/C)TA(T/C)TT-3¢ (I represents an inosine residue) and 5¢-CA(A/G)CA(A/G)TAIATIGG(A/G)TT(A/G)TA CAT-3¢, corresponding to amino-acid sequences RMRTVTNYF (at transmembrane domain II of mamma- lian tachykinin receptors) and MYNPIIYC (at transmem- brane domain VII), respectively. These PCR experiments were performed with five cycles, consisting of 94 °C for 30 s, 40 °C for 30 s and 72 °C for 3 min, followed by 35 cycles, consisting of 94 °C for 15 s, 50 °C for 30 s, and 72 °C for 3 min. The first-round PCR products were reamplified using the degenerate primers 5¢-AI(A/C)GIATG(A/C)GIA CIGTIACIAA(T/C)TA(T/C)TT-3¢ and 5¢-TG(A/G)(A/T) AIGGIA(A/G)CCA(A/G)CAIATIGC-3¢ corresponding to the sequences RMRTVTNYF and AICWLP(F/Y)H (trans- membrane domains II and VI, respectively). The PCR was performed with five cycles of 94 °C for 30 s, 37 °C for 1min,and72°C for 2 min, followed by 15 cycles of a 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 2 min and a final extension at 72 °C for 10 min. The resultant PCR product was purified using the Qiaquick Gel Extraction kit (Qiagen, Valencia, CA, USA) and subcloned into the pCR2.1 vector using a TA cloning kit (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. Subcloned inserts were sequenced on an ABI PRISMTM 310 Genetic Analyzer (PE-Biosystems) using a Big-Dye sequencing kit (PE-Biosytems) and universal primers (M13 or T7 primers). 3¢ RACE of UTKR cDNA First-strand cDNA was amplified using the oligo-dT primer and a gene-specific primer (5¢-CTTGGCCTGTGCGTATT CGATGG-3¢, complementary to nucleotides 1041À63), and the first-round PCR products were reamplified using the anchor primer for 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 3 min (10 min for the last cycle). The products were subcloned and sequenced as described above. 5¢ RACE of UTKR cDNA The template cDNA was synthesized using a primer complementary to nucleotides 752À730 (5¢-ACGGACGCT GCAATAGTGCATGG-3¢), followed by dA-tailing of the cDNA using dATP and terminal transferase (Roche). The first cDNA was amplified using an oligo-dT anchor primer and a gene-specific primer (5¢-GTGAACTTGCAGAATG GTAGCTCG-3¢; complementary to nucleotides 716À693), and the first-round PCR products were amplified using the Table 1. Amino-acid sequences of Uru-TK peptides. The conserved amino acids are shown in bold. Peptide Sequence Uru-TK I LRQSQFVGAR-NH 2 Uru-TK II AAGMGFFGAR-NH 2 Uru-TK III AAPSGFFGAR-NH 2 Uru-TK IV AAYSGFFGAR-NH 2 Uru-TK V APSMGFFGAR-NH 2 Uru-TK VII APKMGFFGAR-NH 2 Ó FEBS 2002 An Urechis tachykinin-related peptide receptor (Eur. J. Biochem. 269) 4239 PCR anchor primer and a primer (5¢-CGAACACCCAG TGGTTATTCAAC-3¢, complementary to nucleotides 693À672), followed by reamplification using the anchor primer and a primer (5¢-GATATCAAAGCGTCAGCAA CTGC-3¢, complementary to nucleotides 638À616). PCRs were performed as described for 3¢ RACE, and the final PCR products were subcloned and sequenced as described above. Determination of the exon/intron structure of the UTKR gene The genomic DNA of echiuroid worms was extracted using the MagExtractor (Toyobo) and the UTKR gene was amplified using the Genomic PCR with Expand TM Long Template PCR System (Roche). The reaction was per- formed with primers corresponding to the 5¢-and 3¢-terminal regions of UTKR cDNA according to the manufacturer’s instructions. The amplified products were subcloned and sequenced using several gene-specific pri- mers. To sequence intron 1, the subcloned PCR products containing the full-length intron 1 were digested with EcoRI, HindIII, HpaIandXhoI, and each fragment was re-subcloned and sequenced. Peptide synthesis and purification Uru-TKs and their analogs were synthesized by a solid- phase peptide synthesizer (Model 433 A, PE-Biosystems, Tokyo, Japan) using the FastMoc TM method and were purified by a C18 reversed-phase HPLC column (Model UG 80, 5 lm, size 20 mm ø · 250 mm, Shiseido, Tokyo, Japan). The peptide sequences were confirmed by a peptide sequencer (Model PSQ-1, Shimadzu, Kyoto, Japan). Expression of UTKR in Xenopus oocytes The ORF region of UTKR cDNA was amplified and inserted into the Xenopus expression vector pSPUTK (Stratagene, La Jolla, CA, USA). The plasmid was linea- rized with HpaI, and cRNA was prepared using SP6 RNA polymerase (Ambion, Texas, USA). 50 nL of the cRNA solution (0.05 lgÆlL )1 ) were injected into oocytes. The oocytes were incubated for 2À4daysat17°C and trans- ferredtoND96buffer[96m M NaCl, 2 m M KCl, 1.8 m M CaCl 2 ,1m M MgCl 2 and 5 m M Hepes (pH 7.6)]. The oocytes were voltage-clamped at )80 mV. The doseÀ response data and the EC50 values of the experiment were analyzed using ORIGIN 6.1 software (Microcal Software Inc.). RESULTS Cloning of a Uru-TK receptor cDNA Comparative analysis of amino-acid sequences of mamma- lian tachykinin receptors and insect tachykinin-related peptide receptors showed that the second, sixth, and seventh transmembrane domains are highly conserved among all receptors. To identify a tachykinin-related peptide receptor of the echiuroid worm, we first performed RT-PCR experiments using degenerative primers corresponding to the conserved regions (see Materials and methods). An amplified cDNA product of 628 bp was subcloned and sequenced. The putative amino-acid sequence was shown to encode a partial transmembrane domain of a GPCR. Moreover, we determined the full-length cDNA sequence encoding the putative GPCR using the 5¢-and3¢ RACE method. Figure 1A shows the 2533 bp putative receptor cDNA containing a 1293 bp ORF flanked by a 306 bp 5¢-untranslatedregion(UTR)anda924bp3¢-UTR. The ORF begins with the ATG codon at position 307, which is supported by the Kozak rule [17], and terminates with a TGA stop codon at position 1602. Only one potential polyadenylation signal AATAAA was found to be located 19 bases upstream of a poly(A) tail. The deduced receptor protein is composed of 431 amino- acid residues (Fig. 1). The sequence showed the presence of the seven hydrophobic transmembrane regions that are the most typical characteristic of GPCRs. The common Cys residues (Cys134 and Cys214) responsible for the disulfide bridge between the first and second extracellular loops are found at corresponding positions of known tachykinin receptors. N-linked glycosylation sites (Asn-X-Ser/Thr, Asn28, Asn39, and Asn223) are also located at the N-terminal and second extracellular domains. The GPCR sequence were also found to contain potential phosphory- lation sites by protein kinase A (Arg/Lys-X-(X)-Ser/Thr, Ser173, Thr262, Ser365, Ser381, Thr389, and Ser396), by protein kinase C (Ser/Thr-X-Arg/Lys, Thr273 and Ser276), and by casein kinase 2 (Ser/Thr-X-(X)-Asp/Glu, Thr262, Ser381, Thr389, Thr400, and Ser404) in the second and third intracellular loop and C-terminal region. Further- more, the Asp/Glu-Arg-Tyr motif (Asp158ÀTyr160) in the second intracellular loop and the Lys/Arg-Lys/Arg-X-X- Lys/Arg motif(Arg278ÀLys282) in the third intracellular loop which are often shown in most GPCRs are also present (Fig. 1), whereas a cysteine residue utilized as a palmityla- tion site in the C-terminal region was not found, given that the Trp/Cys-Cys palmitylation site in tachykinin receptors was replaced with Trp356ÀLeu357 at the corresponding positions of the putative Urechis GPCR (Fig. 1). The lack of this site was not the result of a PCR error or an artifact, as all clones obtained using different polymerases encoded the identical sequence. Comparative study of amino-acid sequences verified that the putative Urechis GPCR sequence including the transmembrane domains and intracellular and extracellular regions displayed high identity to those of mammalian tachykinin receptors and insect tachykinin- related peptide receptors (Fig. 2 and Table 2). In addition, the sequence of this region was shown to be closer to those of tachykinin-related peptide receptors than tachykinin receptors (Table 2). Furthermore, the homology-searching showed no significant similarity of UTKR to any other GPCR. Taken together, these results revealed that the putative Urechis GPCR possesses the essential properties of tachykinin receptors and tachykinin-related peptide recep- tors. Consequently, we concluded that this GPCR is a putative Urechis tachykinin-related peptide receptor and designated the receptor as the Uru-TK receptor, UTKR. Functional expression of UTKR in Xenopus oocytes It is well established that the binding of tachykinins and tachykinin-related peptides to their receptors results in the activation of PLC followed by the production of the 4240 T. Kawada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 intracellular second messengers, InsP 3 and calcium [13À16,18À20]. In Xenopus oocytes, the interaction of an agonist with its GPCR, inducing an elevation of intracel- lular calcium, leads to the activation of a calcium-dependent chloride channel, which is evaluated by direct observation of the resultant inward chloride current. This system has been employed for functional analyses of tachykinin receptors and tachykinin-related peptide receptors [18À21], and thus, we examined whether the UTKR expressed in Xenopus oocytes was activated by its putative endogenous ligands, Uru-TKs. After UTKR cRNA was injected into oocytes followed by incubation at 17 °C for 2À4 days, the receptor-expres- sing oocytes were voltage-clamped at )80 mV. Subse- quently, Uru-TK I was added to an oocyte every 20 min at indicated concentrations in order to prevent desensitization of the receptor. As shown in Fig. 3(A), application of Uru- TK I to the UTKR-expressing Xenopus oocytes evoked a clear response, whereas no signal was observed in the absence of the UTKR cRNA (data not shown). A maximal response was observed at more than 20 n M ,andthehalf- maximal response value (EC50) was calculated to be approximately 1 n M by a doseÀresponse curve of current shift (Fig. 3B). These results confirmed that UruÀTK I is an endogenous ligand of UTKR. In a previous study, we showed that six Uru-TK peptides (Uru-TK IÀV and VII, as summarized in Table 1) were yielded from the single Uru-TK precursor in the nervous tissue of echiuroid worms [10]. To examine whether other Uru-TKs are also endogenous agonists of UTKR, the activities of Uru-TKs IIÀVandVIIonUTKRwere observed by the voltage-clamp method. As shown in Fig. 3B, all EC50 values of Uru TKs IIÀVandVIIwere showntobe0.62À3.15 n M , demonstrating that the effects of all Uru-TKs on UTKR were as potent as that of Uru-TK I. These results indicate that Uru-TKs IIÀVandVIIalsoserve as endogenous agonistic ligands of UTKR with equivalent activity to Uru-TK I. Furthermore, no marked difference in the activity of Uru-TKs on UTKR suggested that UTKR possessed no significant selective affinity with any Uru-TK. Fig. 1. A cDNA and deduced amino-acid sequence of Uru-TK receptor, UTKR. Seven putative transmembrane domains are underlined. The conserved N-glycosylation sites (Asn28, Asn39, and Asn223) are boxed. Potentially phosphorylated serines or threonines (Ser173, Thr262, Thr273, Ser276, Ser365, Ser381, Thr389, Ser396, Thr400, and Ser404) are marked by circles. Cysteines in a disulfide bridge (Cys134 and Cys214) are indicated in black. The Asp-Arg-Tyr and Lys/Arg-Lys/Arg-X-X-Lys/Arg characteristic sequences in G-coupled receptors are written in italic (Asp158-Tyr160 and Arg278-Lys282). Arrows indicate introns-inserted positions. Ó FEBS 2002 An Urechis tachykinin-related peptide receptor (Eur. J. Biochem. 269) 4241 StructureÀactivity relationships of Uru-TKs and mammalian tachykinins Most invertebrate tachykinin-related peptides contain a common Phe-X-Gly-Y-Arg-NH 2 sequence at their C-termini, whereas the C-terminal consensus motif of vertebrate tachykinins is Phe-X-Gly-Leu-Met-NH 2 .More- over, we demonstrated in our previous study that conver- sion of Arg-NH 2 to Met-NH 2 in all Uru-TKs resulted in the loss of the contractile activity of Uru-TKs on the cockroach hindgut, although the peptides and tissues used in these studies were derived from different species [10,22]. To confirm whether the C-terminal Arg-NH 2 is critical for activation of the UTKR, an Uru-TK I analog ([Met10]Uru- TK I), in which the C-terminal Arg-NH 2 is replaced with Met-NH 2 , was synthesized and applied in the voltage-clamp experiment. As shown in Fig. 4A, the [Met10]Uru-TK I analog exhibited no activity on UTKR at concentrations comparable to those of Uru-TK I. This result clearly showed that the Phe-X-Gly-Y-Arg-NH 2 is essential for Fig. 2. Alignment of the amino-acid sequence of receptor core region. Four invertebrate tachykinin-related peptide receptors (UTKR, STKR, NKD and DTKR) and three rat tachykinin receptors (NK1À3R) are aligned. Conserved residues are shadowed and shown in bold. Seven putative transmembrane regions (TM1-7) are indicated above the corresponding sequence part. 4242 T. Kawada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 activation of the receptor. Similarly, SP was shown to fail to activate the UTKR (Fig. 4B). On the other hand, [Arg11]SP showed a potent activity on the UTKR with an EC50 of approximately 6 n M (Fig. 4B). In addition, coapplication of [Met10]Uru-TK I or SP with Uru-TKs had no effect on the activity of Uru-TKs (data not shown), indicating that [Met10]Uru-TK I and SP most likely fail to bind to the UTKR, not exert an antagonistic activity at physiological concentrations. Taken together, these results also supported the notion that the consensus motif Phe-X-Gly-Y-Arg-NH 2 in tachykinin-related peptides plays an essential role in the activation of tachykinin-related peptide receptors. Genomic organization of the UTKR gene Subsequently, we determined the intron/exon structure of the UTKR. Genomic PCR was performed with several primer sets encoding the 5¢-or3¢-terminal region of the UTKR cDNA. All genomic PCR products were subcloned and sequenced, revealing that the UTKR gene consists of five exons and four introns with 3069 bp, 146 bp, 469 bp, and 119 bp, respectively (Fig. 5). The introns were inserted at positions 782, 992, 1143, and 1340 in the UTKR cDNA sequence (Figs 1 and 5). Interestingly, the locations of introns in the UTKR gene are in complete agreement with those of mammalian tachykinin receptor genes [6,23], and this finding is supported by the fact that a typical GT/AG splicing signal is present in all exon/intron junctions (Table 3). This result suggested that the exon/intron struc- ture of tachykinin receptors and tachykinin-related peptide receptors is conserved between vertebrates and inverte- brates. Fig. 3. Activation of UTKR by Uru-TKs. (A) Current shift is evoked by adding 10 n M Uru-TK I for 30 s to the oocytes expressing UTKR. (B) DoseÀresponse curve of the assay using Uru-TKs IÀV and VII. Maximum membrane currents elicited by ligands are plotted. The current caused by 10 )7 M Uru-TKs was taken as 100%. Error bars denote SEM (n ¼ 5). Fig. 4. A comparison of the activities of Uru-TK I, SP, and their analogs. (A) DoseÀresponse curve of Uru-TK I (circles) and [Met10]Uru-TK I (squares). (B) DoseÀresponse curve of SP (stars) and [Arg11]-SP (triangles). Table 2. The identity of sequence encoding the intracellular, extracel- lular, and transmembrane domains of UTKR to those of tachykinin receptors and TRP receptors. Receptor Identity (%) NK1R 46 NK2R 39 NK3R 47 NKD 54 DTKR 54 STKR 55 Ó FEBS 2002 An Urechis tachykinin-related peptide receptor (Eur. J. Biochem. 269) 4243 DISCUSSION Tachykinin-related peptide receptors have been so far characterized exclusively from several insects, although a number of tachykinin-related peptides are widely distri- buted among invertebrates. Consequently, the biological functions of tachykinin-related peptides and their receptors in invertebrate remain unclear. Moreover, the molecular evolution and/or phylogenetic correlation of tachykinin- related peptide receptors have yet to be understood. Thus characterization of a tachykinin-related peptide receptor from other invertebrates is expected to enable us to investigate further common and/or species-specific bio- chemical features and biological roles of tachykinin-related peptides and their receptor. In the present study, we have characterized a novel tachykinin-related peptide receptor, UTKR. This is the first report on tachykinin-related peptide receptors from a noninsect invertebrate species, and also on the genomic analysis of tachykinin-related peptide receptor. The UTKR sequence was shown to be highly similar to tachykinin receptor sequences (Fig. 2 and Table 2), and possesses all regions and motifs typical for tachykinin receptors (Fig. 1) except for a palmitylation site, which is present in all other tachykinin receptors. It is proposed that a palmityl lipid covalently bound to a GPCR may be involved in stabilizing the conformation of a GPCR [24]. However, UTKR, like other tachykinin-related peptide receptors [13À16], were shown to evoke a calcium-dependent chloride influx upon addition of its endogenous and synthetic agonists (Figs 3A,B and 4A,B). These results support the notion that the palmityl group is not requisite for the essential function of tachykinin-related peptide receptors. Some tachykinin-related peptides occasionally showed different activities on tachykinin-related peptide receptors. For example, the locust tachykinin-related peptides, Lom-TKs IÀIV, activated the stable fly tachykinin-related peptide receptor, STKR to a similar degree [16], whereas the Drosophila tachykinin-related peptide receptor, NKD, was shown to respond to LomTK II but not to LomTK I [13]. Furthermore, STKR failed to be activated by Uru-TK II [15], while Uru-TK II not only activated UTKR (Fig. 3B) but also exhibited the contractile activity on the cockroach hindgut and the echiuroid circular body wall muscle [8,10]. These phenomena can be interpreted in two ways. First, tachykinin-related peptide receptors have selective binding affinity to their endogenous ligands. Alternatively, such different reactivities may be caused simply by utilization of heterogenous tachykinin-related peptides and their recep- tors in the functional analyses and biological assays. To address these questions, we evaluated for the first time the effect of tachykinin-related peptides on their receptor using Uru-TKs and UTKR which were characterized from a single invertebrate species, and the echiuroid endogenous ligands, Uru-TKs IÀV and VII, exhibited an equivalent activity on UTKR expressed in Xenopus oocytes (Fig. 3A,B). The possibility that heterologously expressed UTKR possesses some different features from naturally occurring UTKR cannot be entirely excluded. However, many mammalian GPCRs including tachykinin receptors that are expressed in Xenopus oocytes are known to exhibit the same activity and ligand-selectivity as receptors expres- sed in homologous tissues or cultured cells [6,18À20]. Taken together, tachykinin-related peptides, at least Uru- TKs, are highly likely to exhibit no binding selectivity for a homogenous tachykinin-related peptide receptor, unlike mammalian tachykinins SP, NKA, and NKB which have distinctly selective affinity with NK1, NK2, and NK3 receptors, respectively [2]. In addition, the difference in the activities of tachykinin-related peptides on their receptors may be attributed to the utilization of peptides and receptors from different species rather than to the biologically significant binding selectivity of tachykinin-related peptide receptors. To further confirm this possibility, the physiolo- gical characteristics of naturally occurring UTKR are now being investigated using the echiuroid central nervous system. Also of interest is whether tachykinin-related peptide receptor subtypes exist in a single species, like mammalian tachykinin receptors. Three invertebrate tachykinin-related peptide receptors, namely, NKD [13], STKR [16], and UTKR (this study) have so far been shown to interact with tachykinin-related peptides. DTKR that was also isolated from Drosophila has been shown to interact with SP [21], but whether DTKR can bind to Drosophila tachykinin-related peptides [25] remains unclear. Therefore, only one tachykinin-related peptide receptor that can be activated by tachykinin-related peptides has ever been characterized from each invertebrate species. Further investigation is required in order to examine whether some tachykinin-related peptide receptors have subtypes and/or show selective binding to their ligand(s). Table 3. Sequences around the splicing sites in the UTKR genome. Capital and small letters represent exon and intron sequences, respectively. The consensus splicing sites are shown in bold. All entire intoron sequences were deposited in the DDBJ/EMBL/GenBank databases under accession number AB081457. Intron 1 CGACAGgtgagt)3069 bpÀcaacagGTATAT Intron 2 TTTTGTgtaaat)146 bpÀcaacagGTATAA Intron 3 AGACGGgtatga)469 bpÀtttcagGTAGTG Intron 4 TGCCAGgtatgt)119 bpÀttccagATTCCG Fig. 5. Schematic representation of the UTKR cDNA and intron/exon organization of its gene. (A) UTKR cDNA. The transmembrane regions are shadowed. (B) Organization of the UTKR gene. The introns are shown as i1Ài4. 4244 T. Kawada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [Arg11]SP, a SP analog containing Arg-NH 2 ,also activated the UTKR, while SP and [Met10]Uru-TK I, an Uru-TK I analog carrying Met-NH 2 , were devoid of any activity on UTKR (Fig. 4A,B). These results are in good agreement with our previous study, showing that [Met10]Uru-TKs and SP failed to have any effect on the cockroach hindgut, while Uru-TKs exerted contractile activity [10,22]. In combination, these data confirmed that the presence of the -Arg-NH 2 residue in the Phe- X-Gly-Y-Arg-NH 2 consensus motif is critical for the activation of a tachykinin-related peptide receptor and that the binding site of tachykinin-related peptide recep- tors including UTKR discriminates between Arg-NH 2 and Met-NH 2 residues. The amino-acid residues in tachykinin receptors that are involved in binding to ligands and some models of interaction of the binding sites of mammalian receptors with agonists have been proposed [26À28], but the molecular basis of the tachykininÀreceptor interaction remains little understood. Moreover, no information on the recognition of ligands by the binding sites of receptors has been obtained from tachykinin-related peptide receptors. To investigate the binding mode for Uru-TKs and UTKR, site-directed and deleted mutagenesis analyses of UTKR are currently in progress. The UTKR gene has been found to be composed of five exons interrupted by four introns (Fig. 5 and Table 3). Of particular significance is that all introns are present at exactly the same locations as the mammalian tachykinin receptor genes [6,23]. Combined with the findings that UTKR shares the typical features of tachykinin receptors, including the activation of the PLC-InsP 3 -calcium signal transduction pathway, these results lead to the presumption that the tachykinin-related peptide receptors of inverte- brates and the tachykinin receptors of vertebrates evolved from a common ancestral gene. Interestingly, vertebrate tachykinins and invertebrate tachykinin-related peptides are thought to originate from distinct ancestral genes, in contrast to tachykinin-related peptide receptor genes, given that the amino-acid sequences of invertebrate tachykinin- related peptide precursors display no significant similarity to vertebrate preprotachykinins [9,25] and that the architecture of a tachykinin-related peptide precursor is obviously different from that of a tachykinin precursor; multiple tachykinin-related peptide sequences are encoded in a single tachykinin-related peptide precursor [9,25], whereas pre- protachykinin A encodes at most SP and NKA, and only NKB is present in preprotachykinin B [29,30]. These findings are in contrast with other neuropeptides such as the vasopressin/oxytocin superfamily, as the essential amino-acid sequences and the gene architectures of both the vasopressin/oxytocin superfamily peptides and their receptors are well conserved between vertebrates and invertebrates [31À38]. Consequently, the difference in the molecular evolution and/or diversity between tachykinin/ tachykinin-related peptide genes and their receptor genes is raised as a new question. This is also interesting in regard to the functional evolution and conservation of an invertebrate tachykinin-related peptide ligandÀreceptorpairanda vertebrate tachykinin ligandÀreceptor pair. In conclusion, we identified the structure, the genomic organization, and the function of a novel tachykinin- related peptide receptor, UTKR. 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(1999) Neuropeptide families and their receptors: evolutionary perspectives. Brain. Res. 848,1À25. 4246 T. Kawada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . 1 CGACAGgtgagt)3069 bpÀcaacagGTATAT Intron 2 TTTTGTgtaaat)146 bpÀcaacagGTATAA Intron 3 AGACGGgtatga)469 bpÀtttcagGTAGTG Intron 4 TGCCAGgtatgt)119 bpÀttccagATTCCG Fig reactions (RT-PCRs) and rapid amplifications of cDNA ends were performed using Taq Ex polymerase (Takara, Kyoto, Japan) or rTaq DNA polymerase (Toyobo, Osaka, Japan) and a thermal