A novel tachykinin-related peptide receptor of Octopus vulgaris – evolutionary aspects of invertebrate tachykinin and tachykinin-related peptide Atsuhiro Kanda, Kyoko Takuwa-Kuroda, Masato Aoyama and Honoo Satake Suntory Institute for Bioorganic Research, Osaka, Japan Keywords evolution; Octopus vulgaris; oct-TKRPR; tachykinin-related peptide receptor; tachykinin Correspondence A Kanda, Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamotocho, Mishima-gun, Osaka 618-8503, Japan Fax: +81 75 962 2115 Tel: +81 75 962 3743 E-mail: kanda@sunbor.or.jp Database Nucleotide sequence data are available in the DDBJ ⁄ EMBL ⁄ GenBank databases under the accession number AB096700 (Received 16 December 2006, revised 17 February 2007, accepted 28 February 2007) doi:10.1111/j.1742-4658.2007.05760.x The tachykinin (TK) and tachykinin-related peptide (TKRP) family represent one of the largest peptide families in the animal kingdom and exert their actions via a subfamily of structurally related G-protein-coupled receptors In this study, we have identified a novel TKRP receptor from the Octopus heart, oct-TKRPR oct-TKRPR includes domains and motifs typical of G-protein-coupled receptors Xenopus oocytes that expressed oct-TKRPR, like TK and TKRP receptors, elicited an induction of membrane chloride currents coupled to the inositol phosphate ⁄ calcium pathway in response to Octopus TKRPs (oct-TKRP I–VII) with moderate ligand selectivity Substance P and Octopus salivary gland-specific TK, oct-TK-I, completely failed to activate oct-TKRPR, whereas a Substance P analog containing a C-terminal Arg-NH2 exhibited equipotent activation of oct-TKRPs These functional analyses prove that oct-TKRPs, but not oct-TK-I, serve as endogenous functional ligands through oct-TKRPR, although both of the family peptides were identified in a single species, and the importance of C-terminal Arg-NH2 in the specific recognition of TKRPs by TKRPR is conserved through evolutionary lineages of Octopus Southern blotting of RT-PCR products revealed that the oct-TKRPR mRNA was widely distributed in the central and peripheral nervous systems plus several peripheral tissues These results suggest multiple physiologic functions of oct-TKRPs as neuropeptides both in the Octopus central nervous system and in peripheral tissues This is the first report on functional discrimination between invertebrate TKRPs and salivary gland-specific TKs Tachykinins (TKs) are vertebrate multifunctional brain ⁄ gut peptides involved in various central and peripheral functions, including smooth muscle contraction, vasodilatation, inflammation, and the processing of sensory information in a neuropeptidergic or endocrine ⁄ paracrine fashion [1–4] The major mammalian TK family peptides are Substance P (SP), neurokinin (NK) A (NKA), NKB, and hemokinin-1 ⁄ endokinins The vertebrate TKs share a common motif, FXGLM-NH2, at their C-termini [1,5] Three mammalian TK receptors (TKRs), NK1, NK2 and NK3 receptors (NK1R, NK2R, NK3R), have so far been identified These receptors belong to the class I G-protein-coupled receptor (GPCR) family, and have been shown to trigger the phospholipase C–inositol triphosphate–calcium signal transduction cascade via coupling to Gq Abbreviations GPCR, G-protein coupled receptor; inv-TK, invertebrate tachykinin; NK, neurokinin; NKR, neurokinin receptor; oct-TK, Octopus tachykinin; oct-TKRP, Octopus tachykinin-related peptide; oct-TKRPR, Octopus tachykinin-related peptide receptor; SP, Substance P; TK, tachykinin; TKR, tachykinin receptor; TKRP, tachykinin-related peptide; TKRPR, tachykinin-related peptide receptor; TM, transmembrane domain FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2229 Octopus tachykinin-related peptide receptor A Kanda et al protein upon binding to TK peptides [5,6] In the ascidian Ciona intestinalis, TK family peptides, namely Ci-TK-I and Ci-TK-II, were identified [7] Moreover, Ci-TK receptor (Ci-TK-R) displays high amino acid sequence homology to NK1-3R and harbors an intron–exon organization typical of the receptor genes [7] These findings have established that tachykinergic systems are essentially conserved in chordates (vertebrates and ascidians) [1–5,7,8] In protostomes, two TK-type peptides, namely invertebrate TKs (inv-TKs) and tachykinin-related peptides (TKRPs), have been identified Inv-TKs bear a vertebrate TK common motif at their C-termini, and their cDNAs encode a single copy of inv-TK [5,8–10] These peptides were found to be expressed exclusively in the salivary gland, and are devoid of any biological activity on the cognate tissues, despite their various TK-typical activities on vertebrate tissues [5,8–10] TKRPs were isolated from nervous systems or guts of various protostomes [1,5,6,8] Of particular importance is that TKRPs share the C-terminal sequence FX1(G ⁄ A)X2RNH2, analogously to those of vertebrate TKs and inv-TKs, and that multiple copies of TKRPs are encoded by a single precursor of each species, in contrast to TKs [11] In insects and several other invertebrates, a variety of biological activities of TKRPs, such as the contraction of the hindgut and oviduct, depolarization or hyperpolarization of several neurons, and induction of adipokinetic hormone release, have been documented, supporting the view that TKRPs are functional counterparts of vertebrate TKs [8] Such biological actions are believed to be mediated by endogenous TKRP receptors (TKRPRs) To date, DTKR (Drosophila melanogaster), NKD (Drosophila melanogaster), STKR (Stomoxys calcitrans), and UTKR (Urechis unitinctus) have been identified as TKRPRs [12–16] UTKR, STKR, and DTKR, like mammalian TKR, activate the phospholipse C–inositol triphosphate–calcium signal transduction cascade in response to TKRPs but not to any TKs [6,8,16,17], and the genomic structures of UTKR, DTKR and NKD genes were found to basically coincide with those of mammalian TKR genes [6,16] Consequently, TKRs and TKRPRs share the common original GPCR gene In addition, STKR-transfected and DTKR-transfected cells also exhibited dose-dependent increases in cAMP level in response to several insect TKRPs [17–20] The common octopus, Octopus vulgaris, is the first invertebrate species in which both inv-TKs (oct-TK-I and -II) and TKRPs (oct-TKRP I–VII) were identified, as shown in Table [10] However, whether oct-TKs or oct-TKRPs serve as brain ⁄ gut peptides remains to be elucidated Moreover, the large diversity of neuropeptides such as the TK and TKRP family is correlated with the evolution and divergence of the nervous system and their biological roles, and thus, functional characterization of oct-TKs and oct-TKRPs is expected to provide fruitful insights into the evolutionary implications of the TK family within organisms, given that octopuses possess the most advanced intelligence and physiologic systems of invertebrates [21] In this study, we identified a novel TKRPR in Octopus, octTKRPR Sequence identity, ligand selectivity, signal transduction and tissue distribution of oct-TKRPR provided evidence that oct-TKRPR is the Octopus homolog of TKRPRs for oct-TKRPs but not for Table Amino acid sequence of Octopus tachykinin-related peptides and invertebrate tachykinins Tachykinin-related peptides from the brain of Octopus vulgaris oct-TKRP I oct-TKRP II oct–TKRP III oct-TKRP IV oct-TKRP V oct-TKRP VI oct-TKRP VII Val-Asn-Pro-Tyr-Ser-Phe-Gln-Gly-Thr-Arg-NH2 Leu-Asn-Ala-Asn-Ser-Phe-Met-Gly-Ser-Arg-NH2 Thr-Val-Ser-Ala-Asn-Ala-Phe-Leu-Gly-Ser-Arg-NH2 Ser-Asp-Ala-Leu-Ala-Phe-Val-Pro-Thr-Arg-NH2 Met-Asn-Ser-Leu-Ser-Phe-Gly-Pro-Pro-Lys-NH2 Tyr-Ser-Pro-Leu-Asp-Phe-Ile-Gly-Ser-Arg-NH2 Ala-Ser-Leu-His-Asn-Thr-His-Phe-Ile-Pro-Ser-Arg-NH2 Invertebrate tachykinins from the salivary gland of Octopus vulgaris oct-TK-I oct-TK-II Lys–Pro–Pro–Ser–Ser–Ser–Glu–Phe–Ile–Gly–Leu–Met–NH2 Lys–Pro–Pro–Ser–Ser–Ser–Glu–Phe–Val–Gly–Leu–Met–NH2 Substance P and SP-(Arg11) Substance P SP-(Arg11) 2230 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met–NH2 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Arg–NH2 FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS A Kanda et al oct-TKs, and that oct-TKRPR is involved in the regulation of various physiologic functions, including neuronal and contractile processes, in Octopus Results and Discussion Primary structure of the putative oct-TKRPR The second, sixth and seventh transmembrane domains (TMs) are highly conserved among the known TKRPR family To identify receptors for oct-TKRPs in Octopus, four degenerate primers were designed on the basis of the conserved regions, and were used for RT-PCR of first-strand cDNA prepared from the Octopus heart blast searches of the PCR product sequence showed a high level of homology with mouse NK1-3R, Drosophila DTKR, and stable fly STKR A full-length cDNA sequence (1392 bp) encoding the putative oct-TKRPR was determined, by 5¢ ⁄ 3¢-RACE methods, from the Octopus heart (GenBank accession number, AB096700) oct-TKRPR has an ORF of 430 amino acids flanked by 33 bp of 5¢-UTR and 66 bp of 3¢-UTR Multiple sets of clones in every PCR were analyzed, and gave identical nucleotide sequence The sequence showed the presence of the seven hydrophobic TMs that are the most typical characteristic of GPCRs As shown in Fig 1, oct-TKRPR contains several potential sites for N-linked glycosylation and phosphorylation, as follows: two sites of consensus sequences for N-linked glycosylation sites (N-X-S ⁄ T) in the extracellular N-terminal domain; four sites of consensus sequences for phosphorylation by protein kinase A (K ⁄ R-X1-(X2)-S ⁄ T); one site of phosphorylation by protein kinase C (S ⁄ T-X-K ⁄ R); and three casein kinase II sites (S ⁄ T-X1-X2-D ⁄ E) The phosphorylation sites that are involved in the modulation of G protein coupling and receptor function were located exclusively in the third intracellular loop and the C-terminus, suggesting that phosphorylation is involved in the modulation of G protein coupling and receptor function [22] The Asp110 and Asn337 in TM2 and TM7, the consensus tripeptide motif (E ⁄ D-R-Y, DRY in oct-TKRPR) at the interface of TM3, and the K ⁄ R-X1-X2-K ⁄ R site in the third intracellular loop, both of which are believed to play a pivotal role in functions of the class I GPCR family [23], were also conserved Two Cys residues responsible for a disulfide bridge in most GPCRs were present (at positions 137 and 215) in the first and second extracellular loops in oct-TKRPR These results indicated that oct-TKRPR belongs to the class I GPCR family The total amino acid sequence of the oct-TKRPR is 26.8–43.6% homologous to the sequences of TKR and Octopus tachykinin-related peptide receptor the TKRPR family (Table 2) Molecular phylogenetic analysis of TKR and TKRPR sequences showed that oct-TKRPR belongs to the clade of protostome TKRPRs (Fig 2) These results indicated that the cloned receptor is a novel homolog of the TKRPR Functional analysis of oct-TKRPR in Xenopus oocytes TKRP cDNAs are known to bear multiple copies of TKRP sequences [6] The oct-TKRP cDNA (GenBank accession number AB037112) also encodes seven putative TKRP sequences, oct-TKRP I–VII, and the amino acid sequences showed similarity to the TKRP C-terminal common sequence FX1(G ⁄ A)X2R-NH2 (Table 1), suggesting that oct-TKRPs are novel members of the TKRP family To evaluate the activities of oct-TKRPs at oct-TKRPR, oct-TKRPR was expressed in Xenopus oocytes, as functional assays using Xenopus oocytes have been widely used to investigate the ligand–receptor affinity and selectivity of various neuropeptides, including TKRPs, and the in vitro results are actually consistent with in vivo results [7,16,24,25] The voltage-clamped oocytes expressing oct-TKRPR displayed typical inward membrane currents upon application of oct-TKRP II (Fig 3A) EC50 values of oct-TKRP I–IV and VII were shown to be 9.35– 19.3 nm, but oct-TKRP V and VI exhibited relatively low activity, with EC50 values of 230 and 92.5 nm, respectively (Fig 4A–G; Table 3) These results provided undoubted evidence that oct-TKRPs are endogenous ligands of oct-TKRPR The mammalian and Ciona TKRs possess moderate ligand selectivity [6,7], whereas all Uru-TKs, TKRPs of the echiuroid worm U unitinctus, exhibited almost equivalent activity on UTKR, which is in good agreement with the results of physiologic assays [16] Recently, DTKR was shown to elicit an equipotent elevation in intracellular calcium in response to DTK I–V [17] Therefore, our results lead to the conclusion that the moderate ligand–receptor selectivity of oct-TKRPR was established in the evolutionary pathway specific to octopuses Moreover, the possibility cannot be absolutely excluded that octopuses have other oct-TKRPR subtypes that octTKRP V and VI activate more potently, given that oct-TKRP VI, V and VII not completely conserve the TKRP C-terminal common sequence (Table 1) Production of another second messenger, cAMP, was stimulated by STKR and DTKR [17,18], and many GPCRs are coupled to multiple second messengers [26] However, cAMP production was not observed in HEK293 cells expressing oct-TKRPR upon addition of any oct-TKRPs (data not shown) FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2231 Octopus tachykinin-related peptide receptor A Kanda et al Fig Sequence alignment of the oct-TKRPR, TKRPR and TKR family The amino acid sequence of oct-TKRPR was aligned with those of the TKRPRs (UTKR, NKD, STKR, and DTKR), and TKRs (mouse NK1R, NK2R and NK3R, and Ci-TK-R) using CLUSTALW Amino acid residues conserved in all homologs are indicated by an asterisk, and reduced identity is indicated by a colon and dot N-linked glycosylation sites are underlined Potential phosphorylated serine or threonine residues are marked by open circles Bars indicate the seven putative TM domains Amino acid residues in boxes are believed to play a pivotal role in GPCR activation 2232 FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS A Kanda et al Octopus tachykinin-related peptide receptor Table Total amino acid sequence identity scores of oct-TKRPR to the TKR and TKRPR family oct-TKRPR versus Percentage indentity UTKR DTKR STKR NKD Mouse NK1R Mouse NK2R Mouse NK3R Ci-TK-R 43.6 37.2 28.6 30.8 31.3 29.3 31.2 26.8 It is well established that invertebrate TKRPRs are responsive to TKRPs containing the C-terminal FX1(G ⁄ A)X2R-NH2 consensus sequence but not to TKs containing FXGLM-NH2 [6,8,16] Likewise, oct-TKRPR did not show activation upon application of SP, whereas SP-(Arg11), in which the C-terminal Met-NH2 is replaced by Arg-NH2, displayed potent activity on oct-TKRPR (Fig 4H; Table 3) These results are consistent with our previous finding that UTKR was activated by SP-(Arg11) as potently as Uru-TKs, whereas Uru-TK-I-(Met10) completely abolished the ability to activate UTKR [16] Moreover, we tested whether oct-TKRPR was activated by an Octopus inv-TK, oct-TK-I (Table 1), which was isolated from the Octopus salivary gland, and shared the vertebrate TK common motif at the C-terminus [10] However, oct-TK-I failed to trigger the inward current even at levels higher than 10)6 m (Fig 3B), revealing that oct-TKRPR react specifically with oct-TKRPs but not with oct-TKs, although both of them were identified in a single species Altogether, these results revealed that the importance of C-terminal Arg-NH2 Fig Molecular phylogenetic tree of the TKR and TKRPR family oct-TKRPR is boxed A phylogenetic tree was inferred from the amino acid sequences by the neighbor-joining method One thousand booststrap trials were run The numbers at each branch node represent the percentage values given by booststrap The mouse oxytocin receptor was used as an outgroup TKRs: mouse NK1-3R, neurokinin receptors 1–3; Ci-TK-R, C intestinalis TKR TKRPRs: DTKR, D melanogaster; NKD, D melanogaster; STKR, S calcitrans; UTKR, U unitinctus FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2233 Octopus tachykinin-related peptide receptor A A Kanda et al B C Fig Functional expression of oct-TKRPR in Xenopus oocytes (A–C) Traces of membrane current induced by oct-TKRP II at 10)8 M, and oct-TK-I and SP at 10)6 M, in oocytes expressing oct-TKRPR in the specific recognition of TKRPs by TKRPR is conserved in Octopus, and that oct-TKRPs, but not oct-TKs, function as endogenous factors Localization of oct-TKRPR mRNA in Octopus To verify the tissue distribution of oct-TKRPR mRNAs in the central and peripheral nervous systems, and in several peripheral tissues of Octopus, we performed Southern blot analysis of RT-PCR products for oct-TKRPR oct-TKRPR mRNA was expressed in the nervous system and peripheral tissues, including various smooth muscles, whereas b-actin genes were shown to be expressed to a similar degree in all tissues (Fig 5) The distribution of oct-TKRPR mRNA is consistent with biological data showing that octTKRP II or III stimulate spontaneous contractile action (e.g esophagus, aorta, stomach, crop, and oviduct) in Octopus (H Minakata et al., unpublished results) oct-TKRPR was also abundantly expressed in the brain, buccal ganglion, gastric ganglia, olfactory and reduncle lobes, and optic lobe Mammalian NK13Rs were widely distributed in the central and peripheral nervous systems plus several peripheral tissues, such as the brain, heart, gastrointestinal and genitourinary tract, respiratory organs, and muscle [27–30] In keeping with such extensive expression of the receptors, TKs play an important role in smooth muscle contraction, vasodilatation, inflammation, the processing of sensory information in a neuropeptidergic or endocrine ⁄ paracrine fashion, and the release of neurotransmitters in the tachykinergic nerve fiber [8,31,32] Most TKRPs have been shown to stimulate spontaneous contraction of the visceral muscles of insects, 2234 such as the foregut and oviduct [6,33] DTKs were detected in endocrine cell-like bodies of Drosophila posterior midgut as well as in the brain and nervous system, and exhibited myoactivity on the midgut [34] DTKR was localized in Drosophila brain neuropils and ganglion, and the expression profile of DTKR corresponds with immunostaining of DTKs, suggesting the involvement of DTKs in the control of hormone release, and modulation of chemosensory and visual processing in the nervous systems [17] These findings, combined with expression of oct-TKRPR, support the idea that oct-TKRPs have multiple biological roles in not only contraction of smooth muscles but also autonomic functions, feeding, internal secretion, visual sensation, and movement, via oct-TKRPR, as neurotransmitters, neuromodulators, and hormone-like factors In particular, oct-TKRPR mRNA was also detected in the ovary and eggs (Fig 5), and NKR and Ci-TK-R mRNA was localized in the reproductive organs of mammals and C intestinalis [7,28,29], suggesting that oct-TKRPs also control sexual behavior in Octopus Detailed localization of oct-TKRPR in the nervous system and peripheral tissues by in situ hybridization and immunohistochemistry is now being examined Further functional analysis of oct-TKRP is expected to provide a clue to the understanding of the dioecism of octopuses, which is extremely rare in mollusks In addition to the dioecism, octopuses are endowed with several exceptional properties among protostomes: highly advanced nervous and endocrine systems [21] Such advanced characteristics are anticipated to be correlated with molecular and functional evolution of neuropeptides and hormones; for instance, two oxytocin ⁄ vasopressin superfamily peptides and their three receptors were characterized from Octopus in our previous study, whereas other protostomes have been shown to possess only one oxytocin ⁄ vasopressin superfamily peptide [33,34] Moreover, we revealed that the ligand selectivities of octopus oxytocin ⁄ vasopressin receptors are different from those of their vertebrate counterparts [24,35] Therefore, structural and functional identification of octopus neuropeptides and hormones is expected to contribute a great deal to our understanding of the biological mechanism underlying the advanced behavior of Octopus and evolutionary aspects of neuropeptides and hormones The TK and TKRP family represent one of the largest peptide families in the animal kingdom, and O vulgaris is the first species shown to possess both inv-TKs and TKRPs Octopus inv-TK, oct-TKs, were found to be expressed exclusively in the salivary gland, and are devoid of any biological activity on the cognate tissues, despite their FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS A Kanda et al Octopus tachykinin-related peptide receptor A B 100 80 80 60 60 [%] [%] 100 40 40 20 20 0 10-11 10-10 10-9 10-8 Conc.[M] 10-7 10-11 10-10 10-9 10-8 Conc.[M] 10-7 10-6 10-11 10-10 10-9 10-8 Conc.[M] 10-7 10-6 10-10 10-9 10-8 Conc.[M] 10-7 10-6 10-9 10-8 Conc.[M] 10-7 10-6 10-6 C D 100 80 80 60 60 [%] [%] 100 40 40 20 20 0 10 -11 10 -10 -9 10 10 Conc.[M] -8 10 -7 10 -6 E F 100 80 80 60 60 [%] [%] 100 40 40 20 20 0 10 -10 10 -9 -8 10 10 Conc.[M] -7 10 -6 10 10-11 -5 G H 100 80 60 SP-[Arg11] SP 80 60 [%] [%] 100 40 40 20 20 10 -11 10 -10 -9 10 10 Conc.[M] -8 10 -7 10 -6 10-11 10-10 Fig Dose–response curves for oct-TKRPR in Xenopus oocytes (A–G) Dose–response curve over the concentration range 10)11)10)6 M oct-TKRP I–VII with oct-TKRPR Maximum membrane currents elicited by the ligands are plotted Error bars denote SEM (n ¼ 5) (H) Dose– response curve over the concentration range 10)11)10)6 M SP and SP-(Arg11) with oct-TKRPR FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2235 Octopus tachykinin-related peptide receptor A Kanda et al Table EC50 values for oct-TKRPR in Xenopus oocytes Peptide EC50 (nM) oct-TKRP I oct-TKRP II oct-TKRP III oct-TKRP IV oct-TKRP V oct-TKRP VI oct-TKRP VII SP SP-(Arg11) 18.5 9.35 9.5 19.3 230 92.5 14.5 – 35.2 ± ± ± ± ± ± ± 0.18 0.59 0.6 2.86 3.65 5.48 1.4 ± 0.38 various TK-typical activities on vertebrate tissues [10], and completely failed to activate endogenous TKRPR, although it is expressed in the salivary gland Altogether, these findings lead to the conclusion that Octopus acquired oct-TKs as toxin-like substance for use against vertebrates such as fishes, which are prey animals or natural enemies of octopuses, whereas oct-TKRPs and TKRPR were employed as pivotal endogenous factors in evolutionary lineages distinct from oct-TKs Two possible scenarios concerning evolutionary aspects of oct-TKs and oct-TKRPs can be assumed: (a) the oct-TKRP gene and the oct-TK gene might have diverged from the common ancestral gene during the evolution of Octopus species; and (b) the oct-TK gene might have been acquired through gene transfer However, the former scenario is less likely than the latter First, if the oct-TKRP gene and the oct-TK gene had occurred via molecular evolution of the common ancestral gene, other invertebrates, in particular other mollusks, should possess an inv-TK gene Nonetheless, inv-TKs have been identified only in the salivary gland of octopuses (oct-TKs and eledoisin) and female mosquitoes (sialokinins), and not in other mollusks or insects that possess TKRPs [6,8] Moreover, we could not find any inv-TK genes by searching the Drosophila genomic database In contrast, the oxytocin ⁄ vasopressin superfamily peptides have been isolated from diverse mollusks and annelids [24,35–39] Second, there is great difference in gene organization between oct-TK gene and oct-TKRP gene If octopuses had independently evolved oct-TK gene, e.g by duplication and modification of oct-TKRP gene, the organization of the resulting oct-TK gene should display higher similarity to that of the oct-TKRP gene, which has multiple copies of TKRP sequences [6,8] However, the oct-TK and sialokinin genes, like vertebrate NKB genes, encode only the single peptide sequence [9,10] Consequently, these findings allow us to assume that oct-TKs might have been acquired as toxin-like compounds via horizontal transfer of a TK gene after the occurrence of ancestral vertebrate species, rather than oct-TKs evolving from the common antecedent of TKs and TKRPs in octopuses No horizontal gene transfer from vertebrates to invertebrates has so far been confirmed Nevertheless, several vertebrate neuropeptide orthologs have been characterized from lower invertebrates, although not in species closely related to octopuses For instance, an angiotensin-like peptide and opioid peptides have been identified in the blood-sucking leech Erpobdella octoculata, whereas no homologs have ever been found in the closely related annelids or other invertebrates [39] These findings, combined with our present data, indicate the possibility that some vertebrate neuropeptides, e.g the oxytocin ⁄ vasopressin superfamily, are interphyletically conserved in most invertebrate species, but other neuropeptides, including Fig Tissue distribution of oct-TKRPR (upper) and b-actin (lower) in Octopus 2236 FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS A Kanda et al oct-TKs, might have been acquired via horizontal gene transfer from vertebrates to invertebrates after ancestral vertebrate species emerged Conservation of the sequence similarity (Table 2) and exon–intron structure between TKRPR and TKR [16] suggests that they share a common ancestral receptor gene, and that TKRPRs and TKRs have coevolved with peptide and then acquired the ligand selectivity for TKRPs and TKs, respectively, as TKRPRs are not capable of binding to TKs at physiologic concentrations, and vice versa (Table 3) [6] Here, a question is raised regarding the gene structure and C-terminal amino acid residue of a common tachykinin ancestral gene: (a) TKRP genes would have been generated from the ancestor via multiple duplications of the peptide sequence region through evolution of protostome species, but TK and inv-TK genes have conserved the essential original structural organization; or (b) truncation of multiple sequences in the original gene might have resulted in the appearance of inv-TK and TK genes, whereas such multiple sequences have been basically conserved in TKRP genes However, whether the C-terminal Arg- or Met-containing sequence was present in such a putative ancestral gene remains unclear In conclusion, we have presented the primary sequence, reactivity and tissue distribution of an Octopus TKRP receptor, oct-TKRPR Our data provide fruitful insights into evolutionary and interphyletic relationships among TKRPs, inv-TKs, and TKs Octopus tachykinin-related peptide receptor (G ⁄ A)CCA(G ⁄ A)CAIATIGC(G ⁄ A)AA-3¢] under the following conditions: 94 °C for min, and 30 cycles of 30 s at 94 °C, 30 s at 50 °C, and 90 s at 72 °C (7 for the last cycle) The method for cloning was the same as those previously described [25] 3¢-RACE and 5¢-RACE 3¢-RACE was performed as follows The first PCR used the PCR anchor primer and TKRPR-3¢-1F (5¢-CCATCCAG CAACAAAGAGTC-3¢) under the following conditions: at 94 °C, and 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 150 s at 72 °C (5 for the last cycle) The second PCR used the PCR anchor primer and TKRPR3¢-2F (5¢-TAAAATGATGATTGTCGTGGTG-3¢) under the following conditions: at 94 °C, and 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 150 s at 72 °C (5 for the last cycle) The second PCR products were subcloned and sequenced as described above The 5¢-ends of the cDNAs were determined as follows: first-strand cDNA from lg of total RNA using TKRPR-5¢-1R (5¢-GTG TAAACACACTGGCAGAC-3¢) and the 5¢ ⁄ 3¢ RACE kit (Roche Applied Science); first PCR using oligo(dT)-anchor primer and TKRPR-5¢-2R (5¢-GAATAGAGTGTTCCAG ACGG-3¢); second PCR using PCR-anchor primer and TKRPR-5¢-3R (5¢-ATAAGGGCATCTGCCAATGC-3) Both amplifications were performed under the following conditions: at 94 °C, and 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 150 s at 72 °C (5 for the last cycle) Molecular phylogenetic analysis Experimental procedures Animals Adult octopuses (body weight, approximately kg), Octopus vulgaris (common octopus), were purchased from a local fish shop, and kept in artificial seawater at 18 °C Cloning of the partial-length cDNA Total RNA was extracted from Octopus tissues using Sepasol-RNA I Super (Nacalai tesque, Kyoto, Japan) according to the manufacturer’s instructions First-strand cDNA was synthesized with the oligo(dT)-anchor primer supplied in the 5¢ ⁄ 3¢-RACE kit (Roche Applied Science, Indianapolis, IN) The first PCR was performed using TKRPR-Fw1 [5¢-ATG(C ⁄ A)GIACIGTIACIAA(C ⁄ T)TA(C ⁄ T)TT-3¢] and TKRPR-Rv1 [5¢-CA(G ⁄ A)TAIATIATIGG(G ⁄ A)TT(G ⁄ A) TACAT-3¢] under the following conditions: at 94 °C, and 30 cycles of 30 s at 94 °C, 30 s at 45 °C, and 90 s at 72 °C (5 for the last cycle) The second PCR was performed using TKRPR-Fw2 [5¢-TT(C ⁄ T)GCIATITG(C ⁄ T) TGG(C ⁄ T)TICCIT-3¢] and TKRPR-Rv2 [5¢-AIGGIA The amino acid sequences encoding the intracellular, extracellular and TM domains of oct-TKRPR were aligned with the corresponding amino acid sequence of TKRs and TKRPRs and related GPCRs from other animals using the clustalw program The amino acid sequence of Mus musculus (mouse) oxytocin receptor (P97926) was included in the alignment as one group A neighbor-joining tree was constructed on the basis of alignment by the clustalw program The evolutionary distances were estimated using Kimura’s empirical method The sequences used were as follows: mouse NK1R, NP_033339; Homo sapiens (human) NK1R, P25103; mouse NK2R, NP_033340; human NK2R, P21452; mouse NK3R, NP_067357; human NK3R, P29371; C intestinalis (ascidian) Ci-TKR, AB175739; D melanogaster (fruit fly) NKD, P30974; fruit fly DTKR, CAA44595; S calcitrans (stable fly) STKR, AAB07000; and U unitinctus (echiuroid worm) UTKR, AB050456 RT-PCR Southern blot analysis The total RNAs (1 lg) extracted from various tissues were reverse-transcribed by Superscript III (Invitrogen, Carlsbad, FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2237 Octopus tachykinin-related peptide receptor A Kanda et al CA) using oligo(dT)12)18 primer The PCR was performed using TKRPR-Fw3 (5¢-AGATTTTTTCTAAGAACCGCC3¢) and TKRPR-Rv3 (5¢-CTGTCATTTTCTTCCCTGT CG-3¢) under the following conditions: at 94 °C, and 30 cycles of 30 s at 94 °C, 30 s at 50 °C, and 90 s at 72 °C (4 for the last cycle) The PCR products were separated by 1.5% agarose gel electrophoresis, and then transferred onto Hybond-N+ membranes (GE Healthcare, Piscataway, NJ) and crosslinked by UV irradiation Hybridization and detection were processed using the digoxigenin DNAlabeling kit (Roche Applied Science) according to the manufacturer’s instruction A digoxigenin-labeled probe, DIG-TKRPR-5¢-2R, was used for Southern blotting The method for detection was the same as those previously described [24] As a negative control, the extracted total RNAs, which were not reverse transcribed, were used as templates for PCR Thus, we confirmed that there was no amplification of traces of the genomic DNA (data not shown) Expression of the cloned receptor in Xenopus oocytes The ORF region of the novel receptor cDNA was amplified and inserted into a pSP64 poly(A) vector (Promega, Madison, WI) The plasmid was linearized with EcoRI cRNA was prepared using SP6 RNA polymerase (Ambion, Austin, TX) The assay methods were the same as those previously described [25] The methods used for peptide synthesis and purification were the same as those previously described [25] 10 11 12 Acknowledgements We thank Dr Hiroyuki Minakata for providing some information concerning oct-TKRPs 13 References 14 Otsuka M & Yoshioka K (1993) Neurotransmitter functions of mammalian tachykinins Physiol Rev 73, 229–308 Cao YQ, Mantyh PW, Carlson EJ, Gillespie AM, Epstein CJ & Basbaum AI (1998) Primary afferent tachykinins are required to experience moderate to intense pain Nature 392, 390–394 Kramer MS, Cutler N, Feighner J, Shrivastava R, Carman J, Sramek JJ, Reines SA, Liu G, Snavely D, Wyatt-Knowles E et al (1998) Distinct mechanism for antidepressant activity by blockade of central Substance P receptors Science 281, 1640–1645 Page NM, Woods RJ, Gardiner SM, Lomthaisong K, Gladwell RT, Butlin DJ, Manyonda IT & Lowry PJ (2000) Excessive placental secretion of neurokinin B 2238 15 16 17 during the third trimester causes pre-eclampsia Nature 405, 797–800 Severini C, Improta G, Falconieri-Erspamer G, Salvadori S & Erspamer V (2002) The tachykinin peptide family Pharmacol Rev 54, 285–322 Satake H, Kawada T, Nomoto K & Minakata H (2003) Insight into tachykinin-related peptides, their receptors, and invertebrate tachykinins: a review Zool Sci 5, 533–549 Satake H, Ogasawara M, Kawada T, Masuda K, Aoyama M, Minakata H, Chiba T, Metoki H, Satou Y & Satoh N (2004) Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family J Biol Chem 279, 53798–53805 Satake H & Kawada T (2006) Overview of the primary structure, tissue-distribution, and functions of tachykinins and their receptors Curr Drug Targets 7, 963–974 Beerntsen BT, Champagne DE, Coleman JL, Campos YA & James AA (1999) Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti Insect Mol Biol 8, 459–467 Kanda A, Iwakoshi-Ukena E, Takuwa-Kuroda K & Minakata H (2003) Isolation and characterization of novel tachykinins from the posterior salivary gland of the common octopus Octopus vulgaris Peptides 24, 35–43 Nassel DR (1999) Tachykinin-related peptides in invertebrates: a review Peptides 20, 141–158 Li XJ, Wolfgang W, Wu YN, North RA & Forte M (1991) Cloning, heterologous expression and developmental regulation of a Drosophila receptor for tachykinin-like peptides EMBO J 10, 3221–3229 Monnier D, Colas JF, Rosay P, Hen R, Borrelli E & Maroteaux L (1992) NKD, a developmentally regulated tachykinin receptor in Drosophila J Biol Chem 267, 1298–1302 Guerrero F (1997) Cloning of a cDNA from stable fly which encodes a protein with homology to a Drosophila receptor for tachykinin-like peptides Ann NY Acad Sci 814, 310–311 Guerrero FD (1997) Transcriptional expression of a putative tachykinin-like peptide receptor gene from stable fly Peptides 18, 1–5 Kawada T, Furukawa Y, Shimizu Y, Minakata H, Nomoto K & Satake H (2002) A novel tachykininrelated peptide receptor Sequence, genomic organization, and functional analysis Eur J Biochem 269, 4238–4246 Birse RT, Johnson EC, Taghert PH & Nassel DR (2006) Widely distributed Drosophila G-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptides J Neurobiol 66, 33–46 FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS A Kanda et al 18 Torfs H, Shariatmadari R, Guerrero F, Parmentier M, Poels J, Van Poyer W, Swinnen E, De Loof A, Akerman K & Vanden Broeck J (2000) Characterization of a receptor for insect tachykinin-like peptide agonists by functional expression in a stable Drosophila Schneider cell line J Neurochem 74, 2182–2189 19 Poels J, Van Loy T, Franssens V, Detheux M, Nachman RJ, Oonk HB, Akerman KE, Vassart G, Parmentier M, De Loof A et al (2004) Substitution of conserved glycine residue by alanine in natural and synthetic neuropeptide ligands causes partial agonism at the stomoxytachykinin receptor J Neurochem 90, 472–478 20 Poels J, Nachman RJ, Akerman KE, Oonk HB, Guerrero F, De Loof A, Janecka AE, Torfs H & Vanden Broeck J (2005) Pharmacology of stomoxytachykinin receptor depends on second messenger system Peptides 26, 109–114 21 Young JZ (1995) Multiple Matrices in the Memory System of Octopus Oxford University Press, Abbott, NJ 22 Bockaert J & Pin JP (1999) Molecular tinkering of G protein-coupled receptors: an evolutionary success EMBO J 18, 1723–1729 23 Donnelly D, Maudsley S, Gent JP, Moser RN, Hurrell CR & Findlay JB (1999) Conserved polar residues in the transmembrane domain of the human tachykinin NK2 receptor: functional roles and structural implications Biochem J 339(1), 55–61 24 Kanda A, Satake H, Kawada T & Minakata H (2005) Novel evolutionary lineages of the invertebrate oxytocin ⁄ vasopressin superfamily peptides and their receptors in the common octopus (Octopus vulgaris) Biochem J 387, 85–91 25 Kanda A, Takahashi T, Satake H & Minakata H (2006) Molecular and functional characterization of a novel gonadotropin-releasing-hormone receptor isolated from the common octopus (Octopus vulgaris) Biochem J 395, 125–135 26 Krsmanovic LZ, Mores N, Navarro CE, Arora KK & Catt KJ (2003) An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion Proc Natl Acad Sci USA 100, 2969–2974 27 Page NM, Bell NJ, Gardiner SM, Manyonda IT, Brayley KJ, Strange PG & Lowry PJ (2003) Characterization of the endokinins: human tachykinins with cardiovascular activity Proc Natl Acad Sci USA 100, 6245–6250 28 Patak E, Candenas ML, Pennefather JN, Ziccone S, Lilley A, Martin JD, Flores C, Mantecon AG, Story ME Octopus tachykinin-related peptide receptor 29 30 31 32 33 34 35 36 37 38 39 & Pinto FM (2003) Tachykinins and tachykinin receptors in human uterus Br J Pharmacol 139, 523– 532 Pinto FM, Almeida TA, Hernandez M, Devillier P, Advenier C & Candenas ML (2004) mRNA expression of tachykinins and tachykinin receptors in different human tissues Eur J Pharmacol 494, 233– 239 Tsuchida K, Shigemoto R, Yokota Y & Nakanishi S (1990) Tissue distribution and quantitation of the mRNAs for three rat tachykinin receptors Eur J Biochem 193, 751–757 Evangelista S (2001) Involvement of tachykinins in intestinal inflammation Curr Pharm Des 7, 19–30 Holzer P & Holzer-Petsche U (1997) Tachykinins in the gut Part I Expression, release and motor function Pharmacol Ther 73, 173–217 Schoofs L, Holman GM, Hayes TK, Kochansky JP, Nachman RJ & De Loof A (1990) Locustatachykinin III and IV: two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family Regul Pept 31, 199–212 Siviter RJ, Coast GM, Winther AM, Nachman RJ, Taylor CA, Shirras AD, Coates D, Isaac RE & Nassel DR (2000) Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A J Biol Chem 275, 23273–23280 Takuwa-Kuroda K, Iwakoshi-Ukena E, Kanda A & Minakata H (2003) Octopus, which owns the most advanced brain in invertebrates, has two members of vasopressin ⁄ oxytocin superfamily as in vertebrates Regul Pept 115, 139–149 van Kesteren RE, Smit AB, Dirks RW, de With ND, Geraerts WP & Joosse J (1992) Evolution of the vasopressin ⁄ oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis Proc Natl Acad Sci USA 89, 4593–4597 Hoyle CH (1998) Neuropeptide families: evolutionary perspectives Regul Pept 73, 1–33 Satake H, Takuwa K, Minakata H & Matsushima O (1999) Evidence for conservation of the vasopressin ⁄ oxytocin superfamily in Annelida J Biol Chem 274, 5605–5611 Salzet M (2006) Molecular aspect of annelid neuroendocrine system In Invertebrate Neuropeptides and Hormones: Basic Knowledge and Recent Advances (Satake H, ed.), pp 17–35 Transworld Research Network, Kerala, India FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2239 ... receptors, and invertebrate tachykinins: a review Zool Sci 5, 53 3–5 49 Satake H, Ogasawara M, Kawada T, Masuda K, Aoyama M, Minakata H, Chiba T, Metoki H, Satou Y & Satoh N (2004) Tachykinin and. .. peptides and their receptors in the common octopus (Octopus vulgaris) Biochem J 387, 8 5–9 1 25 Kanda A, Takahashi T, Satake H & Minakata H (2006) Molecular and functional characterization of a novel. .. Lys–Pro–Pro–Ser–Ser–Ser–Glu–Phe–Ile–Gly–Leu–Met–NH2 Lys–Pro–Pro–Ser–Ser–Ser–Glu–Phe–Val–Gly–Leu–Met–NH2 Substance P and SP-(Arg11) Substance P SP-(Arg11) 2230 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met–NH2 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Arg–NH2