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
Tachykinins (TKs) are vertebrate multifunctional brain ⁄
gut peptides involved in various central and peripheral
functions, including smooth muscle contraction, vaso-
dilatation, 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-NH
2
,
at their C-termini [1,5]. Three mammalian TK recep-
tors (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 trig-
ger the phospholipase C–inositol triphosphate–calcium
signal transduction cascade via coupling to Gq
Keywords
evolution; Octopus vulgaris; oct-TKRPR;
tachykinin-related peptide receptor;
tachykinin
Correspondence
A. Kanda, Suntory Institute for Bioorganic
Research, 1-1-1 Wakayamadai, Shimamoto-
cho, Mishima-gun, Osaka 618-8503, Japan

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.
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
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 (verte-
brates and ascidians) [1–5,7,8].
In protostomes, two TK-type peptides, namely inver-
tebrate TKs (inv-TKs) and tachykinin-related peptides
(TKRPs), have been identified. Inv-TKs bear a verte-
brate 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

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 1 [10]. However, whether oct-TKs
or oct-TKRPs serve as brain ⁄ gut peptides remains to
be elucidated. Moreover, the large diversity of neuro-
peptides such as the TK and TKRP family is correla-
ted with the evolution and divergence of the nervous
system and their biological roles, and thus, functional
characterization of oct-TKs and oct-TKRPs is expec-
ted 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, oct-
TKRPR. 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 1. Amino acid sequence of Octopus tachykinin-related peptides and invertebrate tachykinins.
Tachykinin-related peptides from the brain of Octopus vulgaris
oct-TKRP I Val-Asn-Pro-Tyr-Ser-Phe-Gln-Gly-Thr-Arg-NH
2
oct-TKRP II Leu-Asn-Ala-Asn-Ser-Phe-Met-Gly-Ser-Arg-NH

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 an-
alyzed, and gave identical nucleotide sequence.
The sequence showed the presence of the seven
hydrophobic TMs that are the most typical characteris-
tic of GPCRs. As shown in Fig. 1, oct-TKRPR con-
tains 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 con-
sensus sequences for phosphorylation by protein kin-
ase A (K ⁄ R-X
1
-(X
2
)-S ⁄ T); one site of phosphorylation
by protein kinase C (S ⁄ T-X-K ⁄ R); and three casein
kinase II sites (S⁄ T-X
1

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 FX
1
(G ⁄ A)X
2
R-NH
2
(Table 1), suggesting that oct-TKRPs are novel mem-
bers 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 neuro-
peptides, 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 dis-
played typical inward membrane currents upon appli-
cation of oct-TKRP II (Fig. 3A). EC
50
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 EC
50

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.
Octopus tachykinin-related peptide receptor A. Kanda et al.
2232 FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS
It is well established that invertebrate TKRPRs
are responsive to TKRPs containing the C-terminal
FX
1
(G ⁄ A)X
2
R-NH
2
consensus sequence but not to
TKs containing FXGLM-NH
2
[6,8,16]. Likewise,
oct-TKRPR did not show activation upon application
of SP, whereas SP-(Arg11), in which the C-terminal
Met-NH
2
is replaced by Arg-NH
2
, 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 abo-
lished the ability to activate UTKR [16]. Moreover,
we tested whether oct-TKRPR was activated by an

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. mel-
anogaster; NKD, D. melanogaster; STKR,
S. calcitrans; UTKR, U. unitinctus.
A. Kanda et al. Octopus tachykinin-related peptide receptor
FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2233
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 per-
formed 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 oct-
TKRP II or III stimulate spontaneous contractile
action (e.g. esophagus, aorta, stomach, crop, and ovi-
duct) 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 NK1-
3Rs were widely distributed in the central and periph-
eral nervous systems plus several peripheral tissues,

gesting that oct-TKRPs also control sexual behavior in
Octopus. Detailed localization of oct-TKRPR in the
nervous system and peripheral tissues by in situ hybrid-
ization and immunohistochemistry is now being exam-
ined. 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 protos-
tomes: 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 oxy-
tocin ⁄ vasopressin superfamily peptides and their three
receptors were characterized from Octopus in our pre-
vious study, whereas other protostomes have been
shown to possess only one oxytocin ⁄ vasopressin super-
family 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 func-
tional identification of octopus neuropeptides and hor-
mones 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 fam-
ilies in the animal kingdom, and O. vulgaris is the first
species shown to possess both inv-TKs and TKRPs.

-8
10
-7
10
-6
100
80
60
40
20
0
[%]
10
-11
10
-10
10
-9
10
-8
10
-7
10
-6
100
80
60
40
20
0

10
-6
100
80
60
40
20
0
[%]
10
-10
10
-9
10
-8
10
-7
10
-6
10
-5
100
80
60
40
20
0
[%]
10
-11

80
60
40
20
0
[%]
10
-11
10
-10
10
-9
10
-8
10
-7
10
-6
Conc.[M]
Conc.[M]
Conc.[M]Conc.[M]
Conc.[M]
Conc.[M]
Conc.[M]
Conc.[M]
SP-[Arg
11
]
SP
HG

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 ⁄ vasopres-
sin 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 independ-
ently 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 simi-
larity 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 com-
pounds 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

Octopus tachykinin-related peptide receptor A. Kanda et al.
2236 FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS
oct-TKs, might have been acquired via horizontal gene
transfer from vertebrates to invertebrates after ances-
tral 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 recep-
tor 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 concentra-
tions, 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 spe-
cies, but TK and inv-TK genes have conserved the
essential original structural organization; or (b) trunca-
tion 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 basic-
ally conserved in TKRP genes. However, whether the
C-terminal Arg- or Met-containing sequence was pre-
sent 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 pro-
vide fruitful insights into evolutionary and interphy-

55 °C, and 150 s at 72 °C (5 min for the last cycle). The
second PCR used the PCR anchor primer and TKRPR-
3¢-2F (5¢-TAAAATGATGATTGTCGTGGTG-3¢) under
the following conditions: 5 min at 94 °C, and 30 cycles of
30 s at 94 °C, 30 s at 55 °C, and 150 s at 72 °C (5 min 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 2 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: 5 min at 94 °C, and 30 cycles of 30 s at 94 °C,
30 s at 55 °C, and 150 s at 72 °C (5 min for the last cycle).
Molecular phylogenetic analysis
The amino acid sequences encoding the intracellular, extra-
cellular 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,

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, Madi-
son, 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].
Acknowledgements
We thank Dr Hiroyuki Minakata for providing some
information concerning oct-TKRPs.
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