Tài liệu Báo cáo khoa học: A novel tachykinin-related peptide receptor of Octopus vulgaris – evolutionary aspects of invertebrate tachykinin and tachykinin-related peptide ppt

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

Fax: +81 75 962 2115
Tel: +81 75 962 3743
E-mail:
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 repre-
sent 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 mem-
brane 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-NH
2
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-NH
2
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.
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

activities on vertebrate tissues [5,8–10]. TKRPs were
isolated from nervous systems or guts of various pro-
tostomes [1,5,6,8]. Of particular importance is that
TKRPs share the C-terminal sequence FX
1
(G ⁄ A)X
2
R-
NH
2
, analogously to those of vertebrate TKs and
inv-TKs, and that multiple copies of TKRPs are enco-
ded 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 induc-
tion of adipokinetic hormone release, have been docu-
mented, 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 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

2
oct–TKRP III Thr-Val-Ser-Ala-Asn-Ala-Phe-Leu-Gly-Ser-Arg-NH
2
oct-TKRP IV Ser-Asp-Ala-Leu-Ala-Phe-Val-Pro-Thr-Arg-NH
2
oct-TKRP V Met-Asn-Ser-Leu-Ser-Phe-Gly-Pro-Pro-Lys-NH
2
oct-TKRP VI Tyr-Ser-Pro-Leu-Asp-Phe-Ile-Gly-Ser-Arg-NH
2
oct-TKRP VII Ala-Ser-Leu-His-Asn-Thr-His-Phe-Ile-Pro-Ser-Arg-NH
2
Invertebrate tachykinins from the salivary gland of Octopus vulgaris
oct-TK-I Lys–Pro–Pro–Ser–Ser–Ser–Glu–Phe–Ile–Gly–Leu–Met–NH
2
oct-TK-II Lys–Pro–Pro–Ser–Ser–Ser–Glu–Phe–Val–Gly–Leu–Met–NH
2
Substance P and SP-(Arg11)
Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met–NH
2
SP-(Arg11) Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Arg–NH
2
Octopus tachykinin-related peptide receptor A. Kanda et al.
2230 FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS
oct-TKs, and that oct-TKRPR is involved in the regu-
lation of various physiologic functions, including neur-
onal 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 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

-X
2
-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, sug-
gesting that phosphorylation is involved in the modula-
tion of G protein coupling and receptor function [22].
The Asp110 and Asn337 in TM2 and TM7, the consen-
sus tripeptide motif (E ⁄ D-R-Y, DRY in oct-TKRPR)
at the interface of TM3, and the K ⁄ R-X
1
-X
2
-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 resi-
dues 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
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 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

values of 230 and 92.5 nm,
respectively (Fig. 4A–G; Table 3). These results pro-
vided undoubted evidence that oct-TKRPs are endo-
genous 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 evolu-
tionary pathway specific to octopuses. Moreover, the
possibility cannot be absolutely excluded that octo-
puses have other oct-TKRPR subtypes that oct-
TKRP V and VI activate more potently, given that
oct-TKRP VI, V and VII do 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).
A. Kanda et al. Octopus tachykinin-related peptide receptor
FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2231
Fig. 1. 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.
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

Octopus inv-TK, oct-TK-I (Table 1), which was isola-
ted 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 identi-
fied in a single species. Altogether, these results
revealed that the importance of C-terminal Arg-NH
2
Table 2. Total amino acid sequence identity scores of oct-TKRPR
to the TKR and TKRPR family.
oct-TKRPR versus Percentage indentity
UTKR 43.6
DTKR 37.2
STKR 28.6
NKD 30.8
Mouse NK1R 31.3
Mouse NK2R 29.3
Mouse NK3R 31.2
Ci-TK-R 26.8
Fig. 2. 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-join-
ing 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. 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,

such as the brain, heart, gastrointestinal and genitouri-
nary tract, respiratory organs, and muscle [27–30]. In
keeping with such extensive expression of the recep-
tors, TKs play an important role in smooth muscle
contraction, vasodilatation, inflammation, the process-
ing of sensory information in a neuropeptidergic or
endocrine ⁄ paracrine fashion, and the release of neuro-
transmitters in the tachykinergic nerve fiber [8,31,32].
Most TKRPs have been shown to stimulate sponta-
neous contraction of the visceral muscles of insects,
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 corres-
ponds 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 neuro-
transmitters, neuromodulators, and hormone-like fac-
tors. 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], sug-

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.

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
C
B
A
Fig. 3. 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.
Octopus tachykinin-related peptide receptor A. Kanda et al.
2234 FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS
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
-11
10
-10
10
-9
10
-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
-11
10
-10
10
-9
10
-8
10
-7

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

10
-10
10
-9
10
-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
-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

FE
DC
BA
Fig. 4. 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.
A. Kanda et al. Octopus tachykinin-related peptide receptor
FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2235
various TK-typical activities on vertebrate tissues [10],
and completely failed to activate endogenous TKRPR,
although it is expressed in the salivary gland. Alto-
gether, these findings lead to the conclusion that Octo-
pus acquired oct-TKs as toxin-like substance for use
against vertebrates such as fishes, which are prey ani-
mals 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 ⁄ 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

from vertebrates to invertebrates has so far been con-
firmed. Nevertheless, several vertebrate neuropeptide
orthologs have been characterized from lower inverte-
brates, although not in species closely related to octo-
puses. For instance, an angiotensin-like peptide and
opioid peptides have been identified in the blood-suck-
ing 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 ver-
tebrate neuropeptides, e.g. the oxytocin ⁄ vasopressin
superfamily, are interphyletically conserved in most
invertebrate species, but other neuropeptides, including
Table 3. EC
50
values for oct-TKRPR in Xenopus oocytes.
Peptide EC
50
(nM)
oct-TKRP I 18.5 ± 0.18
oct-TKRP II 9.35 ± 0.59
oct-TKRP III 9.5 ± 0.6
oct-TKRP IV 19.3 ± 2.86
oct-TKRP V 230 ± 3.65
oct-TKRP VI 92.5 ± 5.48
oct-TKRP VII 14.5 ± 1.4
SP –
SP-(Arg11) 35.2 ± 0.38
Fig. 5. Tissue distribution of oct-TKRPR
(upper) and b-actin (lower) in Octopus.

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-

letic relationships among TKRPs, inv-TKs, and TKs.
Experimental procedures
Animals
Adult octopuses (body weight, approximately 2 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 Sepa-
sol-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: 5 min at 94 °C,
and 30 cycles of 30 s at 94 °C, 30 s at 45 °C, and 90 s at
72 °C (5 min for the last cycle). The second PCR was per-
formed using TKRPR-Fw2 [5¢-TT(C ⁄ T)GCIATITG(C ⁄ T)
TGG(C ⁄ T)TICCIT-3¢] and TKRPR-Rv2 [5¢-AIGGIA
(G ⁄ A)CCA(G ⁄ A)CAIATIGC(G ⁄ A)AA-3 ¢] under the fol-
lowing conditions: 94 °C for 5 min, and 30 cycles of 30 s at
94 °C, 30 s at 50 °C, and 90 s at 72 °C (7 min for the last
cycle). The method for cloning was the same as those previ-
ously 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:
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 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,

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,
A. Kanda et al. Octopus tachykinin-related peptide receptor
FEBS Journal 274 (2007) 2229–2239 ª 2007 The Authors Journal compilation ª 2007 FEBS 2237
CA) using oligo(dT)
12)18
primer. The PCR was performed
using TKRPR-Fw3 (5¢-AGATTTTTTCTAAGAACCGCC-
3¢) and TKRPR-Rv3 (5¢-CTGTCATTTTCTTCCCTGT
CG-3¢) under the following conditions: 5 min at 94 °C, and
30 cycles of 30 s at 94 °C, 30 s at 50 °C, and 90 s at 72 °C
(4 min 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 DNA-
labeling 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 des-
cribed [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, 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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