Anoveltachykinin-relatedpeptide 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 atachykinin-related peptide. In this paper, we
report the identification of anoveltachykinin-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-relatedpeptide 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 peptidereceptor from other species than
insects and also of the genomic structure of a tachykinin-
related peptidereceptor 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-relatedpeptide 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-relatedpeptide 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-relatedpeptide 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 anoveltachykinin-relatedpeptide 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 peptidereceptor 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-relatedpeptidereceptor (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 atachykinin-relatedpeptide 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-relatedpeptide 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-relatedpeptide recep-
tors. Consequently, we concluded that this GPCR is a
putative Urechis tachykinin-relatedpeptidereceptor 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-relatedpeptide 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-relatedpeptidereceptor (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-relatedpeptide 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-relatedpeptide 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-relatedpeptidereceptor (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 atachykinin-relatedpeptide 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 anoveltachykinin-relatedpeptide 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-relatedpeptide 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-relatedpeptide 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-relatedpeptide receptors.
Some tachykinin-related peptides occasionally showed
different activities on tachykinin-relatedpeptide 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-relatedpeptide 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-relatedpeptide 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-relatedpeptidereceptor 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 atachykinin-relatedpeptidereceptor and
that the binding site of tachykinin-relatedpeptide 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-relatedpeptide 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-relatedpeptidereceptor 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 atachykinin-relatedpeptide 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 anovel tachykinin-
related peptide receptor, UTKR. Our data not only
confirmed the characteristics of UTKR as a noninsect
tachykinin-related peptide receptor, but also indicated
unprecedented possibilities that atachykinin-related pep-
tide receptor possesses no significant selective affinity to its
endogenous ligand and that tachykinin-related peptide
receptors and tachykinin receptors originated from the
common ancestral gene.
ACKNOWLEDGEMENTS
We would like to thank Prof Osamu Matsushima for discussion and
encouragement.
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. 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