Báo cáo khoa học: A novel tachykinin-related peptide receptor

A novel tachykinin-related peptide receptor

Sequence, genomic organization, and functional analysis

Tsuyoshi Kawada

, Yasuo Furukawa

, Yoriko Shimizu

, Hiroyuki Minakata

, Kyosuke Nomoto

and Honoo Satake

Suntory Institute for Bioorganic Research, Osaka, Japan;

Department of Biological Science, Faculty of Science,

Hiroshima University, Japan;

Faculty of Life Sciences, Toyo University, Gunma, Japan

Structurally tachykinin-related peptides have been isolated

from various invertebrate species and shown to exhibit their

biological activities through a G-protein-coupled receptor

(GPCR) for a tachykinin-related peptide. In this paper, we

report the identiﬁcation of a novel tachykinin-related pep-

tide receptor, the urechistachykinin receptor (UTKR) from

the echiuroid worm, Urechis unitinctus. The deduced UTKR

precursor includes seven transmembrane domains and typ-

ical sites for mammalian tachykinin receptors and inver-

tebrate tachykinin-related peptide receptors. A functional

analysis of the UTKR expressed in Xenopus oocytes dem-

onstrated that UTKR, like tachykinin receptors and

tachykinin-related peptide receptors, activates calcium-

dependent signal transduction upon binding to its endo-

genous ligands, urechistachykinins (Uru-TKs) IV 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

instead of Arg-NH

. Furthermore, the genomic

analysis revealed that the UTKR gene, like mammalian

tachykinin receptor genes, consists of ﬁve exons interrupted

by four introns, and all the intron-inserted positions are

completely compatible with those of mammalian tachykinin

receptor genes. These results suggest that mammalian

tachykinin receptors and invertebrate tachykinin-related

peptide receptors were evolved from a common ancestral

GPCR gene. This is the ﬁrst identiﬁcation of an invertebrate

tachykinin-related peptide receptor from other species than

insects and also of the genomic structure of a tachykinin-

related peptide receptor gene.

Keywords: tachykinin-related peptide; Uru-TK; UTKR;

Urechis unicinctus; G-protein-coupled receptor.

Tachykinins are vertebrate multifunctional brain/gut pep-

tides that play crucial roles not only in the various peripheral

activities but also in the functions of the central nervous

system including the processing of sensory information

[15]. 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

) 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 IIIVII) as well as Uru-TKs I and II,

and that six of seven Uru-TKs (Uru-TKs IV 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

, which is ana-

logous to the mammalian tachykinin consensus sequence

Phe-X-Gly-Leu-Met-NH

. In addition, no tachykinin-rela-

ted peptides containing the Phe-X-Gly-Y-Arg-NH

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 6188503, 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

, 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, 42384246 (2002) 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

[1114],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

-calcium signal transduc-

tion cascade [15,16]. These ﬁndings imply that tachykinin-

related peptides are the invertebrate functional counterparts,

at least partially, for vertebrate tachykinin family peptides.

However, only a few tachykinin-related peptide receptors

have been characterized from several insects as mentioned

above. Furthermore, tachykinin-related peptides and their

receptors from different species have so far been employed

for studies of tachykinin-related peptide activity on insect

tachykinin-related peptide receptors. Therefore, the bio-

chemical characteristics of tachykinin-related peptides and

their receptors such as the binding selectivity still need to be

fully elucidated, and the interphyletic relationships and

molecular evolution of tachykinin-related peptide receptors

have not been investigated. To further study the biological

functions and evolutionary and phylogenetic relationship of

tachykinin-related peptide receptors and tachykinin recep-

tors, we identiﬁed a novel tachykinin-related peptide recep-

tor, UTKR from the echiuroid worm Urechis unicinctus.

In this paper, we present a UTKR sequence, an exon/intron

structure of the UTKR gene, and the response of the UTKR

to Uru-TKs. To the best of our knowledge, this is the ﬁrst

characterization of a noninsect tachykinin-related peptide

receptor and the structural organization of the tachykinin-

related peptide receptor gene.

MATERIALS AND METHODS

Preparation of RNA from echiuroid worms

Echiuroid worms were purchased from a ﬁshing-bait shop.

Total RNA was prepared from ventral nervous tissues using

TRIzol reagent (Gibco, Gaithersburg, MD, USA),

and mRNA was puriﬁed using OligotexTM-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

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 104163), 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 752730 (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 716693),

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

Uru-TK II AAGMGFFGAR-NH

Uru-TK III AAPSGFFGAR-NH

Uru-TK IV AAYSGFFGAR-NH

Uru-TK V APSMGFFGAR-NH

Uru-TK VII APKMGFFGAR-NH

FEBS 2002 An Urechis tachykinin-related peptide receptor (Eur. J. Biochem. 269) 4239

PCR anchor primer and a primer (5¢-CGAACACCCAG

TGGTTATTCAAC-3¢, complementary to nucleotides

693672), followed by reampliﬁcation using the anchor

primer and a primer (5¢-GATATCAAAGCGTCAGCAA

CTGC-3¢, complementary to nucleotides 638616). 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 ExpandTM 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 FastMocTM 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

) were injected into oocytes. The

oocytes were incubated for 24daysat17C and trans-

ferredtoND96buffer[96m

NaCl, 2 m

KCl, 1.8 m

CaCl

,1m

MgCl

and 5 m

Hepes (pH 7.6)]. The

oocytes were voltage-clamped at )80 mV. The dose

response data and the EC50 values of the experiment were

analyzed using

ORIGIN

6.1 software (Microcal Software

Inc.).

RESULTS

Cloning of a Uru-TK receptor cDNA

Comparative analysis of amino-acid sequences of mamma-

lian tachykinin receptors and insect tachykinin-related

peptide receptors showed that the second, sixth, and seventh

transmembrane domains are highly conserved among all

receptors. To identify a tachykinin-related peptide receptor

of the echiuroid worm, we ﬁrst performed RT-PCR

experiments using degenerative primers corresponding to

the conserved regions (see Materials and methods). An

ampliﬁed cDNA product of 628 bp was subcloned and

sequenced. The putative amino-acid sequence was shown to

encode a partial transmembrane domain of a GPCR.

Moreover, we determined the full-length cDNA sequence

encoding the putative GPCR using the 5¢-and3¢RACE

method. Figure 1A shows the 2533 bp putative receptor

cDNA containing a 1293 bp ORF ﬂanked by a 306 bp

5¢-untranslatedregion(UTR)anda924bp3¢-UTR. The

ORF begins with the ATG codon at position 307, which is

supported by the Kozak rule [17], and terminates with a

TGA stop codon at position 1602. Only one potential

polyadenylation signal AATAAA was found to be located

19 bases upstream of a poly(A) tail.

The deduced receptor protein is composed of 431 amino-

acid residues (Fig. 1). The sequence showed the presence of

the seven hydrophobic transmembrane regions that are the

most typical characteristic of GPCRs. The common Cys

residues (Cys134 and Cys214) responsible for the disulﬁde

bridge between the ﬁrst and second extracellular loops are

found at corresponding positions of known tachykinin

receptors. N-linked glycosylation sites (Asn-X-Ser/Thr,

Asn28, Asn39, and Asn223) are also located at the

N-terminal and second extracellular domains. The GPCR

sequence were also found to contain potential phosphory-

lation sites by protein kinase A (Arg/Lys-X-(X)-Ser/Thr,

Ser173, Thr262, Ser365, Ser381, Thr389, and Ser396), by

protein kinase C (Ser/Thr-X-Arg/Lys, Thr273 and Ser276),

and by casein kinase 2 (Ser/Thr-X-(X)-Asp/Glu, Thr262,

Ser381, Thr389, Thr400, and Ser404) in the second and

third intracellular loop and C-terminal region. Further-

more, the Asp/Glu-Arg-Tyr motif (Asp158Tyr160) in the

second intracellular loop and the Lys/Arg-Lys/Arg-X-X-

Lys/Arg motif(Arg278Lys282) 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 Trp356Leu357 at the corresponding

positions of the putative Urechis GPCR (Fig. 1). The lack of

this site was not the result of a PCR error or an artifact, as

all clones obtained using different polymerases encoded the

identical sequence. Comparative study of amino-acid

sequences veriﬁed that the putative Urechis GPCR sequence

including the transmembrane domains and intracellular and

extracellular regions displayed high identity to those of

mammalian tachykinin receptors and insect tachykinin-

related peptide receptors (Fig. 2 and Table 2). In addition,

the sequence of this region was shown to be closer to those

of tachykinin-related peptide receptors than tachykinin

receptors (Table 2). Furthermore, the homology-searching

showed no signiﬁcant similarity of UTKR to any other

GPCR. Taken together, these results revealed that the

putative Urechis GPCR possesses the essential properties of

tachykinin receptors and tachykinin-related peptide recep-

tors. Consequently, we concluded that this GPCR is a

putative Urechis tachykinin-related peptide receptor and

designated the receptor as the Uru-TK receptor, UTKR.

Functional expression of UTKR in

Xenopus

oocytes

It is well established that the binding of tachykinins and

tachykinin-related peptides to their receptors results in the

activation of PLC followed by the production of the

4240 T. Kawada et al. (Eur. J. Biochem. 269)FEBS 2002

intracellular second messengers, InsP

and calcium

[1316,1820]. In Xenopus oocytes, the interaction of an

agonist with its GPCR, inducing an elevation of intracel-

lular calcium, leads to the activation of a calcium-dependent

chloride channel, which is evaluated by direct observation of

the resultant inward chloride current. This system has been

employed for functional analyses of tachykinin receptors

and tachykinin-related peptide receptors [1821], 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 24 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

,andthehalf-

maximal response value (EC50) was calculated to be

approximately 1 n

by a doseresponse curve of current

shift (Fig. 3B). These results conﬁrmed that UruTK I is

an endogenous ligand of UTKR.

In a previous study, we showed that six Uru-TK peptides

(Uru-TK IV 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 IIVandVIIonUTKRwere

observed by the voltage-clamp method. As shown in

Fig. 3B, all EC50 values of Uru TKs IIVandVIIwere

showntobe0.623.15 n

, 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 IIVandVIIalsoserve

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.

FEBS 2002 An Urechis tachykinin-related peptide receptor (Eur. J. Biochem. 269) 4241

Structureactivity relationships of Uru-TKs

and mammalian tachykinins

Most invertebrate tachykinin-related peptides contain a

common Phe-X-Gly-Y-Arg-NH

sequence at their

C-termini, whereas the C-terminal consensus motif of

vertebrate tachykinins is Phe-X-Gly-Leu-Met-NH

.More-

over, we demonstrated in our previous study that conver-

sion of Arg-NH

to Met-NH

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

is critical for

activation of the UTKR, an Uru-TK I analog ([Met10]Uru-

TK I), in which the C-terminal Arg-NH

is replaced with

Met-NH

, 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

is essential for

Fig. 2. Alignment of the amino-acid sequence of receptor core region. Four invertebrate tachykinin-related peptide receptors (UTKR, STKR, NKD

and DTKR) and three rat tachykinin receptors (NK13R) 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)FEBS 2002

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