A novel tachykinin-related peptide receptorSequence, genomic organization, and functional analysis Tsuyoshi Kawada1, Yasuo Furukawa2, Yoriko Shimizu2, Hiroyuki Minakata1, Kyosuke Nomoto3
Trang 1A novel tachykinin-related peptide receptor
Sequence, genomic organization, and functional analysis
Tsuyoshi Kawada1, Yasuo Furukawa2, Yoriko Shimizu2, Hiroyuki Minakata1, Kyosuke Nomoto3
and Honoo Satake1
1
Suntory Institute for Bioorganic Research, Osaka, Japan;2Department of Biological Science, Faculty of Science,
Hiroshima University, Japan;3Faculty 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) IV 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-NH2instead of Arg-NH2 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 GPCRgene 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
[15] 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
(InsP3) 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 IIIVII) as well as Uru-TKs I and II, and that six of seven Uru-TKs (Uru-TKs IV 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-NH2, which is ana-logous to the mammalian tachykinin consensus sequence Phe-X-Gly-Leu-Met-NH2 In addition, no tachykinin-rela-ted peptides containing the Phe-X-Gly-Y-Arg-NH2 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 exerexpec-ted 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 6188503, 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)
Trang 2[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
[1114], and NKD and STKR, which were cloned from the
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-InsP3-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
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
M A T E R I A L S A N D M E T H O D S
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 ofUTKR cDNA All reverse transcription polymer chain reactions (RT-PCRs) and rapid amplifications of cDNA ends were performed using TaqExpolymerase (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 55Cfor
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 94Cfor 30 s,
40Cfor 30 s and 72 Cfor 3 min, followed by 35 cycles, consisting of 94Cfor 15 s, 50 Cfor 30 s, and 72 Cfor
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 94Cfor 30 s, 37 Cfor
1 min, and 72Cfor 2 min, followed by 15 cycles of a 94 C for 30 s, 45Cfor 30 s, and 72 Cfor 2 min and a final extension at 72Cfor 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 ofUTKR cDNA
First-strand cDNA was amplified using the oligo-dT primer and a gene-specific primer (5¢-CTTGGCCTGTGCGTATT CGATGG-3¢, complementary to nucleotides 104163), and the first-round PCR products were reamplified using the anchor primer for 30 cycles of 94Cfor 30 s, 55 Cfor 30 s, and 72Cfor 3 min (10 min for the last cycle) The products were subcloned and sequenced as described above 5¢ RACE ofUTKR cDNA
The template cDNA was synthesized using a primer complementary to nucleotides 752730 (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 716693), 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.
Trang 3PCR anchor primer and a primer (5¢-CGAACACCCAG
TGGTTATTCAAC-3¢, complementary to nucleotides
693672), followed by reamplification using the anchor
primer and a primer (5¢-GATATCAAAGCGTCAGCAA
CTGC-3¢, complementary to nucleotides 638616) 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 theUTKR 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, HpaI and XhoI, 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 inXenopus 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 24 days at 17 Cand
trans-ferred to ND96 buffer [96 mM NaCl, 2 mMKCl, 1.8 mM
CaCl2, 1 mM MgCl2 and 5 mM 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 ORIGIN6.1 software (Microcal Software
Inc.)
R E S U L T S
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¢- and 3¢ RACE method Figure 1A shows the 2533 bp putative receptor cDNA containing a 1293 bp ORF flanked by a 306 bp 5¢-untranslated region (UTR) and a 924 bp 3¢-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 (Asp158Tyr160) in the second intracellular loop and the Lys/Arg-Lys/Arg-X-X-Lys/Arg motif(Arg278Lys282) 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 Trp356Leu357 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 inXenopus oocytes
It is well established that the binding of tachykinins and tachykinin-related peptides to their receptors results in the activation of PLCfollowed by the production of the
Trang 4intracellular second messengers, InsP3 and calcium
[1316,1820] 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 [1821], 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 17Cfor 24 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 UUru-TKR-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 nM, and the half-maximal response value (EC50) was calculated to be approximately 1 nMby a doseresponse curve of current shift (Fig 3B) These results confirmed that UruTK I is
an endogenous ligand of UTKR
In a previous study, we showed that six Uru-TK peptides (Uru-TK IV 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 IIV and VII on UTKR were observed by the voltage-clamp method As shown in Fig 3B, all EC50 values of Uru TKs IIV and VII were shown to be 0.623.15 nM, 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 IIV and VII also serve
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.
Trang 5Structureactivity relationships of Uru-TKs
and mammalian tachykinins
Most invertebrate tachykinin-related peptides contain a
common Phe-X-Gly-Y-Arg-NH2 sequence at their
C-termini, whereas the C-terminal consensus motif of
vertebrate tachykinins is Phe-X-Gly-Leu-Met-NH2
More-over, we demonstrated in our previous study that
conver-sion of Arg-NH2to Met-NH2in 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-NH2 is critical for activation of the UTKR, an Uru-TK I analog
([Met10]Uru-TK I), in which the C-terminal Arg-NH2is replaced with Met-NH2, 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 (NK13R) are aligned Conserved residues are shadowed and shown in bold Seven putative transmembrane regions (TM1-7) are indicated above the corresponding sequence part.
Trang 6activation 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 nM(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-NH2
in tachykinin-related peptides plays an essential role in the activation of tachykinin-related peptide receptors
Genomic organization of theUTKR gene Subsequently, we determined the intron/exon structure of the UTKR Genomic PCR was performed with several primer sets encoding the 5¢- or 3¢-terminal region of the UTKRcDNA 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) Dose response curve of the assay using Uru-TKs IV and VII Maximum membrane currents elicited by ligands are plotted The current caused by 10)7M 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) Doseresponse curve of Uru-TK I (circles) and [Met10]Uru-TK I (squares) (B) Dose response 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.
Trang 7D I S C U S S I O N
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 a tachykinin-related peptide 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 a novel tachykinin-related peptide 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-related peptide 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-related peptide receptors
[1316], 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-related peptide receptors
Some tachykinin-related peptides occasionally showed
different activities on tachykinin-related peptide receptors
For example, the locust tachykinin-related peptides,
Lom-TKs IIV, activated the stable fly tachykinin-related
peptide receptor, STKR to a similar degree [16], whereas the
Drosophilatachykinin-related peptide 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 IV 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 [6,1820] Taken together, tachykinin-related peptides, at least Uru-TKs, are highly likely to exhibit no binding selectivity for a homogenous tachykinin-related peptide 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 Drosophilatachykinin-related peptides [25] remains unclear Therefore, only one tachykinin-related peptide receptor 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 bpcaacagGTATAT
Intron 2 TTTTGTgtaaat )146 bpcaacagGTATAA
Intron 3 AGACGGgtatga)469 bptttcagGTAGTG
Intron 4 TGCCAGgtatgt)119 bpttccagATTCCG
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 i1i4.
Trang 8[Arg11]SP, a SP analog containing Arg-NH2, also
activated the UTKR, while SP and [Met10]Uru-TK I, an
Uru-TK I analog carrying Met-NH2, 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-NH2 residue in the
Phe-X-Gly-Y-Arg-NH2 consensus motif is critical for the
activation of a tachykinin-related peptide receptor and
that the binding site of tachykinin-related peptide
recep-tors including UTKR discriminates between Arg-NH2
and Met-NH2 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 [2628], but the molecular basis of the
tachykininreceptor 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-InsP3-calcium signal
transduction pathway, these results lead to the presumption
that the tachykinin-related peptide receptors of
inverte-brates and the tachykinin receptors of verteinverte-brates 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-related peptide receptor 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 a tachykinin-related peptide 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 [3138] 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 ligandreceptor pair and a
vertebrate tachykinin ligandreceptor pair
In conclusion, we identified the structure, the genomic
organization, and the function of a novel
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 a tachykinin-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
A C K N O W L E D G E M E N T S
We would like to thank Prof Osamu Matsushima for discussion and encouragement.
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