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

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 cascad

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-NH2,

at their C-termini [1,5] Three mammalian TK recep-tors (TKRs), NK1, NK2 and NK3 receprecep-tors (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: kanda@sunbor.or.jp

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-NH2 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-NH2 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.

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 FX1(G⁄ A)X2

R-NH2, 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

Invertebrate tachykinins from the salivary gland of Octopus vulgaris

Substance P and SP-(Arg11)

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-X1-(X2)-S⁄ T); one site of phosphorylation

by protein kinase C (S⁄ T-X-K ⁄ R); and three casein

kinase II sites (S⁄ T-X1-X2-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 funcmodula-tion [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-X1-X2-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 FX1(G⁄ A)X2R-NH2 (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) EC50 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 EC50 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 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)

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.

It is well established that invertebrate TKRPRs

are responsive to TKRPs containing the C-terminal

FX1(G⁄ A)X2R-NH2 consensus sequence but not to

TKs containing FXGLM-NH2 [6,8,16] Likewise,

oct-TKRPR did not show activation upon application

of SP, whereas SP-(Arg11), in which the C-terminal Met-NH2 is replaced by Arg-NH2, 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)6m (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-NH2

Table 2 Total amino acid sequence identity scores of oct-TKRPR

to the TKR and TKRPR family.

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 melmel-anogaster; STKR,

S calcitrans; UTKR, U unitinctus.

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 sevperiph-eral periphperiph-eral 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)8M , and oct-TK-I and SP at 10)6M , in oocytes expressing

oct-TKRPR.

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Conc.[M]

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SP-[Arg 11 ] SP

H G

F E

D C

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Fig 4 Dose–response curves for oct-TKRPR in Xenopus oocytes (A–G) Dose–response curve over the concentration range 10)11)10 )6M

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 )6MSP and SP-(Arg11) with oct-TKRPR.

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.

Fig 5 Tissue distribution of oct-TKRPR (upper) and b-actin (lower) in Octopus.

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 18C

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 94C, 30 s at 45 C, and 90 s at

72C (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: 94C for 5 min, and 30 cycles of 30 s at

94C, 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 94C, and 30 cycles of 30 s at 94 C, 30 s at

55C, 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 94C, and 30 cycles of

30 s at 94C, 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 94C, and 30 cycles of 30 s at 94 C,

30 s at 55C, 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,

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 94C, 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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