British Journal of Pharmacology (2003) 139, 555–566& 2003 Nature Publishing Group All rights reserved 0007 – 1188/03 $25.00
www.nature.com/bjp

Molecular cloning and characterization of avian bombesin-like
peptide receptors: new tools for investigating molecular basis forligand selectivity
1,5

Maiko Iwabuchi, 2Kumiko Ui-Tei, 3Kazuhiko Yamada, 3Yoichi Matsuda, 4Yasushi Sakai,
Kohichi Tanaka & *,1,5Hiroko Ohki-Hamazaki

1,5

1Department of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental University, Japan;
Undergraduate Program for Bioinformatics and Systems Biology, Department of Biophysics and Biochemistry, Graduate
School of Science, University of Tokyo, Japan; 3Laboratory of Animal Cytogenetics, Center for Advanced Science andTechnology, Hokkaido University, Japan and 4Division of Physiology, Department of Occupational Therapy, School of Nursing
and Rehabilitation Sciences, Showa University, Japan; 5Laboratory of Molecular Neuroscience, School of Biomedical Science andMedical Research Institute, Tokyo Medical and Dental University
2

Keywords:
Abbreviations:

1 Bombesin (BN), originally isolated from amphibians, is structurally related to a family of BN-like
peptides found in mammals, which include gastrin-releasing peptide (GRP) and neuromedin B(NMB). These peptides have important effects on secretion, smooth muscle contraction, metabolism
and behavior. Here we report cloning and characterization of two subtypes of BN-like peptide
receptors in Aves.
2 The amino-acid sequence of chick GRP-R (chGRP-R) is highly identical with mammalian andamphibian GRP-R, and this receptor showed high affinity for GRP, BN and synthetic bombesin
agonist, [D-Phe6, b-Ala11, Phe13, Nle14]bombesin(6 – 14) ([FAFNl]BN(6 – 14)). The chGRP-R gene was
localized to chicken chromosome 1q23distal-q24proximal, where chick homologs of other human Xlinked genes have also been mapped.
3 ChBRS-3.5, having sequence similarities to both mammalian bombesin-like peptide receptor
subtype-3 and amphibian bombesin-like peptide receptor subtype-4, showed high affinity for[FAFNl]BN(6 – 14), moderate affinity for BN, but low affinity for both GRP and NMB.
4 Expression of both receptors was detected in brain, but only chGRP-R was expressed in
gastrointestinal (GI) tissues.
5 When expressed in Chinese hamster ovary K1 cells, these receptors mediate intracellular calcium
mobilization upon agonist stimulation. These results suggest that a novel BN peptide may occur in
Aves as an endogenous ligand for chBRS-3.5.
6 The receptor sequences responsible for ligand selectivities were discussed and this knowledge about
avian BN-like peptide receptors will help us to understand the molecular basis for agonist sensitivitiesof BN-like peptide receptors.
British Journal of Pharmacology (2003) 139, 555 – 566. doi:10.1038/sj.bjp.0705282
Bombesin; Aves; chick; BRS-3; BB4; gastrin-releasing peptide; GRP; NMB
BB4, bombesin-like peptide receptor subtype-4; BN, bombesin; BRS-3, bombesin-like peptide receptor subtype-3;
CHO-K1, Chinese hamster ovary K1 cells; DMEM, Dulbecco’s modified Eagle’s medium; [FAFNl]BN(6 – 14),
[D-Phe6, bAla11, Phe13, Nle14]bombesin(6 – 14); FISH, fluorescence in situ hybridization; GI, gastrointestinal;
GRP, gastrin-releasing peptide; NMB, neuromedin B; PBSK, pBluescriptIISK(); PCR, polymerase chain
reactions; RACE, rapid amplifications of cDNA ends; RT, reverse transcription; TM, transmembrane domain;[YAFNl]BN(6 – 14), [D-Tyr6, b-Ala11, Phe13, Nle14]bombesin(6 – 14)

Introduction
Bombesin (BN) is an amidated tetradecapeptide originallyisolated from skin of the European frog, Bombina bombina, as
an agent that has an ability to induce smooth muscle
contraction (Anastasi et al., 1971). Two peptides, gastrinreleasing peptide (GRP) and neuromedin B (NMB), which*Author for correspondence at; Department of Molecular Neuroscience, Medical Research Institute, Tokyo Medical and Dental
University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan;
E-mail: hamazaki.aud@mri.tmd.ac.jp

have similarities with BN, have been purified subsequently
from the porcine stomach and spinal cord, and these two
peptides represent the only mammalian BN-related peptides
cloned so far (McDonald et al., 1979; Minamino et al., 1983).
GRP and NMB are widely distributed in the brain, lung,
exocrine and endocrine organs and gastrointestinal (GI) tract,
and mimic various physiological effects induced by BN (for areview, see Lebacq-Verheyden et al., 1990). These include
smooth muscle contraction, exocrine and endocrine secretion,induction of grooming and scratching, anorexia, hyperglycemia and hypothermic effects.

556

M. Iwabuchi et al

The high-affinity receptors for these peptides (GRP-R and
NMB-R) as well as a structurally related receptor, bombesin
receptor subtype-3 (BRS-3), have been cloned in mammals(Spindel et al., 1990; Wada et al., 1991; Fathi et al., 1993).
These receptors are G-protein coupled and expressed in thebrain and other organs, notably in GI tract, with the exceptionof BRS-3 which is expressed in the central nervous system(Wada et al., 1992; Ohki-Hamazaki et al., 1997a) and theuterus of guinea pig (Gorbulev et al., 1992). Production andanalysis of mice lacking GRP-R, NMB-R or BRS-3 revealed
that these receptors have overlapping as well as distinct
physiological roles. BRS-3, and to a less significant extent
GRP-R, play a role in the regulation of metabolism andadiposity (Ohki-Hamazaki et al., 1997b; Hampton et al.,1998). Although the hypothermic effect is regulated by both
GRP-R and NMB-R, contraction of smooth muscle in thestomach is controlled exclusively by GRP-R (Wada et al.,
1997; Ohki-Hamazaki et al., 1999). In addition, NMB-R isshown to be involved in stress response through modulation of
the 5-HT system (Yamano et al., 2002).
GRP and BN are structurally similar, but are distinct
peptides (Nagalla et al., 1992). Frogs have GRP and several
forms of BN in the brain or in GI tract. A high-affinity BNlike peptide receptor to one type of BN, [Phe13] BN, was cloned
from amphibian brain and designated as bombesin-like
peptide receptor subtype-4 (BB4) (Nagalla et al., 1995). These
results suggested that mammals might have additional BN-like
peptide receptors that have high affinity for a certain form ofBN, yet to be identified. In fact, the endogenous ligand for
BRS-3 has not yet been discovered. To address this question,
we attempted to isolate BN-like peptide receptors from chickbrain. Phylogenetically, amphibians and amniotes are includedin the same taxon of Tetrapoda and birds and mammals are
evolutionary descendants of the ancestral amniote lineage.
Therefore, knowledge of the BN-like peptide receptors in chickwill provide useful information about the evolution of BN-like
peptide receptors as well as about the molecular basis forligand selectivity of each receptor subtype by comparing theamino-acid sequences.

Methods
Cloning of chick BN-like peptide receptors
Oligo (dT) primed cDNA was synthesized from 200 ng chickbrain poly-A+RNA (BD Biosciences Clontech, Palo Alto, CA,
U.S.A.) using the Superscript II Preamplification system(Gibco-BRL Life Technologies, Gaithersburg, MD, U.S.A.)
and used as a template for polymerase chain reactions (PCR).
To isolate chick BN-like peptide receptors, two degenerateprimers, BNF11 (50 -CCT CGG T(GCT)G GGG T(TGA)TC(GATC) G T(GC)T TCA C-30 ) and BNR10 (50 -GAG CAA
AGG GGT TTA C(AG)C A(AG)G A(GA)T T-30 ), whichcorrespond to nucleotides at the third and seventh transmembrane domains (TM) of BN-like peptide receptors of various
species were designed. PCR was carried out in a 50 ml reaction
mixture containing 3 ml of template, 0.4 mM of each primer,
0.2 mM dNTPs and 2.5 U KOD Dash DNA polymerase(TOYOBO, Osaka, Japan), with the GeneAmp PCR System
9700 (Perkin-Elmer Applied Biosystems, Foster City, CA,
U.S.A.). Thermocycling conditions consist of 941C for 10 s,
British Journal of Pharmacology vol 139 (3)Avian bombesin-like peptide receptors

followed by 30 cycles of 941C for 30 s, 531C for 2 s, and 741C
for 2 min, then a final extension at 721C for 7 min.
A resulting PCR product (550 bp) was excised and purified
from agarose gel, and fragment termini were made blunt andphosphorylated using a BKL Kit (TAKARA, Tokyo, Japan)
and then ligated to the EcoRV site of pBluescriptIISK- (pBSK:
Stratagene, La Jolla, CA, U.S.A.). Competent cells weretransformed with the ligated vectors. Resultant colonies wereisolated and cultured overnight in 3 ml LB medium. PlasmidDNAs were purified with a Quantum Prep Plasmid Miniprep
Kit (BIO-RAD, Hercules, CA, U.S.A.). Sequencing reactions
were performed using the dideoxy chain termination method
according to the protocol followed by the ABI Prism Big Dye
Terminator Reaction Kit (Perkin-Elmer Applied Biosystems)with M13 forward (50 -GTT TTC CCA GTC ACG AC-30 ) andreverse primers (50 -CAG GAA ACA GCT ATG AC-30 ). The
ABI377 automated sequencer (Perkin-Elmer Applied Biosystems) was used. Partial sequences of two candidates, named
chGRP-R and chBRS3.5, were obtained.
Based on these partial sequences, 50 - and 30 -rapid amplifications of cDNA ends (RACE) were performed with the aid of
the SMARTt RACE cDNA Amplification Kit (BD Biosciences Clontech). First-strand cDNA for 50 - and 30 -RACE
were synthesized from 100 ng chick brain polyA+ RNA (BD
Biosciences Clontech) according to the manufacturer’s protocol. The primers used for 50 - and 30 -RACE reactions were
GRP201 (50 -GCA TGG GAT GCT TGG ATC TCC ATT
GGT-30 ) and GRP203 (50 -CAT TTC ATT GCC AGC ATCTGT GCT CGG-30 ), respectively for chick GRP-R (chGRPR), and BRS46 (50 -ATA CCA TGG AGA CAA TCC AGA
CA-30 ) and BRS49 (50 -ACG GTC ATG CCC GTA AGC
AGA TTG AAT C-30 ), respectively, for chick BRS-3.5(chBRS-3.5). The reaction mixture was prepared as indicatedby the manufacturer. The cycling parameters were as follows:
20 cycles of 941C for 5 s, 681C for 10 s and 721C for 3 min,followed by the second reaction consisting of 20 cycles of 941C
for 5 s, 601C for 10 s and 721C for 3 min. These products were
purified by agarose gel electrophoresis, extracted from gel
slices and directly sequenced. All of the sequence data obtained
were aligned and sequences in the coding regions of bothchGRP-R and chBRS-3.5 were determined. GENETYX
software was used for genetic information processing.

Phylogenetic analysis
Predicted receptor amino-acid sequences were aligned using
Clustal W and were used to construct phylogenetic trees
according to the UPGMA method. Phylogenetic and evolutionary analyses were conducted using MEGA version 2.1.

Chromosome preparation and fluorescence in situ
hybridization (FISH)
The fluorescence in situ hybridization (FISH) method was used
to determine chromosomal location of the chicken GRP-R
gene. Splenocyte culture and chromosome preparation were
performed as described by Matsuda & Chapman (1995) andSuzuki et al. (1999). Mitogen-stimulated splenocyte culture
was synchronized by thymidine block, and the incorporationof 5-bromodeoxyuridine during the late replication stage wasmade for differential replication staining after the release from

M. Iwabuchi et al

excessive thymidine. The chromosomal slides were exposed to
UV light after staining with Hoechst 33258.
The chromosome slides were hardened at 651C for 2 h andthen denatured at 701C in 70% formamide/2  SSC anddehydrated in a 70 – 85 – 100% ethanol series at 41C. The
1.4 kb cDNA fragment inserted in pBSK was labeled with biotin
16-dUTP (Roche Diagnostics) following the manufacturer’s
protocol. The labeled DNA fragment was ethanol-precipitatedwith salmon sperm DNA and E. coli tRNA, and then denatured
at 751C for 10 min in 100% formamide. The denatured probe
was mixed with an equal volume of hybridization solution
resulting in a final concentration of 50% formamide, 2  SSC,
10% dextran sulfate, and 2 mg ml1 bovine serum albumin (BSA)(Sigma-Aldrich Co., St Louis, MO, U.S.A.). In all, 20 ml of thishybridization buffer containing 250 ng labeled DNA was added
to the denatured slide, covered with parafilm and incubatedovernight at 371C. The slides were washed for 20 min in 50%
formamide/2  SSC at 371C, and in 2  SSC and 1  SSC for
20 min each at room temperature. After rinsing in 4  SSC, theslides were incubated under coverslips with goat anti-biotin
antibodies (Vector Laboratories) at a 1 : 500 dilution in 1%
BSA/4  SSC for 1 h at 371C. The slides were washed with
4  SSC, 0.1% Nonidet P-40 in 4  SSC and 4  SSC for 5 mineach and then stained with fluorescein-coupled donkey anti-goat
IgG (Nordic Immunology) at a 1 : 500 dilution for 1 h at 371C.
After washing with 4  SSC, 0.1% Nonidet P-40 in 4  SSC,
4  SSC for 10 min on the shaker, the slides were rinsed with
2  SSC and stained with 0.50 mg ml1 propidium iodide. The
chromosome slides were analyzed with a Nikon fluorescencemicroscope using Nikon filter sets B-2A and UV-2A. Kodak
Ektachrome ASA100 films were used for microphotography.

Reverse Transcription (RT) PCR
Total RNAs from embryonic and hatched chick tissues wereprepared using TRIzol (Invitrogen, Carlsbad, CA, U.S.A.).
For day 4 embryos (i.e., 4 days after the beginning of
incubation at 37.81C; E4), the brain and the body regions
were dissected and processed. For E8 and E12 embryos, thewhole brain and GI tract were removed separately. Tissues
from E16 and E18 embryos were dissected and processed for
RNA preparation. Single-stranded cDNA was synthesizedfrom 3 mg of total RNA with the Superscript Preamplification
System (Invitrogen). The cDNA was subjected to 35 cycles of
PCR (941C 30 s, 611C 2 s, 741C 30 s) using 2.5 U of KOD Dash(TOYOBO), 1  KOD buffer, 0.2 mM dNTPs and specificprimers for either chGRP-R (GRP205: CAT GAG AAA CGTCCC CAA TTT GTT and GRP207: GAA TGC CAG GATCCG AGC AC) or chBRS-3.5 (BRS412: CCT TTC CAGGAT TAG AAA TAC TGT GCA C and BRS413: CAA TCTGCT TAC GGG CAT GAC C). A control reaction was
performed using two primers for chick b-actin gene (forward:
GTG ATG AAG CCC AGA GCA AAA G and reverse: AGG
GTC CGG ATT CAT CGT ACT C) using the same PCR
condition except for the annealing temperature, which was 631C
instead of 611C. A volume of 10 ml of each reaction mixture wasresolved by agarose gel electrophoresis and photographed.

Stable cell lines
The chGRP-R cDNAs were obtained from chick brain cDNA
by PCR using the high-fidelity polymerase KOD-plus

Avian bombesin-like peptide receptors

557(TOYOBO) and two primers located just up- and downstream of the open reading frame, chGRPR-1 (50 -TAT GAC
TTT TAA ACT CTG GCG ACT G-30 ) and chGRPR-2 (50 TCT GAG TGC ATC AGA CTT CCT TGC-30 ). Similarly,chBRS-3.5 was amplified with chBRS3.5-2 (50 -GCA AGC
TAA ATA TTA GAC AAT TTT TTC ATC-30 ) andchBRS3.5-3 (50 -GGC AGT AGT GAC TTG ACT GCA
AAC GC-30 ). The resulting PCR products were cloned into the
SmaI site of pBSK. Both strands of each clone were sequenced
at least two times.
To generate eukaryotic expression vectors, the chGRP-R
fragment was cloned into pBSK. The cloned cDNA plasmids
were digested with HindIII and NotI, and the insert fragment
was ligated into pcDNA3 (Invitrogen). The chBRS-3.5
fragment was similarly digested with BamHI and EcoRI, andinserted into pcDNA3. The sequences of the inserts wereverified and the plasmids were purified with the Endofree
Qiagen Maxi Kit (Qiagen, Hilden, Germany) prior totransfection.
Chinese hamster ovary K1 (CHO-K1) cells were seeded in a
six-well plate at a density of 3  105 per well and grownovernight in Dulbecco’s modified Eagle’s medium (DMEM;
Sigma-Aldrich Co.) supplemented with 10% (v v1) fetal calf
serum (Mitsubishi Chemical corporation, Tokyo, Japan),penicillin (100 U ml1, Sigma) and streptomycin (100 mg ml1,
Sigma). All cells were maintained at 371C in a 5% CO2
atmosphere. The following morning cells were transfected withthe plasmids (10 mg) containing either pcDNA3-chGRP-R or
pcDNA3-chBRS-3.5 using 4 ml LipofectAMINETM (Invitrogen) in Opti-MEM (Gibco-BRL) and DMEM for 5 h. At theend of the incubation period, the medium was replaced withDMEM containing 20% fetal calf serum, penicillin andstreptomycin. A 24 h after transfection, cells were cultivatedin DMEM with fetal calf serum, penicillin, streptomycin and1000 mg ml1 Geneticin (Gibco-BRL) to select for stabletransfectants. Selection continued for about 3 weeks. Selectedclones were expanded and split (1 : 30 – 50) every 5 – 7 days at
confluence. Individual clones were screened for high receptor
expression using a binding assay described below.

Binding assay
The procedure has already been reported (Kris et al., 1987).
Briefly, cells were seeded into 24-well tissue culture plates
coated with fibronectin (5 mg cm2, Sigma) and grown toconfluence. The cells were washed three times with binding
buffer that consists of DMEM containing 20 mM HEPES(Sigma), 100 mg ml1 bacitracin (Sigma) and 0.2% BSA(Sigma). For saturation analysis, various concentrations (from50 pM to 1 nM) of [125I][D-Tyr6, b-Ala11, Phe13, Nle14]bombesin(6 – 14) ([125I][YAFNl]BN(6 – 14); Perkin-Elmer Life
Sciences, Boston, MA, U.S.A.) were added to binding buffer
with or without 1 mM unlabeled [D-Phe6, b-Ala11, Phe13,
Nle14]bombesin(6 – 14) ([FAFNl]BN(6 – 14); PHOENIX Pharmaceuticals, Belmont, CA, U.S.A.) and then incubated for
30 min at 371C. The cells were washed twice with ice-coldbinding buffer and three times with ice-cold PBS (Sigma) andthen solubilized with 1 N NaOH. Total cell-associated radioactivity was determined in a g-counter. Nonspecific binding
was defined as the amount of [125I][YAFNl]BN(6 – 14) boundto CHO-K1-transfected cells in the presence of 1 mM
[FAFNl]BN(6 – 14), and was subtracted from the total binding
British Journal of Pharmacology vol 139 (3)558

M. Iwabuchi et alBNchick GRP
GRP(14-27)[FAPNl]BN(6-14)
porcine NMB10

Avian bombesin-like peptide receptors

pGluGlnArgLeuGlyAsnGlnTrpAlaValGlyHisLeuMet
AlaProLeuGlnProGlyGlySerProAlaLeuThrLysIleTyrProArgGlySerHisTrpAlaValGlyHisLeuMet
MetTyrProArgGlyAsnHisTrpAlaValGlyHisLeuMet
DPheGlnTrpAlaValβAlaHisPheNle
GlyAsnLeuTrpAlaThrGlyHisPheMet

Figure 1 Amino-acid sequences of amphibian BN, chGRP, C-terminal portion (14 – 27) of pig and human GRP, synthetic BN
analog ([FAPNl]BN(6 – 14)) and porcine NMB10.

for determination of the specific binding. All binding values
represented saturable binding. Binding data were analyzed
using a radioligand binding analysis program (GraphPad
PRISM) and Kd values were determined. For displacement
analysis, cells were incubated with 40 pM [125I][YAFNl]BN(6 –14) for 30 min at 371C without or with various (from 1  1010
to 5  106 M) concentrations of unlabeled peptides; Bombesin(BACHEM California, Torrance, CA, U.S.A.), GRP(14 – 27)(BACHEM California), porcine NMB10 (PENINSULA
Laboratories, San Carlos, CA, U.S.A.) or [FAFNl]BN(6 –14) (Figure 1). The dose – inhibition curves assessing thepotency of peptides to inhibit binding of [125I][YAFNl]BN(6 –14) to CHO-K1 expressing chick BN-like peptide receptors
were analyzed by a curve-fitting program (SOFTmax Pro) andfound to be best fit to four logistic parameters. IC50 were
calculated using this program, and Ki values were obtained
from the IC50 value using the Cheng – Prusoff equation(Ki ¼ IC50/(1+[ligand]/Kd)).

Measurement of intracellular calcium ([Ca2+])
To measure changes in cytosolic calcium, transfected cells wereplated on 35-mm glass-bottomed plastic dishes (MatTek,
Ashland, MA, U.S.A.) at a density of 1  105 cells per well.
After 24 h of culture, the cells were washed with buffer(145 mM NaCl, 2 mM CaCl2, 3 mM KCl, 1 mM MgCl2, 5 mM
glucose, 10 mM HEPES, pH 7.4) and incubated with 10 mM
fluo-3/AM (Molecular Probes, Eugene, OR, U.S.A.) in thebuffer for 15 min at 371C under reduced light condition. Afterwashing with the buffer, the dish was mounted on the stage ofconfocal laser scanning microscope (FLUOVIEW 500; Olympus, Tokyo, Japan). Fluo-3 was excited at 488 nm with anargon laser and fluorescence was detected at 526 nm. Agonist
diluted in the buffer was directly added to the glass-bottom
area of the dish.

Results
Identifying and cloning of chick BN-like peptide receptors
Chick brain poly A+ RNA was reverse transcribed andamplified with degenerate primers. The primers were designed
based on the published sequences of mammalian andamphibian BN-like peptide receptors. We chose the mostconserved regions, TM3 and 7, to determine the sequence ofthe degenerate primers. The 550 bp PCR product was cloned
into the pBSK and sequenced. Two different sequences
showing identities to known BN-like peptide receptors wereobtained. One candidate clone showed high identity to GRP-R
and we named it chGRP-R. Another one having significant
identities to both BRS-3 and BB4 was designated as chBRS3.5. Based on these partial sequences, primers for both 50 and
30 RACE were designed. For chGRP-R, a 1 kb single band was
British Journal of Pharmacology vol 139 (3)obtained in each 50 and 30 RACE and the putative chGRP-R
sequence in its coding region was determined (Figure 2). 50 and
30 RACE for chBRS-3.5 generated 0.8 and 1 kb band,
respectively, and the sequence of the whole coding region
was determined (Figure 3). The chGRP-R and chBRS-3.5
cDNAs were 1164 and 1191 bp long, encoded a protein of 387
and 396 amino acids with a calculated molecular mass of 43.7
and 44.2 kDa, respectively, in the absence of any posttranslational modifications.
Hydropathy analysis of the predicted chGRP-R and chBRS3.5 proteins exhibited seven hydrophobic regions with an
extracellular amino (N-) terminal end followed by threeinterdigitated intracellular and extracellular loops and acarboxy (C-) terminal intracellular domain, consistent with aseven transmembrane structure typical of members of the Gprotein-coupled receptor superfamily. As shown in Figures 2
and 3, there are four sites of potential N-linked glycosylation(Asn-X-Ser/Thr). Three of them are present within theextracellular N-terminal domain of both chGRP-R (Asn20,
Asn24 and Asn28; arrowed) and chBRS-3.5 (Asn10, Asn18
and Asn29; arrowed), and one site within the second
extracellular loop (Asn192 and Asn196, respectively; arrowed).
These two receptors contain three Cys residues that arehighly conserved among G-protein-coupled receptors: one in
the first extracellular loop (Cys115 and Cys119, respectively;
double-lined) and another in the second extracellular loop
(Cys198 and Cys202, respectively; double-lined), and finallywithin the intracellular C-terminal domain (Cys341,342 andCys346,347, respectively; boxed). Consensus sequences of apotential protein kinase C (PKC) phosphorylation site
(Ser/Thr-X-Arg/Lys) are well conserved in both proteins at
the site between TM5 and 6 (Ser260 and Ser264, respectively;
starred) and within the C-terminal cytoplasmic region(Ser330,362, Thr357 of chGRP-R and Ser335 of chBRS-3.5,
respectively; starred).
When the predicted amino-acid sequences are comparedwithin two avian receptors, the homology between chGRP-R
and chBRS-3.5 is 60%’ higher than that among mouse
receptors (48% between moGRP-R and moBRS-3, 55%
between moGRP-R and moNMB-R and 46% between
moNMB-R and moBRS-3). As depicted in Figure 4, aminoacids are well conserved in the TM domains, especially inTM2, 3, 6 and 7. In contrast, the N- and C-terminal domains
are less preserved. The putative chGRP-R amino-acidsequence is also compared with the mouse, rat and human
GRP-R. As shown in Figure 5, sequence similarity is highexcept for the N-terminal region and the overall amino-acid
identity is more than 80% between chick and mouse, rat or
human (Table 1). The similarity between chBRS-3.5 andmammalian BRS-3 is 66 – 69% (Table 1), and the sequence
disparities are evident in the N-, C-terminal regions and in thethird extracellular loop (Figure 6). The putative chBRS-3.5
protein has 74% identity to frog BB4, higher than the identity
between chBRS-3.5 and mammalian BRS-3.

M. Iwabuchi et al

Avian bombesin-like peptide receptors

ATGGCATCTGGAGAGTGCCTGCTGCTGGACCTGGAAACTGATAACTTCATCCTCTACAACATCTCTGCCAACCAGAGTGCAAAT
MetAlaSerGlyGluCysLeuLeuLeuAspLeuGluThrAspAsnPheIleLeuTyrAsnIleSerAlaAsnGlnSerAlaAsn↑↑↑
CTCTCCGTCCTCAGCGATGAGTGGTTCTACCCAGCCTTCCTCTATGCCATCCCCACCATTTACGGGATCATCATTTTGATAGGC
LeuSerValLeuSerAspGluTrpPheTyrProAlaPheLeuTyrAlaIleProThrIleTyrGlyIleIleIleLeuIleGly
TM1
CTTATCGGTAACATCACTTTGATTAAGATCTTCTGTACTGTAAAATCCATGAGAAACGTCCCCAATTTGTTCATCTCCAGCCTG
LeuIleGlyAsnIleThrLeuIleLysIlePheCysThrValLysSerMETArgAsnValProAsnLeuPheIleSerSerLeu
GCTCTGGGAGACCTGCTGCTGTTAGTGACCTGTGTGCCCGTGGATGCCAGCAGGTACCTTGCTGATGAGTGGCTGTTTGGCAGG
AlaLeuGlyAspLeuLeuLeuLeuValThrCysValProValAspAlaSerArgTyrLeuAlaAspGluTrpLeuPheGlyArg
TM2
ATCGGATGTAAGCTCATTCCCTTCATACAGCTCACTTCTGTAGGGGTGTCAGTCTTCACTCTCACTGCTCTCTCAGCTGACAGG
IleGlyCysLysLeuIleProPheIleGlnLeuThrSerValGlyValSerValPheThrLeuThrAlaLeuSerAlaAspArg
TM3
TATAAAGCTATAGTAAGACCAATGGAGATCCAAGCATCCCATGCACTGATGAAAATTTGTGTGAGAGCTGCTATAATCTGGATT
TyrLysAlaIleValArgProMETGluIleGlnAlaSerHisAlaLeuMETLysIleCysValArgAlaAlaIleIleTrpIle
GCATCCATGCTGCTTGCCATCCCTGAAGCAGTGTTTTCAGATTTACACCCTTTCCATGACAAAGGGACTAATAAAACCTTCATC
AlaSerMETLeuLeuAlaIleProGluAlaValPheSerAspLeuHisProPheHisAspLysGlyThrAsnLysThrPheIle↑
TM4
AGCTGTGCTCCTTACCCACATTCTGATGGGTTGCATCCCAAAATCCACTCAATGGCATCATTTCTGATCTTTTACATCATTCCT
SerCysAlaProTyrProHisSerAspGlyLeuHisProLysIleHisSerMETAlaSerPheLeuIlePheTyrIleIlePro
CTGTCTGTCATTTCAGTATATTATTATTTCATTGCTAAGAACTTGATCCGGAGTGCTTACAACATCCCTGTGGAAGGAAATGTA
LeuSerValIleSerValTyrTyrTyrPheIleAlaLysAsnLeuIleArgSerAlaTyrAsnIleProValGluGlyAsnVal
TM5
CACGTAAGGAAACAGATTGAATCCCGTAAACGACTGGCCAGGACAGTGCTGGTCTTTGTTTGCCTCTTTGCCTTCTGCTGGCTT
HisValArgLysGlnIleGluSerArgLysArgLeuAlaArgThrValLeuValPheValCysLeuPheAlaPheCysTrpLeu
TM6
CCCACTCATATAATCTATTTATACCGGTCCTACCACTACTCAGAGGTGGACACTTCAGTGCTGCATTTCATTGCCAGCATCTGT
ProThrHisIleIleTyrLeuTyrArgSerTyrHisTyrSerGluValAspThrSerValLeuHisPheIleAlaSerIleCys

559

28

56

84

112
140

168

196

224

252

280
308

GCTCGGATCCTGGCATTCACTAACTCTTGTGTCAACCCATTTGCACTCTACTTGCTCAGCAAGAGCTTTCGGAAACAGTTCAAC
AlaArgIleLeuAlaPheThrAsnSerCysValAsnProPheAlaLeuTyrLeuLeuSerLysSerPheArgLysGlnPheAsn 336
TM7
AACCAGCTATTCTGCTGCAGAGCTCGCCTCCTCATCCGCTCCCAGAGCATGGCTAGGAGCACCACACGGATGACCTCCCTCAAG
AsnGlnLeuPheCysCysArgAlaArgLeuLeuIleArgSerGlnSerMETAlaArgSerThrThrArgMETThrSerLeuLys 364
AGCACCAACCACTCCCTGGCCACCTTCAGCCTTATCAATGGCAACCATGTCTGCCATGAAGGCTATGTCTAA
SerThrAsnHisSerLeuAlaThrPheSerLeuIleAsnGlyAsnHisValCysHisGluGlyTyrVal***387

Figure 2 Nucleotide and predicted amino-acid sequences of chGRP-R (Database accession: AY225966). Putative TM domains areunderlined and numbered. Location of potential N-linked glycosylation sites (arrowed) and PKC phosphorylation sites (starred) are
shown. Boxed cysteine residues indicate a putative palmitoylation site. Two cysteine residues possibly forming disulfide bonds aredouble-lined.

Phylogenetic analysis of all BN-like peptide receptors foundin the database demonstrated three main branches: GRP-R,
NMB-R and BB4/BRS-3 families (Figure 7). ChGRP-R is acomponent of the vertebrate BN-R family, together with the
GRP-R subtype. ChBRS-3.5, although a member of BN-R
family, is not a member of GRP-R or NMB-R subtype, but amember of BRS-3/BB4 subtype. This analysis also revealed
that chBN-R genes are more closely related to frog BN-R
rather than mammalian BN-R.

Chromosomal localization of the chGRP-R gene
Chromosomal location of the chGRP-R gene was determinedby FISH (Figure 8). The chGRP-R was localized to chicken
chromosome 1q23distal – q24proximal (ChickGBASE; http://
www.thearkdb.org/browser?species ¼ chicken, 2002). This region, chicken chromosome 1q had been shown to contain fivechicken homologs of human X-linked genes, MAOA, OTC,NROB1, ZFX and SCML2 (Schmid et al., 2000; Burt, 2002).

Expression pattern of chBN-R
Tissue distribution and developmental changes in expressionof chBN-Rs are analyzed by the RT – PCR method (Figure 9).
Striking differences are found between the tissue distribution

patterns of these two receptors. Significant expression ofchGRP-R mRNA was detected in the brain, GI tissue andliver, but not in skeletal muscle. The expression could be
detected after E4 until at least at P1. In contrast, chBRS-3.5
expression is limited to the brain, and expression could not be
detected in the early E4 embryo.

Functional analysis of chBN-R
In order to study the pharmacological properties of the chickBN-like peptide receptors, two independent cell lines stably
expressing chGRP-R or chBRS-3.5 were established. CHO-K1
cells lacking expression of endogenous BN-like peptide
receptors (Katsuno et al., 1999) were transfected withchGRP-R or chBRS-3.5 expression vector using lipofection,
and G418-resistant colonies were expanded. To select transfected cell lines expressing high levels of receptors, clones werescreened by comparing their ability to bind 40 pM[125I][YAFNl]BN(6 – 14), which has high affinity for rat
GRP-R, rat NMB-R, human BRS-3 as well as for frog BB4(Pradhan et al., 1998). [125I][YAFNl]BN(6 – 14) bound to bothchGRP-R and chBRS-3.5 in a saturable manner. In thepresence of 1 mM of non-radiolabeled ligand, only nonspecificbinding was observed, which was proportional to the ligand
concentration and lower than 10% of the total binding.
British Journal of Pharmacology vol 139 (3)560

M. Iwabuchi et al

Avian bombesin-like peptide receptors

ATGTCTCAAGTATACTTGCATCCATCTAATCAGACGTTGTGTGCAGCTACAAATGGTACAGAACTGAAATCAATCCTTGATAATMetSerGlnValTyrLeuHisProSerAsnGlnThrLeuCysAlaAlaThrAsnGlyThrGluLeuLysSerIleLeuAspAsn↑↑↑
GAAACAACAAATGAAAAATGGACCGAAGACTCCTTTCCAGGATTAGAAATACTGTGCACAATTTATGTTACATATGCTGTGATC
GluThrThrAsnGluLysTrpThrGluAspSerPheProGlyLeuGluIleLeuCysThrIleTyrValThrTyrAlaValIle
TM1
ATTTCAGTGGGTCTCCTTGGAAATGCTATACTCATCAAAGTCTTTTTCAAGATTAAATCAATGCAAACGGTTCCAAACATCTTC
IleSerValGlyLeuLeuGlyAsnAlaIleLeuIleLysValPhePheLysIleLysSerMETGlnThrValProAsnIlePhe
ATCACCAGCTTGGCATTTGGAGACCTGCTGCTTTTATTAACTTGCGTGCCTGTAGATGCAACACGGTACATTGTGGATACCTGG
IleThrSerLeuAlaPheGlyAspLeuLeuLeuLeuLeuThrCysValProValAspAlaThrArgTyrIleValAspThrTrp
TM2
ATCTTCGGAAGGATTGGGTGCAAGCTGCTGTCTTTCATCCAGTTAACATCAGTTGGAGTATCAGTGTTCACACTGACTGTTCTC
IlePheGlyArgIleGlyCysLysLeuLeuSerPheIleGlnLeuThrSerValGlyValSerValPheThrLeuThrValLeu
TM3
AGTGCTGACAGGTACAGAGCCATTGTTAAGCCCCTGGAGCTGCAGACCTCGGACGCACTCCTGAAGACCTGCTGTAAAGCTGGC
SerAlaAspArgTyrArgAlaIleValLysProLeuGluLeuGlnThrSerAspAlaLeuLeuLysThrCysCysLysAlaGly
TGTGTCTGGATTGTCTCCATGGTATTTGCTATCCCCGAGGCTGTTTTTTCAGATTTGTATTCGTTCAGCAATCCTGAGAAAAAT
CysValTrpIleValSerMETValPheAlaIleProGluAlaValPheSerAspLeuTyrSerPheSerAsnProGluLysAsn↑
TM4
GTAACTTTTGAGGCATGTGCTCCCTATCCTGTATCTGAGAAGATACTGCAGGAAGTGCACTCCTTGGTTTGCTTCCTCGTGTTC
ValThrPheGluAlaCysAlaProTyrProValSerGluLysIleLeuGlnGluValHisSerLeuValCysPheLeuValPhe
TACATTGTGCCATTGGCTGTTATTTCTGTCTATTATTTTCTTATCGCCAGAACTTTATACAAAAGTACATTCAACATGCCAGCA
TyrIleValProLeuAlaValIleSerValTyrTyrPheLeuIleAlaArgThrLeuTyrLysSerThrPheAsnMETProAla
TM5
GAAGAACACGGTCATGCCCGTAAGCAGATTGAATCCCGCAAGAGAGTTGCAAAAACAGTGCTGGTGCTTGTTGCTTTGTTTGCC
GluGluHisGlyHisAlaArgLysGlnIleGluSerArgLysArgValAlaLysThrValLeuValLeuValAlaLeuPheAla

28

56

84

112
140

168

196

224

252

280

TTTTGCTGGTTGCCCAACCACATCCTCTACCTATACCGCTCTTTTACATACCATACTTCTGTAGATGCTTCTACCTTTCATCTG
PheCysTrpLeuProAsnHisIleLeuTyrLeuTyrArgSerPheThrTyrHisThrSerValAspAlaSerThrPheHisLeu 308
TM6
ATAGTTACTATTTTTTCCCGTGCCTTGGCCTTCAGCAATTCCTGTGTCAATCCTTTTGCTCTTTATTGGCTGAGTAAAAGTTTC
IleValThrIlePheSerArgAlaLeuAlaPheSerAsnSerCysValAsnProPheAlaLeuTyrTrpLeuSerLysSerPhe 336
TM7
CGCCAACATTTTAAGAAGCAAGTCTCGTGCTGCAAGGCCAAGCTTTGCACAAAGCCTCCCAGCGCTCCCCACAGCAATTCACCC
ArgGlnHisPheLysLysGlnValSerCysCysLysAlaLysLeuCysThrLysProProSerAlaProHisSerAsnSerPro 364
CCCAGAGCTCTGTCAGTCACAGGCAGCACACACGGCTCAGAAATCAGCGTTACGCTGCTGACAGATTACAGCATCACCAAAGAAProArgAlaLeuSerValThrGlySerThrHisGlySerGluIleSerValThrLeuLeuThrAspTyrSerIleThrLysGlu

392

GAGGAAAGTGTTTAG
GluGluSerVal***396

Figure 3 Nucleotide and predicted amino-acid sequences of chick BRS-3.5 (Database accession: AY225967). For an explanationof symbols, see Figure 1 legend.

chGRP-RchBRS-3.5

chGRP-RchBRS-3.5

chGRP-RchBRS-3.5

chGRP-RchBRS-3.5

chGRP-RchBRS-3.5

1 : MASGECL--- --LLDLETDN FILYNISANQ SANLSVLSDE WFYPAFLYAI PTIYGIIILI GLIGNITLIK IFCTVKSMRN
1 : M-SQVYLHPS NQTLCAATNG TELKSILDNE TTNEKWTEDS FPGLEILCTI YVTYAVIISV GLLGNAILIK VFFKIKSMQT
* * *
* *
* * *
*
*
* *
* ** ** ** *** * ***TM1
76 : VPNLFISSLA LGDLLLLVTC VPVDASRYLA DEWLFGRIGC KLIPFIQLTS VGVSVFTLTA LSADRYKAIV RPMEIQASHA
80 : VPNIFITSLA FGDLLLLLTC VPVDATRYIV DTWIFGRIGC KLLSFIQLTS VGVSVFTLTV LSADRYRAIV KPLELQTSDA*** ** *** ****** ** ***** ** * * ****** ** ****** ********* ****** *** * * * * *TM2
TM3
156 : LMKICVRAAI IWIASMLLAI PEAVFSDLHP FHDKGTNKTF ISCAPYPHSD GLHPKIHSMA SFLIFYIIPL SVISVYYYFI
160 : LLKTCCKAGC VWIVSMVFAI PEAVFSDLYS FSNPEKNVTF EACAPYPVSE KILQEVHSLV CFLVFYIVPL AVISVYYFLI* * * *** ** ** ******** ** ** ***** ***** *** ** ****** *TM4TM5
236 : AKNLIRSAYN IPVEGNVHVR KQIESRKRLA RTVLVFVCLF AFCWLPTHII YLYRS--YHY SEVDTSVLHF IASICARILA
240 : ARTLYKSTFN MPAEEHGHAR KQIESRKRVA KTVLVLVALF AFCWLPNHIL YLYRSFTYHT S-VDASTFHL IVTIFSRALA* * * * * * * * ******** * **** * ** ****** ** ***** ** * ** * * * * * **TM6
314 : FTNSCVNPFA LYLLSKSFRK QFNNQLFCCR ARL--LIRSQ SMARSTTRMT S-LKSTNHS- LATFSLINGNH VCHEGYV
320 : FSNSCVNPFA LYWLSKSFRQ HFKKQVSCCK AKLCTKPPSA PHSNSPPRAL SVTGSTHGSE ISVTLLTDYS ITKEEESV* ******** ** ****** * * ** * *** * * ** *
*
* *
TM7

Figure 4 Comparison of predicted amino-acid sequences of two BN-R subtypes in chick. Asterisks designate identical amino-acidresidues in the two sequences.

Analysis of these data indicated that the ligand possessed ahigher affinity for chBRS-3.5 (Kd ¼ 0.73 nM) than chGRP-R(Kd ¼ 2.57 nM) (Figure 10, insets).
Using these two cell lines, the affinity of chick BN-like
peptide receptors to the BN and BN-like peptides was
British Journal of Pharmacology vol 139 (3)determined in the competition experiments measuring thedisplacement of [125I][YAFNl]BN(6 – 14) in the presence of
unlabeled peptides. ChGRP-R showed high affinity for[FAFNl]BN(6 – 14), BN and GRP, with Ki values of 1.7, 1.8
and 2.3 nM, respectively. But this receptor showed more than

Avian bombesin-like peptide receptors

M. Iwabuchi et al561

chGRP-R
huGRP-R
moGRP-R
raGRP-R

1:MASGECLLLDLETDNFILYNISANQSANLSVLSDEWFYPAFLYAIPTIYGIIILIGLIGN 60
1:MALNDCFLLNLEVDHFMHCNIS-SHSADLP-VNDDWSHPGILYVIPAVYGVIILIGLIGN 58
1:MAPNNCSHLNLDVDPFLSCNDTFNQSLSPP-KMDNWFHPGFIYVIPAVYGLIIVIGLIGN 59
1:MDPNNCSHLNLEVDPFLSCNNTFNQTLSPP-KMDNWFHPGIIYVIPAVYGLIIVIGLIGN 59

chGRP-R
huGRP-R
moGRP-R
raGRP-R

61:ITLIKIFCTVKSMRNVPNLFISSLALGDLLLLVTCVPVDASRYLADEWLFGRIGCKLIPF 120
59:ITLIKIFCTVKSMRNVPNLFISSLALGDLLLLITCAPVDASRYLADRWLFGRIGCKLIPF 118
60:ITLIKIFCTVKSMRNVPNLFISSLALGDLLLLVTCAPVDASKYLADRWLFGRIGCKLIPF 119
60:ITLIKIFCTVKSMRNVPNLFISSLALGDLLLLVTCAPVDASKYLADRWLFGRIGCKLIPF 119

TM1

TM2

chGRP-R 121:IQLTSVGVSVFTLTALSADRYKAIVRPMEIQASHALMKICVRAAIIWIASMLLAIPEAVF 180
huGRP-R 119:IQLTSVGVSVFTLTALSADRYKAIVRPMDIQASHALMKICLKAAFIWIISMLLAIPEAVF 178
moGRP-R 120:IQLTSVGVSVFTLTALSADRYKAIVRPMDIQASHALMKICLKAALIWIVSMLLAIPEAVF 179
raGRP-R 120:IQLTSVGVSVFTLTALSADRYKAIVRPMDIQASHALMKICLKAALIWIVSMLLAIPEAVF 179
TM4

TM3

chGRP-R 181:SDLHPFHDKGTNKTFISCAPYPHSDGLHPKIHSMASFLIFYIIPLSVISVYYYFIAKNLI 240
huGRP-R 179:SDLHPFHEESTNQTFISCAPYPHSNELHPKIHSMASFLVFYVIPLSIISVYYYFIAKNLI 238
moGRP-R 180:SDLHPFHVKDTNQTFISCAPYPHSNELHPKIHSMASFLVFYVIPLAIISVYYYFIARNLI 239
raGRP-R 180:SDLHPFHVKDTNQTFISCAPYPHSNELHPKIHSMASFLVFYIIPLSIISVYYYFIARNLI 239
TM5

chGRP-R 241:RSAYNIPVEGNVHVRKQIESRKRLARTVLVFVCLFAFCWLPTHIIYLYRSYHYSEVDTSV 300
huGRP-R 239:QSAYNLPVEGNIHVKKQIESRKRLAKTVLVFVGLFAFCWLPNHVIYLYRSYHYSEVDTSM 298
moGRP-R 240:QSAYNLPVEGNIHVKKQIESRKRLAKTVLVFVGLFAFCWLPNHVIYLYRSYHYSEVDTSM 299
raGRP-R 240:QSAYNLPVEGNIHVKKQIESRKRLAKTVLVFVGLFAFCWLPNHVIYLYRSYHYSEVDTSM 299
TM6

chGRP-R 301:LHFIASICARILAFTNSCVNPFALYLLSKSFRKQFNNQLFCCRARLLIRSQSMARSTTRM 360
huGRP-R 299:LHFVTSICARLLAFTNSCVNPFALYLLSKSFRKQFNTQLLCCQPGLIIRSHSTGRSTTCM 358
moGRP-R 300:LHFVTSICARLLAFTNSCVNPFALYLLSKSFRKQFNTQLLCCQPGLMNRSHSTGRSTTCM 359
raGRP-R 300:LHFITSICARLLAFTNSCVNPFALYLLSKSFRKQFNTQLLCCQPSLLNRSHSTGRSTTCM 359
TM7

chGRP-R 361:TSLKSTNHSLATFSLINGNHVCHEGYV
huGRP-R 359:TSLKSTNPSVATFSLIN-GNICHERYV
moGRP-R 360:TSFKSTNPS-ATFSLIN-RNICHEGYVraGRP-R 360:TSFKSTNPS-ATFSLIN-GNICHEGYV

387
384
384
384

Figure 5 Alignment of predicted GRP-R amino-acid sequences of chick, human, mouse and rat. Identical residues in the four
sequences are boxed.

Table 1ChGRP-RChBRS-3.5

Predicted amino-acid identity (%) of BN-like peptide receptor subtypes
BB4
fr

BRS-3.5
Chick

mo

ra

BRS-3
gp

sh

hu

Chick

GRP-R
mo
ra

hu

mo

NMB-R
ra

hu

60
74

60
F

59
67

60
67

61
66

60
68

63
69

F
60

80
58

82
58

60
52

60
52

60
52

80
59

Receptor sequences have been reported as follows: Frog (fr) BB4 (Nagalla et al., 1995), mouse (mo) BRS-3 (Ohki-Hamazaki et al., 1997a),
rat (ra) BRS-3 (Liu et al., 2002), guinea pig (gp) BRS-3 (Gorbulev et al., 1992), sheep (sh) BRS-3 (Whitley et al., 1999), human (hu) BRS-3(Fathi et al., 1993), mouse GRP-R (Spindel et al., 1990), rat GRP-R (Wada et al., 1992), human GRP-R (Corjay et al., 1991), mouse
NMB-R (Ohki-Hamazaki et al., 1997a), rat NMB-R (Wada et al., 1991), human NMB-R (Corjay et al., 1991).

1000-fold lower affinity for NMB (Figure 10a). In contrast,
rank affinities for chBRS-3.5 were [FAFNl]BN(6 – 14)(Ki ¼ 2.0 nM)4BN (Ki ¼ 19.1 nM)4GRP (Ki ¼ 318 nM)4NMB (Ki ¼ 678 nM), demonstrating that this receptor had
high affinity for [FAFNl]BN(6 – 14), moderate affinity for BN,
but lower affinity for both GRP and NMB (Figure 10b).
We further evaluated the functional activity of chGRP-R
and chBRS-3 in the calcium mobilization assay. When CHO
cells stably expressing chGRP-R were incubated with calcium
indicator Fluo-3 and stimulated with either 10 nM GRP or
10 nM [FAFNl]BN(6 – 14), a transient three-fold increase in
intracellular calcium concentration was observed (Figure 11a).
Similarly, both 10 nM BN and 10 nM [FAFNl]BN(6 – 14)
elicited strong response in CHO cells expressing chBRS-3.5(Figure 11b). Since the cells are rapidly desensitized after
agonist challenge, we could not perform dose – response
analysis in detail by using a confocal microscope. However,

we confirmed that GRP failed to elicit calcium response at
10 nM (Figure 11b), although 1 mM GRP was able to increase
intracellular calcium (data not shown). These results showedthat these receptors when expressed on the cell surface caninitiate an appropriate signal transduction cascade.

Discussion
In this paper, we reported for the first time, the cloning andcharacterization of two BN-like peptide receptors in birds. By
exploiting the PCR technology using degenerative primers,chick GRP-R and BRS-3.5 were cloned. The amino-acidsequence of chGRP-R was highly homologous to mammalian
and amphibian GRP-R and in addition, its pharmacologicalproperties, which were characterized by high affinity for[FAFNl]BN(6 – 14) (Ki ¼ 1.7 nM), BN (Ki ¼ 1.8 nM) and GRP
British Journal of Pharmacology vol 139 (3)562

Avian bombesin-like peptide receptors

M. Iwabuchi et alachBRS-3.5
gpBRS-3
huBRS-3
moBRS-3
raBRS-3
shBRS-3

1:MSQVYLHPSNQTLCAATNGTEL-KSILDNETTNEKWTEDSFPGLEILCTIYVTYAVIISV 59
1:MSQKQPQSPNQTLISITNDTESSSSVVSNDTTNKGWTGDNSPGIEALCAIYITYAVIISV 60
1:MAQRQPHSPNQTLISITNDTESSSSVVSNDNTNKGWSGDNSPGIEALCAIYITYAVIISV 60
1:MSQRQSQSPNQTLISITNDTETSSSVVSNDTTHKGWTGDNSPGIEALCAIYITYAGIISV 60
1:MSQRQPQSPNQTLISITNDTETSSSAVSNDTTPKGWTGDNSPGIEALCAIYITYAVIISV 60
1:MSQRQPQSPNQTLISTTNDTESSSSVVPNDSTNKRRTGDNSPGIEALCAIYITYAVIISV 60

chBRS-3.5
gpBRS-3
huBRS-3
moBRS-3
raBRS-3
shBRS-3

60:GLLGNAILIKVFFKIKSMQTVPNIFITSLAFGDLLLLLTCVPVDATRYIVDTWIFGRIGC 119
61:GILGNAILIKVFFKTKSMQTVPNIFITSLALGDLLLLLTCVPVDATHYLAEGWLFGRIGC 120
61:GILGNAILIKVFFKTKSMQTVPNIFITSLAFGDLLLLLTCVPVDATHYLAEGWLFGRIGC 120
61:GILGNAILIKVFFKTKSMQTVPNIFITSLAFGDLLLLLTCVPVDATHYLAEGWLFGKVGC 120
61:GILGNAILIKVFFKTKSMQTVPNIFITSLAFGDLLLLLTCVPVDATHYLAEGWLFGKVGC 120
61:GILGNAILIKVFFKTKSMQTVPNIFITSLAFGDLLLLLTCVPVDVTHYLAEGWLFGRIGC 120

TM1

TM2

chBRS-3.5 120:KLLSFIQLTSVGVSVFTLTVLSADRYRAIVKPLELQTSDALLKTCCKAGCVWIVSMVFAI 179
gpBRS-3
121:KVLSFIRLTSVGVSVFTLTILSADRYKAVVKPLERQPSNAILKTCAKAGCIWIMSMIFAL 180
huBRS-3
121:KVLSFIRLTSVGVSVFTLTILSADRYKAVVKPLERQPSNAILKTCVKAGCVWIVSMIFAL 180
moBRS-3
121:KVLSFIRLTSVGVSVFTLTILSADRYKAVVKPLERQPPNAILKTCAKAGGIWIVSMIFAL 180
raBRS-3
121:KVLSFIRLTSVGVSVFTLTILSADRYKAVVKPLERQPSNAILKTCAKAGGIWIMAMIFAL 180
shBRS-3
121:KVLSFIRLTSVGVSVFTLTILSADRYKAVVKPLERQPPNAILKTCAKAGCIWIMSMIIAL 180
TM4

TM3

chBRS-3.5 180:PEAVFSDLYSFSNPEKNVTFEACAPYPVSEKILQEVHSLVCFLVFYIVPLAVISVYYFLI 239
gpBRS-3
181:PEAIFSNVHTLRDPNKNMTSEWCAFYPVSEKLLQEIHALLSFLVFYIIPLSIISVYYSLI 240
huBRS-3
181:PEAIFSNVYTFRDPNKNMTFESCTSYPVSKKLLQEIHSLLCFLVFYIIPLSIISVYYSLI 240
moBRS-3
181:PEAIFSNVYTFQDPNRNVTFESCNSYPISERLLQEIHSLLCFLVFYIIPLSIISVYYSLI 240
raBRS-3
181:PEAIFSNVYTFQDPNRNVTFESCNSYPISERLLQEIHSLLCFLVFYIIPLSIISVYYSLI 240
shBRS-3
181:PEAIFSNVYTFQDPDKNVTFKACASYPVSERLLQEIHSLLCFLVFYIIPLSIISVYYSLI 240
TM5

chBRS-3.5 240:ARTLYKSTFNMPAEEHGHARKQIESRKRVAKTVLVLVALFAFCWLPNHILYLYRSFTYHT 299
gpBRS-3
241:ARTLYKSTLNIPTEEQSHARKQVESRKRIAKTVLVLVALFALCWLPNHLLNLYHSFTHKA 300
huBRS-3
241:ARTLYKSTLNIPTEEQSHARKQIESRKRIARTVLVLVALFALCWLPNHLLYLYHSFTSQT 300
moBRS-3
241:ARTLYKSTLNIPTEEQSHARKQIESRKRIAKTVLVLVALFALCWLPNHLLYLYHSFTYES 300
raBRS-3
241:ARTLYKSTLNIPTEEQSHARKQIESRKRIAKTVLVLVALFALCWLPNHLLYLYHSFTYES 300
shBRS-3
241:ARTLYKSTLNIPTEEQRHARKQIESRKRIAKTVLVLVALFALCWLPNHLLYLYRSFTSQT 300
TM6

chBRS-3.5 300:SVDASTFHLIVTIFSRALAFSNSCVNPFALYWLSKSFRQHFKKQVSCCKAKL-CTKPPSA 358
gpBRS-3
301:YEDSSAIHFIVTIFSRVLAFSNSCVNPFALYWLSKTFQKQFKAQLFCCKGELPEPPLAAT 360
huBRS-3
301:YVDPSAMHFIFTIFSRVLAFSNSCVNPFALYWLSKSFQKHFKAQLFCCKAERPEPPVADT 360
moBRS-3
301:YANHSDVPFVIIIFSRVLAFSNSCVNPFALYWLSKTFQQHFKAQLCCLKAEQPEPPLGDI 360
raBRS-3
301:YAEPSDVPFVVTIFSRVLAFSNSCVNPFALYWLSKTFQKHFKAQLCCFKAEQPEPPLGDT 360
shBRS-3
301:YMDSSTVHLFVTIISRILAFSNSCVNPFALYWLSNTFQQHFKAQLFCCKAGRPDPTAANT 360
TM7

chBRS-3.5 359:PHSNSPPRA-LSVTGSTHGSEISVTLLTDYSITKEEESVgpBRS-3
361:PLNSLAVMGRVSGTENTHISEIGVASFIGRPMKKEENRVhuBRS-3
361:SLTTLAVMGTVPGTGSIQMSEISVTSFTGCSVKQAEDRF
moBRS-3
361:PLNNLTVMGRVPATGSAHVSEISVTLFSGSSAKKGEDKVraBRS-3
361:PLNNLTVMGRVPATGSAHVSEISVTLFSGSTAKKGEDKVshBRS-3
361:PLDNLAVMGRVPGAASTQMSEISVSPFTGCSVKKEDDRV

b

396
399
399
399
399
399

chBRS-3.5
frBB4

1:MSQVYLHPSNQTLCAATNG-TELKSILDNETTNEKWTEDSFPGLEILCTIYVTYAVIISV 59
1:MPEGF-QSLNQTLPSAISSIAHLESLNDSFILGAKQSEDVSPGLEILALISVTYAVIISV 59

chBRS-3.5
frBB4

60:GLLGNAILIKVFFKIKSMQTVPNIFITSLAFGDLLLLLTCVPVDATRYIVDTWIFGRIGC 119
60:GILGNTILIKVFFKIKSMQTVPNIFITSLAFGDLLLLLTCVPVDASRYIVDTWMFGRAGC 119

TM1

TM2

chBRS-3.5 120:KLLSFIQLTSVGVSVFTLTVLSADRYRAIVKPLELQTSDALLKTCCKAGCVWIVSMVFAI 179
frBB4
120:KIISFIQLTSVGVSVFTLTVLSADRYRAIVKPLQLQTSDAVLKTCGKAVCVWIISMLLAA 179
TM3

TM4

chBRS-3.5 180:PEAVFSDLYSFSNPEKNVTFEACAPYPVSEKILQEVHSLVCFLVFYIVPLAVISVYYFLI 239
frBB4
180:PEAVFSDLYEFGSSEKNTTFEACAPYPVSEKILQETHSLICFLVFYIVPLSIISAYYFLI 239
TM5

chBRS-3.5 240:ARTLYKSTFNMPAEEHGHARKQIESRKRVAKTVLVLVALFAFCWLPNHILYLYRSFTYHT 299
frBB4
240:AKTLYKSTFNMPAEEHTHARKQIESRKRVAKTVLVLVALFAVCWLPNHMLYLYRSFTYHS 299
TM6

chBRS-3.5 300:SVDASTFHLIVTIFSRALAFSNSCVNPFALYWLSKSFRQHFKKQVSCCKAKLCTKPPSAP 359
frBB4
300:AVNSSAFHLSATIFARVLAFSNSCVNPFALYWLSRSFRQHFKKQVYCCKTEPPASQQSPT 359
TM7

chBRS-3.5 360:HSNSPPRALSVTGSTHGSEISVTLLTDYSITKEEESV
frBB4
360:HSSTITGITAVKGNIQMSEISITLLSAYDVKKE----

396
392

Figure 6 Comparison of deduced amino-acid sequences of chick BRS-3.5 with mammalian BRS-3 (a), or with frog (fr) BB4 (b). gp,
guinea pig; hu, human; mo, mouse; ra, rat; sh, sheep. Identical residues are boxed. References for each sequence are indicated inTable 1.

British Journal of Pharmacology vol 139 (3)raGRP-R
85
99

frGRP-R-fragment

68

frNMB-R-fragment
huNMB-R
raNMB-R

100
100
67

moNMB-RchBRS-3.5
frBB4
gpBRS-3
huBRS-3

99

shBRS-3

81
62

moBRS-3[125I] [YAFNl] BN(6-14) bound (%)

chGRP-R

99

amoGRP-R
huGRP-R

100

100

Avian bombesin-like peptide receptors

563

100
90
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[FAFNl]BN(6-14)

70

BN

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GRP(14-27)
NMB

50

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40

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M. Iwabuchi et al

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20

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200
100
0
0

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250 500 750 1000 1250
Free (pM)

99 raBRS-3

0
10-10

b
[125I] [YAFNl] BN(6-14) bound (%)

Figure 7 Phylogenetic analysis of BN-like peptide receptors.
Putative amino-acid sequences were aligned and the alignments
were used to construct phylogenetic trees according to the UPGMA
criteria. Numbers next to branch points are bootstrap values basedon 1000 resamplings. The evolutionary distance was described interms of substitutions per site. Amino-acid sequences of fr (frog)
GRP-R and NMB-R proteins are from Nagalla et al. (1995). Other
references and abbreviations are explained in Table 1.

Figure 9 Tissue-specific expression of two chick BN-like peptide
receptors are analyzed by RT – PCR. E, embryonic day; P,
posthatching day.

10 -6

10 -8
10-7
Concentration (M)

100

10-5

400

90
80

300
200
100
0

70

0

250 500 750 1000 1250
Free (pM)

60
50
40
30
20
10
0
10-10

Figure 8 Chromosomal localization of the chGRP-R using a
cDNA clone as a biotinylated probe. Arrows indicates the
hybridization signals. The metaphase spreads were photographedwith Nikon B-2A (a, c) and UV-2A (b) filters. R- and G-banded
patterns are demonstrated in (a, c) and (b), respectively.

10-9

Bound (pM)

0.05

10-9

10 -8
10-7
Concentration (M)

10 -6

10-5

Figure 10 Abilities of BN-like peptides to inhibit binding of[125I][YAFNl]BN(6 – 14) to chick GRP-R (a) or chick BRS-3.5 (b)expressed in CHO-K1 cells. Cells expressing either receptor wereincubated with 40 pM [125I][YAFNl]BN(6 – 14) and the indicatedconcentrations of peptides for 30 min at 371C. The mean of at least
two experiments is shown. Each experiment includes two replicates.
[125I][YAFNl]BN(6 – 14) binding affinity was determined for eachcell line in saturation analysis as described under Methods.
Saturation binding curves are shown in the insets.

(Ki ¼ 2.3 nM), resembled GRP-Rs cloned and characterized todate. For example, rat pancreatic acinar cells expressing GRPR show a Ki value of 0.99 nM for [FAFNl]BN(6 – 14) (Pradhan
et al., 1998), mouse GRP-R-transfected BALB3T3 has Ki
values of 0.9 and 3.1 nM for BN and GRP, respectively (Benyaet al., 1994), and these values for human GRP-R transfected
BALB3T3 are 1.4 and 6.2 (Benya et al., 1995). ChGRP-R was
distributed in the brain and GI tissue, which is a common
feature of GRP-R of mammals, amphibians and Aves (Battey
et al., 1994; Nagalla et al., 1995). Chick GRP-R gene wasmapped to chicken chromosome 1q23distal – q24proximal, theregion where the similarity to the human X chromosome had
been determined in previous studies (Schmid et al., 2000; Burt,
2002). In contrast, chBRS-3.5 had high similarity to amphiBritish Journal of Pharmacology vol 139 (3)

564

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M. Iwabuchi et al

3.5

GRP
3

[FAFNl]BN(6-14)

2.5
2
1.5
1
0.5
0

20

40

60

80

100

120

Time (sec)

b
3.5

BN
3

[FAFNl]BN(6-14)
GRP

2.5
2
1.5
1
0.5
0

20

40

60

80

100

120

Time (sec)Figure 11 Intracellular calcium mobilization in CHO-K1 cells
expressing chick GRP-R (a) or chick BRS-3.5 (b). Cells werelabeled with calcium indicator Fluo-3/AM, stimulated with 10 nM of
the indicated agonists and fluorescence was measured over 120 s.

bian BB4 and to a lesser extent, to mammalian BRS-3.
ChBRS-3.5 had high affinity for the synthetic peptide[FAFNl]BN(6 – 14) (Ki ¼ 2.0 nM), moderate affinity for BN(Ki ¼ 19.1 nM) and low affinity for both GRP (Ki ¼ 318 nM)
and NMB (Ki ¼ 678 nM). These pharmacological properties ofchBRS-3.5 were reminiscent of BB4 (Ki values are 0.41 nM for[FAFNl]BN(6 – 14), 14 nM for BN, 79 nM for GRP and 11 nM
for NMB) rather than BRS-3 (Ki values are 8.9 nM for[FAFNl]BN(6 – 14), 410,000 nM for BN and GRP and
4800 nM for NMB in human BRS-3-transfected BALB3T3),
suggesting that the chBRS-3.5 belonged to the BB4 family(Mantey et al., 1997; Katsuno et al., 1999). This hypothesis
was further supported by phylogenetic analysis based onamino-acid sequence similarity. Expression of chBRS-3.5
mRNA was limited to the brain, a characteristic shared byboth BB4 and BRS-3 (Nagalla et al., 1995; Ohki-Hamazaki
et al., 1997a). Upon binding of agonist to both receptors, weobserved mobilization of intracellular calcium, which showedthat these receptors were able to initiate a signal transduction
cascade.
After the discovery of BN in frog, antiserum against this
peptide was applied to the tissues from other species including
British Journal of Pharmacology vol 139 (3)Avian bombesin-like peptide receptors

birds, revealing immunopositive cells in the endocrine tissue ofchick. These studies indicated that the avian proventriculus
was an abundant source of BN (Erspamer et al., 1978; Timson
et al., 1979; Vaillant et al., 1979). Subsequently, McDonald
et al. (1980) succeeded in purifying GRP from chicken
proventricular tissue. Its sequence showed the expected Cterminal homology with BN, and identical N and C termini (18identities out of 27 residues) with porcine GRP (Figure 1).
Following intravenous or intracerebroventricular injections,avian GRP had a satiety effect on chick and turkey (Savory &Hodgkiss, 1984; Denbow, 1989). The effectiveness of GRP
upon intracerebroventricular injection suggested that this
peptide localized not only in the peripheral endocrine organ,as demonstrated earlier by immunohistochemistry, but also inbrain. In fact, Vaillant et al. (1979) found substantial
quantities of BN-like immunoreactivity in turkey brain;
Cassone & Moore (1987) reported the existence of BN-likereactivity in the suprachiasmatic region of house sparrow and
Berk & Smith (1994) described projection of BN-immunoreactive cells to the vagal complex of the pigeon. Morerecently, Dubbeldam & den Boer-Visser (2000) studied indetail the distribution of GRP immunoreactivity in the brainof the dove. These investigations suggested that receptors for
BN-like peptides, in particular GRP, should exist in the avian
brain, and here we provide evidence for GRP-R and BRS-3.5
expressions in the brain.
ChGRP-R and chBRS-3.5 retain many structural featuresthat are signatures for members of the G-protein-coupled
receptor, and which are common in BN-like peptide receptors.
First, three possible N-linked glycosylation sites are present
within the extracellular N-terminal domain and also within thesecond extracellular loop. Second, Cys residues that may serveto stabilize the structure of the receptor are conserved: two Cys
residues in the first and second extracellular loops, which canform an intramolecular disulfide bridge constraining theconformation of extracellular domain, and a Cys residue
located in the intracellular C-terminal region, which may
be important in anchoring the receptor to the plasma
membrane. And finally, potential PKC phosphorylation sites
are located between TM5 and 6, and within the C-terminal
cytoplasmic region. These latter sites along with other Ser and
Thr residues that could be phosphorylated with other kinases
may be important in desensitization and internalization of thereceptor upon agonist binding (Benya et al., 1993; Kroog et al.,1995).
The human Grpr is linked to chromosome Xp22.2 – p22.13(reference or NCBI database: http://www.ncbi.nlm.nih.gov/).
Five chicken homologs of human X-linked genes, MAOA,
OTC, NROB1, ZFX and SCML2, have been mapped tochicken chromosome 1q (Schmid et al., 2000; Burt, 2002), theregion where the chGRP-R was localized by FISH. Therefore,
the chGRP-R is mapped to the chromosomal region whereconserved linkage homology with human chromosome X has
been demonstrated (Schmid et al., 2000). This result confirmsthat the GRP-R is phylogenically conserved and provides
additional genetic evidence to confirm that the Z chromosome,one of the avian sex chromosome, and the mammalian X
chromosome had not evolved from a common ancestral
linkage group. Concerning chBRS-3.5, we tried to elucidate
its chromosomal location using the same method, but
unfortunately, we could not define it because of the highbackground.

M. Iwabuchi et al

When comparing the pharmacological properties of chGRPR and chBRS-3.5, an important difference should be noted.
Although the chGRP-R has high affinities for BN,[FAFNl]BN(6 – 14) and also for GRP, chBRS-3.5 has highaffinity only for [FAFNl]BN(6 – 14), and affinities for BN and
GRP are 10- and 100-folds lower, respectively. These results
demonstrate that chBRS-3.5 can discriminate BN and GRP,
despite the remarkable structural similarity between these two
peptides. This implies that the endogenous ligand for chBRS3.5 may be some form of BN, probably the chick type, like frog
BB4 having [Phe13]BN for the endogenous ligand. In supportof this notion, the similarity between frog GRP-R and BB4 is
59% (Nagalla et al., 1995), the value almost equivalent to thesimilarity between chGRP-R and chBRS-3.5 (60%).
Akeson et al. (1997) reported four amino acids required forhigh-affinity BN binding. All of these residues, Gln121, Pro199,
Arg288 and Ala308 of mouse GRP-R are conserved in equivalent
position in chGRP-R, and in chBRS-3.5 except for Ala308.
These results coincide with the high affinity of chGRP-R andmoderate affinity of chBRS-3.5 for BN. Moreover, two
residues, Phe185 and Ala198, have been shown to be indispensable for the selectivity of mouse GRP-R for GRP (Tokita
et al., 2002). These residues are conserved in both chGRP-R
and chBRS-3.5 that showed selectivity for GRP rather than
NMB. Affinity for NMB was not significant in both chGRP-R
and chBRS-3.5, consistent with the lack of Ile216 of rat NMB-R
in equivalent positions of these receptors (Fathi et al., 1993).
However, it should be noted that the low affinity for NMB
cannot be explained solely by this fact because BB4 has a highaffinity for NMB in spite of the lack of this amino-acid residue

Avian bombesin-like peptide receptors

565(Katsuno et al., 1999). We have not yet succeeded in isolating
chick NMB-R, and it is not known whether the birds have
NMB as well as NMB-R.
Recently, cloning and characterization of rat BRS-3
revealed that this receptor does not have high affinity for[FAFNl]BN(6 – 14) (Liu et al., 2002). The amino acids locatedin the third extracellular loop were divergent between humanand rat BRS-3 and by constructing chimeric molecules, theyreported that the amino-acid difference of this domain was
indeed the main cause of the poor affinity. Alignment ofchBRS-3.5 with mammalian BRS-3 revealed major amino-aciddifferences in the N-terminal domain and in the third
extracellular loop. Since the difference in the N-terminal
region is conspicuous between chBRS-3.5 and human BRS-3,
this domain is probably not the main factor controlling theaffinity for [FAPNl]BN(6 – 14). Therefore, it may be the third
extracellular loop that is important for high-affinity binding to[FAPNl]BN(6 – 14).
The phylogenetic tree indicates that frog BB4, chick BRS3.5 and mammalian BRS-3 had a close evolutionary history,
but the group for frog BB4 and chick BRS-3.5, andmammalian BRS-3 group, evolved differently after a certain
point in time. Our results suggest that the endogenous ligand
for BRS-3 may have an amino-acid sequence significantly
different from BN.

This work was supported by Grants-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology of Japan and theJapan Society for the Promotion of Science.

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(Received December 17, 2002
Revised March 11, 2003
Accepted March 19, 2003)