Molecular and Cellular Endocrinology 277 (2007) 51–60

Cloning and activation of the bullfrog apelin receptor:



coupling and high affinity for [Pro



Mi Jin Moon

Ju Yeon Lee

b a

, Da Young Oh

, Jae Il Kim

b a , d

, Jung Sun Moon

, Sehyung Cho

c a

, Dong-Ki Kim

, Hyuk Bang Kwon

d a

, Jong-Ik Hwang

, Jae Young Seong

a a

, ∗



Laboratory of G Protein-Coupled Receptors, Korea University College of Medicine, Seoul 136-705, Republic of Korea b

Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea c

Kyung Hee Institute of Age-Related and Brain Diseases, Kyung Hee University, Seoul 130-701, Republic of Korea d

Hormone Research Center, School of Biological Sciences and Technology,

Chonnam National University, Gwangju 500-757, Republic of Korea

Received 2 October 2006; received in revised form 7 July 2007; accepted 27 July 2007


In mammals, apelin and its G protein-coupled receptor, APJ, regulate blood pressure, intake of food and water, and cardiac contractility. In this study, we report the cloning and functional characterization of APJ in the bullfrog, Rana catesbeiana . Bullfrog APJ (bfAPJ) cDNA contains an open reading frame of 1083 nucleotides encoding a protein of 360 amino acid residues. Sequence alignment reveals 75% amino acid identity with Xenopus , 63% identity with zebrafish and 40–42% identity with mammalian APJs. RT-PCR analysis and tissue binding assay reveal high expression of bfAPJ mRNA in the brain, particularly in the hypothalamus, and moderate expression in the pituitary, testis, adrenal gland and lung.

Whereas [pGlu 1 ]apelin-13 did not induce CRE-luc (protein kinase A-specific reporter) and SRE-luc (protein kinase C-specific reporter) activity in cells expressing bfAPJ, this apelin-13 decreased forskolin-induced CRE-luc activity and cAMP accumulation in a pertussis toxin-sensitive manner.

This study indicates that bfAPJ may couple to G i/o

. [Pro 1 ]apelin-13, a synthetic apelin based on the sequence of the putative apelin gene from many non-mammalian species, activates bfAPJ with 5–10-fold greater sensitivity/affinity than mammalian apelin-13. Collectively, this study expands our understanding of the physiological roles of this receptor system in non-mammalian species.

© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: APJ; Apelin; Bullfrog; G i/o


1. Introduction

The G protein-coupled receptor (GPCR) APJ, also known as angiotensin II receptor-like 1 (ATGRL1), was first isolated

from human genomic DNA ( O’Dowd et al., 1993 ). The amino

acid sequence of human APJ (hAPJ) is 31% homologous to angiotensin-type 1 receptor (AT1), but hAPJ does not specifically bind angiotensin II. APJ mRNA is expressed throughout the central nervous system (CNS) such as the hypothalamus, hippocampus, pyriform cortex and spinal cord. It is also present in peripheral tissues including the pituitary, spleen, heart and lung


De Mota et al., 2000; Hosoya et al., 2000; Medhurst et al., 2003 ).

Treatment of hAPJ-expressing CHO cells with the endogenous

Corresponding author. Tel.: +82 2 920 6090; fax: +82 2 921 4355.

E-mail address:

(J.Y. Seong).

0303-7207/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.

doi: 10.1016/j.mce.2007.07.008

APJ ligand, apelin, reduces forskolin-induced cAMP production (

Habata et al., 1999 ). In addition, activation of mouse

APJ activates extracellular-regulated kinases (ERK) in a pertussis toxin-sensitive fashion (

Masri et al., 2002 ). Together, these

results suggest that APJ preferentially couples to G

( Masri et al., 2006 ).

i/o protein

The apelin peptide was initially isolated from bovine stom-

ach extracts ( Tatemoto et al., 1998 ). Subsequent studies have

shown that the apelin mRNA is expressed in the hypothalamus, thalamus, spinal cord, pituitary, heart, lung, kidney and mammary gland (

Lee et al., 2000; Kawamata et al., 2001;

Kleinz et al., 2005 ). Secretion of apelin has also been demon-

strated from the colostrums and adipocytes ( Habata et al., 1999;

Boucher et al., 2005 ). An active apelin form, apelin [42–77]

(also known as apelin-36), is processed from a 77-amino acid preproapelin peptide (

Tatemoto et al., 1998 ). However, there

are also 13- and 17-amino acids apelin peptides, apelin [65–77]


2. Materials and methods

M.J. Moon et al. / Molecular and Cellular Endocrinology 277 (2007) 51–60 and apelin [61–77] (also known as apelin-13 and apelin-17, respectively) that share identical C-terminals (

Hosoya et al.,

2000; Kawamata et al., 2001; Medhurst et al., 2003 ). The Gln

residue at the N-terminus of apelin-13 may be pyroglutamylated, producing [pGlu


]apelin-13, which is more potent than apelin-36 in competitive binding and cAMP production inhibi-

tion assays ( Hosoya et al., 2000; Kawamata et al., 2001 ). The

C-terminal 13 amino acids of apelin are highly conserved across species, suggesting this region is critical for receptor binding and activation.

The physiological roles of apelin and APJ are not well understood, however, their co-localization in the heart, lung and various CNS regions suggests a paracrine or autocrine mechanism of action (

Masri et al., 2005 ). Consistent with hypothalamic

expression, apelin signaling may be involved in food and water

intake ( Lee et al., 2000; Taheri et al., 2002; Sunter et al., 2003 ).

Apelin and APJ mRNA are present in vasopressinergic neurons of the supraoptic nucleus, where apelin may negatively regu-

late activity of vasopressinergic neurons ( Reaux et al., 2001;

De Mota et al., 2004 ). As such, apelin administration induces a transient decrease in blood pressure in rats and mice ( Lee et al., 2000, 2005; Tatemoto et al., 2001; Ishida et al., 2004 ).

This effect is abolished in apj -deficient mice, indicating that the apelin/APJ receptor system may counterregulate vasopressor stimulation (

Ishida et al., 2004 ). In the heart, apelin, acting

via APJ, increases cardiac contractility in rodents in vivo and in vitro (

Szokodi et al., 2002; Ashley et al., 2005 ). Recently, the

role of apelin and APJ in Xenopus cardiovascular development has been demonstrated (

Cox et al., 2006; Inui et al., 2006 ). In

addition to these physiological roles, APJ is also known to be a coreceptor for entry of several HIV-1 and SIV strains, and apelin

peptides may be able to block the entry of HIV ( Edinger et al.,

1998; Zou et al., 2000 ).

APJ has been cloned in a number of species including human, chimpanzee, rat, mouse and zebrafish. A Xenopus APJ homolog was cloned and named X-msr (mesenchyme-associated serpentine receptor) (

Devic et al., 1996 ). However, ligand selectivity

and intracellular signaling capability of non-mammalian APJs remains poorly defined. Amino acid sequences for the APJ ligand apelin have been identified in mammalian species (

Masri et al., 2005 ) and in

Xenopus (

Cox et al., 2006; Inui et al., 2006 ).

Using BLAST searches, we have identified apelin-like genes in a number of non-mammalian species and found that these putative apelin peptides differ significantly from mammalian apelin.

We have also isolated a full-length cDNA for APJ in the bullfrog,

Rana catesbeiana , and determined its ligand selectivity and signaling pathway. Our results indicate that bullfrog APJ (bfAPJ) is likely G i/o

-coupled and responds better to [Pro


]apelin-13, a synthetic apelin peptide based on non-mammalian apelin genes, than mammalian [pGlu



2.1. Materials

Mammalian apelin-13s ([pGlu


]apelin-13, [Gln


]apelin-13) and



]apelin-13 were synthesized by AnyGen Co. (Gwangju, Korea). 12O tetradecanoylphenol-13-acetate (TPA) and forskolin were purchased from

Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and OPTI-MEM were obtained from Life Technologies

(Grand Island, NY, USA). SuperFect, Effectene and DNA Maxi-prep kit were purchased from Qiagen (Valencia, CA, USA).

2.2. Plasmids

The pcDNA3 expression vector and the pCMV

-gal vector were obtained from Invitrogen (San Diego, CA, USA) and Clontech (Palo Alto, CA, USA), respectively. The signaling specific reporter vectors, CRE-luc, SRE-luc and AP-

1-luc were purchased from Stratagene (La Jolla, CA, USA). The c-fos-luc vector, containing the

711 to +45 sequence of the human c-fos promoter constructed in the pFLASH vector, was a kind gift from Dr. R. Prywes (Columbia University,

USA). The cDNA for human APJ (hAPJ) was obtained from Neurogenex Co.

(Seoul, Korea).

2.3. Animals and tissue preparation

Adult male and female bullfrogs (100–120 g body weight) were obtained from a local supplier (BCPC, Taein, Korea). They were housed in glass tanks and supplied with tap water under simulated natural conditions. Frogs were killed by decapitation and tissues were quickly dissected, immediately frozen in liquid nitrogen and stored at


C for further use. Animal manipulations were performed according to the Regulation of Animal Care of Korea University

College of Medicine.

2.4. RNA isolation and RT-PCR

Total RNA was extracted from either the forebrain or the pituitary of the bullfrog using Trireagent (Sigma) according to the manufacturer’s instructions.

Poly (A)


RNA was purified from total RNA using Oligotex mRNA kit (Qiagen, Valencia, CA, USA). RNAs from different tissues were reverse transcribed using the random hexamer and MMLV reverse transcriptase (Promega, Madison,

WI, USA). The cDNAs served as templates for subsequent PCR amplification of partial clones using several sets of forward (DGF1–DGF6) and reverse

(DGR1–DGR8) degenerate primers ( Table 1 ). PCR conditions were as follows:

denaturation at 95


C for 5 min, followed by 35 cycles at 95

C for 1 min and 72

C for 1 min,

C for 1 min. PCR products of expected sizes were excised, purified and subcloned into pGEM-T easy vector (Promega). Positive clones were isolated and purified using a Plasmid Miniprep Kit (Qiagen). The

DNA of plasmids containing the proper inserts was sequenced with the dideoxy chain-termination method using an automated sequencer.

To determine the tissue distribution of the APJ mRNAs, RT-PCR was performed using the primer pair APJT-F and APJT-R (

Table 1 ). Tissues were

collected from five male bullfrogs (five females for the ovary) and stored at


C. The first-strand cDNA was isolated using the random hexamer and

MMLV reverse transcriptase (Promega). PCR conditions were as follows: denaturation at 95 and 72

C for 5 min, followed by 30 cycles at 95

C for 40 s.

C for 30 s, 57

C for 30 s

2.5. Cloning of the full-length cDNAs by rapid amplification of cDNA ends (RACE)

To obtain the full-length APJ cDNA, two gene-specific primers (5 -GSP1, 5 -

GSP2 and 3 -GSP1, 3 -GSP2) and two nested-gene-specific primers (5 -NGSP1,

5 -NGSP2 and 3 -NGSP1, 3 -NGSP2) for 5 - and 3 -RACE were designed


Table 1 ). Poly (A)-rich RNAs purified from the forebrain, hypothalamus and

pituitary were used to synthesize adapter-ligated double-stranded cDNA using the Marathon cDNA Amplification Kit (Clontech). 5 - and 3 -RACE was performed using GSPs and NGSPs in combination with adapter primers AP1 and

AP2, respectively. RACE products were cloned in the pGEM-T easy vector

(Promega). After obtaining the proper 5 - and 3 -RACE products, gene specific forward and reverse primers were designed to obtain the full-length cDNAs. We finally obtained four individual clones of full-length bfAPJ with an identical sequence. These clones were then inserted into the pcDNA3 expression vector at Hind III and Xho I enzyme sites.

M.J. Moon et al. / Molecular and Cellular Endocrinology 277 (2007) 51–60 53

Table 1

Oligonucleotide sequences of primers


Amplification of partial cDNA

5 -RACE of APJ

3 -RACE of APJ

Cloning of APJ ORF

APJ tissue distribution




5 -GSP1

5 -GSP2

5 -NGSP1

5 -NGSP2

3 -GSP1

3 -GSP2

3 -NGSP1

3 -NGSP2





Oligonucleotide sequence















2.6. Luciferase assays

HEK-293T cells were maintained in DMEM in the presence of 10% fetal bovine serum. For luciferase assays, cells were plated in 24-well plates one day before transfection with SuperFect reagent (Qiagen). Approximately, 48 h after transfection, cells were treated with the apelin ligands for 6 h. Cells were harvested after ligand treatment and luciferase activity in cell extracts was measured in a Multi-label counter according to a standard luciferase assay system

(Perkin-Elmer, Wellesley, MA, USA). The luciferase values were normalized to

-galactosidase values. Transfection experiments were performed in triplicate and repeated at least three times.

2.7. Tissue binding assay

Tissue binding assay was performed using




]apelin-13 (Amersham Pharmacia Biotech, UK). Tissues were collected from five male bullfrogs

(five females for the ovary) and stored at


C. Tissues were thawed on ice and homogenized in 1 ml cold homogenizing buffer containing 0.25 M sucrose.

The membrane fractions were precipitated by centrifugation at 12,000

× g for

15 min at 4

C and resuspended in 400

␮ l of resuspending buffer containing

10 mM Tris–HCl (pH 7.6). Protein concentration was measured using BCA


Protein assay Kit (Pierce, Bonn, Germany). One hundred nanograms of membrane samples were incubated on ice for 80 min with





Non-specific binding was determined in the presence of 1

M unlabeled (cold)



]apelin-13. After centrifugation at 12,000

× g for 20 min at 4

C, radioactivity of pellet samples was determined using Wallac 1480 Wizard gamma counter (Perkin-Elmer, Wellesley, MA, USA).

2.8. Cell binding assay

One day before transfection, 1



5 cells were seeded in 12-well plates.

Forty-eight hours after transfection, cells were washed with PBS twice and incubated for 1 h in 400

␮ l of serum-free DMEM containing 0.1% BSA and




]apelin-13 in the presence of various concentrations of cold apelin-

13 peptides. Cells were washed with ice cold DPBS twice. Radioactivity was determined by resolving the cells in 1% SDS and 0.2 M NaOH.

2.9. cAMP accumulation assay

Twenty-four hours before transfection, HEK-293T cells were seeded into

12-well plates. Cells were transfected with Effectene reagent (Qiagen), and

1 day later were labeled with 2

Ci/ml of [


H]adenine (NEN Life Science

Products, Boston, MA) for 24 h. Cells were washed with PBS twice, then incubated at 37

C for 20 min in serum-free DMEM containing 1 mM 1-methyl-

3-isobutylxanthine (IBMX) and stimulated by apelin-13 peptides in combination with forskolin (FKN) and pertussis toxin (PTX) for 15 min. The reactions were terminated by replacing medium by ice-cold 5% trichloroacetic acid containing 1 mM ATP and 1 mM cAMP. [


H]cAMP and [


H]ATP were separated on AG 50W-X4 resin (BioRad, Hercules, CA) and alumina columns as pre-

viously described ( Oh et al., 2005 ). The cAMP accumulation was expressed as





H]ATP + [





2.10. Data analysis

Dose–response curves and ligand concentration inducing half maximal inhibition (IC


) were obtained using GraphPad PRISM3 software (GraphPad, San

Diego, CA). All data are presented as mean


S.E.M. The data were analyzed by one-way ANOVA followed by the Bonferroni Test.

p < 0.05 was considered statistically significant.

3. Results

3.1. Cloning of bullfrog APJ cDNA

Degenerate primers (DGF5 and DGR2, see

Table 1 ) were

used to amplify a partial bfAPJ cDNA (616 bp). Then, the entire open reading frame (ORF) of bfAPJ was obtained by 5 and 3 -RACE PCR using several bfAPJ gene-specific primers.

Nucleotide and deduced amino acid sequences of the bfAPJ cDNA are shown in

Fig. 1 . The cDNA for full-length bfAPJ

consists of 148 bp (

148 to

1), 789 bp (1084–1872) of untranslated regions (UTR) and 1083 nucleotides that encode a protein of 360 amino acids. Hydropathy analysis of bfAPJ reveals seven stretches of hydrophobic amino acid residues corresponding to the seven transmembrane domains (TMDs). Amino acid sequence alignment of bfAPJ with human, chimpanzee, mouse, rat, Xenopus

and zebrafish APJ ( Fig. 2 ) reveals 75% sequence

identity with Xenopu s, and other species also share some amino acid identity: zebrafish (63%), human (42%) and rat (40%)

( Table 2 ). The TMDs share a relatively high degree of amino

acid sequence identity across species, while the extracellular Nterminus, extracellular loop 3 (ECL3) and cytoplasmic C-tail share little (data not shown). Like most non-mammalian APJ, bfAPJ has a relatively short cytoplasmic C-tail. bfAPJ contains

54 M.J. Moon et al. / Molecular and Cellular Endocrinology 277 (2007) 51–60

Fig. 1. Nucleotide and deduced amino acid sequence of bfAPJ cDNA. Numbers on the right refer to nucleotide and the amino acid positions. A start codon (ATG) and a stop codon (TAA) are indicated by +1 and stop, respectively. The transmembrane domains are underlined and indicated by TMD I–VII.

an Asp-Arg-Tyr (DRY) motif at the boundary between TMD III and intracellular loop 2 (ICL2) as well as an Asn-Pro-x-x-Tyr

(NPxxY) motif in TMD VII, typical sequences of rhodopsin-like

GPCRs. Cys residues in the ECL1 and ECL2 are well conserved among the APJs. The bfAPJ contains two putative N-linked glycosylation sites in the extracellular N-terminal domain, while mammalian APJs have only one N-linked glycosylation site. In the intracellular C-terminal tail, bfAPJ has several putative phosphorylation sites for protein kinase C (PKC), casein kinase 1 ␣

(CK1 ␣ ) and G protein-coupled receptor kinase (GRK) (

Fig. 2 ).

M.J. Moon et al. / Molecular and Cellular Endocrinology 277 (2007) 51–60 55

Fig. 2. Sequence alignment of bullfrog APJ with multiple other species. Conserved amino acid residues are shaded. Numbers on the right side indicate the position of amino acid residues. The predicted seven transmembrane domains (TMDs) are indicated with a black line above the aligned sequences. The gray, dotted black and black lines indicate a CK1

␣ phosphorylation site,

-arrestin binding site and PKC phosphorylation site, respectively. Single or double asterisks denote an

N-glycosylation site and a GRK phosphorylation site, respectively.

3.2. Tissue distribution of bfAPJ

To determine tissue distribution of the bfAPJ mRNA, RT-

PCR analysis was performed using a variety of central and peripheral tissues. PCR products with 457 nucleotides were detected in the whole brain, hypothalamus, pituitary, testis, adrenal gland and lung. Spleen and ovary did not express detectable levels of APJ mRNA. RT-PCR for glyceraldehyde 3phosphate dehydrogenase (GAPDH) was performed under the

same conditions as a control ( Fig. 3 A).

To confirm bfAPJ expression at the protein level, tissue binding assay was performed using




]apelin-13. Nonspecific binding was determined in the presence of 1 ␮ M unlabeled (cold) [pGlu


]apelin-13. Like bfAPJ mRNA expres-

Table 2

Amino acid identity between bullfrog APJ and other species

Bullfrog Xenopus Zebrafish Rat Mouse Chimpanzee Human





























Sequences were retrieved from GeneBank: Xenopus APJ (CAA63612), zebrafish APJ (AAH56308), rat APJ (NP 112639), mouse APJ (AAH39224), chimpanzee

APJ (XP 521984) and human APJ (NP 005152).

56 M.J. Moon et al. / Molecular and Cellular Endocrinology 277 (2007) 51–60 pertussis toxin (PTX, 100 ng/ml), a specific G i/o inhibitor, in cells expressing hAPJ (

Fig. 4 C) or bfAPJ ( Fig. 4 D), sug-

gesting that APJs are coupled to G i/o

. In mock-transfected cells, [pGlu


]apelin-13 had no effect on FKN-activated CRE-

luc activity ( Fig. 4 E), indicating that HEK-293T cells do

not naturally express the endogenous receptors for this ligand.

To further confirm G i/o coupling of bfAPJ, cAMP accumulation was measured in HEK 293T cells expressing bfAPJ.



]apelin-13 alone did not induce cAMP accumulation, however, [pGlu 1 ]apelin-13 was able to reduce FKN-induced cAMP accumulation and this inhibitory effect was blocked by


Fig. 4 F).

Fig. 3. Tissue distribution of bfAPJ. RT-PCR (A) was performed in various tissues, with 30 rounds of amplification, to determine bfAPJ mRNA distribution.

As a control, RT-PCR amplification was performed under the same conditions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers. Tissue binding assay (B) was performed to determine APJ expression at the protein level. Membrane fractions (100 ng) of several tissues were obtained and binding reaction was carried out using




]apelin-13. Non-specific binding was determined in the presence of 1

M [pGlu


]apelin-13. Marker (M), brain (B), hypothalamus (H), pituitary (P), testis (T), adrenal gland (A), heart (He), kidney

(K), liver (Li), lung (Lu), spleen (Sp), stomach (St) and ovary (O) are shown.

sion, the brain, hypothalamus, pituitary and testis showed a specific binding to

ligand ( Fig. 3 B).




]apelin-13, while the spleen, ovary, liver and kidney did not show a specific binding to the

3.3. Activation and signaling of APJs by apelin-13

The open reading frames of hAPJ and bfAPJ were subcloned into pcDNA3, a mammalian expression vector. To investigate intracellular signaling by APJs, HEK-293T cells were co-transfected with either hAPJ or bfAPJ cDNA in combination with signaling specific luciferase (luc) vectors such as CRE-luc, SRE-luc, c-fos-luc or AP-1-luc. We previously demonstrated that CRE-luc and c-fos-luc reporter systems are useful tools for discriminating adenylyl cyclase/protein kinase

A (AC/PKA) and phospholipase C/protein kinase C (PLC/PKC) signaling pathways, respectively (

Oh et al., 2003; Seong et al., 2003 ). The serum response element (SRE) is activated by

stimulation of MAP Kinase via G q

-coupled receptors results mainly through PKC activation. The activator protein 1 (AP-

1) is also activated by G q

-coupled GPCRs following PKC stimulation (

Hill et al., 2001 ). Forty-eight hours after trans-

fection, cells were treated with [pGlu


]apelin-13, an adenylyl cyclase activator forskolin (FKN, 5

M) or a protein kinase C activator 12O -tetradecanoylphenol-13-acetate (TPA, 200 nM).



]apelin-13 did not induce CRE-luc, SRE-luc, c-fosluc or AP-1-luc activities in hAPJ or bfAPJ-expressing cells

( Fig. 4 A and B). However, when APJ-expressing cells were

treated with [pGlu 1 ]apelin-13 in the presence of FKN, FKN-

induced CRE-luc activity was significantly decreased ( Fig. 4 C

and D). These inhibitory effects were completely blocked by

3.4. Sequence alignment of non-mammalian apelins

Using a BLAST search, putative apelin sequences were identified in a number of non-mammalian species, including two amphibian species ( Xenopus laevies (BC099044) and

Xenopus tropicalis (CX330889)), a reptilian species (Gekko

(CO439015)), three fish species (pufferfish (CAAE01014998), zebrafish (CO924544) and pimephales (DT229932)) and an avian species (chicken (CR522896)). The amino acid sequence corresponding to mammalian apelin-13 is highly conserved in non-mammalian species except for the first position which is a Gln residue in mammalian and Xenopus species and a Pro residue in other non-mammalian species. The sequences that correspond to mammalian apelin [42–64] are highly variable across the vertebrate species (

Fig. 5 ).

3.5. Ligand sensitivity/affinity of bfAPJ

Since apelin-13 is more potent than apelin-36, we synthesized [pGlu


]apelin-13, [Gln


]apelin-13 and [Pro


]apelin-13 and compared potencies of these peptides at hAPJ and bfAPJ.

These peptides induced a concentration-dependent decrease of

FKN-induced CRE-luc activity in HEK-293T cells transfected

with hAPJ ( Fig. 6 A) or bfAPJ ( Fig. 6 B). [pGlu


]apelin-13 and [Gln




]apelin-13 showed a slightly higher potency than

]apelin-13 at hAPJ as revealed by IC




0.31 nM for [pGlu 1 ]apelin-13, 1.45

± 0.45 nM for [Gln 1 ]apelin-13 and


± 0.60 nM for [Pro


]apelin-13, respectively) ( Fig. 6 A).

For bfAPJ, however, [Pro


]apelin-13 was approximately 10fold more potent than [pGlu


]apelin-13 and [Gln




: 7.76


2.00 nM for [pGlu

1 for [Glu


]apelin-13 and 0.97


]apelin-13, 9.56



0.25 nM for [Pro


3.38 nM


respectively) ( Fig. 6 B). The cAMP assay also revealed

that [Pro


]apelin-13 is more potent than [pGlu


]apelin-13 and [Gln


]apelin-13 at bfAPJ. IC tide are 5.24


2.01 nM for [pGlu


50 values for each pep-

]apelin-13, 5.92


2.47 nM for [Gln


]apelin-13 and 0.80


0.35 nM for [Pro



respectively ( Fig. 6 C).

Ligand affinity at bfAPJ was determined using displacement binding assay. [Pro


]apelin-13 showed a higher binding affinity than [pGlu 1 ]apelin-13 to bfAPJ. The log IC

50 are 4.98

± 0.84 nM for [Pro




]apelin-13, respectively ( Fig. 6 D).


]apelin-13 and 20.65

± 3.07 nM for

M.J. Moon et al. / Molecular and Cellular Endocrinology 277 (2007) 51–60 57

Fig. 4. [pGlu


]apelin-13-induced APJ signaling. Plasmids containing hAPJ or bfAPJ cDNA were cotransfected with signaling specific luciferase gene vectors into

HEK-293T cells. Forty-eight hours after transfection, cells were treated with [pGlu or G i/o protein inhibitor, PTX (100 ng/ml) for 6 h. [pGlu

(B). [pGlu




]apelin-13 (1

M), a signaling specific activator (i.e. FKN (5

M), TPA (200 nM)

]apelin-13 did not induce signaling specific reporter gene activities in cells expressing hAPJ (A) and bfAPJ

]apelin-13 inhibited FKN-induced CRE-luc activity and PTX blocks this effect in cells expressing hAPJ (C) and bfAPJ (D). The apelin effect was not determined in mock-transfected cells (E). [pGlu


]apelin-13 suppressed the FKN-induced cAMP accumulation in bfAPJ-expressing cells (F). Data shown are the means


S.E.M. of three independent experiments (

* p < 0.05).

Fig. 5. Sequence alignment of preproapelin amino acids. Conserved amino acid residues are shaded. Sequences were retrieved from GeneBank: human (NM 017413), mouse (NM 013912), chicken (CR522896), gekko (CO439015), Xenopus tropicalis (CX330889), Xenopus laevis (BC099044), zebrafish (CO924544), pufferfish

(CAAE01014998) and pimephales (DT229932). Asterisks denote conserved basic amino acids, potential protease cleavage sites.

58 M.J. Moon et al. / Molecular and Cellular Endocrinology 277 (2007) 51–60

Fig. 6. Differential activity of [Pro


]-, [pGlu


]- and [Gln


]apelin. [pGlu


]apelin-13 ( ), [Gln


]apelin-13 ( ) and [Pro


]apelin-13 ( ) induced a concentrationdependent decrease of FKN-induced CRE-luc activity in HEK-293T cells expressing either hAPJ (A) or bfAPJ (B). Three peptides decreased the FKN-induced cAMP accumulation in HEK-293T cells expressing bfAPJ in a dose-dependent manner (C). Cell binding assay was performed using




]apelin-13 in the presence of various concentrations of [Pro


]apelin-13 and [pGlu


]apelin-13. [Pro


]apelin-13 showed a higher binding affinity than [pGlu

(D). Data shown are the means


S.E.M. of three independent experiments.


]apelin-13 in bfAPJ-expressing cells

4. Discussion

The role of apelin and the APJ receptor in non-mammalian systems is poorly understood. Recently, the role of apelin and

APJ in angiogenesis during Xenopus embryonic development has been demonstrated (

Cox et al., 2006; Inui et al., 2006 ).

APJ sequences for Xenopus

( Devic et al., 1996 ) and zebrafish

(AAH56308) were described previously, however, ligand selectivity, G protein coupling and adult tissue distribution were not determined. In this study, we have cloned APJ from the bullfrog and described its basic characteristics.

The structure of bfAPJ is typical of the rhodposin-like GPCR family. bfAPJ shares a high degree sequence identity with nonmammalian species like Xenopus (75%) and fish (63%). In contrast, sequence identity with mammalian APJs is rather low

(40–42%), particularly in the extracellular N-terminus, extracellular loop 3 (ECL3) and cytoplasmic C-terminal tail. The

C-terminal tail of bfAPJ is relatively short in comparison with mammalian APJs, and it differs significantly even from Xenopus and fish APJs. Such structural differences often affect the ligand selectivity and G protein coupling of receptors that

have evolved from the same ancestor gene ( Wang et al., 2001;

Acharjee et al., 2004; Oh et al., 2005 ). Ligand selectivity of

bfAPJ and hAPJ does indeed differ. bfAPJ responds better to

[Pro 1 ]apelin-13 than [pGlu 1 ]apelin-13, while hAPJ responds slightly better to [pGlu


]apelin-13 than [Pro


]apelin-13. However, G protein coupling is the same for both human and bullfrog receptors.

BLAST search revealed genes encoding putative APJ ligands (apelin-like peptides) in many non-mammalian species.

Sequence alignment of mammalian and non-mammalian preproapelin peptide sequences highlights variability at the

N-terminal region. In contrast, the C-terminus is highly conserved, suggesting that these residues may be responsible for receptor binding and activation. A number of basic amino acid residues are also conserved among non-mammalian and mammalian species. Since these residues are potential cleavage sites for peptidases, it is likely that there are multiple apelin forms within a given species. Based on propeptide sequences, [Pro


]apelin-13 is a putative ligand in all nonmammalian species including fish, reptilian and avian species, except for Xenopus . It is can be postulated that the bullfrog has



]apelin-13 as a natural ligand since bfAPJ shares 75% identity with Xenopus APJ. However, there are a number of reports that neuropeptide sequences in amphibians vary among species. For instance, Xenopus glucagon is identical to human glucagon but differs from bullfrog glucagon by one amino acid


Irwin et al., 1997 ). The amino acid sequences of the neu-

rotensin peptide from four different amphibian species differ from each other (

Shaw et al., 1992; Desrues et al., 1998; Warner et al., 1998; Klein et al., 2002 ). A gonadotropin-releasing hor-

mone peptide from Rana dybowskii differs by one amino acid from those of Xenopus

and mammals ( Yoo et al., 2000 ). One

interesting finding of this study is that [Pro


]apelin-13 is 10fold more potent than [pGlu


]apelin-13 and [Gln


]apelin-13 at bfAPJ. Variations of neuropeptide sequences in amphib-

M.J. Moon et al. / Molecular and Cellular Endocrinology 277 (2007) 51–60 ian species, together with high conservation of [Pro


]apelin-13 in non-mammalian species and a high affinity/sensitivity of



]apelin-13 at bfAPJ, increase the possibility that the bullfrog may have [Pro


]apelin-13 as an endogenous ligand, instead of [Gln


]apelin-13 or [pGlu


]apelin-13. However, the sequence of the bullfrog apelin remains to be identified.

The mammalian APJ is known to couple to G i/o protein as apelin reduces FKN-induced cAMP production and stimulates

ERK phosphorylation in a PTX-dependent manner (

Habata et al., 1999; Masri et al., 2002, 2006 ). Like mammalian APJ,

bfAPJ may preferentially couple to a G cascade. Both [Pro

1 i/o

]apelin-13 and [pGlu


-mediated signaling

]apelin-13 induce a concentration-dependent decrease of either FKN-induced CREluc activity or FKN-induced cAMP accumulation in HEK-293T cells expressing bfAPJ. This inhibitory effect was blocked by

PTX. The apelin ligands failed to activate other reporter genes, indicating that bfAPJ is coupled to G i/o

. The sequence identity between bfAPJ and hAPJ is generally low (40–42%), but the intracellular loops, which most likely mediate receptor to

G-protein coupling, are relatively well conserved. Thus, receptor signaling has been conserved through evolutionary selection pressure.

In mammals, apelin regulates release of neurohormones and pituitary hormones. APJ mRNA is highly expressed in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of rat hypothalamus, as well as the anterior and intermediate lobes of rat pituitary (

De Mota et al., 2000; Reaux et al., 2001 ). Arginine

vasopressin (AVP) release from AVP neurons, as well as its neuronal activity, is inhibited by apelin, indicating that this peptide

is a potent diuretic factor that can counteract AVP action ( Reaux et al., 2001; De Mota et al., 2004 ). Furthermore, administration

of an apelin peptide intracerebroventricularly increases plasma

ACTH levels and decreases plasma prolactin and gonadotropin levels (

Taheri et al., 2002 ). In addition to control of pituitary

hormone release centrally, the apelin mRNA is also present in the pituitary (

Lee et al., 2000 ), suggesting that it may also

act in an autocrine or paracrine fashion to regulate pituitary hormone release. In adult bullfrogs, the APJ mRNA is highly expressed in the CNS (particularly the hypothalamus), pituitary and adrenal grand. Therefore, the endogenous bullfrog apelin peptide and bfAPJ may play an important role in the bullfrog neuroendocrine system much as it does in mammals. In fact, role for apelin/APJ in the hypothalamus–pituitary–adrenal gland axis is likely given the presence of APJ mRNA in bullfrog adrenal gland. Expression of bfAPJ mRNA in the testis and lung indicates that APJ-mediated signaling may control reproductive and respiratory processes in the bullfrog as well.

In contrast to mammals, adult bullfrogs contain very low levels of APJ mRNA in the heart and spleen, reflecting a potential functional difference between mammalian and non-mammalian systems.

In conclusion, we have cloned and functionally characterized bullfrog apelin receptors. This study helps to elucidate the phylogenetic history of apelin and its receptor in the vertebrate lineage, while also expanding our understanding of the physiological role(s) of this receptor system in non-mammalian species.



This work was supported by a grant (KRF-2004-

C00141) to H.B.K. from the Korea Research Foundation, a grant (M103KV010005-06K2201-00510) to J.Y.S. from Brain

Research Center of the 21st Century Frontier Research Program.


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