斜带石斑鱼(Epinephelus coioides)两种生长激素受体的克隆及其特性和组织分布研究

grouper (Epinephelus coioides)

Yun Li1#, Xiaochun Liu1#, Yong Zhang1, Pei Zhu1, Haoran Lin1, 2*

1. State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, Sun Yat-Sen University, Guangzhou,

China (510275)

2. College of Ocean, Hainan University, Haikou, China (570228)

E-mail：ls32@zsu.edu.cn

# These two authors contributed equally to this work

Abstract

The cDNAs encoding two distinct growth hormone receptors were firstly cloned and sequenced from the liver of orange-spotted grouper (Epinephelus coioides). The cDNA of grouper GHR1 consisted of 2673-bp and encoded 658 amino acids and that of grouper GHR2 consisted of 29-bp and encoded 577 amino acids. The two cDNAs had 78.6% identity in nucleotide sequence and 37.8% identity in deduced amino acid sequence. Northern blot analyses demonstrated a single GHR1 transcript of approximately 4.3Kb in liver and a single GHR2 transcript of approximately 3.9Kb in the liver and muscle. In the Real-time PCR assay, grouper GHR1 and GHR2 were expressed in all tissues tested, and the expression of GHR2 was significantly higher than GHR1 in telencephalon, cerebellum, pituitary, heart and white muscle, whereas the expression of GHR1 was significantly higher in liver. The grouper GHR1 had high homology to somatolactin receptor (SLR) and the grouper GHR2 might play a role of modulator of grouper GHR1. These results indicated that there were two types of GHR existing in orange-spotted grouper and they had different structural features and physiological functions. However the general functional differences between GHR1 and GHR2 of orange-spotted grouper remained further research.

Keywords: Growth hormone receptor; tissues expression; orange-spotted grouper

Introduction

Growth hormone (GH) plays important roles in regulating many aspects of physiology, including growth (Cavari et al., 1993), metabolism (Copeland and Nair, 1994), osmoregulation (Sakamoto et al., 1997; Tatsuya and Hirano, 1993), immune functions (Yada et al., 1999) and reproduction (McLean et al., 1993).

The interaction of GH and its specific receptor (growth hormone receptor, GHR), which involves a binding of single GH molecule with two GHR and then a cascade of tyrosine and protein phosphorylation, rousts up the potential effects of GHR (Argetsinger and Carter-Su, 1996). Based on the conserved structural features, the GH receptors belong to class I cytokine receptor superfamily (Kopchick and Andry, 2000), which has some common characteristics, such as the FGEFS ligand-binding domain, a single transmembrane domain, extracellular cysteine residues, two conserved cytoplasmic domains called Box 1 and Box 2 which are very important for signal transduction of the receptor (VanderKuur et al, 1994) and cytoplasmic tyrosine residues.

GHR cDNA squences of some high vertebrates have already been cloned (Adams et al, 1990; Hauser et al, 1990; Takashi et al, 1998). The deduced amino acid sequences of GHR show a low identity between Aves and mammals (Takashi et al, 1998). In fish, the GHR cDNA squences of several teleost species have recently been cloned, such as goldfish (Carassius auratus) (Lee et al., 2001), turbot (Scophthalmus maximusi) (Calduch-Giner et al., 2001), Japanese eel (Anguilla japonica) (Ozaki et al., 2001), black seabream (Acanthopagrus schlegeli) (Tse et al., 2003) and gilthead seabream (Sparus aurata) (Calduch-Giner et al., 2003; Saera-Vila et al., 2005).Recent studies have proved that two types of GHR exist in one teleost species. Several authors have postulated a divergent evolution of teleost GHRs. In this scenario, more than one GHR gene is maintained by selection through the radiation of teleosts (Saera-Vila et al., 2005). The classical GHRs of most teleosts (non-salmonid fish), containing 6 to 7 extracellular cysteine residues, should be called GHR1. The GHRs of salmonids, containing 5 extracellular cysteine residues, would be called GHR2.

Orange-spotted grouper is a commercially important fish and popularly cultured in Southeast Asia as well as China. However, low survival rate of larval and slow growth of fingerings limited the amplification of grouper’s mariculture.In the former research, our lab had accomplished the molecular cloning, tissue distribution, and ontogeny of growth hormone in orange-spotted grouper (Li et al., 2005).Basing on the achievement on grouper GH, the aim of this study was to clone and characterize two types of orange-spotted grouper GHRs (gGHRs) and compare the tissue expression of two types of gGHRs and attempt to elucidate the function of gGHRs.

Materials and methods

Experimental animals and sampling

Orange-spotted groupers (Female, 2-year-old) were obtained from Guangdong Daya Bay Fishery Development Center, Huizhou, PR China. Fish were anesthetized and sacrificed by decapitation. The liver samples for gGHRs cloning and various tissue samples for distribution study of gGHRs mRNA were removed and frozen immediately in liquid nitrogen and stored at − 80 °C until RNA extraction.

Growth hormone receptor cDNAs cloning

RNA extraction

Total RNA was extracted from the liver using Trizol® reagent (Fermentas, USA). RNA concentration was determined by optical density reading at 260 nm, and integrity was verified by ethidium bromide staining of 28 s and 18 s ribosomal bands on a 1% agarose gel. RNA samples were then stored at −80°C until further analysis.

Degenerate oligodeoxynucleotide primers and partial clone of gGHR cDNAs

One microgramme of total RNA was used to synthesize the first strand cDNA using the ReverTra Ace-α-TM First Strand cDNA Synthesis Kit (TOYOBO, Janpan) following the manufacturers’ instruction and the reverse-transcription (RT) products were stored at −20 °C. According to the conserved sequences between the reported GH receptor cDNAs, particularly of those from fishes, two couples of degenerate oligodeoxynucleotide primers, R1R , R1F , R2R and R2F (details in Table 1), were designed. In the PCR amplification for gGHR1 partial fragment, 35 cycles of amplification were performed using a cycle profile of 95 °C for 3 min, 95 °C for 15s, 55 °C for 15s, and 72 °C for 1 min with 0.5µl RT product as template and R1R and R1F as primers. In the PCR amplification for gGHR2 partial fragment, the same cycle profile used for gGHR1 was performed with 0.5µl RT product as template and R2R and R2F as primers.

TTTT Rapid amplification of cDNA ends (RACE)

To obtain the sequences of full-length gGHR cDNAs, nested 3′ and 5′ RACE PCR was performed. After determining the nucleotide sequences of the partial clone of gGHRs cDNAs,gGHRs gene-specific primers (GSP) were designed for 3′-RACE and 5′-RACE, respectively (Table 1).

Table 1 Nucleotide sequences of the primers used

Primers Sequences

R1F 5′ GTR CAC ATC MGC TGC AGG ATG 3′

R1R 5′ TGA GTT GCY GKC CAG GAG AC 3′

R2F 5′ GRW SAC KTT CCG CTG CAG ATG 3′

R2R 5′ TCR ATG AAY TCC ACC CAG GG 3′

Primers for 3′ RACE PCR

AP 5′ GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T 3′AUAP 5′ GGC CAC GCG TCG ACT AGT AC 3′

GSP:

GHR1-F1 5′ CGG AGA GAA GGA GGA CAA CCA G 3′

GHR1-F2 5′ GGG TCA ACA CAG ACT TCT ATG C 3′

GHR2-F1 5′ CCA AAG TGA GCG GGA AGG TAG AC 3′

GHR2-F2 5′ GAT CCC TGG GTG GAA TTC ATC G 3′

Primers for 5′ RACE PCR

AAP 5′ GGC CAC GCG TCG ACT AGT ACG GGG GGG GGG 3′

AUAP 5′ GGC CAC GCG TCG ACT AGT AC 3′

GSP:

GHR1-R1 5′ CCA AAG TGA GCG GGA AGG TAG AC 3′

GHR1-R2 5′ TCT GTG CTG CTG AGA GAC GAC 3′

GHR2-R1 5′ CCA TCT CCA CAT CTG CCG ACT G 3′

GHR2-R2 5′ CTC ATT GAA AAA GCA CTC ATT GGG 3′

β-Actin:

β-Actin-F 5′ GGT GGG TAT GGG TCA GAA AGA 3′

β-Actin-R 5′ GAT GAG GAA GTG CTG TCG 3′

Primers for Realtime PCR

GHR1-Real-R 5′ GCA TCC TCA GCA TCC ACC 3′

GHR1-Real-F 5′ GCG ACT CCA TCT TCA TTC A 3′

GHR2-Real-R 5′ ACC CGA ACC TCG TGA ATG 3′

GHR2-Real-F 5′ GAC GCT GCT GAA TGT GA 3′

Mixed Bases: Y:C/T R:A/G M:A/C S:G/C W:A/ T K:G/T

One microgramme total RNA from the liver was used to synthesize the first strand cDNA using the ReverTra Ace-α-TM First Strand cDNA Synthesis Kit (TOYOBO, Janpan) following the manufacturers’ instruction and the reverse-transcription (RT) products were stored at −20 °C.

The first round of gGHR1 3′ RACE PCR reaction was carried out with GHR1-F1 and AUAP as the GSP (Table 1) using PCR reaction mixture with Taq DNA Polymerase (MBI Fermentas, USA) following the manufacturer’s instruction. Thirty-five cycles of amplification were completed using a cycle profile of 95 °C for 3 min, 95 °C for 15s, 58°C for 15 s, and 72 °C for 1 min and 30 s. Then, 1µl of 100-fold diluted first-round PCR product was subjected to a nested PCR with the same reaction mixture except that the 3′-GSP1 (GHR1-F1) was replaced by 3′-GSP2 (GHR1-F2), and the PCR profile was as same as that for the first round PCR. In the 5’RACE, totalRNA was treated with DNase and

Ⅰthe RT reaction was performed using gGHR1 5′-GSP1 (GHR1-R1) as primer. The gGHR1 5′ fragment was obtained using nest PCR with GHR1-R1 and AAP (GeneRacer™ Kit, Invitrogen, USA) for first round primers and GHR1-R2 and AUAP for second round primers. In the same way, 3’RACE and 5’RACE of gGHR2 were performed using GHR2-F1 and GHR2-F2 as 3′-GSP and GHR2-R1 and GHR2-R2 as 5′-GSP, respectively. Sequence analysis

Amplification products were separated by agarose gel electrophoresis, and the band of desired size was excised and purified using E.Z.N.A.® Gel Extraction Kit (Omega BioTek, USA). The purified amplification products were then subcloned to pTZ57R/T vector (Fermentas, USA). The positive clones were sequenced. The cDNAs sequences and the deduced amino acid sequences were compared with the sequences in the GenBank database using BLAST program available for the NCBI internet website. Alignments of amino acid sequences were achieved using the programs of DNAstar and Clustalx 1.8. Phylogenetic tree was constructed using Clustalx 1.8 and Mega 2.0 for unrooted analysis.

Northern blot analysis

Each 20µg total RNA isolated from liver, muscle and ovary was electrophoresed in a 1% denaturing agarose gel containing 0.66% of formaldehyde using 1× TBE buffer as the running buffer. The RNA was blotted from the gel by electric transfer and cross-linked to Hybond-N nylon paper (Northern Max™-Gly Kit, Ambion, USA). The partial gGHRs cDNAs fragments were used as probes and were labeled using PCR Dig Labeling System (Northern Max™-Gly Kit, Ambion, USA) with the gene-specific primers (GHR1: GHR1-Real-R and GHR1-Real-F; GHR2: GHR2-Real-R and GHR2-Real-F, Table 1). After overnight hybridization at 46.5 °C, the blots were washed twice with washing buffer for 5 min at room temperature and twice with blocking buffer for 20 min at 42 °C, respectively. After the membrane was submerged into the detection buffer with CDP-Star substrate, the chemiluminescent signals from the membrane were detected by Syngene Bio Imaging (Synoptics, UK). The analysis of two gGHRs shared a same nylon paper that cross-linked with RNA samples.

Real-time PCR assay for tissue expression of GHRs mRNA transcripts in grouper

Real-time reverse transcription PCR

Total RNA of various tissues was extracted using Trizol® reagent (Fermentas, USA). 1µg total RNA of each tissue was treated with DNase I, and reverse transcribed with oligo-dT. Reactions without reverse transcriptase were included as negative controls. No amplification was detected in negative controls.

mRNA levels of two gGHRs were determined by Real-time RT-PCR using SYBR® Green I and an ABI PRISM® 7900 Sequence Detection System (Applied Biosystems). Orange-spotted grouper β-actin (NCBI GenBank Accession No. AY510710) was chosen as housekeeping gene to correct potential variations in RNA loading. Real-time Specific primers for each gene of interest were designed (GHR1: GHR1-Real-R and GHR1-Real-F; GHR2: GHR2-Real-R and GHR2-Real-F; β-actin: β-Actin-R and β-Actin-F, Table 1). Plasmid constructs (pTZ57R/T vector) were used as external standards for each target gene. The concentration of purified plasmids was quantified by optical measurements at 260 nm. The 20µl Real-time PCR reactions contained 10µl SYBR®Green Realtime PCR Master Mix (TOYOBO, Janpan), 1.00µl of RT product, and 0.4 µMof each forward and reverse primer. Primer concentration was optimized according to the manufacturer’s instructions. Briefly, the cDNA denatured by preincubation for 60 s at 95 °C; the template was amplified for 40 cycles of denaturation for 15 s at 95 °C, annealing for 15 s at 58.5°C and extension for 23 s at 72°C.

Data analysis

The concentration of aim gene was based on threshold cycle number (CT). The CT for each sample was determined by the ABI Sequence Detector Program. Sample concentration was determined by relating CT to a gene-specific standard curve (Fig.1). Each transcript level was normalized on the basis of the quantification of the β-actin gene. The level of each GHR was described with relative concentration (RCGHR/RCβ-actin).

Fig.1. Standard curves used for quantification of mRNAs in Real-time PCR.

Statistical analyses

Quantitative data were expressed as means±S.E.M. Statistical differences were estimated by one-way ANOVA followed by Duncan's multiple range test, a probability level of 0.05 was used to indicate significance. All statistics were performed using SPSS 13.0 (SPSS, Chicago, IL, USA).

ResultsCloning and characterization of two gGHR cDNAs

Two unique cDNA fragments, each approximately 750～800 bp in size, were amplified by RT-PCR from total RNA isolated from the liver of orange-spotted grouper using degenerate oligodeoxynucleotide primers. Further investigation using 3′-RACE PCR revealed products of approximately 1100 bp and 1900 bp, respectively. 5′RACE PCR of two types of gGHR supplyed products of approximately 1500 bp and 600 bp, respectively. Assembly of the cDNA fragments resulted in a 2673-bp cDNA for gGHR1 (NCBI GenBank Accession No. EF052273) and a 29-bp cDNA for gGHR2 (NCBI GenBank Accession No. EF052274), which had a nucleotide identity of 78.6%. The gGHR1 cDNA encoded 658 amino acids that contained a putative signal peptide of 46 amino acids, a 273-amino acid extracellular domain, a single transmembrane domain of 21 amino acids, and a 3-amino acid intracellular domain (Fig.2). gGHR2 cDNA encoded 577 amino acids that contained a putative signal peptide of 19 amino acids, a 244-amino acid extracellular domain, a single transmembrane domain of 20 amino acids, and a 314-amino acid intracellular domain (Fig.3). The two types of gGHRs shared a 37.8% amino acid identity.

1 GGC TGA AGT GGA GAA GCC GGA CTT TTT TCG TGC CTG TTG GTG CTC ACG CTG GCC ATC AGA 60

61 TGA CCA AGT TGT GAA AAG TCG ATG TGC GCT GTC TTT GCA GTT TTT CTC CCC AGC TTG AGA 120

1 M C A V F A V F L P S L R 13

121 CTT CGA AAC AAC ATC ATG ACT GTC TCG TCC TCC TCC TCC AAT GTC GTA GTC CTT CTC CTA 180

14 L R N N I M T V S S S S S N V V V L L L 33

181 ATT TCC TCC CTG GAT TGG CTG TCC ACT CCT GGA TCA GCG TTT CTC ATG GGC CGG GAC CAC 240

34 I S S L D W L S T P G S A F L M G R D H 54

241 GTG ACG TCA CCA GCT CCC GTG GGA CCT CAC ATC ACA GAG TGC ATA TCG AGG GAC CTG GAG 300

55 V T S P A P V G P H I T E ○C I S R D L E 74

301 ACA TTC CGG TGT TGG TGG AGT CCA GGT GAC TTC CAC AAC CTG TCC TCC CCT GGA GCA CTC 360

75 T F R ○C W W S P G D F H □N L S S P G A L 94

361 AGA GTC TTC TAC CTG AAG AAA AAC TTG CCC ACC AGT GAA TGG AAA GAG TGT CCA GAG TAC 420

95 R V F Y L K K N L P T S E W K E ○C P E Y 114

421 CTT CAT TCA AAT AGG GAG TGC TTC TTC GAT GGA AAC CAT ACA TCG GTT TGG GTC CCC TAC 480

115 L H S N R E ○C F F D G □N H T S V W V P Y 134

481 TGC ATG CAG CTC CGC GGC CAA AAC AAC ATC ACA TAT TTC AAC GAG GAC GAC TGT TTC ACT 540

135 ○C M Q L R G Q □N N I T Y F N E D D ○C F T

154

541 GTG GAG AAT ATC GTA CGG CCT GAC CCA CCA GTG TCT CTA AAC TGG ACT CTG CTG AAT ATA 600

155 V E N I V R P D P P V S L □N W T L L N I 174

601 AGT CCC TCC GGG CTC AGC TAT GAT GTC ATG GTC AAC TGG GAG CCC CCA CCC TCT GCA GAC 660

175 S P S G L S Y D V M V N W E P P P S A D 194

661 GTC GGG GCG GGC TGG ATG CGC ATT GAA TAC GAG ATC CAG TAC AGA GAG AGA AAC ACC ACA 720

195 V G A G W M R I E Y E I Q Y R E R □N T T 214

721 AAC TGG GAA GCA TTG GAG ATG CAG CCG CAC ACC CTG CGC ACA ATC TAC GGT CTG CAC ATA 780

215 N W E A L E M Q P H T L R T I Y G L H I 234

781 GGA AAA GAG TAT GAG GTG CAC ATC CGC TGT AGG ATG CAG GCC TTC ACT AAG TTT GGA GAG 840

235 G K E Y E V H I R ○C R M Q A F T K F G E 254

841 TTC AGC GAC TCC ATC TTC ATT CAA GTG ACT GAG ATT CCC AGC AAA GAG TCT ACC TTC CCG 900

255 F S D S I F I Q V T E I P S K E S T F P 274

901 CTC ACT TTG GTT CTT ATT TTT GGG ATT GTG GGC ATC CTC ATA CTC ATC ATG CTA ATT GTC 960

275 L T L V L I F G I V G I L I L I M L I V 294

961 GTC TCT CAG CAG CAC AGA TTG ATG ATG ATT CTG TTG CCA CCA GTT CCC GCA CCC AAA ATT 1020

295 V S Q Q H R L M M I L L P P V P A P K I 314

1021 AAG GGC ATC GAT CCA GAG CTG TTA AAG AAG GGG AAG CTG GAT GAG CTG AAT TTT ATC CTG 1080

315 K G I D P E L L K K G K L D E L N F I L 334

1081 AGT GGT GGA GGT ATG AGC GGC CTG CCC ACA TAC GCG CCA GAT TTC TAC CAA GAC GAG CCA 1140

335 S G G G M S G L P T ◇Y A P D F ◇Y Q D E P 354

1141 TGG GTG GAG TTC ATC GAG GTG GAT GCT GAG GAT GCA GAT ACC GGA GAG AAG GAG GAC AAC 1200

355 W V E F I E V D A E D A D T G E K E D N 374

1201 CAG GGC TCA GAC ACC CAG AGG CTC CTC AGT CTG TCC CAG CCC GTC AGC CAC CAT ATG AAC 1260

375 Q G S D T Q R L L S L S Q P V S H H M N 394

1261 ATA GGC TGC TCC AAT GCC GTC AGC TTC CCT GAT GAT GAC TCA GGC CGG GCC AGC TGTTAC 1320

395 I G C S N A V S F P D D D S G R A S C ◇Y

414

1321 GAC CCA GAC CTG CCT GAC CAA GAC ACC CTA ATG CTG ATG GCC ACC CTG CTG CCA GGC CAA 1380

415 D P D L P D Q D T L M L M A T L L P G Q 434

1381 CCC GAG GAC GGA GAA GCA TCC TTC GAT GTT GTT GAA AGA GCC CCA GCC CCA GAG AGA GGC 1440

435 P E D G E A S F D V V E R A P A P E R G 454

1441 GAG AGG CCC CTT GTT CAA ACC CAA ACT GGA GGG CCC CAG ACT TGG GTC AAC ACA GAC TTC 1500

455 E R P L V Q T Q T G G P Q T W V N T D F 474

1501 TAT GCC CAA GTC AGC AAT GTA ATG CCC TCT GGG GGT GTG GTA CTA TCT CCT GGC CAG CAA 1560

475 ◇Y A Q V S N V M P S G G V V L S P G Q Q 494

1561 CTC AGA ATC CAG GAG AAT ACG TTA GCC ACA GAG GAG GAG ACA CAG AAG AAG GGA AAA GAG 1620

495 L R I Q E N T L A T E E E T Q K K G K E 514

1621 CAC AAA GGC AAC GAG GAC ACT GAG GAG AAC AAG CAG AAA GAG CTG CAG TTT CAG CTG CTG 1680

515 H K G N E D T E E N K Q K E L Q F Q L L 534

1681 GTC GTG GAT CCT GAA GGC AGC GGC TAC ACC ACA GAG AGC AAC GCC CGG CAG ATC AGC ACT 1740

535 V V D P E G S G ◇Y T T E S N A R Q I S T 554

1741 CCC CCC AGC TCC CCC ATG CCT GGC GAG GGT TAT CAA ACC ATA CAC CCT CAG CCA GTG GAG 1800

555 P P S S P M P G E G ◇Y Q T I H P Q P V E 574

1801 ACC AAA CCT ACC CCC ACA GCA GAG GAT AAT CAA TCC CCT TAC ATT CTT CCT GAC TCT CCC 1860

575 T K P T P T A E D N Q S P ◇Y I L P D S P 594

1861 CAA TCC CAG TTC TTT GCC CCT GTT GCA GAC TAC ACA GTG GTA CAG GAG GTG GAC AGT CAA 1920

595 Q S Q F F A P V A D ◇Y T V V Q E V D S Q 614

1921 CAC AGT CTG CTC CTA AAC CCG CCT CCC CGC CAG TCT CCC CCT CCC TGC GTG CCA CAG CAC 1980

615 H S L L L N P P P R Q S P P P C V P Q H1981 CCA CTC AAG GCC CTA CCT GCA ATG CCA GTG GGG TAC ATC ACC CCA GAC CTC CTG

GGG AAC 2040

635 P L K A L P A M P V G ◇Y I T P D L L G N

654

2041 CTC TCA CCA TGA AAT GAC AAT GCC ATC AGG CCT TTA AAG AGT AAG GTT GAT CGA

CCA GGA 2100

655 L S P * 658

2101 AGT CCC CTG CAG TCA TAA AAG CCT GTT TCT ACC TGA TTC CTG CTG GAA ACC AAA TAA

CCC 2160

2161 GCA GCG CAC AGT GCG GAT GAG AGT GTA CAG GTG TTT GTT TTC AGA GGG CGC CGA

AAG GGA 2220

2221 GAC GTG GAA AGC TAC CAA GCT GTA TTC TTG ACT CTT TTA CAT CTG CTA CAT CAG TTA

ATT 2280

2281 TGA TGC AAA GGC AGC CAG AAT TAA ACA TCG CAA ACC AAA TGC ACA GTT CTG CTC

TAT ATG 2340

2341 TCG ACA TCA ATG CAC AAG ATA CAC GCA GAT ATA AGG CTG AGA CTG TGA TTG TAA

TGA GTC 2400

2401 AAA AAA GCA CCA GCT CAC TGC TTA ACT GAC ATC TCT AGC TAA GTG TGT GTA GAG

CAG TGG 2460

2461 CAT TGT TGA AGT CTT AAT CTA ACA AAG AGA TGT CAG TGT TTG ATT GAC TGA GAG AAG

ACG 2520

2521 AGC AGA TTC TAG TCA TGC TTT TCA CAC CAC ACC AGG AAG AAG CTG TCG TTG ATA CCT

TTT 2580

2581 GCT GGC AGA GAT GTT TTG CCG CTC AAT CGT GGC TTA TTT TGT ATT ATT ACA TTT ATA

TTT 20

21 CAT GCT ATC CTA TAG CAC CTA AAA AAA AAA AAA 2673

Fig.2. Nucleotide sequences of orange-spotted grouper GHR1 and the deduced amino acid sequences.

Transmembrane region is shaded with dark. Putative signal peptide is underlined. Box1 and box2 motifs are

squared in an open rectangle. Conserved cysteine residues are denoted by a Ο around the amino acid. Potential

N-linked glycosylation sites are denoted by a around the amino acid. The FGEFS motif is in bold and

◇

underlined with broken line. Conserved tyrosine residues are indicated by a around the amino acid. The stop

sequence is denoted by *.

1 AGT CTC GGT TAG ACA CAT TCC CGG TTG GTT TTC AGA GGC TGT CCG CGG ACT GTA CGG

ACC 62

63 TAT CGT GGA CTT CAT CTG TGT GTG AGA GAT TTC GAT GCT GTT GCT GCT TTA TCA AGC

AAA 122

123 CCT GAG AGA GCT TTA GGA CGC TGG AAG CAC ACT GCT GTC CAC TTG TTC TCC ACA GCG

CTT 182

183 AAC GCC ATG GCT GCC GCT TTC ACC ATG CTC TTC TTC TTT CTT CAC ATC TTC ACT GCC

TCT 242

1 M A A A F T M L F F F L H I F T A S

18

243 GCG CTG GAA TCG GCC TCT GAG CAA GTC CTC CCC GAT GCA CAC CCA CAC CTC ACT GGC

TGT 302

19 A L E S A S E Q V L P D A H P H L T G ○C

38

303 GTC TCT GCC AAC ATG GAG ACT TTC CGC TGC AGA TGG AAT GTC GGC ACT tCC CAG AGC

CTC 362

39 V S A N M E T F R ○C R W N V G T S Q S L363 TCT GAG CCG GGA GCT CTC CGC TTA TTC TAC ATC AAC AAA AAA TCA CCT CAT GCT CCT CCC 422

59 S E P G A L R L F Y I N K K S P H A P P

78

423 AAA GAG TGG AGC GAG TGT CCT CAC TAC AGC ACC GAC AGG CCC AAT GAG TGC TTT TTC AAT 482

79 K E W S E ○C P H Y S T D R P N E ○C F F N

98

483 GAG AAC CAC ACA TCC ATC TGG ACG TCT TAC CTT GTC CAG CTC AGC TCG AGG GAT CAA GCC 542

99 E □N H T S I W T S Y L V Q L S S R D Q A 118

543 ATC CTC TAT GAT GAG AAC AGC TTC AAC GTT CAA GAC ATT GTG CAA CCA GAT CCT CCA TTT 602

119 I L Y D E N S F N V Q D I V Q P D P P F 138

603 GGT GTG AAC TGG ACG CTG CTG AAT GTG AGT TTG ACC GGC ACT CAC TAT GAC ATC ATT GTG 662

139 G V □N W T L L □N V S L T G T H Y D I I V 158

663 AAC TGG AAG CCG CCT CAG TCG GCA GAT GTG GAG ATG GGA TGG ATG AGG CTG CAG TAC GAG 722

159 N W K P P Q S A D V E M G W M R L Q Y E 178

723 GTC CAG TAC CGA GAA GTC AAC TCT GAC CTA TGG GAA GTG CTC GAC CTT GTG ACG AGC ACA 782

179 V Q Y R E V N S D L W E V L D L V T S T 198

783 TAT CGC TCC ATT TTT GGG CTT CAA ACT AAT GTC ATT CAC GAG GTT CGG GTC CGG TGC AAA 842

199 Y R S I F G L Q T N V I H E V R V R ○C K 218

843 ATG TTC GGT GGG AAA GAG TTC GGA GAG TTC AGC GAC TCT GTG TTT GTG CAC GTT CCA TCG 902

219 M F G G K E F G E F S D S V F V H V P S 238

903 AAA GTG TCG AGA TTC CCA GTG GTG GCC TTG CTC ATC TTC GGT GCC TTG TGT CTT GTA ACC 962

239 K V S R F P V V A L L I F G A L C L V T 258

963 ATC TTG ATG TTA GTC ATC GTA TCG CAG CAG GAA AAG TTG ATG GTG ATT CTT TTG CCT CCT 1022

259 I L M L V I V S Q Q E K L M V I L L P P 278

1023 GTT CCT GGA CCT AAA ATC AGA GGG ATT GAC CCT GAA CTA CTC AAG AAA GGG AAG CTG AGG 1082

279 V P G P K I R G I D P E L L K K G K L R 298

1083 GAG TTG ACA TCC ATC TTG GGC GGC CCC CCT GAT CTG AGG CCG GAG CTG TAC AAC AAC GAT 1142299 E L T S I L G G P P D L R P E L ◇Y N N D 318

1143 CCC TGG GTG GAA TTC ATC GAC CTG GAC ATC GAG GAG CAG AGT GAC AGA CTC ACA GAC CTG 1202

319 P W V E F I D L D I E E Q S D R L T D L 338

1203 GAC ACG GAT TGC CTC ATG GAG CGC TCT TTG TCT TCT AAC TGC TCC CCC CTG TCC ATC GGC 1262

339 D T D C L M E R S L S S N C S P L S I G 358

1263 TTC AGA GAC GAC GAC TCG GGC CGG GCC AGC TGC TGC GAC CCA GAT CTC CCC AGC GAC CCT 1322

359 F R D D D S G R A S C C D P D L P S D P 37

1323 GAA CCA TCC CCT TTC ATT CCT CTC ATC CCC AAT CAA ATC CAC AGT AAG GAA CCT GCA TGC 1382

379 E P S P F I P L I P N Q I H S K E P A C 398

1383 CTG ACA CCC TGC GAG CCG AAC TCC CCG GCC CAG AGC CCC ACA GCT GGG GAG CCT TTT TCT 1442

399 L T P C E P N S P A Q S P T A G E P F S 418

1443 GTA GCG CCA GGC AGA GAG GCA ATG TAC ACC AAG GTG AGT GAG GTG AGG TCA TCA GGC AAG 1502

419 V A P G R E A M ◇Y T K V S E V R S S G K

438

1503 GTA CTG CTG TCG CCG GAA GAG CAG ACT GAG GAA CCC ACT AGC AAA GAC ACA GAG AAA GAG 1562

439 V L L S P E E Q T E E P T S K D T E K E 458

1563 AAG ATG GCG GAG AAA GAG AAA GAA AAG AAA GAG TTT CAG CTC CTG GTG GTG AAT CCA GAA 1622

459 K M A E K E K E K K E F Q L L V V N P E 478

1623 CAT GGA GGT TAC ACC TCA GAG CTC AAC GCA GGA AAA ATG AGC CCA AGA TCG TCC TCA GGA 1682

479 H G G ◇Y T S E L N A G K M S P R S S S G

498

1683 GAC ATG AGT GAA CCC TGC CAG ACA GGA GGA GAC TCC CCT TAC CAC GAA TCA GAT CCC ACC 1742

499 D M S E P C Q T G G D S P ◇Y H E S D P T

518

1743 CCC ATG TCC CCT CTT TCC CCT GCA CCT GTC TAC ACC GTG GTC GAA GGT GTT GAC ATG CAG 1802

519 P M S P L S P A P V ◇Y T V V E G V D M Q

538

1803 AAC AGC CTC TTA CTT ACA CCA AAC TCA ACG CCT GCT CCC CAG CTG ATA ATC CCG AAAACC 1862

539 N S L L L T P N S T P A P Q L I I P K T

558

1863 GTG CCA ACA CCA GAC GGC TAC CTA ACC CCT GAC CCT TTG GGA AGC ATC ACA CCA

TAG ATC 1922

559 V P T P D G ◇Y L T P D P L G S I T P *

577

1923 AGC GCT GAC TCT TCA CAG CTA AGA CTA TTC TCA AAG GCA CAT AAA AGC TTG AAA

ACA GTC 1982

1983 TTG GAG ATC GAC TAT TGA GGG TCC TAT GGT TGA GTG TTG AAT ATC AGG TCT CCT AAA

ATA 2042

2043 GCT GTG TCT GTA CTG CCA CAA CCT CTG ACC TCC CGC TGC TCC CTC TGA CAG AGC TGG

AAG 2102

2103 TTT GGT ATG AGG GAG GAA GAG CAC ATC AGA CTT TGT TTC ACC GCA GTT GTG AAA

AGG GCA 2162

2163 AAG GGA AAC TTT ATC ATT CAT TTG GAG TCG TCT TCA ATG TTC ACT CGC CTG TAA CTC

CTC 2222

2223 TGG TTT GGT GGT CTG GTC CAT CAG CTC CTG AGG AAA ATC TCT GGC TTT TCA GCA GCT

TCA 2282

2283 CCG TCT TCA CCA ACT TGT CTC TGA GTT TGT GCA TCT GTC AAA AGA GGT GTT AAA GTT

CTG 2342

2343 TAG CGC TGC AGA GTT GGT GGT AAT TCC CTG CAG TTT TGT CAC TAT GAG TAA CCT CTT

CGA 2402

2403 CAT CAC AGG TAG TCA TTT CAT TCA GTG TTA AAA TAA GAG TAT TGG ATT TAA ATA AGA

GTG 2462

2463 CAT TTA ATT TAC TGT CGC ATG CTT CAT AAC GGT GAT TTT GTA CAT ATC AGA AGC TTC

AGG 2522

2523 TCA TCA TGA TAT ACT CTC GTC TTT TTA TGA ATC CAA CAT TGT TAT GTT TTT TCG TAA

CAT 2582

2583 TCC TTT TCA GGC TTT AAG ATA AAA ATG GCC ATG AGG AAA ATG TTA CTT GCC AAT CAG

CCT 22

23 TTA TTG GGG TAT TGC TGC CAA CAT ATG ATT AAA ATA AAG CAT GAG CTG TTC GCA TGT

AAG 2702

2703 AGA ATC AAA TGG AAC ATA CAA TAG TGT CTT ATT TTT GTT CTA AAT TGT GAA ACA TTT

TCA 2762

2763 GGG CTT ACA GCC AAA ATA ATT TGG ATT GCG AGT CAG TAT TTC TTT TTG AGT GCA TGG

ACT 2822

2823 ATT AAA GTT TGT ATG TTT TAG GTT GGT TTC TGG TTG ACA GTG CAT ATG ATA CAG ATT

TAA 2882

2883 AGT TGG GGG TAC ATG GTC ATC AGT GTT GAA TTT GTA CGG TTA TTG CAT TTT AAC TGT

TCA 2942

2943 GGT GTT ACA AAT AAA CAA CTT TCA TAC CTT TTA AAA AAA AAA AAA AA 29

Fig.3. Nucleotide sequences of orange-spotted grouper GHR2 and the deduced amino acid sequences.

Transmembrane region is shaded with dark. Putative signal peptide is underlined. Box1 and box2 motifs are

squared in an open rectangle. Conserved cysteine residues are denoted by a Ο around the amino acid. Potential

N-linked glycosylation sites are denoted by a around the amino acid. The FGEFS motif is in bold and

underlined with broken line. Conserved tyrosine residues are indicated by a around the amino acid. The stop

◇

sequence is denoted by *.

Northern blot analysis showed a single band of approximately 4.3Kb with gGHR1 probe from the liver RNA sample (Fig.4) and a single band of approximately 3.9Kb with gGHR2 probe from the liver and muscle RNA samples (Fig.4).

Fig.4. Northern blot analysis of the orange-spotted grouper GHRs precursor mRNA in various tissues. Total RNA were isolated from the liver (right), muscle (middle), ovary (left).A prominent single GHR1 transcript of 4.3 Kb in liver and GHR2 transcript of 3.9Kb in liver and white muscle were shown.

Using unrooted analysis with Clustalx 1.8 and Mega 2.0, phylogenetic tree via comparison of the amino acid sequences of gGHRs with other teleostean species was constructed (Fig.5). The amino acid sequences of other species were downloaded from GenBank database. It showed that the gGHRs were evolutionary more related to the Perciform species (seabream and tilapia) and Pleuronectiform species (flounder and turbot) but less related to four other fish groups, Cyprinid, Silurid, Salmonid and Anguillid.

Fig.5. Phylogenetic tree based upon the alignment of amino acid sequences of grouper growth hormone receptors and reported GHRs in other teleostean species. The tree was generated by ClustalX1.8 and Mega 2.0. Numbers indicate bootstrap values from 100 replications. The GHRs sequences used in analysis and their GenBank accession numbers are: orange-spotted grouper (Epinephelus coioides) GHR1, EF052273; orange-spotted grouper GHR2, EF052274; turbot (Scophthalmus maximusi) GHR, AF352396; Japanese flounder (Paralichthys olivaceus) GHR, AB058418; black seabream (Acanthopagrus schlegeli) GHR1, AF502071; black seabream GHR2,

AY662334; gilthead seabream (Sparus aurata) GHR1, AF438176; gilthead seabream GHR2, AY573601; rainbow trout (Oncorhynchus mykiss) GHR1, AY861675; rainbow trout GHR2, AY751531; Southern catfish (Silurus meridionalis) GHR1, AY336104; Southern catfish GHR2, AY973231; goldfish (Carassius auratus) GHR,

AF293417; grass carp (Ctenopharyngodon idella) GHR1, AY283778; coho salmon (Oncorhynchus kisutch) GHR1, AF403539; coho salmon GHR2, AF403540; bastard halibut (Paralichthys olivaceus) GHR, AB058418; cherry salmon (Oncorhynchus masou) GHR, AB071216; Japanese ell (Anguilla japonica) GHR1, AB180476; Japanese ell GHR2, AB180477; Mozambique tilapia (Oreochromis mossambicus) GHR1, AB115179; Nile tilapia (Oreochromis niloticus) GHR2, AY973233; Atlantic salmon (Salmo salar) GHR1, AY462105.

Tissue expression of gGHRs mRNA

RNAs from various tissues were extracted and reverse transcribed. The resulting cDNAs encoding gGHR1 and gGHR2 were quantified by Real-time RT-PCR (Fig.6), which enabled an assessment of the distribution of GHR mRNAs among tissues. Both gGHRs were found in all tissues tested; however, the amount of each gGHR mRNA varied by tissue. In the brain regions, the highest level of gGHR mRNA expression was observed in the pituitary for gGHR1 and in the pituitary, telencephalon and cerebellum for gGHR2 respectively，much lower levels of expression were detected in the cerebellum and hypothalamus for gGHR1 and in the hypothalamus for gGHR2 respectively. In the peripheral regions, expression was highest in the liver for both gGHRs, high levels were found in the white muscle and adipose tissue. The levels of two types of gGHRs were much lower, but detectable in other viscera tissues. Quantitation of gGHRs mRNAs by Real-time RT-PCR also allowed for comparison of the abundances of the two receptor mRNAs within tissues (Fig.6). Under normal physiological conditions, gGHR2 mRNA levels were

significantly higher than gGHR1 mRNA levels in most tissues tested, such as telencephalon,cerebellum, pituitary, heart and white muscle. By contrast, gGHR1 predominated in spinal cord and liver.

Fig.6. Expression of GHR1 and GHR2 mRNA in various tissues of orange-spotted grouper. mRNAs level were quantified by Real-time RT-PCR. Data are presented as mean±SEM. For a given a receptor type, groups with different letters are significantly (P<0.05) different; * indicates a significant (P<0.05) difference between receptors

types within the same tissue.

Discussion

Former studies have proved that two types of GHRs exist in one teleost species. Two distinct GHRs have been reported in gilthead seabream (Josep et al., 2003; Saera-Vila et al., 2005) and black seabream (Tse et al., 2003; Jiao et al., 2006). Two types of GHR possess different structural features and physiological functions.

The amino acid sequence deduced by gGHR1 cDNA contained 7 extracellular cysteines, which formed 3 disulfide bonds with C68, C78, C111, C121, C135 and C152, and the impaired cysteine (C244) could form disulfide linkage during GH-induced GH receptor dimerization(Zhang et al., 1999). It also conserved five potential extracellular N-glycosylation sites alike that reported in gilthead seabream (Josep et al., 2003), and the FGEFS motif for ligand-binding. In the intracellular domain, the gGHR1 has 9 tyrosine residues which provid docker sites for signaling molecules after phosphorylation (Wang et al., 1996) and Box 1 and Box 2 domains. gGHR2 lacked one pair of extracellular cysteines and three intracellular tyrosines that conserved in gGHR1. Two disulfide bonds could be formed between C38, C48, C84 and C95. The absence of one disulfide bond in GHR2 would result in less astriction in structure, and the decrease of some intracellular tyrosines, which were related to STAT phosphorylation in signal transduction (Hansen et al., 1997; Herrington et al., 2000), might induce low efficiency of signal transduction. For the identical structural features, some authors suggested the GHR2 as salmonid-like GHR.

Interestingly, recent research in Japanese Medaka (Oryzias latipes) suggested that most of the genes reported as GH receptor from other fish species (especially the non-salmonid fishes) might be, at least phylogenetically, receptors for somatolactin (SLRs) (Fukamachi et al., 2005). Comparing amino acid sequence of gGHRs with some fish SLRs, we found that the new cloned gGHR1 had high amino acid identity of 73.3% to the SLR of Japanese Medaka and similar sequence features to the SLR of Japanese medaka of 7 extracellular cysteines and 8 intracellular tyrosine residues except the absence of the first tyrosine residue in medaka SLR (Fig.7), while the gGHR2 had low identity of 40.4%, and it was similar to the comparisons between SLRs and GHRs in other non-salmonid fishes (Fukamachi et al., 2005). It supported the orthologous relationship between SLR and non-salmonid GHR (GHR1) (Fukamachi et al., 2005). But the physiological functional homology between GHR1 and SLR remained to be further investigated. Orange-spotted grouper GHR1 (1)

MCA VFA VFLPSLRLRNNIMTVSSSSSNVVVLLLISSLDWLSTPGSAFLMGRDHVTSPAP- 59

Gilthead seabream GHR1 (2)

----MA VFSSSSSSSSSSSSSSSSTSNLLLLLLVSSLDWLSTRGSVFVM--DHMTSSAP- 53

Japanese medaka SLR (3)

-------------------MADAFSPGLLLMLMMSTLDWLSTPGSAFLFDRDRAKS-AP- 39

Cherry salmon SLR (4) ----------------------MAAPSFLLLFLLG---LLSA VRSSSLMDPGSMTSSDPS

35

Orange-spotted grouperGHR2 (5) ----------------------MAAAFTMLFFFLH---IFTASALES-ASEQVLPD---- 30 Gilthead seabream GHR2 (6) ----------------------MAAALT-LLFCLY---ILTSSALES-ASEQVHPQ---- 29 Coho salmon GHR1 (7) ----------------------MAISHILFICLVL---ILPVLSQEPPTSKQALFQ---- 31 Coho salmon GHR2 (8) ----------------------MATSRILFICLLL---ILTVVSQEPPTSEQALPQ---- 31

1

-VGPHITECISRDLETFRCWWSPGDFHNLSSPGALRVFYLKKNL---PTSEWKECPEYLHSNR-ECFFDGNHT SVWVPYCMQLRGQNN 142

2

-VGPHFTECISREQETFR C WWSPGGFHNLSSPGALRVFYLKKDS---PNSEWKECPEYSHLKR-ECFFDVNHT SVWIPYCMQLRGQNN 136

3

-LEPHFTECVSRDLETFRCWWSPGSFQNLSSPGALRVFYLKKDP---PLSEWKECPEYNHLNR-ECFFDTNHT SVWLPYCVQLRSQNN 122

4

VQAPHLTGCKSREQETFRCWWSPGSFQNLTEPGALQIQYWKKND---LTKEWKECPDYSSSVKNECFFNKN NTVIWIKYCVRLHSESQ 119

5

-AHPHLTGCVSANMETFRCRWNVGTSQSLSEPGALRLFYINKKSPHAPPKEWSECPHYSTDRPNECFFNEN HTSIWTSYLVQLSSRDQ 1166

-RDPHLTGCVSANMETFRCRWNVGTLQNLSKPGELRLFYINKLSPLDPPKEWTECPHYSIDRPNECFFNKN HTSVWTPYKVQLRSRDE 115

7

-IRPQITGCVSHDMNTFRCRWNVGVFQNLTEPRDLRIFYYINDRNISP-KEWGECPRY-ADRTNECFFNESYT KVWMTYSVQLRSGDQ 115

8

-IRPQITGCVSHDMNTFRCRWNVGTFQNLTEPRDLRMFYYINNKNISP-KEWSECPNYMADRIDECFFNESY TKVWITYSVQLRSGDQ 116

* * * * **** * * * * * * ** *** * **** * * * *

1 ITYFNEDDCFTVENIVRPDPPVSLNWTLLNISPSGLSYDVMVNWEPPPSADVGAGWMRIEYEIQYRERNTT NWEALEMQPHTLRTIYG 230

2 VTYLDEDYCFTVENIVRPDPPVSLNWTLLNISPSGLSYDVMVNWEPPPSADVGAGWMRIEYEIQYTERNT TNWEALEMQPHTQQTIYG 224

3 VTYFNEEDCFTVENIVRPDPPVSLNWTLLNVSPSGLSYDVMVSWEPPPSADVQAGWMRIEYEVQYRERN TTNWEALEMQPHTQQTIYG 210

4

NKTYDT-LCFELQDIVHPDPPV ALSWTLLNISRSGLNYDIMASWEPPPSADVSVGWLTLVYEVQYRRRNSS HWKVLEHEFGTQQSIYG 206

5

AILYDE-NSFNVQDIVQPDPPFGVNWTLLNVSLTGTHYDIIVNWKPPQSADVEMGWMRLQYEVQYREVNS DLWEVLDLVTSTYRSIFG 203

6

STLYDE-NTFTVDAIVQPDPPVDLTWTTLNESLSGTYYDIILSWKPPQSADV AMGWMTLQYEVQYRSASSD LWHA VEPVTVTQRSLFG 202

7

DILYXE-VIFTVEDIVEPDPPIALNWTLLNVGLTGSHFDIMLSWEPPHSADVSMGWMTLQYEVQYREVNST LWRTVDLEKGMQRSLYG 202

8

DNLYDE-VIFTVEDIVEPDPPIALNWTLLNVGLTGSHFDIMVSWEPPHSADVSMGWMTLQYEVQYREVNS TLWRMVNLEKGRQRSLYG 203

* ** **** ** ** * * * ** **** ** ** ** * *

1 LHIGKEYEVHIRCRMQAFTKFGEFSDSIFIQVTEIPSKESTFPLTLVLIFGIVGILILIMLIVVSQQHRLMMILLP PVPAPKIKGIDP 318

2 LQIGKEYEVHIRCRMQAFVKFGEFSDSVFIQVTEIPSQDSNFPFKLALIFGVLGILILILLIGISQQPRLMMILL PPVPAPKIKGIDP 312

3

LQIGKEYEVHIRCRMQAFTK FGEF SDSIFMQVTEIPSTDSTAPLTLVLIFGTVGILIVIMLIVISQQQRLMMIL LPPVPAPKIKGIDS 298

4

LQTGEAYEVRAHCAMRAFNNFGEFSDVIFVHVPEIPNKESTFPVTLVLIFGA VGV AILLMLIIFSQQQRLMVI LLPPVPAPKIKGIDP 294

5

LQTNVIHEVRVRCKMFGGKEFGEFSDSVFVH---VPSKVSRFPVV ALLIFGALCLVTILMLVIVSQQEKLMVI LLPPVPGPKIRGIDP 288

6

LKHNVNHEVRVRCKMLAGKEFGEFSDSIFVH---IPAKVSSFPVV ALLLFGALCLV AILMLVIISQQEKLMFILLPPVPGPKIRGIDP 287

7

LRSNTDNEVRVRCKTLASRNFGEFSDSIFIH---IPTKESRLPVTVLLVFAALGLA VILMLVIYSQQQKLMVILL PPIPGPKIKGIDP 287

8

LRTNTDNEVRVRCKTLASRNFGEFSDPIVIH---IPTKESRFPVTVLLVFAALGLA VILILVIYSQQQKLMMILL PPIPGPKIKGIDP 288

* ** * ****** * * * * * * *** ** ***** * *** ***

1 ELLKKGKLDELNFILSGGGMSGLPTYAPDFYQDEPWVEFIEVDAEDADTGEKEDNQGSDTQRLLSLSQPV SHHMNIGCSNA VSFPDDD 406

2 ELLKKGKLDELNFILSGGGMGGLSTYAPDFYQDEPWVEFIEVDAEDADAAEKEENQGSDTQRLLDPPQPV SHHMNTGCANA VSFPDDD 400

3 DLLKKGKLEELNFILNGGSIGGLQTFSPDFYQDEPWVEFIEVDEEDADSREKEDDQASDTQKLLGPPQPIS DHRTIRCSETIRHPEEV 386

4

ALLKKGKLDELNFILSGAGMGALHSYPPDLYQDVPWVEFIELDADEPEPGEK-DNQSSDLQRLLGHN---N HHTNHGCFHGLSIPDDD 382

5

ELLKKGKLRELTSILGG-----PPDLRPELYNNDPWVEFIDLDIEEQSDRLT----DLDTDCLMERS-----LSSNCSP LSIGFRDDD 362

6

ELLKKGKLRELTSILGG-----PPNLRPELYNNDPWVEFIDLDIEEQSDKLT----DLDTDCLMHRS-----LSSNCTP VSIGFRDDD 361

7

ELFKKGKLAELTSILGG-----IPDLRPDLYSDDPWVEFIELDMEEPNDRLT----VLDTQCLMDHC-----ASSDCP PITIGFRDDD 361

8

ELFKKGKLAELTSILGD-----HPDLRPELYGEDPWVEFIELDMEEPNDRLT----ELDTQCLMDRS-----PSSDCPP LTIGFGDDD 362

* ***** ** ** * * ****** * * *

1

SGRASCYDPDLPDQDTLMLMATLLPGQPEDGE-----------ASFDVVER------APAP--ERGERPLVQTQTGGPQ TWVN---TD 472

2

SGRASCYDPDLHDQDTLMLMATLLPGQPEDGE-----------DSFDVVER------APVI--ERSERPLVQTQTGGPQ TWLN---TD 466

3

FGHGSCYNADRPEEDSKVQMDAPPPGQAGDKE-----------SSTEFAER------TPAL--DRCRAPPVQTQTGTPQ TWVN---TD 452

4 SGRASSYDPELPDQETPMLMAALLSCQPNKVEPCLGNCSHSSTPSLDVLEVPCPGLQAPDLPPEGGERHLV QTQLGGPQSWVN---MD 467

5

SGRASCCDPDLPSDPEPSPFIPLIPNQIHSKEP----------ACLTPCEP--------------N—SPAQSPTAGEPFSV APGRE A 424

6

SGRASCCDPDLPSDPEASPFHPLIPNQTLSKEV----------SCQTASEP--------------S—SPVQSPASGEPPFAALG REA 423

7

SGRASCCDPDLH-DPEA-PFHSLLPNTSHALEP----------SCLASTKA--------------S—SPVQTPTTEDSPWAAPG RED 421

8

SGRASCCDPDLP-EPEAFPFHPLLSNTSPSLVP----------SG-ASTEA--------------S—SPVQTPTTGMILWA VPGR ED 423

* * *

1

FYAQVSNVMPSGGVVLSPGQQLRIQENTLATEEETQKKGKEHK-GNEDTEENKQKELQFQLLVVDPEGSG YTTESNARQISTPPSSPM 559

2

FYAQVSNVMPSGGVVLSPGQQLRFQESTSAAEDEAQKKGK----GSEDSEEKTQKELQFQLLVVDPEGSGY TTESNARQISTPPSTPM 550

3

FYAQVSNVMPSGGVVLSPGQQLRTQESTPASEHGAQTKGKQRD-ESGDMDGQRQKEPQFHLLVVDPEGS GYTTESNCWQNSTPSNSPN 539

4 FYAQVSDVTPTGGVVLSPGQQVGAPENTPTTKKDKKGKMEGLEEESEEEEERRTDKLMFQLLVV APEAG GYTTESSGRQMSTPDPS-T 554

5

MYTKVSEVRSSGKVLLSPEEQTEEP----TSKDTEKEKMA----------EKEKEKKEFQLLVVNPEHGGYTSELNA GKMSPRSSS-- 496

6

MYTQVSEVRSSGKVLLSPEEQTEVEK--TTGKDTEKDIMA----------EKEKAKKEFQLLVVNADHGGYTSEL NAGKMSPRLSI-- 497

7

LYTQVNEVGPTGEVLLTNEEQRNVEE---NSEKDEKENE------------KEKKKKEFQLLVVNADVGGYTSELD AGKMSARLPT-- 493

8

LYTQVSEVRPTGEVLLMPEEQSKVEK---DAEEKAKEEMG-----------KKKPRKEFQLLVVNADRGGYTTELD AGKMSAKLPT-- 495

* * * * * * * * **** *** * *

1

PGEGYQTIHPQPVETKPTPTAE--------DNQSPYILPDSPQ------SQFFAPV ADY-TVVQEVDSQHSLLLNPPPRQ SPPPCVPQ 632

2

PGSGYQTIHPQPVETKPAATAE--------NNQSPYILPDSPQ------SQFFAPV ADY-TVVQEVDSQHSLLLNPPPRQ SPPPCLPH 623

3

PGAGYQTIPPPPVDIKPAASAD--------ANQLPYILPDCP--------QLV APV ADY-TVVQELDTHHSLLLDPTPIQS PPPCPSQ 610

4

PGEVYHTFPPPSAESKPHQEAYLPATTLLGDYQSPYILPDSPP------AQFLPRVFGLPLWVQDVDFQHSLLLN PPSPQRSPTCSPQ 636

5

---GDMSEPCQTG------GD------------SPYHESDP------TPMSPLSPAPVY-TVVEGVDMQNSLLLTPNSTPAPQLI IPK 557

6

---GDQSEPGLTGDLSPLPPA------------SPYHESDT------TA VSPLPPAPVY-TVVEGVDRQNSLLLTPNSTPAPQL IIPK 5

7

---GRASQPAPTEDSSLVQGQPF------GEYQSLYFEAEMPPIPPASPVSPLPPVSAY-TMVEGVGRQNSLLLKPG PTPAPQPVLTK 4568

---GRGSQPAPTEDSRLVQGQPF------GEYQSLYFEAEMPPIPPTSPVSPLLPLSVY-TMVEGVDRQNSLLLKPS PPPAPHPVLTK 544

* * **** *

1 HPLKALPAMPVGYITPDLLGNLSP 656

2 HPTKALAAMPVGYVTPDLLGNLSP 7

3 PPLKTQ--IPVDYITPDLLGNLLP 632

4 PPSKPLPVMPIGYLTPDLLGNLSP 660

5 T---VP--TPDGYLTPDPLGSITP 576

6 T---MP--TPGGYLTPDLLGSITP 583

7 LPLPTP--TPEGYLTPDLLGNITP 593

8 LPLPTP--TPEGYLTPDLLGNITP 595

* * *** ** *

Fig.7. Multiple alignment of some teleost SLRs and GHRs. Identical amino acid sequences are indicated by asterisks under the sequences. Conserved cysteine residues and tyrosine residues are shaded with dark. The FGEFS

motifs are in bolds and italics

Phylogenetic tree analysis showed that GHRs in teleost fish could be segregated into two clades. The GHRs of non-salmonid fishes belonged to GHR1 or GHR2 clade, but the two GHRs of all salmonids tested (Coho salmon, Atlantic salmon and Rainbow trout) belonged to the GHR2 clade (Fig.5). It suggested that the GHRs reported in salmonids should be the different isoforms or splicings of GHR2. However, no different isoform or splicing of two gGHRs was found by the northern blot analysis.

Evolution by Gene Duplication (Ohno, 1970)——the old hypothesis had been proved and accepted. The vertebrates got evolution by the whole genome duplications but the fishes (especially the teleost) had undergone an additional duplication as the ancient fish-specific genome duplication hypothesis (Wittbrodt, et al., 1998), and more than one GHR genes were maintained by selection through the radiation in the teleosts. On the other hand, ancestral existence and consequent loss of GHR in the higher vertebrates seems likely (Fukamachi et al., 2005). So the concurrence of two GHRs existed only in some teleost species. It seemed that GHR1 is the retained form through the evolution of all vertebrate species. However, whether the GHR1 existed in salmonids needed more evidence and further reassessments.

Phylogenetic analysis and homology analysis further indicated that the gGHRs were evolutionary more related to the Perciform species and Pleuronectiform species. gGHR1 showed high homology of 80% approximately to Japanese flounder GHR，turbot GHR，black seabream GHR1，Nile tilapia (Oreochromis niloticus) GHR1 and gilthead seabream GHR1 (Table 2), and the gGHR2 had higher identities to black seabream GHR2 (79.2%), gilthead seabream GHR2 (80.4%) and Nile tilapia GHR2 (71.8%). The results were similar to those found in grouper GH (Li et al., 2005), which accorded with the co-evolution of ligand and receptor.

gGHR1 and gGHR2 were expressed in all tissues tested. Two types of gGHRs were present at the highest level in some regions of the brain and the liver. The high level of gGHRs in the brain might relate to the various roles of GH in the central nervous system, and the situation that liver is the most important tissue for binding of GHR to GH and signal transduction could explain the high abundance of gGHRs in the liver. The expression of gGHR2 is significantly higher than gGHR1 in most tissues such as telencephalon, cerebellum, pituitary, heart and white muscle. It is similar to the situation reported in black seabream (Jiao et al., 2006). However, the expression of gGHR1 is significantly higher than gGHR2’s in the liver. The higher expression of gGHR1 in liver might be relative to the physiological function of GHR1, which is more functional effective in forming linkage with GH and signal transduction. The higher expression of gGHR2 in most tissues tested might serve to the modulating to gGHR1. As former research reported, one type of receptor could modulate another related type of receptor (Zhang et al., 2000). We postulated that the gGHR2 could be a modulator to gGHR1. For the absence of one disulfide bond, the structure of gGHR2 is less astricted and it could modulate the GH-GHR linkage and gGHR1’s dimerization and signal transduction. In addition, GHR represents different variants including the growth hormone-binding protein (GHBP) or truncated membrane-anchored forms in many higher vertebrate species (Edens and Talamantes, 1998). The truncated GHR isoform can compete for GH binding with the full-length receptor (Ross, 1999) and some GHR could be cleaved to form the GHBP. Therefore, gGHR2 may play the same roles as the variant GHR isoforms do in higher vertebrate species, which modulate the function of the full-length receptor, inhibit signaling, and generate large amounts of GHBP or regulate the production of GHBP (Ross et al., 1997).

In conclusion, different from the situation that only one GHR exists in the high vertebrate,

there are two types of GHRs existing in orange-spotted grouper. Two gGHRs have differentstructural features and physiological functions. gGHR1 has high binding and signaling effect and gGHR2 might modulate the gGHR1 in ligand-binding and signal transduction as the variant GHR isoforms did in higher vertebrate species. However, the general functional differences between gGHR1 and gGHR2 remain further research.

Acknowledgements

This work was supported by the National Marine 863 Projects of China (No. 2001AA621010), the National Natural Science Foundation of China (No. 39970586), Project of Scientific and Technological scheme of Guangdong Province (No. 2001AA621010) and Specialized Research Fund for the Doctoral Program, Ministry of Education, China (No. 20040558012). Thanks to Ma, X.L., Liu, Y. for the assistance.

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