Evolution of Receptors for Growth Hormone and Somatolactin in Fish and Land Vertebrates: Lessons from the Lungfish and Sturgeon Orthologues

Shoji Fukamachi Æ Axel Meyer

Abstract Two cognate hormones, growth hormone (GH) Keywords Somatolactin receptor (SLR) and somatolactin (SL), control several important physio- Growth hormone receptor (GHR) Lungfish Sturgeon logical processes in vertebrates. Knowledge about GH and Fish-specific genome duplication (FSGD) Synteny its receptor (GHR) has accumulated over the last decades. Sub-functionalization However, much less is known about SL and its receptor (SLR). SL is found only in fish (including lungfish), sug- gesting that it was present in the common ancestor of Introduction vertebrates, but was lost secondarily in the lineage leading to land vertebrates after the lungfish branched off. SLR was Somatolactin (SL) is a hormone that belongs to the growth suggested to be a duplicated copy of GHR acquired only in hormone (GH)/prolactin (PRL) family, and is either up- or teleosts via the fish-specific genome duplication (FSGD). down-regulated during various important physiological This scenario (i.e., the existence of SL but not SLR in the activities in fish (Fukada et al. 2005 and references vertebrate ancestors) is intriguing but contested. In this therein). Although direct evidence for the role of SL in study, we first evaluated the plausibility of this scenario these activities is mostly lacking, the recent identification through synteny analyses and found that the loci for GHR of a SL-deficient mutant in medaka (color interfere, ci) and SLR are located in syntenic genomic positions, whereas demonstrated that SL functions in body-color regulation, the loci for GH and SL are not. Next, we cloned GHRsof lipid metabolism, or cortisol secretion in vivo (Fukamachi lungfish and sturgeon, which possess SL but did not et al. 2004, 2005, 2006). Further analyses of the ci phe- undergo the FSGD (i.e., they should not possess SLR). notypes might provide evidence for other potential Their phylogenetic positions in the GHR/SLR tree functions since the SL receptor (SLR) is rather broadly further support the fish-specific scenario for the GHR SLR expressed in various organs (Fukada et al. 2005; Saera-Vila duplication. Interestingly, their sequences share greater et al. 2005; Fukamachi et al. 2005; Jiao et al. 2006; Ozaki similarity with teleost SLRs and reptilian/amphibian GHRs et al. 2006). than with the GHRs of mammals, birds, and teleosts. On The current nomenclature of the fish SLR is controver- the basis of these results, we discuss the validity of the sial and confusing, as is often the case with gene families nomenclature of the teleost-specific copy of GHR as SLR that resulted from complicated gene duplications (Mindell and an ancestral receptor(s) for SL before the evolution of and Meyer 2001). The confusion arose because SLR, which SLR during the FSGD. was first defined in salmon (Fukada et al. 2005), is orthologous to a subset of growth hormone receptors (GHRs, particularly GHR1) of other teleosts (Fukamachi et al. 2005). Several teleost species have two divergent GHR-like that exibit distinctive residual and struc- & S. Fukamachi ( ) A. Meyer tural differences, and whose sequences are less than 40% Lehrstuhl fu¨r Zoologie und Evolutionsbiologie, Department of Biology, University of Konstanz, D 78457 Konstanz, Germany identical. In addition, their in vitro characteristics were e mail: Shoji.Fukamachi@uni konstanz.de shown to be different in salmon (Fukada et al. 2004, 2005); 360 i.e., one of the GHRs binds GH, while the other predom- Materials and Methods inantly binds SL (in this manuscript, we call the former GHR and the latter SLR). Nevertheless, the teleost GHR Synteny and SLR are often assumed to be duplicated copies of an ancestral GHR (Saera-Vila et al. 2005; Jiao et al. 2006; Genomic sequences containing GHR, SLR, GH,orSL, were Ozaki et al. 2006) that were acquired through the fish- retrieved from the following databases: National Center for specific genome duplication (FSGD). Biotechnology Information (NCBI; , http://www. The FSGD, which occurred in the lineage leading to ncbi.nlm.nih.gov/genome/guide/human/), University of modern (teleost) fishes (Hoegg and Meyer 2005; Meyer Tokyo Genome Browser (UTGB; medaka, http://www. and Van de Peer 2005), produced many teleost-specific medaka.utgenome.org/, version 1.0), Joint Genome Insti- duplicated genes (Meyer and Schartl 1999; Postlethwait tute (JGI; fugu, http://www.genome.jgi-psf.org/ et al. 2004; Mitani et al. 2006; and references therein). Takru4/Takru4.home.html, version 4.0), and Ensembl However, the evolutionary scenario, that SLR is a teleost- (zebrafish, http://www.ensembl.org/Danio rerio/index. specific paralogue of GHR, remains an open question. This html, version 6.0). Annotation of human is because lungfish and sturgeon, which did not experience followed that of the NCBI database. We manually rean- the FSGD, and hence should not possess duplicated copies notated other teleost sequences by blastx, and the human of GHR (i.e., SLR), indeed do have SL (Amemiya et al. gene with the lowest e-value was used for the annotation. 1999; May et al. 1999). These findings suggest that the SL In cases where multiple human genes showed similarly low gene already existed in the common ancestor of verte- e-values (e.g., genes in a gene family), all were included. brates, but that SLR was later acquired via the FSGD only We ignored any transposase-like or reverse transcriptase- in teleosts (Fig. 1). like sequences, which represented repetitive sequences. In this study, we used available synteny data to first assess whether or not the teleost SLR is really a duplicated copy of GHR. We then cloned GHRs of lungfish and Fish sturgeon. Because both of these fish are expected to possess the ancestral hormone receptor set (i.e., GH, SL, and one We used lungfish (Protopterus dolloi) and sturgeon (Huso copy of GHR), which is different from that of tetrapods dauricus) samples stored at 80°C in the laboratory. We (i.e., GH and one copy of GHR) or teleosts (i.e., GH, SL, confirmed the species’ identity by sequencing fragments of and two copies of GHR), their GHR sequences might have their 16S rRNA using universal primers (50-CGCCTG unique features, and could be helpful for characterizing the TTTAACAAAAACAT-30 and 50-CCGGTCTGAACTCA evolutionary history of the receptor(s) for SL. GATCACGT-30) and constructing phylogenetic trees with corresponding sequences available on the GenBank data- base (data not shown). ??? SLR SL GHR Cloning GH Tetrapods Lungfish Sturgeon Teleosts We designed four degenerate primers for nested reverse- transcriptase polymerase chain reactions (RT-PCR) on the Loss of SL The FSGD || regions relatively conserved among vertebrate GHR/SLRs. Acquisition of SLR? Primer sequences for the first PCR are f: 50-ATH- GTNCARCCNGAYCCNCC-30 and r: 50-ARYTCDATRA AYTCNACCCA-30, and those for the second PCR are f: 50- 0 0 Presence of SL without SLR? YTNAAYTGGACNYTNYTNAA-3 and r: 5 -TCDATNC CYTTDATYTTNGG-30. Total RNA was extracted from Fig. 1 The hypothesis addressed in this study. Phylogenetic rela frozen muscle samples using TRIZOL (Invitrogen), and tionships among tetrapods, lungfish, sturgeon, and teleosts are shown. template cDNA was synthesized by SuperScript III reverse GH exists in all lineages. GHR has been identified in tetrapods and 0 0 teleosts. SL has been identified so far only in the fish lineages, transcriptase (Invitrogen) using 3 (5 -ATTCTAGAGGCC indicating that SL should have been secondarily lost from land GAGGCGGCCGACATGTTTTTTTTTTTTTTTTTVN-30) vertebrates (arrow on the left). To date, SLR has been found only in and 50 (50-AAGCAGTGGTATCAACGCAGAGTGGCCA teleosts and may be a duplicated copy of GHR acquired via the FSGD TTATGGCCGGG-30) adaptors. RT-PCR parameters were (arrow on the right). This evolutionary scenario, however, assumes a hormone receptor set of GH, SL, and GHR, but not SLR in some fish 94°C for 1 min; 40 cycles of 98°C for 20 s, 40°C for 1 min, lineages and the common vertebrate ancestor (indicated by red lines) 72°C for 1 min; with a final extension at 72°C for 10 min. 361

Amplified bands were excised from an agarose gel, purified undetermined amino acid (X) as a query, three Xs con- using HiBind DNA column (Peqlab), and directly tained within signal peptides of the opossum GHR sequenced using the BigDye Terminator verson 3.1 cycle (NM 001032976) had to be removed prior to this analysis. sequencing kit (Applied Biosystems) on an ABI PRISM1 For the same reason, we could not use coho salmon GHR1 3100 genetic analyzer (Applied Biosystems). (AF403539), since it contains an X in the middle of the To obtain 50 and 30 flanking regions of the cDNAs, we extracellular domain. performed rapid amplification of cDNA ends (RACE) using 50 or 30 primers complementary to the adaptors (50- AAGCAGTGGTATCAACGCAGAGT-30 or 50-ATTCTA Genomic Structure GAGGCCGAGGCGGC-30, respectively) and species-spe- cific nested primers, designed according to the cDNA Genomic DNA of lungfish and sturgeon were extracted sequences obtained above (data not shown). PCR param- from muscle tissue by phenol-chloroform extraction. eters were 94°C for 1 min; 30 cycles of 98°C for 20 s, 60°C Genomic DNA of eel (Anguilla japonica) was kindly for 1 min, 72°C for 3 min; with a final extension at 72°C provided from elsewhere (see the ‘‘Acknowledgements’’). for 10 min. We used TaKaRa LA Taq polymerases (Ta- Species-specific primers were designed to sandwich the kara) for the reactions and reproducible bands were region where the SLR-specific intron presumably exists, identified, excised, and directly sequenced as described and were used for both PCR with template genomic DNA above. We designed additional species-specific primers and RT-PCR. Products were run on 1% agarose gels, that amplified overlapping regions of the entire cDNAs and stained by ethidium bromide, photographed on an ultravi- verified that their sequences were consistent with the olet (UV) transilluminator, and then sequenced. results from the degenerate RT PCR and RACE experiments. Results

Phylogeny Reconstruction SLR is a Teleost-Specific Paralogue of GHR, Whereas SL is Not a Teleost-Specific Paralogue of GH Amino acid sequences of GHR, SLR and PRL receptor (PRLR) of vertebrates were retrieved from the GenBank By taking advantage of the available whole or draft gen- and aligned by ClustalW (http://www.ebi.ac.uk/clustalw/). ome sequences, we investigated genes surrounding the We used Phyml (Guindon and Gascuel 2003; GHR and SLR loci of vertebrates to determine if synteny is http://www.atgc.lirmm.fr/phyml/) and MrBayes (Ronquist conserved. Using a medaka GHR sequence (GenBank and Huelsenbeck 2003; http://www.mrbayes.csit.fsu.edu/) accession number DQ010539) on scaffold (Scf) 627, we software under the JTT+I+G+F substitution model fol- found (by tblastx) that fugu and zebrafish have orthologues lowing the results from ProtTest (Abascal et al. 2005; on Scf 128 and (Chr) 21, respectively. Using http://www.darwin.uvigo.es/software/prottest.html) and the medaka SLR sequence (DQ002886) on Scf 74, we ModelGenerator (Keane et al. 2006; http://www.bioinf. found orthologues on Scf 11 of fugu and Chr 8 of zebrafish. may.ie/software/modelgenerator/). All of these scaffolds/chromosomes contain sequences similar to human genes located on Chr 5p12 13.1 (Fig. 2). This well-conserved synteny suggests that not only the Structure Prediction GHRs, but also the SLRs of medaka, fugu, and zebrafish, are orthologous to human GHR. Therefore, the teleost SLR We used the SOSUIsignal program (Gomi et al. 2004; was likely acquired through a duplication of GHR. Con- http://www.bp.nuap.nagoya-u.ac.jp/sosui/sosuisignal/ sidering that this duplication involved a rather large sosuisignal submit.html) and NetNGlyc 1.0 server (http:// chromosomal region (note that synteny of [ 2Mbofthe www.cbs.dtu.dk/services/NetNGlyc/) to predict N-terminal human chromosome is conserved), the FSGD would most signal peptides, transmembrane regions, and potential N- likely be the cause of this GHR duplication (Fig. 4). glycosylation sites. To predict secondary structures, we We similarly analyzed synteny around the SL and GH used the New Joint method (Ito et al. 1997; loci. GHs of fugu (U63807), medaka (AF134606), and http://www.cbrc.jp/papia-cgi/ssp query.pl?query=seq). As zebrafish (AJ937858) are located on Scf 7980, Scf 697, and the New Joint server does not allow protein sequences Chr 3, respectively. Although the fugu scaffold was not exceeding 500 amino acids as a query, we manually sufficiently long for this analysis, both the medaka and trimmed excess C-terminal amino acids before submitting zebrafish GH loci showed conserved synteny with the sequences. Since it also does not permit a sequence with human GH cluster locus located on Chr 17q23-25 (Fig. 3). 362

RNF180 5q12 2 HTR1A R7BP GADD45G EFNA5 5q11 2 q13 5q12 3 C6 C7 OXCT1 C6 OXCT1 GHR MGC42105 9q22 1 q22 2 5q21 Chr.21 18 5 M 18 1 M GADD45G/GADD45B RNF180 R7BP 9q22 1 q22 2/19p13 3 5q12 2 5q12 3 C6 C7 GHR MGC42105 Medaka Scf.627 145 k 100 k 0 k GADD45G/GADD45B DERL2/DERL3 SMARCB1 R7BP 9q22 1 q22 2/19p13 3 EFNA5/EFNA2/EFNA3 17/22q11 23 22q11 5q12 3 C6 C7 GHR MGC42105 5q21/19p13 3/1q21 q22 Fugu Scf.128 600k 550 k 640 k LOC646887 LOC153684 LOC653791 FLJ21657 LOC643977 LOC389289 LOC644010 MGC42105 LOC646916 LOC644116 OSRF RPL37 CARD6 LOC646824 LOC285636 LOC402213 FLJ10246 HMGCS1 FLJ32363 NNT PTGER4 PRKAA1 C7 FLJ40243 C6 PLCXD3 TCP1L2 OXCT1 FBXO4 GHR SEPP1 ZNF131 CCL28 PAIP1 LOC283412 Human Chr.5 42 0M 43 8 M 40 7M 41 0M 43 0M HTR1A RNF180 R7BP GADD45G/GADD45B 5q11 2 q13 5q12 2 5q12 3 C7 PLCXD3 OXCT1 LOC285636 FBXO4 SLR SEPP1 ZNF131 MGC42105 9q22 1 q22 2/19p13 3 Fugu Scf.11 1 78 M 1 91 M 1 80 M CREB3L3 HTR1A RNF180 R7BP 19p13 3 GADD45G/GADD45B 5q11 2 q13 5q12 2 5q12 3 C7 PLCXD3 OXCT1 LOC285636 FBXO4 SLR SEPP1 ZNF131 MGC42105 9q22 1 q22 2/19p13 3 Medaka Scf.74 1 30 M 1 45 M 1 23 M 1 40 M

R7BP RNF180 5q12 3 NP 5q12 2 C7 PLCXD3 OXCT1 LOC285636 FBXO4 SLR SEPP1 ZNF131 14q13 1 Zebrafish Chr.8 38 5 M 39 0 M 38 4 M

Fig. 2 The conserved synteny between the GHR and SLR loci. chromosomes (if they are nonsyntenic) are indicated on top. Horizontal bars represent partial scaffolds/chromosomes whose Arrowheads indicate 50 30 orientations of each gene. Note that positions are indicated below (bp). Species names and scaffold/ multiple genes surrounding the human GHR locus (C7, C6, PLCXD3, chromosome numbers are indicated on the left. Colored boxes in the OXCT1, LOC285636, FBXO4, SEPP1, ZNF131, and MGC42105) are bars indicate genes we manually annotated (see the ‘‘Materials and found in similar positions/orientations around both the GHR and SLR Methods’’ section); yellow, GHR; green, SLR; blue, syntenic; and loci of these teleost genomes grey, nonsyntenic genes. Gene names and their locations on human

Medaka SL (AY530202) and its fugu orthologue (predicted) We constructed degenerate primers at regions con- are located on Scf 186 and Scf 131, respectively. Zebrafish served in both GHRs and SLRs of vertebrates (in order to SL (SLa; AY376857) is located on Chr 18. Their flanking amplify both, if they exist) and performed nested RT-PCR regions commonly contain sequences similar to human using muscle cDNAs as templates. Muscles are one of the genes on Chr 11q24 (instead of Chr 17q23-25), where no organs in which both GHR and SLR are most strongly apparent trace of SL can be identified (a missing ohnologue; transcribed in at least some teleosts (Fukada et al. 2004, Postlethwait 2006). Importantly, another copy of zebrafish 2005; Saera-Vila et al. 2005; Fukamachi et al. 2005). SL (SLb; AY221126) is located on Chr 10 and its flanking Direct sequencing of the amplified products using regions also contain sequences similar to human genes on degenerate primers resulted in homologous gene sequen- Chr 11q24. These observations support orthology relation- ces in both the lungfish and sturgeon (and also Polypterus ships within, but not between, GHs and SLs. Therefore, senegalus; data not shown), indicating that only a single unlike SLR, SL should have existed prior to the FSGD, copy of GHR was amplified, as expected. We determined which produced SLa and SLb paralogs in teleosts. Whereas entire cDNA sequences by RACE and termed them GHR the presence of SLb hass been reported for some teleost (instead of SLR), according to the evolutionary consider- species other than zebrafish (Zhu et al. 2004), we could not ations we have described. find SLb in the genomes of medaka and fugu. The lungfish GHR (EF158850) consists of 602 606 amino acids (we found possible allelic polymorphisms that cause several amino acid substitutions and insertions/ Isolation of Lungfish and Sturgeon GHRs deletions; data not shown) and phylogenetic trees using PRL receptors (PRLRs) as an outgroup placed it basal to The teleost-specific GHR duplication suggests that the tetrapod GHRs (Fig. 4). The extracellular (hormone-bind- hormone receptor set of GH, SL, and GHR, but not SLR, ing) domain (ECD) of the lungfish GHR is most similar to actually existed in the common vertebrate ancestor. For those of tetrapod GHRs, with 39% (guinea pig) to 47% further characterization of the receptor evolution, we (pigeon) shared sequence identities, followed by teleost determined the GHR sequences of lungfish and sturgeon, SLRs, with 35% (halibut) to 41% (eel/carps) identities, and both of which, due to their phylogenetic position, are teleost GHRs, with 31% (catfish) to 36% (eel) identities. It expected to have retained this ancestral hormone receptor is intriguing to note that the lungfish GHR is always more set (Fig. 1). similar to SLRs than to GHRs in all teleost species from 363

ACLY MYL3 SCN4A/SCN8A/SCN3A/SCN9A LOC201175 ARHGAP27 WNK1 SCN10A MYO7A 17q12 q21 3p21 3 p21 2 17q23 q25 3/12q13/2q24/2q24 GH 17q21 31 17q21 31 12p13 3 3p22 p21 11q13 5 Zebrafish Chr.3 21 7 M 21 5 M 21 3 M

SCN4A/SCN3A/SCN1AN4A N3A N1 CDC27 17q2317 23 q25 25 3/2q24/2q243/2 24/2 24 3 GH 17q12 17q23 2 Medaka Scf.697 118 k 0 k

GH Fugu Scf.7980 4 6 k 0 k

LOC645966 CD79B CSHL1 CSH2 PSMC5 LOC440455 MGC10986 PECAM1 LOC645993 TEX2 ERN1 ICAM2 SCN4AN4 GH1 CSH1SH1 GH2 SMARCD2 FTSJ3 DDX42 CCDC47 LYK5 MAP3K3 Human Chr.17 59 8 M 59 5 M 59 1 M

H17 SRPR TIRAP FLJ21103 DCPS KCNJ5 P53AIP1 PRUSD4 ST3GAL4 KIRREL3 PRR10 LOC646131 LOC387820 ETS1 FLI1 KCNJ1 RICS Human Chr.11 125 5M 126 0 M 127 0 M 128 0 M 128 4 M ILVBL 19p13 1 RBM7 BARX2 TIRAP FAM118B B3GAT1 11q23 1 q23 2 11q25 KIRREL3IRREL SL ETS1 FLI1 KCNJ1 KCNJ5 ST3GAL4 11q24 3 11q25 Fugu Scf.131 0 8 M 0 5 M 0 2 M

H PK3 LOC644196 11p13 4q12 KIRREL3IRREL SL ETS1 FLI1 KCNJ1 KCNJ5 STAL 3G 4 Medaka Scf.186 0 3 M 0 5 M 1 2 M 1 0 M

KIAA1822/KIAA1822L YIF1B/YIF1A ZFP36L1 RASGRF2 TALDO1 RELN 14q32/1q41 19q13 2/11q13 14q22 q24 SLa ETS1 5q13 KIRREL3 11p15 5 p15 4 7q22 Zebrafish Chr.18 52 6 M 52 0 M 51 7 M 52 5 M CRY2/CRY1 11p11 2/12q23 q24 1 DUSP11 REXO2 KCNJ1 FOXRED1 GTF2 RD2B ASAM LOC283157 2p13 2 11q23 1 q23 2 KIRREL3 SLb DCPS 11q24 2 7q11 23 11q24 1 11q24 1 Zebrafish Chr.10 44 2 M 44 5 M 45 2 M 45 0 M

Fig. 3 The lack of synteny between the GH and SL loci. Horizontal top. Arrowheads indicate 50 30 orientations of each gene. Note that bars represent partial scaffolds/chromosomes whose positions are the syntenic genes around the GH locus (SCN4A) and those around indicated below (bp). Species names and scaffold/chromosome the SL locus (TIRAP, DCPS, STSGAL4, KIRREL3, ETS1, FLI1, numbers are indicated on the left. Colored boxes in the bars indicate KCNJ1, and KCNJ5) are located on different chromosomes in human. genes we manually annotated; yellow, GH; green, SL; blue or pink, On the other hand, conserved synteny is observed between the SLa syntenic; and grey, nonsyntenic genes. Gene names and their and SLb loci of zebrafish (bottom) locations on human chromosomes (if nonsyntenic) are indicated on which both GHR and SLR have been cloned (i.e., salmon, Lineage-Specific Features of the Vertebrate seabream, medaka, tilapia, catfish, and eel). GHR/SLRs: From Their Residual Comparisons The sturgeon GHR (EF158851) consists of 571 amino acids and phylogenetic trees supported its position basal Molecular mechanisms of the GH GHR interaction have to both the GHRs and SLRs of teleosts (Fig. 4). This been extensively studied using mammalian models and result further supports the hypothesis that SLR is a teleost- functionally important amino acids at the ECD have been specific duplicated copy of the ancestral GHR. The ECD structurally, biochemically, and genetically determined. In of the sturgeon GHR is most similar to those of teleost order to further characterize the lungfish and sturgeon GHR SLRs, with 39% (tilapia) to 54% (carp) identities, fol- sequences, we compared their equivalent residues with lowed by tetrapod GHRs with 39% (buffalo/guinea pig) to those of other receptors (Figs. 5 and 6). 48% (turtle) identities, and teleost GHRs, with 33% One apparent difference specifically found in teleost (catfish) to 44% (rainbow trout) identities. The exception GHRs is the lack of two of six disulfide-bond-forming is eel GHR (47%), which exceptionally retained SLR-like cysteines, which are perfectly conserved among tetrapod features (see below). Again, the sturgeon GHR is always GHRs and teleost SLRs. In the eel, however, both GHR more similar to SLRs than to GHRs in all teleost species. (AB180477) and SLR (AB180476) retain all six cysteines, The e-values of the blastp analyses also support that the as do the lungfish and sturgeon GHRs. Therefore, after the lungfish/sturgeon GHRs are more similar to teleost SLRs FSGD and the separation of eels from the other teleost than to teleost GHRs (data not shown). However, signif- lineages, two of the six ancestral cysteines were lost spe- icantly more rapid diversification of GHRs than SLRs in cifically in teleost GHRs. teleosts was not always supported by the relative-rate test The WSXWS-equivalent motif (the Y/FGXFS motif) using the MEGA3 software (Kumar et al. 2004; seems conserved throughout the vertebrates (with the p = 0.000 0.216); the same test did support more rapid exception of AGEFS in the eel GHR). We found that the diversification of both teleost receptors than sturgeon placental-specific Y222 also occurs in lungfish, though the GHR (p \ 0.001; data not shown; Robinson-Rechavi and phenylalanine (found in sturgeon) still seems to represent Laudet 2001). an ancestral state. 364

a Vertebrate PRLRs

Mammalian GHRs

1.00 Chicken GHR (NM001001293) 1.00 1.00 Pigeon GHR (D84308) Turtle GHR (AF211173) 0.74 Frog GHR (AF193799)

Lungfish GHR (EF158850)

Sturgeon GHR (EF158851)

Eel GHR (AB180477) Catfish GHR (AY973231) 1.00 Atlantic salmon GHR1 (AY462105) Coho salmon GHR1 (AF403539) 0.99 1.00 Rainbow trout GHR1 (AY861675) Atlantic salmon GHR2 (DQ163908) Rainbow trout GHR2 (AY573600) 1.00 Coho salmon GHR2 (AF403540) Masu salmon GHR (AB071216) Medaka GHR (DQ010539) 1.00 Tilapia GHR (AY973233) Black seabream GHR (AY662334) Gilthead seabream GHR (AY573601)

Catfish SLR (AY336104) 1.00 Grass carp SLR (AY283778) Mrigal SLR (AY691179) Catla SLR (AY691178) Rohu SLR (AY691177) 0.97 Common carp SLR (AY741100) Goldfish SLR (AF293417) Eel SLR (AB180476) Masu salmon SLR (AB121047) 0.92 Black seabream SLR (AF502071) 1.00 Gilthead seabream SLR (AY699980) Medaka SLR (DQ002886) Tilapia SLR (AY973232) Turbot SLR (AF352396) 0.1 Atlantic halibut SLR (DQ062814) Bastard halibut SLR (AB058418)

Fig. 4 Phylogenetic trees of the vertebrate GHR/SLR/PRLRs as inferred from the (a) MrBayes and (b) PhyMl software. Posterior probabilities or bootstrap values are shown at several representative nodes. Detailed subtrees of vertebrate PRLRs and mammalian GHRs were constructed but are not shown. Numbers in brackets are GenBank accession numbers

GHR dimerizes when being activated, and residues glycosylation sites, suggesting more derived states of tel- involved in the dimerization have also been identified (de eost GHRs. Vos et al. 1992). Several mutations have also been iden- There is an unpaired cysteine (C241) in the human GHR tified from human patients with GH-insensitive dwarfism that forms a disulfide bond during receptor dimerization (Laron syndrome; Laron 2004), indicating that substituted (Zhang et al. 1999). Although this bond was shown to be residues are functional (or substituting residues are mal- dispensable, this cysteine is perfectly conserved among functional). Some amino acids at these sites show strict mammalian, avian, and reptilian GHRs. Equivalent cyste- conservation (e.g., F96 and P131), while others are only ines are not found in amphibian GHRs, teleost SLRs, or conserved within lineages (e.g., R161 and S201). teleost GHRs. Instead, they have an unpaired cysteine Asparagines N97, N138, and N182 (located after the between the 13th and 14th beta strands, with the exceptions fifth beta strand, within the eighth, and after the tenth beta of eel SLR and catfish GHR. Like the receptors of lower strand in Fig. 6) were shown to be glycosylated and vertebrates, the lungfish and sturgeon GHRs also have the important for GH binding in pig (Harding et al. 1994). unpaired cysteine between the 13th and 14th beta strands. Whereas N97 is strongly conserved in all the receptors, Of the approximately 10 structural/functional residues N138 and N182 seem not to be conserved among teleost that actually participate in the GH GHR interaction (Har- GHRs. Lungfish and sturgeon GHRs retain all the three ding et al. 1994; de Vos et al. 1992; Clackson & Wells 365

b Vertebrate PRLRs

Mammalian GHRs 998 Chicken GHR (NM001001293) 997 Pigeon GHR (D84308) Turtle GHR (AF211173) 878 Frog GHR (AF193799)

Lungfish GHR (EF158850)

Sturgeon GHR (EF158851) 1000 Eel GHR (AB180477) Catfish GHR (AY973231) 791 Atlantic salmon GHR1 (AY462105) Coho salmon GHR1 (AF403539) 829 1000 Rainbow trout GHR1 (AY861675) Atlantic salmon GHR2 (DQ163908) Coho salmon GHR2 (AF403540) 1000 Masu salmon GHR (AB071216) Rainbow trout GHR2 (AY573600) Medaka GHR (DQ010539) 999 Tilapia GHR (AY973233) Black seabream GHR (AY662334) Gilthead seabream GHR (AY573601)

Catfish SLR (AY336104) 1000 Grass carp SLR (AY283778) Mrigal SLR (AY691179) Catla SLR (AY691178) Rohu SLR (AY691177) 757 Common carp SLR (AY741100) Goldfish SLR (AF293417) Eel SLR (AB180476) Masu salmon SLR (AB121047) 557 Black seabream SLR (AF502071) 1000 Gilthead seabream SLR (AY699980) Medaka SLR (DQ002886) 0.1 Tilapia SLR (AY973232) Turbot SLR (AF352396) Atlantic halibut SLR (DQ062814) Bastard halibut SLR (AB058418)

Fig. 4 continued

1995; Clackson et al. 1998), those that contribute the most amphibian GHRs and teleost SLRs have glutamine (non- to the binding affinity, W104 and W169, are perfectly charged hydrophilic), and teleost GHRs have methionine conserved in all the GHR/SLRs. This suggests that the (hydrophobic), all of which show strong (though not per- fundamental mechanisms for the hormone receptor bind- fect) lineage-specific conservation. Considering that the ing did not diverge during the receptor evolution. D164 is substitution of R43 dramatically changes the binding also completely conserved, though it has received less specificity of the receptor (Souza et al. 1995; Yi et al. attention to date. R43 is one of the most interesting resi- 2002), this lineage-specific conservation might indicate dues because it determines the binding specificity of human lineage-specific functions of these receptors. Because the GHR, which is restricted only to GHs of Old World pri- lungfish and sturgeon GHRs have a glutamine at R43 as per mates (Souza et al. 1995; Yi et al. 2002). The arginine the and amphibian GHRs and the teleost SLRs, this (positively charged) at R43 interacts with aspartic acid should be interpreted as an ancestral residue. (negatively charged) at D171 of the primate GHs. GHs All these ancestral reconstructions are supported by (and also SLs) of other vertebrates have histidine (posi- PAML analyses (Yang 1997; data not shown). Importantly, tively charged) at D171, which may cause ionic repulsion we found no amino acids unique to lungfish or sturgeon against the R43. Instead of R43, GHRs of nonprimate GHRs at these functional sites, despite their unique hor- mammals and birds have leucine (hydrophobic), reptile and mone receptor set. 366

Fig. 5 Comparison of Disulfide WSXWS Hormone Receptor Missense bonds box binding dimerization mutations Extra functional residues in the Q131 I441 T351 P451 G551 C161 intron 222 622 002 102 S69 83 8 38 49 801 221 301 401 501 601 621 721 461 561 961 341 541 641 741 051 251 extracellular domain of 4 34 44 Human (NM_000163) CCCCCC YGEFS REIWIPDEDIW NSLTHDYS FPVIQVR no vertebrate GHR/SLRs. Positions Baboon (AF150751) |||||| ||||| ||V|||||||| |||||||| ||||||P - Macaque (NM_001042667) |||||| ||||| ||V|||||||| |||||||| ||||L|P - of the compared residues are Squirrel monkey (AF339061) |||||| ||||| L||||||Q||| |||||||| ||I|||P - indicated at the top. Vertical Rabbit (AF015252) |||||| ||||| L|||||E||V| |||||||| ||||||P - Buffalo (AY608917) |||||| ||K|| L|V|T|ED|V| |||||||| ||I|L|P - lines indicate that the equivalent Cattle (NM_176608) |||||| ||K|| L|V|T|ED|V| |||||||| ||I|L|P no residues are identical to those of Sheep (NM_001009323) |||||| ||K|| L|V|T|ED|V| |||||||| ||I|L|P no Dog (NM_001003123) |||||| ||||| L|||||E||V| |||||||| ||I|||P no the human GHR. Missense Tetrapods Panda (AF395535) |||||| ||||| L|||||E||V| |||||||| ||I|||P - Pig (NM_214254) |||||| ||||| L|||||E||V| |||||||| ||I|||P - mutations of E44K, D152H, and Guinea pig (AF247665) |||||| ||||| L|||K|E||V|||A|Y||| ||T|||| - E224D are not shown to avoid Mouse (NM_010284) |||||| |S||| L||||||||V| ||||R||| ||I|||P no Rat (NM_017094) |||||| |S||| L||||||||V| |||PR||| ||I|||P no redundancies. Existence/ Opossum (NM_001032976) |||||| F|||| L|||T||||V| |||||||| ||T|||P - absence of the SLR specific Chicken |||||| F|||| L||||||||V|||Q||||| ||T|||P no intron is shown in the far right Pigeon |||||| F|||| L||||||||V|||Q||||| ||T|||P - Turtle |||||| F|G|| Q||||T|||V| |||||||| ||T|||P - column (see also Fig. 7) Xenopus |||||| F|||| Q|||VS|D|V| ||||R||A ||I||LP no Lungfish Lungfish |||||| ||||| QVV||||||V|||RSY||| ||I|L|P no Sturgeon Sturgeon |||||| FS||| Q||||||||V|||Q||||G ||I|LIP no Eel |||||| A|||| M|||TTEN|V|||QS|||G |||AV|S no Catfish ||||AY F|D|| FVV|FQE||V| SGP|F||G ||LVM|S - Atlantic salmon-1 ||||SI F|||| MNV|MTED|V||G|||||G ||||MLH - Coho salmon-1 ||||SI F|||| MNV|MTED|V||G|||||G ||||MLH - Rainbow trout-1 ||||SI F|||| MNV||TED|V||G|||||G ||||MLH - Teleosts Atlantic salmon-2 ||||SI F|||| MNV|MTED|V||G|||||G ||||M|H - Rainbow trout-2 ||||SI F|||| MNV||TED|V||G|||||G ||||M|H - (GHR) Coho salmon-2 ||||SI F|||| MNV||TED|V||G|||||G ||||M|H - Masu salmon ||||SI F|||| MNV||TE||V||G|||||G ||||M|H - Medaka ||||AF F|G|| M|V|TQQD|V| |||||||G ||||M|D no Tilapia ||||NG F|||| MM||TYND|V|||S||||G ||||LLP - Black seabream ||||RK F|||| M|||TFEA|V| |||SY||G ||EVLLQ - Gilthead seabream ||||KT F|||| M|V|T||A|V| |||SY|FG ||E|ILQ - Catfish |||||| F|D|| LV||TIEN|V|||RS|||G ||I|M|P - Grass carp |||||| F|||| Q|||TSEN|V| |||S|||G ||||L|P - Mrigal |||||| F|||| Q|||TSEN|V|||RS|||G |||VL|P - Catla |||||| F|||| Q|||TSEN|V|||RS|||G |||VL|P - Rohu |||||| F|||| Q|||TSEN|V|||RS|||G |||VL|P - Common carp |||||| F|||| Q|||TSEN|V|||RS|||G |||VL|P - Goldfish |||||| F|||| Q|||TSEN|V|||RS|||G ||||L|P - Teleosts Eel |||||| F|||| Q|V|TQED|V| S|RSY||G ||||MAP no (SLR) Masu salmon |||||| F|||| Q||||KQD|V|||RSN||G ||I|MAP - Black seabream |||||| F|||| Q|V|V|EN|V|||PSS||G ||IVM|P yes Gilthead seabream |||||| F|||| Q|V|||EN|V|||PSS||G ||IVM|P - Medaka |||||| F|||| L|V|L|EN|V|||PSS||G |||VM|P yes Tilapia |||||| F|||| Q||||TEN|V|||PSN||G ||TVMIP - Turbot |||||| F|||| Q|||||EN|V|||PSS|FG ||IVM|P - Atlantic halibut |||||| F|||| Q|V||AEN|V|||PSS||G ||LVM|P - Bastard halibut |||||| F|D|| Q|||||EN|V|||PSS||G ||IVM|P yes

Lineage-Specific Features of the Vertebrate GHR/ c SLRs: From Comparisons of Their Predicted Secondary Fig. 6 Summary of predicted secondary structures of the extracel Structures lular domains of the vertebrate GHR/SLRs. (a) GHRs of 14 placentals, one marsupial, two avian, one reptilian, one amphibian, one lungfish, one sturgeon, and 12 teleosts; and SLRs of 16 teleosts The SOSUIsignal program (Gomi et al. 2004) classified all were analyzed by prediction programs. Each letter represents one the receptors as with N-terminal signal peptides amino acid; light blue H, pink E, or grey C indicates that the residue and single membrane-spanning domains (with the excep- is predicted to form alpha helix, beta sheet, or coil, respectively, by the New Joint program (paler colors indicate that the prediction was tions of seabream GHRs of black seabream and gilthead relatively weakly supported). The amino acid sequences were not seabream and carp SLR; Fig. 6a). This indicates that the aligned; we eliminated all alignment gaps so that the alignment is entire ECDs of the GHR/SLRs have been successfully only ensured at the transmembrane domain (brown boxes on the determined in most (if not all) of the species. The ECD of right). Color codes are as follows; green, signal peptides; purple, conserved (paired and unpaired) cysteines; yellow, potential N placental GHRs is exceptionally long, which may reflect glycosylation sites; indigo, four residue segment connecting two the insertion of a novel exon in this lineage (exon 3; Dos subdomains; and black, the WSXWS equivalent motif. Dark blue Santos et al. 2004). and black bars along the top indicate the positions of 14 beta strands The ECD of human GHR consists of two subdomains, in the human GHR (Somers et al. 1994; and references therein) and the exon specific to placental GHRs. (b) Amino acid alignment of each of which contains seven beta strands that make two GHR/SLRs of representatives from mammal, bird, reptile, amphib beta sheets (de Vos et al. 1992). The New Joint program ian, lungfish, sturgeon, and teleost. Major residues at each site are (Ito et al. 1997) predicted this secondary structure well, indicated in red. Predicted signal peptides, transmembrane domains, though relatively short strands tend to hamper reliable N glycosylation sites, and other conserved residues/subdomains were boxed with the same color used in (a). Functional prolines prediction. In spite of low similarities among the in the box I motif (Goujon et al. 1994) and tyrosines (Hansen et al. ECD sequences of the vertebrate GHR/SLRs (e.g., 1996) are highlighted by gray a Human HHHHHHHHHHHCCCCCCCCCCCCHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEECECCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEEEECCCCCCCCCCCCCCCCCCCCCCCEEEEEEEHHHHEECHCHEEEEEECCCCCCCC 183 Baboon HHHHHHHHHHHCCCCCCCCCCCCCCEEEHEHEEHCEECCCCCCCCCCCCCCCCCCCCCCCCCCECCCCCCCCCCCCCCCCCEEEEEECCCHCCECHHCCCCCCCCCCCCCCCECCCCCCCEEEEEEEEECCCCCCCCCCCCCCCCCCCCCCCEEEEEEEHHHHHCCHCHHHHEHHCCCCCCCC 183 Macaque HHHHHHHHHHHCCCCCCCCCCCCCCEEEHEHEEHCEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCECCCCCCCEEEEEEEEECCCCCCCCCCCCCCCCCCCCCCCHEEEEEEEEEHHCCCCCHEEEHHCCCCCCCC 183 Squirrel monkey HHHHHHHHHHHCCCCCCCCCCCCCHHHHHHHHHHHEEEECCCCCCCCCCCCCCCCCCCCCCCEEHHHHHHHHHCCCCCCCCEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCECCCCCCCEEEEEEEEECCCCCCCCCCCECCCCCCCCCCCEEEEEEEHHEEECCCCCCEEEEHCCCCCCCC 183 Rabbit HHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCEEEECCCCCCCCCCCCCCCCCCCCCCCEEEECCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEEEECCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEEHHHCCCCCCEEEECCCCCCCCC 183 Buffalo HHHHHHHHHHHECCCCCCCCCCCCCCHEEEECCCCHHEEEEECCCCCCCCCCCCCCCCCCCCEEEECECCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCEEECCCCCCCCCCEEEEEECCCCCECECCCCCCCCCCCCCCCCCCEEEHCHHHHHHHHHHHECCCCCCCCCC 179 Mammals Cattle HHHHHHHHHHHECCCCCCCCCCCCCCHEHEHCCCCHHEEEEEEECCCCCCCCCCCCCCCCCCEEEECECCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCEEECCCCCCCEECEEEEECCCCCCECECCCCCCCCCCCCCCCCCCEEEHCHHHHHHHHHHHECCCCCCCCCH 179 Sheep HHHHHHHHHHHCCCCCCCCCCCCCCCHEEEECCCCCEEEECCCCCCCCCCCEEEECCCCCCCEEEECECCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCEEECCCCCCCEECEEEEEECCCCCEEECCCCCCCCCCCCCCCCCCEEEHCHHHHHHHHHHHECCCCCCCCCC 179 Dog HHHHHHHHHHHECCCCCCCCCCCCCCEECCCCCCCEEECCCCCCCCCCCCCCCCCCCCCCCCHHEEHECCCCECCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCEEEECHHHCCCCCEEEEECCCCCCCCC 183 Panda HHHHHHHHHHHHCCCCCCCCCCCCCHEEEECCCCCEECCCCCCCCCCCCCCCCCCCCCCCCCEEECCECCCCCCCCCCCCCEEEEEEECCCCCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEHCCCCCEEEEECCCCCCCCC 183 Pig HHHHHHHHHHHCCCCCCCCCCCCCCHHEEEHCCCCCEEECCCCCCCCCCCCCCCCCCCCCCCEEEECECCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCEEEECEEECCHCCCEEEECCCCCCCCC 183 Guinea pig HHHHHHHHHHHHECCCCCCCCCEHEEEEECHCCHHHHHECCCCCCCCCCCCCCCCCCCCCCCEEEECECCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCEEECCCCCCCCCCEEEEECCCCCCCCCCCECHCEECCCCCCCCEEEEEECCCCCEEECCEEEEECCCCCCCC 183 Mouse HHHHHHHHHHHEEEECCCCCCCCCCCCHHCCCCCCEEECCCCCCCCCCCCCCCCCCCCCCCCEEEEECCCCCCCCCCCCCCEEEEECCCCCCCHHHHHHHHHHCCCCCCCCCCCCCCCCEEECCCCCCEEECEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECHCCCCCCEEEEECCCCCCCHH 192 Rat HHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCECCCCCCCCCCCCCCCCCCCCCCCCCCEEEEECCCCCCCCCCCCCEEEEEEEEECCCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEEEECCCCCCCCCCCHCCCCCCCCCCCCCCCEEEEEECCCCCCCCEEEEECCCCCCCCC 184 Opossum CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCHHHHHHCCCCCCCCCCCCCCCCCCCCEEECCCCCCCECCCCEECCCCCCCHHHHCECCCCCCCCCCCCCEEEEEHCCCHHHHHHCEEECCCCCCCCCCCCC 159

Chicken HHHHHHHHHHHEHCCCCCCCCCCCHCHCCCCCCCCCCCCCEECEEECCCCECCCCCEEEEEECCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEECCCCCCCCHHHCCCCCCCCCCCCCCCCEEEEEHCCCCCCCCCCEEEECCCCCCCCCCC 156 Pigeon HHHHHHHHHHHHHHCCCCCCCCCCHCCCCCCCCCCCCCCCEEEEEECCCCEECCCCCCCEEEEEHCCCCCCCCCCCCCCCCCCCCEEECCCCCCEECCEEEEECCCCCCHHHCCCCCCCCCCCCCCCCEEEEEHCCCCCCCCCCEEEECCCCCCCCCCC 159 Avian-Amphibian Turtle HHHHHHHHHHHEECCCCCCCCCHEEECCCCCCEECCCCCCEEEEEECCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCEEECCCCCEEEEEEEEECCCCCCCCHHCCCCCCCCCCCCCCCCEEEEEHCCHHHHHHCCCEECCCCCCCCCC 157 Frog HHHHHHHHHEEEEEECCCCCCEEECCCCCCEEEECCCCCCEEEEEECCCCCCHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCEEECCCCCEEEEEEEECCCCCCCCCCCHCCCCCCCCCCCCCHCHCEEEEEEECCCEEEEEEEEECCCCC 154

Lungfish CCCCHEHHEEECCCCECCCCCCCCCCCCCCCEEEECCCCCEEEEEEEEECCCCCCCCCCHEEEEECCCCCCCCCCCCCCCCCCCCCEECCCCCCCCCCCCEEECCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEECCCCCEHEEEECHCCCCCC 156 Lungfish & Sturgeon Sturgeon HHHHHHHEHEHECCCEEEECCCCCCCCCCCCCCEEEEECCCCCCEEEEEECCCCCCCCCCCCCEEEEECCCCCCCCCCCCCCCCCCCCEEECCCCCCEEECEEECCCCCCCCEEEECECCCCCCCCCCCCCCCCCEEEEEECCCCCCHEEEEEECCCC 158

Eel HHHHHHHHHHHHHEHCCECCCCCCCCCCCCCCCCCCCECECCCCHCHHEEEECCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCEEHCCCCCCCEEEEEEEEECCCCCEEECCCCCCECCCCCCCCCCCCCEEEEEECCCCCHHHHEECECCCC 163 Catfish CCECCCHEEHEEEECCCCHHHHHHHHHCCCCCCEEECCCCCCEEEEEECCCCCCEECCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCCEEHHCCCCEEEEHHHHHECCCCCCCCCHHHHCHCHHCCCCCCCHHCHEEEEECCCCEEEEEEEEECC 159 Atlantic salmon-1 CHHCCHHHHHHHHHHCECCCCCCCCHHHHHCECCCEEEEECCCCCEEECEECECCECCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEECCCCCCCCCEHCHEEEEECCCCCCCCCHHHEEEEEECCCCCCCECEEEEEC 160 Rainbow trout-1 CHHCCHHHHEHEEHCCCCCCCCCCCCCCCEECCCCCCCEECCCCCEEECEEEECCECCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEEEECCCCCCCCEHCHEEEEECCCCCCCCCHHHHEEEEEECCCCCCEEEEEEEC 160 Atlantic salmon-2 CHHCHHHEEEEECCCEEECCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCHEEEEECCCCCCCCCCCCCCCCCCCCCCCHEEECCCCCEEEEEEEEEECCCCCCCCCCHEEEEECCCCCCCCCHHHHEEEEECCCCCCCEEEEEEEC 161 Teleosts Rainbow trout-2 CHHCCHHHEEHEEEEEECCCCCCCCCCCCCCCCCCCEEEECCCCCEEEEEECCCCCCCCCCCCCCHEEEECCCCCCCCCCCCCCCCCCCCCCCHEEECCCCCEEEEEEEEEECCCCCCCCCCHEEEEECCCCCCCCCEEEEEEEEECCCCCCCECEEEEEC 161 Coho salmon-2 CCCCHHHHHEHEEHHEECCCCCCCCCCCCCCCCCCCEEEECCCCCEEEEEECCCCCCCCCCCCCHEEEEECCCCCCCCCCCCCCCCCCCCCCCHHEECCCCCEEEEEEEEEECCCCCCCCCCHEEEEECCCCCCCCCHHHHEEEEEECCCCCCEEEEEEEC 161 (GHR) Masu salmon CCCCHHHHHEHEEHCEECCCCCCCCCCCCCCCCCCEEEEECCCCCEEEEEECCCCCCCCCCCCCHHEEEECCCCCCCCCCCCCCCCCCCCCCCHHEECCCCCEEEEEEEEEECCCCCCCCCCHEEEEECCCCCCCCCHHHHEEEEECCCCCCCEEEEEEEC 161 Medaka HHHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCEEECCEECCCCCCCCCCCCHEEEEHCCCCCCCCCCCCCCCCCCCCCCCCCEEECCCCCEHHHEEEEEECCCCCCEEEEHHHHCCCCCCCCCCCCCCCEEEEEECCCCCCEEEEEEEC 159 Tilapia CCCHHHHHHHHHHHHHHHHHCCCCCCCHECCCCCCCCCCCCCCEEEECCCCCCCCCCCCCCCHHHHEEECCCCCCCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEEEECCCCCHEHCCCCCEEECCCCCCCCCEEEEEEEEEECCCCCEEEEEEEEC 161 Black seabream HEEEEEHHHHHHHHHHHEEEEECCCCCCCCCCCCCCCCCCCCCCECCCCCCEEEEEECCCCECCCCCCCCHHEEEECCCCCCCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEEEECCCCCCCECCCCCCEEEEEECCCCCCEEEEEEECHCCCCCHEHEEECCC 168 Gilthead seabream HHHHHHHHHHEEECCCCCHHCCCHCCCCCCCCCCCCCCCCCCCEEEECCCCCCECCCCCCCCHEEEEECCCCCCCCCCCCCCCCCCCCCCCCCEEECCCCCCCCCCCEEEECCCCCCCCCCCCCEEEEEEECCCCCCEEEEEEECCCCCCCEEEEEEEEC 160

Catfish HHHHHHHHHHHHHHHCCCCCCCCCCCCCEECCCCCCCCCCCCCCCCEEEEEEEECCCCCCCCCCCCCHHHHHHHHHHCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEEEHCCCCCCCCCCCCCCCECCCCCCCCCCCCCCCEEEEECCCCCEHEEEEECECCCC 168 Grass carp CHCCCCHCEEEECEECCCCCECHCCCEEEECCCCCCCCCCCCCCCCCCEEEEEEECCCCCCCCCCCCEEEEEHCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEECCEEEEEECCCCCCCCCCCCECECCCCCCCCCCCCCEEEEEHCHCCHHEEEEEEECCCC 165 Mrigal CCCCCCECEEEEEEECCCCCEEECCCCCCCCCCCCCCCCCCCCCCCCCEEEEECCCCCCCCCCCCCCEEEEHCCCCCCCCCCCCCCCCECCCCCEHCCCEEEEEECEEEEEECCCCCCCCCCCCECECCCCCCCCCCCCCEEEEECCCCCCCEHEEEECCCCC 163 Catla HHHHHHHHHHHCHEEEEEECCCCCECCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCHEEEEHCCCCCCCCCCCCCCCCECCCCCCCCCCEEEEECCEEEEEECCCCCCCCCCCCECECCCCCCCCCCCEEEEEEECCCCCCCEHEEEECCCCC 167 Teleosts Rohu HHHHHHHHHHHCHEEEEEECCCCCECCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCEEEEECCCCCCCCCCCCCCCCCCCCCEEHCCCEEEEECCEEEEEECCCCCCCCCCCCECECCCCCCCCCEEEEEEEEECCCCCCCEHEEEECCCCC 167 Common carp CCHHHCHCEEEEEEECCCCCCECCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCEEEHEHCCCCCCCCCCCCCCCCECCCCCCCCCCEEEEEEEECEEEECCCCCCCCCCCCECEECCCCCCCCCCEEEEEEECCCCCCCEHEEEECCCCC 163 (SLR) Goldfish HHHHHHHHHHHHCEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCHEEEHHHHCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEEEEEEECECCCCCCCCCCECEECCCCCCCCCEEEEEEEEECCCCCCEEEEEEECCCC 163 Eel CCCHHHEEEEECCCEHECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEECEEEECECCCCCCCCECCHCCCCCCCCCCCCCEEEEEEEEEECCCCCEEEEHCCCCCCC 165 Masu salmon CCCHHHHHHHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEECCCCCCCCCCCCHEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEEECCCCCCCCCCCHHHHHHCCCCCCCCCEEEEEEEEEECCCCCCCEEEEECCCCC 167 Black seabream EEEECCCCCCCCCCCCEEEEEHHHCHHHHHCCCCHHHECHCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCHHEEEECCCCCCCCCCCCCCCCCCCCCEEEECCCCCCECEECECCCCCCCEEEECCCCCECCCCCCCCCCCCCEEEEEEECCCCCCCEEEEEEECCCC 177 Gilthead seabream EEEECCCCCCCCCCCCCCCCCCCEEEEEHHHCHHEHCCCCCEEEECCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCEEEECCCCCCECEECCCCCCCCCEEEECCCCCECCCCCCCCCCCCEEEEEEEECCCCCCCEEEEEEECCCC 184 Medaka CCCCCCCCHHHHHHHHHHCCCCCCCCEEEECCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCHHEEEECCCCCCCCCCCCCCCCCCCCCCEECCCCCCCECCEEEECCCCCCCEEECCCCCCCCCCCCCCCCCCCEEEEEEECCCCCCCEEEEEEECCCC 170 Tilapia ECCCCCCCHHHEEHHCCCCECCCCCCEEEEECCCCCCCHCECCCCCCCCCCCCCEEEEEECCCCCCCCCCCCHEEEEEECCCCCCCCCCCCCCCCCCCCCEEECCCCCCEEEEEEECCCCCCCCEEECCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCEEEEEEEECCCC 171 Turbot EECCCCCCCEEEEEHHCCHHHHCCCCEEEECCCCCCCCCCCCCCCCCHCCCCCCCEEEEEECCCCECCCCCCCCEEEEEECCCCCCCCCCCCCCCCCCCCCCEECCCCCCEEEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCCEEEEEEEEECCCCCCCEEEEEEECCCC 171 Atlantic hal but EEECCCCCHHEEEEEEECCCCCCCCCCEHHHHHCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCHHEEEECCCCCCCCCCCCCCCCCCCCCEEEECCCCCEEEEEEECHCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEECCCCCCCCEEEEEEECCCC 171 Bastard hal but EEECCCCCHHEEEEEECCCCCCCCCCCHHHHCCCCCCCCCCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCCHEEEEEECCCCCCCCCCCCCCCCCCCCCEEEEECCCCCECEECCCCCCCCCCEECCCCCCCCCCCCCCCCCCCCCEEEEECCCCCCCEEEEECCCCCC 171

Human CCCEEEEEHHHHHCCCCCCHCHHCCCCCCCCCCEEEEEECCHHEEEEECCCCCCCCCCCCCEEEEEEECCCCCCCCCCCCCCCHEEEEEHHHCCHHEEEEEEEECCCCHHEEEEECCCCCCCCCCC 309 Baboon CCCHEEEEEHHHHCCCCCCCCCCCCCCCCCCCCEEEEEECCCHEEEEECCCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCHEEEEEHHHCCHHHHHHEEEECCCCHHEEEEECCCCCCCCCCC 309 Macaque CCCEEEEEEHHHHCCCCCCHCHHCCCCCCCCCCEEEEECCCCHHHHHHECCCCCCCCCCCCEEEEEECCCCCCCCCCCCCCCCHEEEEEHHHCCHHHHHHEHHHHCCCHHEEEEECCCCCCCCCCC 309 Squirrel monkey CCCHEEEEEHHHCCCCCCHHHHCCCCCCCCCCCEEEEEECCCHEEEEHCCCCCCCCCCCHHHHHHECCCCCCCCCCCCCCCCCEEEEEEECCCCCEEHEEEHHHCCCCHHEEEEECCCCCCCCCCC 309 Rabbit CCCEEEEEEHHHCCCCCCHHHHHCCCCCCCCCCEEEEEECCCHEEEEHCCCCCCCCCCCCCHEEEEEECCCCCCCCCCCCCCCHEEEEEEHHCCCEEEEEEEEECCCCHHEEEEECCCCCCCCCCC 309 Buffalo CCEEEEEHHCCCCCCCCCHHCHCCCCEEEEECCCECECCCCCHHEEEHECCCCCCCCCCCCHEEEEECCCCCCCCCCCCCCCCHEEEEEHHHHCHHHHHHHHHHHCCCCHEEEEECCCCCCCCCCC 305 Cattle CCEEEEEHHCCCHCCCCCCCCCCCCCCEEEECCCECECCCCCHHEEEHECCCCCCCCCCCCHEEEEECCCCCCCCCCCCCCCCHEEEEEHHHHCHHHHHEEEEECCCCHHEEEEECCCCCCCCCCC 305 Sheep CCEEEEEHHCCCCCCCCCHHHHHCCCCEEEECCCECECCCCCHHEEEHCCCCCCCCCCCCCHEEEEECCCCCCCCCCCCCCCCHEEEEEEHHCCCHEEEEEEEECCCCHHEEEEECCCCCCCCCCC 305 Dog CCCHEEEEEHHHHCCCCCCHCCCCCCCCCCCCEEEEECCCCCHEEEEHCCCCCCCCCCCCCCHEEEEECCCCCCCCCCCCCCCHEEEEEEHHCCCHHHHHHHHHCCCCHHEEEEECCCCCCCCCCC 309 Panda CCCEEEEEEHHHHCCCCCCHCHCCCCCCCCCCCEEEEEECCCHEEEEHCCCCCCCCCCCCCHHHEECCCCCCCCCCCCCCCCCHEEEEEEHHCCCHHHHHHHHHCCCCHHEEEEECCCCCCCCCCC 309 Pig CCCEEEEEEHHHCCCCCCHHHHHCCCCCCCCCCEEEEEECCCHEEEEHCCCCCCCCCCCCCHEEEEECCCCCCCCCCCCCCCCHEEEEEEEHCCCEEHHHHHHHHCCCHHEEEEECCCCCCCCCCC 309 Guinea pig CCCEEEEHHHEEECCCCCCCCCCCCCCCCCCCEEEEECCCCHHHEEEHHCECCCCCCCCCCHEEEEECCCCCCCCCCCCCCCHHHEEEEEHCCCCCHHEHHHHHHCCCCHEEEEECCCCCCCCCCC 309 Mouse CHHEEEEEHHHHCCCCCCCHHECCCEEEEECCCCEECCCCCCEEEEHCCCCCCCCCCHHHHHHEEHCCCCCCHCCCCCCCCCCHEEEEEECCCCHHEEEEEEEHCCCCHEEEEEECCCCCCCCCCC 318 Rat EEHEEEEEHHCHCCCCHCHCCCCCCCCCCCCCEEEEEECCCCHEEEHCCCCCCCCCCHHHHHHEECCCCCCHHHCCCCCCCCCHEEEEEECCCCHHEEEEEEEECCCCHEEEEEECCCCCCCCCCC 310 Opossum EEEEHHHHHHCCCCCCHHHHHEEECCCCCCEEEEECCCCHEEEEECCCCCCCCCCCCCHEEEEECCCCCCCCCCHHHHCHHCHHHEEEEEEHHCCCEEEEEEEEHCCCCHHHEEEECCCCCCCCCC 285

Chicken CHEEEEEHHHHCCCCCCCCCCHCCCCCHECCEECECCCCCEEEEEECCCCCCCCCCECCHEEEEEHCCCCHEEECCCCCCCCCEEEEEEECCCCCCHHHHHHHHCCCCCCEEEEECCCCCCCCCCC 282 Pigeon CCEEEEEEHHHCCCCCCCCCCHCCHHCHHCCEEEEEECCCEEEEEECCCCCCCCCCECCHEEEEHCCCCCEEEECCCCCCHCCEEEEEEECCCCCCEHEEEHHHCCCCHHHEEEECCCCCCCCCCC 285 Turtle CCCHEEEEEEHHHCCCCCCCCHHHHHHCCEHCCCCCECCCCCCEEEEHCCCCCEHECCCCCCEEEEECCCCCEECCCCCCCCCEEEEEEECCCCCHHHHHHHHHHHHHHHHEEEECCCCCCCCCCC 283 Frog CCCCCCCEEEEEEEEECCCCCCHHHHHHHHHCCCCEEEEECCCCCHHHHHHHCCCCCCCCCCCCHHHCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHHHHHHHHHHCCHCEEEECCCCCCCCCCC 280

Lungfish CCCCCHHEHHHHHHCCCCCCCHHHHHHHHHHEECCEEEEECCCCEEEEEHHCCCCCCCCCCCCCCEEEEECCCCCCCCCCCCCEEEEEEECCHCHHHHHHHHHHCCCCHHHEEEECCCCCCCCCCC 282

Sturgeon CCCCCCCEEEEEECCCCCCCCCCCCCCCHHHHHCCCCEECCCCCCCEEEEEHCCCCCCCCCCCCCCCEEEECCCCCCCCCCCCHHEEEEECCCCHEEEEEEEEECCCCCHEEEECCCCCCCCCCCC 284

Eel CCCHHHCHEEEHECCCECCCCCCCCCCCCCCCCCCCCEEEEEECCCCCHHHHHHHHCECCCCCCCCCEEEECCCCCCCCCCCCEEEEEEECCCCHCHHEEEEEECCCCCEEEEEECCCCCCCCCCC 289 Catfish CCCCCCCEHEEEEEECCCCCCCCCCCCCCCCCCCCCCCCCEEEEECCCCCCEEEEHECCCCCCCCCCCCCEEEEEECCCCCCCCHEEEEEHHHHHCHEEEEEEECCCCHHEEEEECCCCCCCCCCC 285 Atlantic salmon-1 CCCCCCHCEEHEEEECCECECECCCCHEHEECCHHCCHHEEECCCCCCCCCEEEHHCCCCCCCCCCCCCCEEEEECCCCCCCCHEHHEHHHHHHHHHHEEEEECCCCCHEEEEEECCCCCCCCCCC 286 Rainbow trout-1 CCCCCCHCEEHEEEECCECECECCCCHEHECCCCCCCCHEEECCCCCCCCCEEEHECCCCCCCCCCCCCCEEEEECCCCCCCCHEHHEHHHHHHHHHHHHHHHHCCCCHEEEEEECCCCCCCCCCC 286 Atlantic salmon-2 CCCCCCCCCCHEEEECCECECEHCCHHHHHHHHHCCCHHEEECCCCCCCCCEEEHCCCCCCCCCCCCCCCEEEEECCCCCCCCHEEEEHHHHHHHHHHEEEEHHCCCCHHHHEHCCCCCCCCCCCC 287 Rainbow trout-2 CCCCCCHCEEHEEEECCEEECEHCCHHHHHEHCCCCCCCEEEECCCCCCCCEEEHECCCCCCCCCCCCCCEEEEECCCCCCCCHEEEEHHHHHHHHHHEEEEHHCCCCHHEEEECCCCCCCCCCCC 287 Coho salmon-2 CCCCCCHCCECEEEECCEEECEHCCHHHHHEHCCCCCCCEEEEECCCCCCCEEEHECCCCCCCCCCCCCCEEEEECCCCCCCCHEEEEHHHHHHHHHHEEEEHHCCCCHHEEEECCCCCCCCCCCC 287 Masu salmon CCCCCCHCCECEEECCCECECEHCCHHHHHEHCCCCCCCEEEECCCCCCCCEEEHECCCCCCCCCCCCCCEEEEECCCCCCCCHEEEEHHHHHHHHHHHEHHHCCCCCHHEEEECCCCCCCCCCCC 287 Medaka CCCCCCHHHEHEEEEEEECCCCCCCCCCEEEEHHCCCCHEEEECEECCCCCHEEHECCCCCCCCCCCCCCHHHHHHCCCCCCCEEEEEHHHHHHHHHHHHHHHHCCCCHHEEEEECCCCCCCCCCC 285 Tilapia CCCCCCCCHHHHHHHHHECCCCCCCCEEEEECCCCCCCECEECCEECCCCCHEEHCHCCCCCCCCCCCCCEEEECCCCCCCCCEEEEEEHHHHHHHCHEEEEEECCCCCHEEEEECCCCCCCCCCC 287 Black seabream CCCCCCCHEEHEEECCCEEECCCCCCCCEECCCEECCCCCHECCCCCCCCCHHEEHHHCCCCCCCCCCCHEEEECCCCCCCCCEEEEHHHHHHHHHHHHHHHHHCHCCCEEEEEECCCCCCCCCCC 294 Gilthead seabream CCCCCHHHHEHEEECEEEEEECCCCHEEEEECCCCECCHCHHHCCCCCCCCHHEHHHHCCCCCCCCCCCCEEEECCCCCCCCCEHEEHHHHHHHHHHHHHHHHHCHCCCEEEEEECCCCCCCCCCC 286

Catfish CCCCCCCHECHCECECEEHCCCCCCCCCCCCCCCCCEEEECCCCCCCEEEEEEECCCCCCCCCCCCCEEEECCCCCCCCCCCCHEEEEHHHHHHHHHHHHHHHHHCCCHEEEEEECCCCCCCCCCC 294 Grass carp CCCCCCEEEEEEEEEEEEECCCCCHEEEHCCCCCCCEEEEECCCCCCEEEEEEECCCCCCCCCCCCCEEEEEHECCCCCCHHHHEHEEEEHEHHHEEEEEHHHHHCCCHEEEEEECCCCCCCCCCC 291 Mrigal CCCHHEEHHHHHHHHEEEHCCCCCHHHHCCCCCCCCHEECCCCCCCHEEEEHEECCCCCCCCCCCCCEEEEEECCCCCCCCCCCEEEEEEHECHEEEEEEHHHHHCCCHHEEEEECCCCCCCCCCC 289 Catla CCCHHEEEEEHEECEEEEEECCCCCCCCCCCCCCCHHEECCCCCCCCHHHHHHHHCCCCCCCCCCCCEEEEEECCCCCCCCCCCEEEEEEHECHEEEEEEHHHHHCCCHEEEEEECCCCCCCCCCC 293 Rohu CCCHHEEEEEHHEHEHEEECCCCCHHHHCCCCCCCCCECCCCCCCCCEEEEHCHCECCCCCCCCCCCEEEEEECCCCCCCCCCCEEEEEEHECHEEEEEEHHHHHCCCHHEEEEECCCCCCCCCCC 293 Common carp CCCHHEEEEEHEECEEEEECCCCCHEEEHCCCCCCCEEEEECCCCCCEEEEEEECCCCCCCCCCCCCEEEEEECCCCCCCCCCCEEEEEEHECHEEEEEEHHHHHCCCHEEEEEECCCCCCCCCCC 289 Goldfish CCCHEEEEECEHCEEEEEECCCCCHEEEHCCCCCCCEEEEECCCCCCEEEEHCCCCCCCCCCCCCCCEEEEEECCCCCCCCCCCEEEEEHHHHHHHHEEEHHHHHCCCHHEEEEECCCCCCCCCCC 289 Eel CCCCCCCCEEEEECECECCCCCCCHCEHCCCCCCCCEEEEEEECCCCCEEECCCCCCEECCCCCCCCEEEEECCCCCCCCCCCCHHEEEEHHHHHCHEEEEEHHHCCCHHEEEEECCCCCCCCCCC 291 Masu salmon CCCECEEEEEEEECEHEECCCCCCEEEHECCCCCCCCEEEEECCCCHHHHHHHHHHHCCCCCCCCCEEEEECCCCCCCCCCCCEEEEEEECCHHHHHHHEEEEECCCCHEEEEEECCCCCCCCCCC 293 Black seabream CCCCCCCEEEEEEHEEECCCCCCCHHHHHCCCCCCCEEEEEEECCCCEEEEHHHHCCEECCCCCCCCEEEEECCCCCCCCCCCEHHHHHHHHHHHHEEEEEHCCCCCCCEEEEEECCCCCCCCCCC 303 Gilthead seabream CCCCCCCEEEEEEHEEECCCCCCCHHHHHCCCCCCCEEEEEEECCCCEEEEHHHHCCEECCCCCCCCEEEEECCCCCCCCCCCCHHHHHHHHHHHHEEEEEHCCCCCCCEEEEEECCCCCCCCCCC 310 Medaka CCCCHCCEEEEEEHEEECCCCCCCCHHHHCCCCCCCEEEEEEECCCCEECHHHHHCCCCCCCCCCCCEEEEECCCCCCCCCCCEEEEEEECCCCEEEEEEEEEHCCCCHHEEEEECCCCCCCCCCC 296 Tilapia CCCEEECEEEEEHHHHECCCCCCCCHHHHHCCCCCCEEEEEEECCCCEEEEHHHHHCECCCCCCCCCEEEEECCCCCCCCEHEEEEEEEECCCCEEEEEEEEHHCCCCHEEEEEECCCCCCCCCCC 297 Turbot CCCCCCCCEEEEEEECCCCCCCCCHHHHCCCCCCCCHEEEEEECCCCEEEEHHCHCCCCCCCCCCCCEEEEECCCCCCCCCCCEEEEEEEHHHCHEEHHEEEHHCHCCHHEEEHCCCCCCCCCCCC 297 Atlantic hal but CCCHHHCHEHHECCEEECCCCCCCHHHEEEECCCCCHEEEEEECCCCEEEEHHHHCCCCCCCCCCCCEEEEECCCCCCCCCCCEEEEEEEHHHCHEEEEHHEHHCCCCHEEEEEECCCCCCCCCCC 297 Bastard hal but CCCHHHHHEEEEEEECCCCCCCCCHHHHCCCCCCCCCEEEEEECCCCEEEEHHCHCCECCCCCCCCCEEEEEEEECCCCCCCCCEEEEEEHHHCHEEEEEEEHHCCCCEEEEEEECCCCCCCCCCC 297 367 368

b Human GHR M-D-LWQ-LLLTLALAGSSDAFSGSEATAAILSRAPWSLQSVNPGLKTNSSKEPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNT-QEWTQ 96 Chicken GHR M-D-LRH-LLFTLALVCANDSLSASDD---LLQW------PQISKCRSPELETFSCYWTDGK---VTTSGTIQLLYMKRSD-----E 67 Turtle GHR M-D-LWQ-LLLILVLVCADSSLSAGEV---ILGR------PHTIKCRSPEQETFSCHWTDKDFHNLTAPGTIQLLYMRRND-----E 70 Frog GHR MA--YWL-FWVTVILLYIDGSTAATDVS---LGK------PRIIKCRSPQQETITCYWTNGEFRNLSLSGLLKLQYKKKTE-----A 70 Lungfish GHR M-D-RSICLLFFVILGQLRCSVTTTEAPV------KKPRFITCRSPEQVTFTCWWTVGSFTNLSESGELKLQYKKGSN-----D 71 Sturgeon GHR MA--TRL-VFLSLVFGAMSVSVGEHLTTSEV------PPKRPYFVDCRSPEQETFSCWWSAGDYQNLTGPGALSVFFQKNRD-----V 74 Medaka SLR MADAFSPGLLLMLMMSTLD--WLSTPGSAFLFDR------DRAKSAPL--EPHFTECVSRDLETFRCWWSPGSFQNLSSPGALRVFYLKKDP---PLS 85 Medaka GHR MAAA------LTVLLCCLY--AIAAAEAD------SKKVQLQKPPHLTGCVSTNMETFRCSWKVGTFHNLSDAGDLRLFYLNQNSLSAAPA 77

EWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSN-GGT-V-DEKCFSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETK 203 DWKECPDYITAGENSCYFNTSYTSIWIPYCVKLANK-DEV-F-DEKCFSVDEIVLPDPPVHLNWTLLNTSQTGIHGDIQVRWDPPPTADVQKGWITLEYELQYKEVNETK 174 EWKECPDYITAGENSCYFNTSYTSIWITYCVKLINK-DDV-L-DEKCFSVDEIVQPDPPVGLNWTLLNTSLTRIHADIQVRWDPPPSADVQKGWITLEYELQYKEVNETK 177 NWTDCPDTITGGENSCYFSKTYTSIWVSYCTKLVSE-DTE-F-DDYCFSVDDIVEPDPPMALNWTVLNISLTRMRIDIQLSWEPPPSADVSSGWISLEYEVHIKEANESQ 177 DWLDCPEYI-QGENGCYFNSSYCNVWIPYCVKLLSK-EDVEY-DKSCFSVDEIVQPDPPVNLNWTLLNISRSGLYADILVSWEAPPSADVLNGWIALEYQLQYKDRNETQ 178 -WRECPDYSTAGENHCYFNSTYTSIWIPYCVQLRSQIQDIVY-HQMCFTVDEIVVPDPPVGLNWTLLNISQTGQHFDILIQWEPPPSADVKKGWISLVYEPQYRAINMSD 182 EWKECPEYNH-LNRECFFDTNHTSVWLPYCVQLRSQNNVTYFNEEDCFTVENIVRPDPPVSLNWTLLNVSPSGLSYDVMVSWEPPPSADVQAGWMRIEYEVQYRERNTTN 194 EWRECPEYSRETPHQCFFNEEHTTVWTQYAVQLRSGDGAVVY-DEVFFNVQDIVEPDPPVGLNWTLLNVSLTGTHFDIMVTWRPPDSADVKMGWMTLSYEVQHRSLSSDH 186

WKMMDPILTTSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLPQ--MSQFTCEEDF-YFPWLLIIIFGIFGLTVMLFVFLFSKQQRIKMLILPPVPVPKIKGID 310 WKELEPRLSTVVPLYSLKMGRDYEIRVRSRQRTSEKFGEFSEILYVSFTQAGIEFVHCAEEI-EFPWFLVVVFGVCGLAVTAILILLSKQPRLKMLIFPPVPVPKIKGID 283 WRELEPMLTTVVPLYSLKLGRDYEIRVRSRQRASEKFGGFSETLYVSLS--SISSVLCDEEI-QFPWLLIIVFGTFGLIVVMFLILFSKHRRLKMLILPPVPAPKIKGID 284 WTILDKVQTKYLPVYALKTGKDYFARVRCKQLSNGKFGEFSDVLHIPLS------ILPEPDLPDVPFLLFLIIGLFGMLLVFIFILVFKKKRLKMIILPPVPVPKIKGID 281 WEQVEHTVMTKLPVYSLVTGREYEIRVRCKQRNFLKYGEFSNAIYVVIPRNVK-----EKEGTAFPMIFVLTFGAIGLTMIILLLMHTKQQRLKLLILPAVPVPKIKGID 283 WKPLEVERARYQSVYGLKTGMEYEIRVRCKQTASEKFSEFSDHLYVYIPKVPT------KE-TLFPFTMILIFGAVGVIIVLMLIIFSQQQRLMVYFLPPVPVPKIMGID 285 WEALEMQPHTQQTIYGLQIGKEYEVHIRCRMQAFTKFGEFSDSIFMQVTEIPSTD------STAPLTLVLIFGTVGILIVIMLIVISQQQRLMMILLPPVPAPKIKGID 297 WEVTDLVSSTHRTLYGLQTNVNHEVRVRCKMHGMTRFGGFSDSAFVH---ILSKV------SRFPVMALLITGALCLVAILMLVIISQQEKLMVVLLPPVPEPKIKGVD 286

PDLLKEGKLEEVNTILAI-----HDSYKPEFHSDDSWVEFIELDIDEPDEKTEE----SDTDRLLSSDHEKSHS----N----LGVKDGDS--GRTSCCEPDILETDFNA 401 PDLLKKGKLDEVNSILAS-----HDNYKTQLYNDDLWVEFIELDIDDSDEKNRV----SDTDRLLSDDHLKSHS----C----LGAKDDDS--GRASCYEPDIPETDFSA 374 PDLLKKGKLDEVNSILAN-----HDSYKPQLYNDNSWVEFIELDIDDPDEKTEG----LDTDRLLGDNHCKSHN----C----LGVKDDDS--GRASCCEPDIPETDFSA 375 PDLLQRGKLDEVNSILAC-----HDHYKQQLYNDDPWVEFIELDLDDTDEKNEG----SDTDRLLGEEHRKNHN----C----LGVKDDDS--GRASCCEPDIPETDFSN 372 PDLLKNGKLDELNSVLAG-----SYASKPILYMDDTWVEFIEVDISDHDEKKHG----SDMEKLLGEDFPNSHN----C----QGIKDDDS--GRDSCYEPDIPDMGDAL 374 PELLKKGKLDELNSILSG-----HHTFKPELYNDDLWVEFIELDIDDQDGRSNH----SDTHKLLGSENLNG-N----C----LNIKDDDS--GRASCYEPELPDSDTLD 375 SDLLKKGKLEELNFILNGGSIGGLQTFSPDFYQDEPWVEFIEVDEEDADSREKEDDQASDTQKLLGPPQPISDHRTIRCS---ETIRHPEEVFGHGSCYNADRPEEDSKV 404 SRLLKKGKLQEFSSVL-----GGHPNLRPELYSD-PWVEFIDLDME----RQNDRLTCLDTDCLVDSSSS-S-N----CSPLAASFRDHDS--GRSSCCEQDLLCDPNPS 378

NDIHEGTSEVAQPQRLKG-EADLLCLDQKNQNNSP--YHDACPATQQPSV-IQAEKNKPQPLPTEGAESTHQAAHIQLSNPSSLSNIDFYAQVSDITPAGSVVLSPGQKN 507 SDTCDAISDIDQFKKVTEKEEDLLCLHRKDDVEAL--QSLANTDTQQPHTSTQSESRESWPPFADSTDSANPSVQTQLSNQNSLTNTDFYAQVSDITPAGSVVLSPGQKS 482 SDTCDGTSDIDQSKKTSEKEEDLLCLDQKDNNESH--ASPDDPTAQKPNTNIQSEDNQSKLPFADSIESTRPPVHTQLSNQSSGANTDFYAQVSNVTPAGSVVLSPGQTN 483 SDTCDGTSDLGQTQNVKENEADLLCLDEKSNSVSP--TNVSVPNTED--GSPKPEAEKTCPVAVSENEPTSLPVSAPISKMRAKPSMDFYALVSDITPAGRLLLSPGQRM 478 DNPCHTTSDQPQEEKIKE---DLLCLKENDNISPSCFTSDSANSGDLLAEKRMPSDLTTGFVVSEESKSNRPPVHSQLSNQSSV-NLDFYTQVSDITPAGGVLLSPGQQS 480 RSQAEA------REEKASLL---DSEGKAAHSSTADPSPPGRSHPGAT------AEGASRSHPQIQSQLSSQSWV-NLDFYAQVSNITPAGGVLLSPGQQS 460 QMDAPPPGQAGDKESSTE------FAERTPALDRC------RAPPVQTQTGTPQTWVNTDFYAQVSNVMPSGGVVLSPGQQL 474 PVHPPISDQI------TPNAPAC------QPDSGAGVGVQSPGRDAT--YTQVCEVR-SGNVMLSPEEHL 433

KAGMSQCDMHP------EMVSLCQENFLMDNAY---FCEADAKKCIPVAPHIKVESHIQPSLNQEDIYITTESLTTAAGRPGTG-EHV------PGSEMPVPDY 595 KVGRAQCESCT------EQ------NFTMDNAY---FCEADVKKCIAVISQEEDEPRVQEQSCNEDTYFTTESLTTTGINLGASMAET------PSMEMPVPDY 565 KRRRTQCEAYT------EPAIPCQPNFTTDYAY---FCEADVKKCIAVTSCDAVEPHAQEQSFNEDVYFTTESLTTTAENPGAAIVEA------SSSEMPVPDY 572 KNENEECN------QPVIQHPANLNPDSPY---ICESAVTAFCAASKPRDTEASVKPNVI-DDSYFTTESLNIPEMNPCFA-EKA------SSYDMPVSDY 562 KVEKIESKPES------REPSKYQMPAAVIGAY---TFEMDVKK-I--NNLSNESKPVLEKAFDQGSYFTAESLNSVNLNSGVRGEYQ------HSSLMPVSDY 566 KIEKTAEAHSS------PKTQKCQLAMTTDSAY---TSEMDAKNISATPSLNGNEPKV------TVSPYTVSEAP------ASSVPPVSDY 530 RTQESTPASEHGAQTKGKQRDESGDMDGQRQKEPQFHLLVVDPEGSGYTTESNCWQNSTPSNSPNPGAG-YQT-IPPPPVDIKPAASADANQLPYILPDCPQLVAPVADY 582 ------DGGSIVELVEEEAGRKEA------VNADHKSYGEE-----HSGPKSPPSATPA-Y------476

TSIHIVQSPQGLILNATALPLPD------KEFLSSCGYVSTDQLNKIMP 638 TSIHIVHSPQGLVLNATALPVPE------KEFNMSCGYVSTDQLNKIMP 608 TSIHIIHSPQGLVLNATALPVPD------KEFMLSCGYVSTDQLNKILP 615 TSVHIINSPQNLVLNTTVLP--N------KEFLAPCGYMTPDQVNKVMK 603 TSIQMIDSQLSLIPKSDTLP--S------RCFSKP-PYVTPDQLSSFMP 606 TTVQAVDSQQSLLLNPSTLP--T------KGTPMPAGYLTPEQLNSIMP 571 TVVQELDTHHSLLLDPTPIQSPPPCPSQPPLKT-QIPVDYITPDLLGNLLP 632 TMVDGVDTHNSLLLTT----SSTPSVHLTPLKQLPTPGSYLIPDLLSSITP 523

Fig. 6 continued approximately 35% identity between GHRs of tetrapods (Brown et al. 2005). Lungfish and sturgeon GHRs have and teleosts), the predicted secondary structures are all linkers of 14 and 12 amino acids, respectively; again similar strikingly similar (Fig. 6a). to the teleost SLRs and amphibian and reptilian GHRs. However, we note that the length of linker residues Taken together, our residual and structural comparisons between the ECD and the transmembrane domain (TMD) did not reveal any unique features specific to the lungfish and appeared to differ in a lineage-specific manner. Between the sturgeon GHRs. Instead, they always share identical residues bottom of the ECD and the top of the TMD (around Y230 and similar structures with some or all of other GHR/SLRs. F247), there is a linker of 16 or 17 amino acids in placental The similarities (e.g., overall sequence similarity, position of GHRs. The linker of teleost SLRs is perfectly conserved for paired/unpaired cysteines, residues at R43, linker length, 12 amino acids, and that of teleost GHRs is only nine amino glycosylation sites, etc.) are more often observed with teleost acids (with one exception, again in the eel, of 12 amino SLRs and poikilothermic-tetrapod GHRs rather than with acids). Marsupial and amphibian GHRs have relatively homeothermic-tetrapod and teleost GHRs. divergent linkers of 13 20 amino acids (longer in more derived species). We suspect that these differences are not trivial because the linker length is rather divergent as a whole Genomic Structures of the Lungfish and Sturgeon (9 20 amino acids), yet is strictly conserved within each GHRs lineage. Furthermore, the length of the linker characterizes structural rigidity/flexibility of the ECD. A recent study In previous studies, orthology relationships of eel GHR/ showed that GH-mediated rotation of the dimerized recep- SLR to other teleost receptors remained ambiguous (Fu- tors at an appropriate angle is necessary for its activation kamachi et al. 2005; Ozaki et al. 2006). Although the 369

species Lungfish Eel primers lGf1/r lGf2/r lGf3/r eSf/r eGf/r templates ggcgcgc g

lGf1 lGf2 lGf3 Lungfish GHR 5´ AAA lGr eSf Eel SLR 5´ AAA eSr eGf Eel GHR 5´ AAA eGr 0.2 kb

Fig. 7 Absence of the SLR specific intron from lungfish/eel GHRs lungfish GHR are shown by black arrows. Templates and primers and also from eel SLR. Expected positions of the intron (gray arrows) used for PCRs are shown above the gel image (c, cDNA; g, genomic and approximate positions of the primers are shown on the left DNA). The same result was obtained from sturgeon (data not shown) diagram. Expected positions of the two conserved introns on the present study clarifies their relationships (Fig. 4), the et al. 2006; Ozaki et al 2006). This nomenclature is basi- phylogenetic position of the eel SLR is unexpectedly high cally (evolutionarily) correct according to our present up in the gene tree and does not conform to its species results, which showed that both the teleost GHR and SLR phylogeny when analyzed by both Bayesian and maxi- are orthologous to the mammalian GHR (Figs. 2 and 4). mum-likelihood methods (compare the tree topologies Also to be reconsidered, however, is that both GHR1 and between the GHRs and SLRs of teleosts). For further GHR2 in salmonids represent GHR (GHR2), which reflects clarification of their evolutionary history, we investigated another (4R) polyploidization (Moghadam et al. 2005; whether the extra intron specifically found in teleost SLRs Fig. 4). GHR1 was defined as SLR in salmon based on (Fukamachi et al. 2005) is found in the eel receptors as functional evidence; SL binds to this receptor more well. We also analyzed the lungfish and sturgeon GHRsin strongly than GH does (Fukada et al. 2005). In order to this regard. Comparisons of RT-PCR and genomic PCR further support this nomenclature which does not reflect the products, however, showed that none of these receptors evolutionary history (Mindell and Meyer 2001), further have the extra intron (Fig. 7). We also found that the intron evidence showing that SLR really functions as a receptor does not exist in the predicted zebrafish receptors (Fig. 2). for SL would be necessary. Therefore, it is likely that the ancestral GHR did not have The clearest evidence would be provided through the this intron and SLRs later acquired it after the evolutionary isolation of mutants or knockouts for SLR as has been done separation of eels, and probably Cypriniformes, from the for human, mouse and chicken GHRs (Agarwal et al. 1994; stem lineage that leads to more derived teleosts. Zhou et al. 1997; Laron 2004). Experiments using medaka would be the most logical, because a medaka mutant for SL has already been isolated and a gene-knockout method Discussion (TILLING) has been established in this species (Fukamachi et al. 2004; Taniguchi et al. 2006). Precise agreements Our present analyses on the GHR/SLR sequences of tet- between the phenotypes of the ci mutant and a SLR rapod and teleosts, and also the newly determined GHR knockout, if obtained, would clearly establish that SLR is a sequences of ‘‘living fossils’’ revealed for the first time that necessary and sufficient receptor for SL. Such in vivo (1) the SLR is a teleost-specific GHR paralogue acquired evidence will also exclude the possibility that the in vitro most likely through the FSGD, (2) there are multiple binding data only reflect a less significant cross-reaction, as lineage-specific residues and structures among tetrapod has been shown between GH and PRLR in human (Somers GHRs, teleost GHRs, and teleost SLRs, and (3) the lungfish et al. 1994). and sturgeon GHRs share similarities more often with teleost SLRs and amphibian and reptilian GHRs than with mammalian, avian or teleost GHRs (Figs. 2, 4, 5, and 6). Ancestral Receptor for SL

Even if such functional evidence would support the validity Clarification of the Nomenclature of Teleost Receptors of the SLR nomenclature, the question would remain: which receptor(s) did SL bind to before the FSGD (Fig. 1)? Currently, GHR and SLR of teleosts are often referred to as The ancestral characteristics that are retained relatively GHR2 and GHR1, respectively (Saera-Vila et al. 2005; Jiao frequently in teleost SLRs (Figs. 5 and 6) and the in vitro 370 binding of not only SL but also GH to the salmon SLR receptor(s) during evolution, this putative receptor would (Fukada et al. 2005) seem to be suggestive in this regard. have been retained in at least some of the available tetrapod Another intriguing result was recently reported for the eel: and teleost genomes but that does not seem to be the while its SLR specifically binds to GH, its GHR binds to case. both GH and SL (Ozaki et al. 2006; personal communi- cation). These examples from two teleost species do not contradict each other in that one of the duplicated GHRs Receptor for SLb and Future Subjects To Be Addressed (i.e., salmon GHR and eel SLR) binds specifically to GH while the other (i.e., salmon SLR and eel GHR) binds to The FSGD duplicated not only GHR but also GH and SL. both GH and SL. Indeed, it appears that the SLR-like (i.e., While the duplicated GH seems to have been lost from the probably more-ancestral) features are exceptionally genomes of medaka, fugu, or zebrafish, the duplicated SL is retained in eel GHR (e.g., overall sequence similarity, retained in zebrafish (Fig. 3) and some other teleost lin- linker length, conserved cysteines, etc.; Figs. 5 and 6). eages (Zhu et al. 2004). The duplicated SLa and SLb have Therefore, the presumed role of GHR in the vertebrate limited sequence similarities (\50%) and the receptor for ancestor was as a receptor for both GH and SL. After the SLb has not been investigated in any species to date. The FSGD, this dual function might have been subfunctional- additional polyploidization in salmonids (Moghadam et al. ized in teleosts: one became the receptor for GH and the 2005) and a possible loss of GHR in turbot (Saera-Vila other for SL. et al. 2005) makes generalizations about the overall hor- This ancestral dual function (i.e., the GHSLR hypothe- mone receptor relationships in teleosts difficult. These sis) may be testable through functional studies on ‘‘living inconsistent hormone receptor sets and their distinctive fossils’’ representatives of basal lineages of the fish and expression patterns and functional diversifications (e.g., tetrapod lineages. However, the lungfish or sturgeon GHRs Fukada et al. 2004; Zhu et al. 2004; Fukada et al. 2005; would not necessarily have retained the ancestral function Fukamachi et al. 2005; Saera-Vila et al. 2005; Very et al. precisely as it was when their lineages arose in the Devo- 2005; Jiao et al. 2006; Ozaki et al. 2006; Zhu et al. 2007) nian period, since their hormone receptor relationships would indicate that the in vivo roles of these orthologous may also have diverged in a lineage-specific manner during genes are not necessarily identical in all teleost species. more than 400 million years of evolution. Therefore, the Our results aid in the clarification of the evolutionary his- results obtained from such experiments could not be tories of these hormones and receptors. Comparative interpreted as strong evidence either for, or against, this studies with divergent species (e.g., medaka and zebrafish) hypothesis. Nevertheless, in vitro binding of their GHRs to using not a part but the entire hormone receptor set of this both GH and SL, as is the case for the salmon SLR and eel gene family, would be necessary to provide reliable clues GHR, and consequent activations of mutually different about past and present functions of these hormones and target genes (if obtained) would most parsimoniously receptors. support the GHSLR hypothesis. The species from which we cloned GHRs (P. dolloi and H. dauricus) and the spe- Acknowledgments The authors would like to thank S. Hoegg of the University of Konstanz and J. G. Inoue of Florida State Uni cies from which GH/SL sequences were reported (P. versity for the lungfish and sturgeon samples, and for advice on the annectens, A. gueldenstaedtii, and A. transmontanus) are phylogenetic reconstruction; Y. Minegishi and J. Aoyama of the unfortunately different. In order to avoid the lineage- and University of Tokyo for the eel genomic DNA; Y. Ozaki of species-specific artifacts (e.g., R43 of primate GHRs dis- National Institute of Genetics and H. Fukada of Kochi University for detailed information about the eel GHR; and D. Gerrard for the cussed in the ‘‘Results’’ section), more cloning work (or idea of the relative rate test and comments on the manuscript. The purification of the hormones from these endangered fish) authors would also like to thank H. Mitani of the University of would be necessary to rule out this possibility. Tokyo and T. Yada of the National Research Institute of Fisheries An alternative hypothesis for the ancestral receptor for Science for comments on the manuscript. This research was sup ported by a long term fellowship (#LT00059/2005 L) from the SL would be that the common vertebrate ancestor had a International Human Frontier Science Program Organization to novel receptor for SL that is not orthologous to the GHR/ S.F., and grants from the Deutsche Forschungsgemeinschaft and the SLRs. 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