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General and Comparative Endocrinology 155 (2008) 201–207

Phylogenetic analysis of the insulin-like growth factor bindingprotein (IGFBP) and IGFBP-related protein gene families

Buel D. Rodgers a,b,1,*, Eric H. Roalson c,1, Cullen Thompson a

a Department of Animal Sciences, Washington State University, Pullman, WA 99164-6351, USAb School of Molecular Biosciences, Washington State University, Pullman, WA 99164-6351, USA

c School of Biological Sciences, Washington State University, Pullman, WA 99164-6351, USA

Received 18 October 2006; revised 7 April 2007; accepted 19 April 2007Available online 27 April 2007

Abstract

Insulin-like growth factor (IGF) activity is regulated by six high affinity binding proteins (IGFBPs) and possibly by some of the nineIGFBP-related proteins (IGFBP-rPs). To determine the phylogenetic relationship of this proposed gene superfamily, we conducted max-imum likelihood (ML) and Bayesian inference analyses on a matrix of amino acid sequences from a diversity of vertebrate species. Asingle most likely phylogram, ML bootstrap, and Bayesian consensus tree of 10,000,000 generations revealed a monophyletic IGFBPlineage independent of the IGFBP-rPs. The IGFBPs segregated into three distinct clades: IGFBP-1, -3, and -6. Subsequent gene dupli-cation events within the IGFBP-1 and -3 clades resulted in the production and divergence of IGFBP-2 and -4 within the IGFBP-1 cladeand IGFBP-5 in the IGFBP-3 clade. By contrast, the IGFBP-rPs were distributed paraphyletically into two clades: IGFBP-rP1, 5, and 6in one clade and the CCN family (IGFBP-rP2–4,7–9) in another. A recently identified IGFBP-3 homolog in rainbow trout localized tothe IGFBP-2 subclade. Subsequence analysis identified a RGD motif common to IGFBP-2 orthologs, but did not identify the nuclearlocalization sequence present in IGFBP-3 and -5 homologs. The putative trout IGFBP-3 was 36–55% identical to different IGFBP-2 pro-teins, but only 24–27% identical to IGFBP-3 proteins. These results suggest that the IGFBPs and IGFBP-rPs are at best distantly relatedand that the limited similarities likely resulted from exon shuffling. They also suggest that rainbow trout, and possibly other salmonids,possess two IGFBP-2 paralogs as the putative trout IGFBP-3 is misannotated.� 2007 Elsevier Inc. All rights reserved.

Keywords: Phylogenetics; IGFBP; IGFBP-rP

1. Introduction

The bioavailability of both insulin-like growth factor(IGF)-I and -II is mediated by six high affinity IGF-bindingproteins (IGFBP-1 to -6). This occurs locally at the cell andtissue level as well as systemically where the IGFBPs signif-icantly enhance the circulating half-life of the IGFs (Hwaet al., 1999b). Several recent studies, however, suggest that

0016-6480/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2007.04.013

* Corresponding author. Address: 124 ASLB, PO Box 646351, Depart-ment of Animal Sciences, Washington State University, Pullman, WA99164-6351, USA. Fax: +1 509 335 4246.

E-mail address: danrodgers@wsu.edu (B.D. Rodgers).1 These authors contributed equally to the studies described herein.

some IGFBPs, in particular IGFBP-3 and -5, can modulatecellular activities without binding to either IGF (Duan andXu, 2005; Firth and Baxter, 2002; Lee and Cohen, 2002;Oufattole et al., 2006). Despite minor differences, theIGFBPs share common amino and carboxy terminaldomains, both of which facilitate IGF binding, althoughonly the former domain is critical, and each IGFBP is wellconserved in the different vertebrate classes. Indeed, con-servation extends beyond primary sequence as all of theIGFBP genes characterized to date are similarly organized(Hwa et al., 1999b). By contrast, the IGFBP-related pro-teins (IGFBP-rP1 to 9) share very little overall homologywith the IGFBPs, although they possess amino terminaldomains similar to those of the IGFBPs and some arecapable of IGF binding, albeit with considerably lower

202 B.D. Rodgers et al. / General and Comparative Endocrinology 155 (2008) 201–207

affinity (Kim et al., 1997). Their ability to influence IGFaction, however, has not been convincingly demonstratedas most of their defined functions are either independentof IGF-binding or have not been thoroughly described(Hwa et al., 1999b; Perbal, 2004).

The cysteine rich amino terminal domains of mostIGFBPs, excluding only IGFBP-6 homologs, andIGFBP-rPs contain a GCGCCXXC or ‘‘IGFBP’’ motifwhose function is poorly understood (Hwa et al., 1999b).The IGFBPs additionally contain a thyroglobulin-type Imotif within their carboxy terminal domains while theIGFBP-rPs contain many different motifs depending uponthe specific protein. For instance, IGFBP-rP1 and -rP5(a.k.a. IGFBP-7/Mac25 and L56, respectively) also possessKazal-type serine proteinase inhibitor (both rP1 and 5),immunoglobulin-like (rP1) and serine protease (rP5) motifs(Hwa et al., 1999b). The CCN (CTGF; Cef10/Cyr61 andNov) sub-family of IGFBP-rPs, which include IGFBP-rP2 (a.k.a. connective tissue growth factor, CTGF), -rP3(NovH), -rP4 (Cyr61), -rP7 (WISP2), -rP8 (WISP1), and-rP9, also possess Von Willebrand factor type C repeatsand thrombospondin type I repeats, none of which arefound in any IGFBP (Rachfal and Brigstock, 2005).Despite the structural differences noted and the low levelof overall homology, previous reviews have suggested acommon ancestral link between the two protein families(Hwa et al., 1999a,b; Kelley et al., 2000). Kelley et al.(2000) suggested that the IGFBP-rPs arose from a thyro-globulin-like ancestor and that a primordial IGFBP subse-quently diverged. Hwa et al. (1999a,b) further suggestedthat the entire IGFBP/IGFBP-rP ‘‘superfamily’’ evolvedfrom an ancestral ‘‘IGF binder’’ into two distinct proteinfamilies with different binding affinities for the IGFs: highaffinity IGF binders (IGFBPs) and low affinity binders(IGFBP-rPs). However, such conclusions may be unsub-stantiated in the absence of a rigorous phylogenetic analy-sis of the putative superfamily.

In order to determine the true phylogenetic relationshipbetween these gene families, we conducted maximum like-lihood (ML) and Bayesian inference analyses on 64 IGFBPand IGFBP-rP amino acid sequences representing mostvertebrate classes. These computational methods generatea ML point estimate of the IGFBP phylogeny and twomeasures of branch support: ML bootstraps and Bayesianposterior probabilities of tree distributions from millions ofgenerations (Huelsenbeck et al., 2002; Huelsenbeck andRonquist, 2001; Huelsenbeck et al., 2001). Our results indi-cate that five independent gene duplication events areresponsible for the divergence of the IGFBPs into three dis-tinct subclades: the IGFBP-6 clade, the IGFBP-1 clade,which also contains IGFBP-2 and -4, and the IGFBP-3clade, which also contains IGFBP-5. They also suggest thatthe two families (IGFBPs and IGFBP-rPs) may not neces-sarily share a common ancestor, but that the similar aminoterminal domains may be a product of exon shuffling. Thelack of clear phylogenetic and functional relationshipsbetween the IGFBPs and the IGFBP-rPs suggests that

the current classification of these two largely unrelatedgroups as a ‘‘superfamily’’ should be revised.

2. Materials and methods

2.1. Phylogenetic analysis

A single matrix composed of homologous amino acid sequences fromspecies in all vertebrate classes except Agnatha and Reptilia (no knownhomologs) was constructed using Vector NTI for the Macintosh. Thisincluded IGFBP sequences from bovine (Bos taurus), zebrafish (Danio

rerio), chicken (Gallus gallus), human (Homo sapiens), little skate (Leuco-

raja erinacea), mouse (Mus musculus), striped bass (Morone saxatilis),sheep (Ovis aries), rainbow trout (Oncorhynchus mykiss) and Africanclawed frogs (Xenopus laevus and Xenopus tropicalus). Human and mousemyostatin (MSTN) sequences were also included as an unrelated out-group. A list of the accession numbers for each IGFBP, IGFBP-rP andMSTN homolog or the matrix itself will be provided upon request.

Maximum likelihood (ML) and ML boootstrap analyses of the IGFBPmatrix were performed using the proml and seqboot modules of PHYLIP3.66 (Felsenstein, 2007). ML phylogram reconstruction used the Jones–Taylor–Thornton probability model, constant rates, global rearrange-ments, and random sequence order. Bootstrap analyses were run on 100replicate data matrices under the same conditions as above, but withoutglobal rearrangements due to time constraints.

Bayesian inference analysis was performed on the matrix using MrBa-yes v.3.0 (Huelsenbeck and Ronquist, 2001). Ten million generations wereperformed with four chains (Markov Chain Monte-Carlo) and a tree wassaved every 100 generations. Priors included a mixed amino acid modelallowing for optimization of the model during the analysis (Minin et al.,2003). Multiple analyses were started from different random locationswithin the tree space in order to test for the occurrence of stationarity,convergence and mixing within the ten million generations. Posteriorprobability distributions from separate replicates were compared for con-vergence to the same posterior probabilities across branches. Majority ruleconsensus trees of the 80,000 sampled during the Bayesian inference anal-yses yielded probabilities indicating monophyletic clades (Lewis, 2001).The trees from the MrBayes analysis were then loaded into PAUP* afterdiscarding the trees generated within the first 2,000,000 generations. Thus,the sampled trees included only those obtained post ‘‘burnin’’ of the chain(Huelsenbeck and Ronquist, 2001) and after stationarity was established.Bayesian posterior probability (pp) values are therefore presented abovebranches when greater than 50% and as a consensus topology.

2.2. BLAST analysis and alignment of IGFBP-3 orthologs

The amino terminal 150 amino acid sequence of human IGFBP-3 wascompared to the zebrafish expressed sequence tag (EST) database usingthe basic local alignment tool for protein sequences (BLASTP). A multiplesequence alignment of human and zebrafish IGFBP-3 and -5 proteins as wellas a putative rainbow trout IGFBP-3 homolog was constructed using VectorAlign X and the BLOSUM 62 matrix (gap penalty 10, extension 0.05).

3. Results

3.1. Phylogenetic analysis

The ML analysis of the IGFBP amino acid matrixresulted in one most likely tree (�lnL = 22742.11243;Fig. 1). ML bootstrap results suggest many strongly sup-ported branches, particularly within IGFBP clades, butsome lack of support for the exact relationships amongthe major clades. Posterior probability (PP) distributionsfrom the Bayesian inference analysis resulted in congruent

Fig. 1. Phylogenetic relationship of IGFBP and IGFBP-rP gene products. Maximum likelihood (ML), ML boootstrap, and Bayesian inference analyseswere performed on a single matrix composed of homologos amino acid sequences from various vertebrate species using PHYLIP 3.66 and MrBayes v.3.0(Bt, Bos taurus; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Le, Leucoraja erinacea; Mm, Mus musculus; Ms, Morone saxatilis; Oa, Ovis aries;Om, Oncorhynchus mykiss; Xl, Xenopus laevus; Xt, Xenopus tropicalus). A total of 100 ML bootstrap iterations and 10,000,000 Bayesian generations wereperformed (trees saved every 100 generations). Trees from the first 2,000,000 generations were discarded as burn-in to assure that stationarity wasestablished and the remaining 80,000 post burn-in trees were used to construct the majority rule consensus tree shown with PAUP*. Two independentanalyses were performed and produced identical results. ML bootstrap and Bayesian posterior probability values are shown above each branch (ML/Bay.PP) when greater than 50%. The major subclades are shaded and the monophyletic IGFBP group and the paraphyletic IGFBP-rP groups are labeled onthe right. (BP, IGFBP; rP, IGFBP-rP; MSTN, myostatin; CCN proteins include CTGF, Cyr61, WISP, NovH and IGFBP-rP2–4,7–9; IGFBP-rP1,5,6include Mac25, HtrA and ESM; * misannotated).

B.D. Rodgers et al. / General and Comparative Endocrinology 155 (2008) 201–207 203

topologies to the ML analyses and similar support forbranches when branches with PP values P95% are com-pared with those with bootstrap values P70% (Fig. 1).

The phylogenetic hypothesis presented here of IGFBP/BP-rP superfamily diversification reveals a monophyleticdistribution of all IGFBPs that was independent of the

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IGFBP-rPs (Fig. 1). The IGFBP family was divided intothree distinct sister clades: the IGFBP-6 clade, theIGFBP-1 clade that also included IGFBP-2 and -4 andthe IGFBP-3 clade that also included IGFBP-5. A totalof five independent gene duplication events were thereforedetected. The first created the IGFBP-1/-3 and -6 sisterclades while the second created the IGFBP-1 and -3 clades.This was followed by two additional duplication events inthe IGFBP-1 clade and one event in the IGFBP-3 clade(Fig. 1).

By contrast, the IGFBP-rPs are distributed paraphylet-ically into two clades: the IGFBP-rP1, 5, and 6 clade, andthe CCN family (IGFBP-rP2, -rP3, -rP4, -rP7, -rP8, and -rP9). None of the IGFBP-rPs segregated within any of theIGFBP subclades or vice versa, suggesting the two ‘‘genefamilies’’ evolved independent from one another and thatany relationship between the two families is distant andoccurred prior to the appearance of protochordates.Indeed, individual IGFBP-rPs are only 13–19% identicalto human IGFBP-3 overall as the limited homology is con-fined to small regions within the carboxy terminal domainscommonly referred to as IGFBP motifs. Similar pair wisecomparisons between representatives of the three differentIGFBP-rP clades reveal very little homology betweenclades. For example, IGFBP-rP1 and -rP5 are 14.3–17.5% and 7.5–16.6% identical to the CCN familymembers, respectively. The same is true for IGFBP-rP6(9.1–15.4%) and an unrelated zebrafish myostatin homolog(Kerr et al., 2005) (zfMSTN-2, 10.1–13.4%). By contrast,the different CCN proteins themselves are up to 48%identical.

Low stringency BLASTP analysis of the zebrafish ESTdatabase with the amino terminal domain of humanIGFBP-3 (first 150 amino acids) retrieved all of the previ-ously identified zebrafish IGFBPs (Duan et al., 1999; Liet al., 2005; Maures and Duan, 2002) and in addition, atankyrase homolog (TANK1; GID, 68397005) that alsocontained a carboxy terminal IGFBP motif (Fig. 2, shadedresidues 60–70). However, the TANK1 protein is only10.9% identical to IGFBP-3 overall and the tankyrase fam-

Fig. 2. Partial amino acid alignment of human (Hs) IGFBP-3 and a novel zebrEST database using a 150 amino acid sequence from the amino terminus of huNo.: XM_682318.1, GI:68397005) that shares motifs common to the IGFBPswhereas residue numbers within a specific sequence are indicated to the left anIGF-binding (Buckway et al., 2001; Yan et al., 2004) are indicated with asteri

ily as a whole is functionally unrelated to either theIGFBPs or IGFBP-rPs. These data together suggest thatthe limited homology shared between all three protein fam-ilies possibly occurred via exon shuffling and that otherproteins may also possess similar domains and motifs.

3.2. Identification of a novel rainbow trout IGFBP-2 paralog

Each IGFBP clade (Fig. 1) contained only orthologs forthat specific IGFBP with one exception. A recently identi-fied rainbow trout IGFBP-3 homolog segregated within theIGFBP-2 subclade rather than with the IGFBP-3 subclade,suggesting that this particular homolog is actually anIGFBP-2 paralog as another rainbow trout IGFBP-2 wasalso characterized (Kamangar et al., 2006). The true iden-tity of the putative IGFBP-3 was therefore determined bycomparative alignments and subsequence analysis.

Paired and multiple sequence alignments of the putativerainbow trout IGFBP-3 with different mammalian and fishorthologs of IGFBP-2, -3, and -5 indicate that the troutIGFBP-3 shares more identities with IGFBP-2 sequencesthan with those of IGFBP-3. Indeed, it is only 24% identi-cal overall to human and zebrafish IGFBP-3 and 27% iden-tical to IGFBP-5s (Fig. 3). By contrast, it is 36%, 53%, and55% identical to human, zebrafish and rainbow troutIGFBP-2, respectively. Comparative subsequence analysisidentified several motifs in the trout IGFBP-3 that are alsofound in IGFBP-2 orthologs including a RGD motifwithin the carboxy-terminal domains (Fig. 3). However,the nuclear localization sequence found in all IGFBP-3and -5 orthologs is not present in the rainbow troutIGFBP-3. The low level of sequence homology withIGFBP-3 proteins, the high level with IGFBP-2s and thepresence and absence of clade-specific motifs all comple-ment the phylogenetic analysis and indicate that the puta-tive rainbow trout IGFBP-3 is actually a second IGFBP-2paralog rather than an IGFBP-3 ortholog. The putativerainbow trout IGFBP-4 sequence used in this study wasobtained from a partial EST clone. Nevertheless, it segre-gated with other IGFBP-4 homologs. This suggests that

afish (Dr) tankyrase homolog (TANK1). BLASTP analysis of the zebrafishman IGFBP-3 identified a novel tankyrase homolog (GenBank Accessionand IGFBP-rPs. Alignment positions are numbered above the sequences,d in parentheses. Conserved regions are shaded and residues necessary forsks.

Fig. 3. Amino acid alignment of human (Hs), zebrafish (Dr), and rainbow trout (Om) IGFBP-3 homologs. Individual sequences were aligned using VectorAlignX. Alignment positions are numbered above the sequences, whereas residue numbers within a specific sequence are indicated to the left and inparentheses. The nuclear localization sequence conserved among all IGFBP-3 (BP3) and IGFBP-5 (BP5) proteins (KGRKR) and the integrin-bindingRGD sequence commonly found in IGFBP-2 proteins are boxed. Conserved residues within the alignment are shaded and residues involved in IGFbinding (Buckway et al., 2001; Yan et al., 2004) are highlighted with asterisks.

B.D. Rodgers et al. / General and Comparative Endocrinology 155 (2008) 201–207 205

unlike the misannotated IGFBP-3, the rainbow troutIGFBP-4 is indeed a true ortholog, although this can onlybe confirmed by isolating a complete clone.

4. Discussion

The monophyletic distribution of the IGFBPs in con-trast to the paraphyletic distribution of the IGFBP-rPsindicates that the IGFBPs are distinct, at best only distantlyrelated to IGFBP-rPs and that members of the putativeIGFBP-rP gene family may not be as closely related as orig-inally presumed. In fact, the extreme low level of homologyshared between the three different IGFBP-rP clades sug-gests that they are as dissimilar as comparisons betweenthe IGFBPs and IGFBP-rPs. Previous attempts to define

the evolutionary relationships between these families werebased either on sequence similarities and associations withhox gene clusters (Kelley et al., 2000) or with a rudimentaryand unrooted tree (Hwa et al., 1999b). Several differenceswere noted when the tree described by Hwa et al. (1999b)was compared to that reported herein (Fig. 1).

Hwa et al. (1999b) presented four models that couldhave explained the evolution of the proposed IGFBPsuperfamily: three based on the divergence of a primitiveIGFBP, IGFBP-rP or common ancestral gene and oneon the shuffling of an amino terminal module. The com-plete lack of any significant amino acid conservationamong the IGFBPs and IGFBP-rPs outside of the IGFBPmotifs in addition to the monophyletic distribution ofIGFBPs strongly suggests that the similar amino terminal

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domains in both protein groups resulted from exon shuf-fling. This model cannot be definitively proved, althoughit was also favored by Hwa et al. and was based in parton the fact that the common domains are encoded by a sin-gle exon in every gene. Shared motifs are not necessarilyindicative of exon shuffling, however, as they could haveevolved convergently. The similarities within the amino ter-minal regions discussed extend beyond the GCGCCXXCIGFBP motif in some, but not all, of the IGFBP-rPs andpossibly other proteins as well. This includes TANK1whose amino terminal domain is far more similar to theIGFBPs than are comparable domains from any of theIGFBP-rPs (Fig. 2). In addition, several residues criticalto IGF binding (Buckway et al., 2001; Yan et al., 2004)are also found in TANK1, although they are lacking inmost of the IGFBP-rPs. It is unknown whether TANK1can or does bind the IGFs. However, Yan et al. (2006)recently determined that the amino terminal domain ofCCN3 (a.k.a. IGFBP-rP3 and NovH) cannot replace thesimilar domain of IGFBP-3 as the IGF affinity of the chi-meric IGFBP-3 was significantly reduced to levels compa-rable of CCN3. This indicates that the IGFBP motifalone does not confer high-affinity binding as other aminoterminal motifs/residues are required. Exon shuffling,therefore, seems likely responsible for the similaritiesnoted, although it was followed by significant genetic diver-gence. The less likely alternative explanation, extreme func-tional divergence of a common ancestor, cannot be entirelyexcluded without further analysis, especially given the veryearly divergence of IGFBPs and IGFBP-rPs.

Kamangar et al. (2006) recently characterized severalcDNA clones for all six rainbow trout IGFBP homologsand IGFBP-rP1. This includes a putative IGFBP-3 thatsegregated along with IGFBP-2 orthologs in our phyloge-netic analysis. Subsequence analysis and sequence align-ments (Fig. 3) also suggest that it is actually anadditional IGFBP-2 paralog rather than the troutIGFBP-3. Duplicate genes are common in the bony fishesdue to a genome wide duplication event that occurred earlyin their evolution (Amores et al., 1998; Postlethwait et al.,1998). An additional genome duplication event specificallywithin the salmonids (Hordvik, 1998) is responsible for upto four unique copies of many genes in some species includ-ing the rainbow trout (Brunelli et al., 2001; Garikipatiet al., 2006, 2007; Kavsan et al., 1993; McKay et al.,2004). Thus, the existence of two IGFBP-2 paralogs inrainbow trout is not necessarily surprising. The possiblelack of an IGFBP-3 ortholog in salmonids (none have beencloned to date) is highly unusual and suggests that, if true,compensatory mechanisms may have evolved specificallywithin these fishes. Indeed, 244,984 ESTs (83,863 unique,see www.TIGR.org) have been sequenced to date from dif-ferent rainbow trout tissues including the liver and noIGFBP-3 homologs have been identified (Rexroad et al.,2003).

Given the lack of a clear ancestral relationship betweenthe IGFBPs and the IGFBP-rPs, and among the three dif-

ferent IGFBP-rP clades as well, it is questionable whetherthe ‘‘IGFBP superfamily’’ classification is justified as itdoes not reflect the phylogenetic relationships of the differ-ent protein families. The physiological significance of lowaffinity binding interactions between IGFBP-rPs and theIGFs remains equally questionable, especially in the pres-ence of the IGFBPs which bind with much higher affinity,as other IGF-independent functions have already been wellestablished for many IGFBP-rPs (Bleau et al., 2005; Per-bal, 2004; Rachfal and Brigstock, 2005). The alternativeIGFBP-rP nomenclature (WISP, NovH, CTGF, CCN,etc.) may therefore be more appropriate in light of futurerevisions. Nevertheless, the IGFBP phylogenies describedwill help in better defining the evolution of this diverse genefamily. Future studies, however, may require the identifica-tion of homologs from more basal groups and from inver-tebrates as the origin of IGFBPs have yet to be described.

Acknowledgments

This work was supported by a Grant from the UnitedStates Department of Agriculture (2004-34468-15199) toBuel D. Rodgers.

References

Amores, A., Force, A., Yan, Y.L., Joly, L., Amemiya, C., Fritz, A., Ho,R.K., Langeland, J., Prince, V., Wang, Y.L., Westerfield, M., Ekker,M., Postlethwait, J.H., 1998. Zebrafish hox clusters and vertebrategenome evolution. Science 282, 1711–1714.

Bleau, A.M., Planque, N., Perbal, B., 2005. CCN proteins and cancer: twoto tango. Front Biosci. 10, 998–1009.

Brunelli, J.P., Robison, B.D., Thorgaard, G.H., 2001. Ancient and recentduplications of the rainbow trout Wilms’ tumor gene. Genome 44,455–462.

Buckway, C.K., Wilson, E.M., Ahlsen, M., Bang, P., Oh, Y., Rosenfeld,R.G., 2001. Mutation of three critical amino acids of the N-terminaldomain of IGF-binding protein-3 essential for high affinity IGFbinding. J. Clin. Endocrinol. Metab. 86, 4943–4950.

Duan, C., Ding, J., Li, Q., Tsai, W., Pozios, K., 1999. Insulin-like growthfactor binding protein 2 is a growth inhibitory protein conserved inzebrafish. Proc. Natl. Acad. Sci. USA 96, 15274–15279.

Duan, C., Xu, Q., 2005. Roles of insulin-like growth factor (IGF) bindingproteins in regulating IGF actions. Gen. Comp. Endocrinol. 142, 44–52.

Felsenstein, J., 2007. PHYLIP (Phylogeny Inference Package) version6.44. Distributed by the author. Department of Genetics, University ofWashington, Seattle.

Firth, S.M., Baxter, R.C., 2002. Cellular actions of the insulin-like growthfactor binding proteins. Endocr. Rev. 23, 824–854.

Garikipati, D., Gahr, S.A., Rodgers, B.D., 2006. Identification, Charac-terization and quantitative expression analysis of rainbow troutmyostatin-1a and -1b genes. J. Endocrinol. 190 (3), 879–888.

Garikipati, D., Gahr, S.A., Roalson, E.H., Rodgers, B.D., 2007. Char-acterization of rainbow trout myostatin-2 genes (rtMSTN-2a & -2b):genomic organization, differential expression and pseudogenization.Endocrinology 148 (5), 2106–2115.

Hordvik, I., 1998. The impact of ancestral tetraploidy on antibodyheterogeneity in salmonid fishes. Immunol. Rev. 166, 153–157.

Huelsenbeck, J.P., Larget, B., Miller, R.E., Ronquist, F., 2002. Potentialapplications and pitfalls of Bayesian inference of phylogeny. Syst. Biol.51, 673–688.

Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference ofphylogenetic trees. Bioinformatics 17, 754–755.

B.D. Rodgers et al. / General and Comparative Endocrinology 155 (2008) 201–207 207

Huelsenbeck, J.P., Ronquist, F., Nielsen, R., Bollback, J.P., 2001.Bayesian inference of phylogeny and its impact on evolutionarybiology. Science 294, 2310–2314.

Hwa, V., Oh, Y., Rosenfeld, R.G., 1999a. Insulin-like growth factor bindingproteins: a proposed superfamily. Acta. Paediatr. Suppl. 88, 37–45.

Hwa, V., Oh, Y., Rosenfeld, R.G., 1999b. The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocr. Rev. 20, 761–787.

Kamangar, B.B., Gabillard, J.C., Bobe, J., 2006. Insulin-like growthfactor-binding protein (IGFBP)-1, -2, -3, -4, -5, and -6 and IGFBP-related protein 1 during rainbow trout postvitellogenesis and oocytematuration: molecular characterization, expression profiles, and hor-monal regulation. Endocrinology 147, 2399–2410.

Kavsan, V., Koval, A., Petrenko, O., Roberts Jr., C.T., LeRoith, D., 1993.Two insulin genes are present in the salmon genome. Biochem.Biophys. Res. Commun. 191, 1373–1378.

Kelley, K.M., Desai, P., Roth, J.T., Haigwood, J.T., Arope, S.A., Flores,R.M., Schmidt, K.E., Perez, M., Nicholson, G.S., Song, W.W., 2000.Evolution of endocrine growth regulation: the insulin like growthfactors (IGFs), their regulatory binding proteins (IGFBPs) and IGFreceptors in fishes and other ectothermic vertebrates. In: Fingerman,M., Nagabhushanam, R. (Eds.), Recent Advances in Marine Biotech-nology. Science Publishers, Inc, Enfield, p. 292.

Kerr, T., Roalson, E.H., Rodgers, B.D., 2005. Phylogenetic analysis of themyostatin gene sub-family and the differential expression of a novelmember in zebrafish. Evol. Dev. 7, 390–400.

Kim, H.S., Nagalla, S.R., Oh, Y., Wilson, E., Roberts Jr., C.T.,Rosenfeld, R.G., 1997. Identification of a family of low-affinityinsulin-like growth factor binding proteins (IGFBPs): characterizationof connective tissue growth factor as a member of the IGFBPsuperfamily. Proc. Natl. Acad. Sci. USA 94, 12981–12986.

Lee, K.W., Cohen, P., 2002. Nuclear effects: unexpected intracellularactions of insulin-like growth factor binding protein-3. J. Endocrinol.175, 33–40.

Lewis, P.O., 2001. Phylogenetic systematics turns over a new leaf. TrendsEcol. Evol. 16, 30–37.

Li, Y., Xiang, J., Duan, C., 2005. Insulin-like growth factor-bindingprotein-3 plays an important role in regulating pharyngeal skeleton

and inner ear formation and differentiation. J. Biol. Chem. 280, 3613–3620.

Maures, T.J., Duan, C., 2002. Structure, developmental expression, andphysiological regulation of zebrafish IGF binding protein-1. Endocri-nology 143, 2722–2731.

McKay, S.J., Trautner, J., Smith, M.J., Koop, B.F., Devlin, R.H., 2004.Evolution of duplicated growth hormone genes in autotetraploidsalmonid fishes. Genome 47, 714–723.

Minin, V., Abdo, Z., Joyce, P., Sullivan, J., 2003. Performance-basedselection of likelihood models for phylogeny estimation. Syst. Biol. 52,674–683.

Oufattole, M., Lin, S.W., Liu, B., Mascarenhas, D., Cohen, P., Rodgers,B.D., 2006. Ribonucleic acid polymerase II binding subunit 3 (Rpb3),a potential nuclear target of insulin-like growth factor binding protein-3. Endocrinology 147, 2138–2146.

Perbal, B., 2004. CCN proteins: multifunctional signalling regulators.Lancet 363, 62–64.

Postlethwait, J.H., Yan, Y.L., Gates, M.A., Horne, S., Amores, A.,Brownlie, A., Donovan, A., Egan, E.S., Force, A., Gong, Z., Goutel,C., Fritz, A., Kelsh, R., Knapik, E., Liao, E., Paw, B., Ransom, D.,Singer, A., Thomson, M., Abduljabbar, T.S., Yelick, P., Beier, D.,Joly, J.S., Larhammar, D., Rosa, F., et al., 1998. Vertebrate genomeevolution and the zebrafish gene map. Nat. Genet. 18, 345–349.

Rachfal, A.W., Brigstock, D.R., 2005. Structural and functional proper-ties of CCN proteins. Vitam. Horm. 70, 69–103.

Rexroad 3rd, C.E., Lee, Y., Keele, J.W., Karamycheva, S., Brown, G.,Koop, B., Gahr, S.A., Palti, Y., Quackenbush, J., 2003. Sequenceanalysis of a rainbow trout cDNA library and creation of a gene index.Cytogenet. Genome Res. 102, 347–354.

Yan, X., Baxter, R.C., Perbal, B., Firth, S.M., 2006. The aminoterminalinsulin-like growth factor (IGF) binding domain of IGF bindingprotein-3 cannot be functionally substituted by the structurallyhomologous domain of CCN3. Endocrinology 147 (11), 5268–5274.

Yan, X., Forbes, B.E., McNeil, K.A., Baxter, R.C., Firth, S.M., 2004.Role of N- and C-terminal residues of insulin-like growth factor(IGF)-binding protein-3 in regulating IGF complex formation andreceptor activation. J. Biol. Chem. 279, 53232–53240.