Medaka receptors for somatolactin and growth hormone: phylogenetic paradox among fish growth hormone receptors

Abstract

Somatolactin (SL) in fish belongs to the growth hormone/prolactin family. Its ortholog in tetrapods has not been identified and its function(s) remains largely unknown. The SL-deficient mutant of medaka (color interfere, ci) and an SL receptor (SLR) recently identified in salmon provide a fascinating field for investigating SL's function(s) in vivo. Here we isolated a medaka ortholog of the salmon SLR. The mRNA is transcribed in variable organs. Triglycerides and cholesterol contents in the ci are significantly higher than those in the wild type, providing the first evidence of SL's function in suppressing lipid accumulation to organs. Interestingly, phylogenetic comparisons between the medaka SLR and growth hormone receptor (GHR), which is also isolated in this study, in relation to GHRs of other fish, suggested that all GHRs reported from nonsalmonid species are, at least phylogenetically, SLRs. An extra intron inserted in medaka and pufferfish SLRs and flounder and sea bream GHRs also supports their orthologous relationship, but not with tetrapod GHRs. These results may indicate lineage-specific diversification of SLR and GHR functions among fish or just an inappropriate naming of these receptors. Further functional and comparative reassessments are necessary to address this question.

Figure 1.

Lateral views of the wild-type and ci medaka. Body color of the ci (top) is less yellow than that of the wild type (bottom) due to reduced xanthophores (orange pigment cells) on the skin. As shown, the ci fish are in good health accompanied by no apparent morphological or behavioral anomaly under normal breeding conditions.

Figure 2.

Cloning of the medaka SLR. (A) Comparative-genomic prediction of medaka and fugu SLR exons. Solid and shaded boxes indicate translated and untranslated regions, respectively. Corresponding exons of medaka and fugu retain identical exon-intron boundaries. Note that their SLR proteins are encoded by nine exons. (B) Nucleotide sequence of the medaka SLR cDNA (GenBank accession no. DQ002886). Arrowheads indicate position of introns inserted. Shaded residues indicate 5′- and 3′-UTRs. Underlining indicates polymorphic residues detected between the HNI (experimentally determined) and Hd-rR (on the genome sequence) alleles.

Figure 3.

Amino acid sequences of the medaka SLR and GHR. (A) Amino acid alignment of the SLRs and GHRs of medaka, fugu, and masu salmon. Those of fugu are putative (see text). Major residues among each group are in red. Asterisks indicate that the major residues for SLRs and GHRs are identical. Boxes indicate residues that contain exon-intron boundaries in their codons or the first residues in each exon. The conserved cysteines (six disulfide linked and one unpaired), FGXFS motif, box 1, box 2, and tyrosines (see text) are highlighted in yellow. (B) A phylogenic tree drawn by the Neighbor program in the PHYLIP package (http://evolution.genetics.washington.edu/phylip.html) using entire sequences of the SLRs and GHRs. All nodes are supported by bootstrap values of 100, demonstrating an orthologous relationship among the SLRs and GHRs of these fish (see also Figure 7).

Figure 4.

Cloning of the medaka GHR. (A) Comparative-genomic prediction of medaka and fugu GHR exons. Solid and shaded boxes indicate translated and untranslated regions, respectively. Corresponding exons of medaka and fugu retain identical exon-intron boundaries. Note that their GHR proteins are encoded by eight exons. (B) Nucleotide sequence of the medaka GHR cDNA contained in OLc08.02d (GenBank accession no. DQ010539). Arrowheads indicate position of introns inserted. Shaded residues indicate 5′- and 3′-UTRs. Another exon in the 5′ upstream region that does not encode protein as reported in tetrapod GHRs might be missing from this EST clone. Underlining indicates polymorphic residues detected between the HNI (experimentally determined by direct sequencing of RT-PCR products) and Hd-rR (contained in OLc08.02d) alleles. Polymorphisms in residues with dashed underlining were not examined.

Figure 5.

Expression of the medaka SLR and GHR. (Bottom) SLR and GHR mRNAs and the approximate positions of primers used in RT-PCR. Vertical solid lines indicate the position of introns.

Figure 6.

Physiological defects in the ci medaka. Results are shown as means ± SEM. Solid bar, wild type; open bar, ci. Significance of differences between two groups was analyzed by ANOVA followed by Student's t-test or Mann-Whitney U-test. (A) Hepatosomatic index and cortisol concentration in plasma were significantly higher and lower in the ci, respectively. *P < 0.05; **P < 0.01. (B) Triglycerides are significantly accumulated in both the muscle and the liver of the ci, while accumulation of cholesterol is apparent only in the liver.

Figure 7.

Phylogenetic relationships among SLRs, GHRs, and PRLRs of vertebrates. (A) A phylogenic tree drawn by clustalW available at EMBL-EBI (http://www.ebi.ac.uk/clustalw/). Numbers indicate bootstrap values calculated using MEGA3 software (Kumar et al. 2004). To avoid disturbances from possible splicing variations reported in tetrapods (e.g., an extra exon in mouse, loss of an exon in opossums and avians), proteins encoded by the last seven exons of the medaka SLR (see Figure 2B) and corresponding regions of other SLRs, GHRs, and PRLRs were used for analysis (a similar tree was actually drawn by using the full length of these proteins). In this analysis, we added SLR and GHR of another pufferfish (Tetraodon nigroviridis) predicted from its genome database (http://www.genoscope.cns.fr/externe/tetraodon/), although several putative sequencing errors on the database were manually corrected (e.g., from TCCCCCT to TCCCCT and from GCCCA to GCCA). A yellow background indicates PRLRs, although the position of the sea bream PRLR (Santos et al. 2001) is arguable (white background). Blue and pink backgrounds indicate GHRs and SLRs, respectively. Considering positions of eel GHR1 and GHR2 (although their bootstrap values, indicated in gray, are relatively low), GHRs in a green background seem more likely to be SLRs. Similar trees were drawn by parsimony, neighbor-joining, or maximum-likelihood methods using the programs (Protpars, Neighbor, or Proml) in the PHYLIP package. (B) The positions of the extra intron inserted at the SLR (GHR) loci reported to date. Red indicates major amino acids at each site among the four species. Their coding sequences are indicated below. The intron is inserted at the positions indicated by arrowheads.