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PLoS One
2013 Jan 01;81:e53482. doi: 10.1371/journal.pone.0053482.
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Structural and functional divergence of growth hormone-releasing hormone receptors in early sarcopterygians: lungfish and Xenopus.
Tam JK
,
Chow BK
,
Lee LT
.
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The evolutionary trajectories of growth hormone-releasing hormone (GHRH) receptor remain enigmatic since the discovery of physiologically functional GHRH-GHRH receptor (GHRHR) in non-mammalian vertebrates in 2007. Interestingly, subsequent studies have described the identification of a GHRHR(2) in chicken in addition to the GHRHR and the closely related paralogous receptor, PACAP-related peptide (PRP) receptor (PRPR). In this article, we provide information, for the first time, on the GHRHR in sarcopterygian fish and amphibians by the cloning and characterization of GHRHRs from lungfish (P. dolloi) and X. laevis. Sequence alignment and phylogenetic analyses demonstrated structural resemblance of lungfish GHRHR to their mammalian orthologs, while the X. laevis GHRHR showed the highest homology to GHRHR(2) in zebrafish and chicken. Functionally, lungfish GHRHR displayed high affinity towards GHRH in triggering intracellular cAMP and calcium accumulation, while X. laevis GHRHR(2) was able to react with both endogenous GHRH and PRP. Tissue distribution analyses showed that both lungfish GHRHR and X. laevis GHRHR(2) had the highest expression in brain, and interestingly, X. laevis(GHRHR2) also had high abundance in the reproductive organs. These findings, together with previous reports, suggest that early in the Sarcopterygii lineage, GHRHR and PRPR have already established diverged and specific affinities towards their cognate ligands. GHRHR(2), which has only been found in xenopus, zebrafish and chicken hitherto, accommodates both GHRH and PRP.
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Figure 2. Functional characterization of lfGHRHR and xGHRHR2.Intracellular cAMP accumulation ([cAMP]i) in response to 100 nM of GHRH and related peptides on CHO-K1 cells transfected with (A) lfGHRHR and (D) xGHRHR2 (*** indicates P<0.001, ** indicates P<0.01, and * indicates P<0.05). Effects of GHRH and related peptides on graded concentrations of peptides on [cAMP]i (B) lfGHRHR- and (E) xGHRHR2-expressing cells. The intracellular calcium mobilization ([Ca2+]i) assays of (C) lfGHRHR- and (F) xGHRHR2-expressing cells. For [cAMP]i, values represent mean ± SEM (n = 4). For ([Ca2+]i, data were expressed in ΔRFU value (maximum changes in the fluorescence signals from baseline) and converted to percentage of the maximum of xGHRH-induced [Ca2+]i elevation. Results are expressed as mean ± SEM from at least 10 independent experiments, cell number = 20 to 50. Peptide species: h, human; x, X. laevis, zf, zebrafish D. rerio; gf, goldfish C. auratus.
Figure 3. Tissue distribution profile of lfGHRHR and xGHRHR2.Gene expression of (A) lfGHRHR in P. dolloi and (B) xGHRHR2 in X. laevis was assessed by quantitative real-time PCR. The expression level of each gene was calculated from respective standard curve. Data are expressed as mean ± SEM (n = 4). Highest expression for both GHRH receptors was detected in the brain, as observed in mammals.
Figure 4. Gene linkage comparisons of GHRHRs in the Osteichthyes lineage.Genes in vicinity of GHRHR were mapped, and syntenic genes were linked by straight lines. Size of the chromosomal region analyzed was given underneath based on the current edition of Ensembl databases. Syntenic genes encoding other secretin GPCR receptors were drawn in grey boxes, and the conserved flanking genes of GHRHR were drawn in closed boxes. (A) Gene environment of GHRHR in the Sarcopterygii lineage represented by human, mouse, lizard, chicken, frog and coelacanth was compared. Despite the syntenic genomic locations of GHRHR and neighbouring genes from human to avians, GHRHR was not located in frog. (B) Gene environment of GHRHR in the Actinopterygii lineage represented by fugu, tetraodon, stickleback, medaka, and zebrafish. Apart from the less conserved genomic region of zebrafish GHRHR, genes in proximity of other teleost GHRHRs were highly syntenic. However, they displayed an entirely different gene environment when compared to the sarcopterygian GHRHRs. (C) Genomic location analysis of xGHRHR characterized in present work and GHRHR2 in zebrafish and chicken. Gene synteny could neither be identified inter-species nor between the two GHRHR genes in the same species. The figures were not drawn to scale.
Figure 5. GHRHR, GHRHR2 and PRPR in Osteichthyes lineage.Identified GHRHR, GHRHR2, PRPR and their ligand specificity were summarized in each taxon group. The Sarcopterygii-Actinopterygii split was marked by a black dot (•) and the timing of TSGD (Teleost-specific genome duplication) was indicated [14]. Abbreviations and terminology: Transcript refers to mRNA transcribed; Genome means identified in genome but not isolated; N.A., not available; N.D., not determined (no reported transcript and not found in genome); N.R., No reactivity; *, not endogenous ligand.
Figure 1. Evolutionary analysis of the osteichthyans secretin GPCR family.The maximum likelihood (ML) optimal tree topology is presented and was constructed with MEGA5. ML bootstrap values higher than 50% are indicated at nodes. To facilitate interpretation, PTHR was used as an outgroup based on the proposed models for secretin GPCR family evolution [7]. The tree supported the identities of lfGHRHR and xGHRHR2 as the orthologs of mammalian GHRHR and chicken GHRHR2 respectively. Accession numbers of the sequences used were listed in Table S2.
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