Evolution of the relaxin-like peptide family

Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Australia.
BMC Evolutionary Biology (Impact Factor: 3.37). 03/2005; 5(1):14. DOI: 10.1186/1471-2148-5-14
Source: PubMed


The relaxin-like peptide family belongs in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; relaxin-1, 2 and 3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. The functions of relaxin-3, INSL4, INSL5, INSL6 remain uncharacterised. The evolution of this family has been contentious; high sequence variability is seen between closely related species, while distantly related species show high similarity; an invertebrate relaxin sequence has been reported, while a relaxin gene has not been found in the avian and ruminant lineages.
Sequence similarity searches of genomic and EST data identified homologs of relaxin-like peptides in mammals, and non-mammalian vertebrates such as fish. Phylogenetic analysis was used to resolve the evolution of the family. Searches were unable to identify an invertebrate relaxin-like peptide. The published relaxin cDNA sequence in the tunicate, Ciona intestinalis was not present in the completed C. intestinalis genome. The newly discovered relaxin-3 is likely to be the ancestral relaxin. Multiple relaxin-3-like sequences are present in fugu fish (Takifugu rubripes) and zebrafish (Danio rerio), but these appear to be specific to the fish lineage. Possible relaxin-1 and INSL5 homologs were also identified in fish and frog species, placing their emergence prior to mammalia, earlier than previously believed. Furthermore, estimates of synonymous and nonsynonymous substitution rates (dN/dS) suggest that the emergence of relaxin-1, INSL4 and INSL6 during mammalia was driven by positive Darwinian selection, hence these peptides are likely to have novel and in the case of relaxin-1, which is still under positive selection in humans and the great apes, possibly still evolving functions. In contrast, relaxin-3 is constrained by strong purifying selection, demonstrating it must have a highly conserved function, supporting its hypothesized important neuropeptide role.
We present a phylogeny describing the evolutionary history of the relaxin-like peptide family and show that positive selection has driven the evolution of the most recent members of the family.

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Available from: Ross Bathgate, Jan 21, 2014
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    • "Relaxin-3 is thought to modify neuronal activity primarily via activation of the 7-transmembrane G i/o -protein-coupled receptor -relaxin family peptide-3 receptor (RXFP3; also known as GPCR135) [6] [21] [22]. Several lines of evidence support this cognate peptide/receptor relationship: relaxin-3 binds to RXFP3 with high affinity (IC 50 /EC 50 0.5 nM) [21] [22] [23]; the genes encoding the peptide and receptor protein have phylogenetically co-evolved and are both highly conserved across species [21] [24]; there is a strong overlap between the distribution of RXFP3 mRNA/binding sites and relaxin- 3 positive fibres within rodent brain [14] [15] [25]; and relaxin-3 is the only member of the relaxin peptide family that can activate RXFP3 [21] (see [6] [26] for review). However, despite strong evidence that relaxin-3/RXFP3 forms a physiologically relevant cognate signalling pair, other data suggest some degree of ligand promiscuity under experimental conditions. "
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    ABSTRACT: Anatomical and pharmacological evidence suggests the neuropeptide, relaxin-3, is the preferred endogenous ligand for the relaxin family peptide-3 receptor (RXFP3) and suggests a number of putative stress- and arousal-related roles for RXFP3 signalling. However, in vitro and in vivo evidence demonstrates exogenous relaxin-3 can activate other relaxin peptide family receptors, and the role of relaxin-3/RXFP3 signalling in specific brain circuits and associated behaviours in mice is not well described. In this study, we characterised the behaviour of cohorts of male and female Rxfp3 gene knockout (KO) mice (C57/B6JRXFP3TM1/DGen), relative to wild-type (WT) littermates to determine if this receptor KO strain has a similar phenotype to its ligand KO equivalent. Rxfp3 KO mice displayed similar performance to WT littermates in several acute behavioural paradigms designed to gauge motor coordination (rotarod test), spatial memory (Y-maze), depressive-like behaviour (repeat forced-swim test) and sensorimotor gating (prepulse inhibition of acoustic startle). Notably however, male and female Rxfp3 KO mice displayed robust and consistent (dark phase) hypoactivity on voluntary home-cage running wheels (∼20-60% less activity/h), and a small but significant decrease in anxiety-like behavioural traits in the elevated plus maze and light/dark box paradigms. Importantly, this phenotype is near identical to that observed in two independent lines of relaxin-3 KO mice, suggesting these phenotypes are due to the elimination of ligand or receptor and RXFP3-linked signalling. Furthermore, this behavioural characterisation of Rxfp3 KO mice identifies them as a useful experimental model for studying RXFP3-linked signalling and assessing the selectivity and/or potential off-target actions of RXFP3 agonists and antagonists, which could lead to an improved understanding of dysfunctional arousal in mental health disorders, including depression, anxiety, insomnia and neurodegenerative diseases.
    Full-text · Article · Sep 2014 · Behavioural Brain Research
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    • "The first hypotheses regarding the origin and diversification of the Rln/Insl-Rxfp signaling systems in vertebrates proposed that three of the peptides (Rln3, Insl5 and Rln) were present prior to the diversification of teleosts, and that the fourth peptide (Insl3) arose in tetrapods (Wilkinson and Bathgate, 2007; Wilkinson et al., 2005a). With the identification of multiple Rln3-and Rxfp3-like molecules in teleosts (Wilkinson et al., 2005a), it was proposed that the signaling of Rln/Insl peptides in teleosts was mediated solely by Rxfp3-like receptors (Wilkinson and Bathgate, Fig. 4. Maximum Likelihood (ML) optimization of the phylogenetic relationship among rln/insl genes across vertebrates, with midpoint rooting. The ML tree is based on DNA sequences including only the first two nucleotides in each codon and employing the miHIV + G model of sequence evolution. "
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    ABSTRACT: Relaxin family peptide receptors (Rxfps) and their ligands, relaxin (Rln) and insulin-like (Insl) peptides, are broadly implicated in the regulation of reproductive and neuroendocrine processes in mammals. Most placental mammals harbour genes for four receptors, namely rxfp1, rxfp2, rxfp3 and rxfp4. The number and identity ofrxfpsin other vertebrates are immensely variable, which is probably attributable to intraspecific variation in reproductive and neuroendocrine regulation. Here, we highlight several interesting, but greatly overlooked, aspects of the rln/insl-rxfp evolutionary history: the ancient origin, recruitment of novel receptors, diverse roles of selection, differential retention and lineage-specific loss of genes over evolutionary time. The tremendous diversity of rln/insl and rxfp genes appears to have arisen from two divergent receptors and one ligand that were duplicated by whole genome duplications (WGD) throughout vertebrate evolution, although several genes, notably relaxin in mammals, were also duplicated via small scale duplications. Duplication and loss of genes have varied across lineages: teleosts retained more WGD-derived genes, dominated by those thought to be involved in neuroendocrine regulation (rln3, insl5 and rxfp 3/4 genes), while eutherian mammals witnessed the diversification and rapid evolution of genes involved in reproduction (rln/insl3). Several genes that arose early in evolutionary history were lost in most mammals, but retained in teleosts and, to a lesser extent, in early diverging tetrapods. To elaborate on their evolutionary history, we provide updated phylogenies of the Rxfp1/2 and Rxfp3/4 receptors and their ligands, including new sequences from early diverging vertebrate taxa such as coelacanth, skate, spotted gar, and lamprey. We also summarize the recent progress made towards understanding the functional biology of Rxfps in non-mammalian taxa, providing a new conceptual framework for research on Rxfp signaling across vertebrates.
    Full-text · Article · Jul 2014 · General and Comparative Endocrinology
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    • "Human relaxin-2 is the only form of circulating relaxin that is substantially increased during pregnancy [4]. Human relaxin-2 is functionally equivalent to relaxin-1 in all other mammals [5]. "
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    ABSTRACT: Background: Although relaxin causes vasodilatation in systemic arteries, little is known about its role in cerebral arteries. We investigated the expression and role of relaxin in basilar arteries after subarachnoid hemorrhage (SAH) in rabbits. Methods: Microarray analysis with rabbit basilar artery RNA was performed. Messenger RNA expression of relaxin-1 and relaxin/insulin-like family peptide receptor 1 (RXFP1) was investigated with quantitative RT-PCR. RXFP1 expression in the basilar artery was investigated with immunohistochemistry. Relaxin concentrations in cerebrospinal fluid (CSF) and serum were investigated with an enzyme-linked immunosorbent assay. Using human brain vascular smooth muscle cells (HBVSMC) preincubated with relaxin, myosin light chain phosphorylation (MLC) was investigated with immunoblotting after endothelin-1 stimulation. Results: After SAH, RXFP1 mRNA and protein were significantly downregulated on day 3, whereas relaxin-1 mRNA was significantly upregulated on day 7. The relaxin concentration in CSF was significantly elevated on days 5 and 7. Pretreatment with relaxin reduced sustained MLC phosphorylation induced by endothelin-1 in HBVSMC. Conclusion: Upregulation of relaxin and downregulation of RXFP1 after SAH may participate in development of cerebral vasospasm. Downregulation of RXFP1 may induce a functional decrease in relaxin activity during vasospasm. Understanding the role of relaxin may provide further insight into the mechanisms of cerebral vasospasm.
    Full-text · Article · Jul 2014 · BioMed Research International
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