Tracey N. Wilkinson

Evolution of the relaxin-like peptide family

BackgroundThe 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.ResultsSequence 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.ConclusionsWe 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.

Coevolution of the Relaxin‐Like Peptides and Their Receptors

Abstract: Currently, four relaxin peptide family receptors are known: LGR7 is the relaxin receptor, although it also interacts specifically with relaxin‐3; LGR8 is the insulin‐like factor 3 (INSL3) receptor; and GPCR135 or the somatostatin‐ and angiotensin‐like peptide receptor (SALPR) and GPCR142 are both specific relaxin‐3 receptors. Because these receptors coevolved together with their relaxin ligands, phylogenetic analysis of these sequences can provide insight into peptide‐receptor interactions and even predict interacting partners for INSL4, INSL5, and INSL6, the receptors for which are unknown.

Identification of the N-Linked Glycosylation Sites of the Human Relaxin Receptor and Effect of Glycosylation on Receptor Function

The relaxin receptor, RXFP1, is a member of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family. These receptors are characterized by a large extracellular ectodomain containing leucine-rich repeats which contain the primary ligand binding site. RXFP1 contains six putative Asn-linked glycosylation sites in the ectodomain at positions Asn-14, Asn-105, Asn-242, Asn-250, Asn-303, and Asn-346, which are highly conserved across species. N-Linked glycosylation is the most common post-translational modification of G-protein-coupled receptors, although its role in modulating receptor function differs. We herein investigate the actual N-linked glycosylation status of RXFP1 and the functional ramifications of these post-translational modifications. Site-directed mutagenesis was utilized to generate single- or multiple-glycosylation site mutants of FLAG-tagged human RXFP1 which were then transiently expressed in HEK-293T cells. Glycosylation status was analyzed by immunoprecipitation and Western blot and receptor function analyzed with an anti-FLAG ELISA, (33)P-H2 relaxin competition binding, and cAMP activity measurement. All of the potential N-glycosylation sites of RXFP1 were utilized in HEK-293T cells, and importantly, disruption of glycosylation at individual or combinations of double and triple sites had little effect on relaxin binding. However, combinations of glycosylation sites were required for cell surface expression and cAMP signaling. In particular, N-glycosylation at Asn-303 of RXFP1 was required for optimal intracellular cAMP signaling. Hence, as is the case for other LGR family members, N-glycosylation is essential for the transport of the receptor to the cell surface. Additionally, it is likely that glycosylation is also essential for the conformational changes required for G-protein coupling and subsequent cAMP signaling.

Evolution of the relaxin-like peptide family: from neuropeptide to reproduction.

Abstract: The relaxin‐like peptide family consists of relaxin‐1, relaxin‐2, and relaxin‐3 and the insulin‐like peptides (INSL)‐3, INSL4, INSL5, and INSL6 (human relaxin‐2 is equivalent to relaxin‐1 in other species). Evolution of this family has been contentious. We therefore sought to clarify the issue by performing phylogenetic analysis of all relaxin‐like peptides from the genomic databases available. Surprisingly, the phylogeny, combined with previous biologic characterizations, suggest that although relaxins original function was likely in the brain, its reproductive role was acquired just prior to the divergence of amphibians. This phylogeny also illuminates inconsistencies in relaxin evolution in invertebrates, chickens, and cows.

Resolving the Unconventional Mechanisms Underlying RXFP1 and RXFP2 Receptor Function

The receptors for relaxin and insulin‐like peptide 3 (INSL3) are now well‐characterized as the relaxin family peptide (RXFP) receptors RXFP1 and RXFP2, respectively. They are G‐protein‐coupled receptors (GPCRs) with closest similarity to the glycoprotein hormone receptors, with both containing large ectodomains with 10 leucine‐rich repeats (LRRs). Additionally, RXFP1 and RXFP2 are unique in the LGR family in that they contain a low‐density lipoprotein class A (LDL‐A) module at their N‐terminus. Ligand‐mediated activation of RXFP1 and RXFP2 is a complex process involving various domains of the receptors. Primary ligand binding occurs via interactions between B‐chain residues of the peptides with specific residues in the LRRs of the ectodomain. There is a secondary binding site in the transmembrane exoloops which may interact with the A chain of the peptides. Receptor signaling through cAMP then requires the unique LDL‐A module, as receptors without this domain bind ligand normally but do not signal. This is an unconventional mode of activation for a GPCR, and the precise mode of action of the LDL‐A module is currently unknown. The specific understanding of the mechanisms underlying ligand‐mediated activation of RXFP1 and RXFP2 is crucial in terms of targeting these receptors for future drug development.

The relaxin peptide family and their novel G-protein coupled receptors

Relaxin-1 is a heterodimeric peptide hormone primarily produced by the pregnant corpus luteum and/or placenta and is involved in many essential physiological processes centered on its action as a potent extracellular matrix (ECM) remodeling agent. Insulin-like peptide 3 (INSL3), also known as relaxin-like factor, is predominantly expressed in the Leydig cells of the testes and is an important mediator of testicular descent. The relaxin-1 equivalent peptide in humans is actually the product of the human RLN2 gene, human 2 (H2) relaxin. Recently identified and thought to be the ancestral relaxin, relaxin-3 is specifically expressed in the nucleus incertus of the mouse and rat brain and is most likely an important neuropeptide. Each of the hormones above act on cell membrane G-protein coupled receptors (GPCRs). The relaxin-1 receptor is leucine-rich repeat-containing GPCR 7 (LGR7) whereas INSL3 acts on the closely related LGR8. These receptors have large extra-cellular domains containing multiple leucine-rich repeats (LRRs) and a unique LDL receptor-like cysteine-rich motif (LDLR-domain). Relaxin-3 will bind and activate LGR7 with 50-fold lower activity than H2 relaxin. Two relaxin-3 selective GPCRs; somatostatin and angiotensin like peptide receptor (SALPR) and GPCR 142 were recently identified, these type I GPCRs are unrelated to LGR7 and LGR8. The discovery and characterisation of these receptors is greatly aiding the quest to unravel the mechanics of these important hormones, however with three other family members, insulin-like peptides 4–6 (INSL4, INSL5 and INSL6) with unknown functions and unidentified receptors, there is still much to be learnt about this hormone family.