Relaxin Family Peptide Research Articles

Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules

Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism.

Development of a scaffold displaying exoloops of RXFP1.

Relaxin family peptide receptor 1 (RXFP1), the cognate receptor for relaxin, is a G-protein coupled receptor (GPCR) possessing a unique extracellular region consisting of a domain of 10 leucine rich repeats (LRRs) linked to an N-terminal low density lipoprotein Class A module. Relaxin binds to its receptor primarily by a high affinity interaction with the LRRs. An additional low-affinity interaction has been proposed to occur between relaxin and the the exoloops (ELs) of the transmembrane domain, however the molecular detail of this interaction remains undefined. While site directed mutagenesis and subsequent functional characterisation of these mutants traditionally allows identification of residues contributing to receptor function, in this case results are complicated by the presence of the high affinity binding site in the LRRs. To create a tool to investigate the low-affinity interaction, a protein scaffold system displaying exoloops 1 and 2 from RXFP1 was designed. This was achieved by inserting RXFP1 exoloops 1 and 2 into the native loops of a thermostabilised 6 kDa GB1 protein creating EL1/EL2-GB1. This protein has been expressed and purified in milligram quantities and used in conjunction with biophysical techniques such as NMR to explore relaxin binding to the exoloops of RXFP1.

Insulin-Like 3 Signaling Is Important for Testicular Descent but Dispensable for Spermatogenesis and Germ Cell Survival in Adult Mice1

Relaxin family peptide receptor 2 (RXFP2) is the cognate receptor of a peptide hormone insulin-like 3 (INSL3). INSL3 is expressed at high levels in both fetal and adult Leydig cells. Deletion of Insl3 or Rxfp2 genes in mice caused cryptorchidism resulting from a failure of gubernaculum development. Using a novel mouse transgenic line with a knock-in LacZ reporter in the Rxfp2 locus, we detected a robust Rxfp2 expression in embryonic and early postnatal gubernaculum in males and in postmeiotic spermatogenic cells in adult testis. To study the role of INSL3/RXFP2 signaling in male reproduction, we produced a floxed Rxfp2 allele and used the Cre/loxP approach to delete Rxfp2 in different tissues. Using Cre transgene driven by retinoic acid receptor beta promoter, conditional gene targeting in gubernacular mesenchymal cells at early embryonic stages caused high intraabdominal cryptorchidism as in males with a global deletion of Rxfp2. However, when the Rxfp2 was deleted in gubernacular smooth or striated muscle cells, no abnormalities of testicular descent or testis development were found. Specific ablation of Rxfp2 in male germ cells using Stra8-icre transgene did not affect testis descent, spermatogenesis, or fertility in adult males. No significant change in germ cell apoptosis was detected in mutant males. In summary, our data indicate that the INSL3/RXFP2 signaling is important for testicular descent but dispensable for spermatogenesis and fertility in adult males.

The Importance of Tryptophan B28 in H2 Relaxin for RXFP2 Binding and Activation

H2 relaxin (relaxin) is a member of the insulin–relaxin superfamily and exhibits several non-reproductive functions in addition to its well-known properties as a pregnancy hormone. Over the years, the therapeutic potential of relaxin has been examined for a number of conditions. It is currently in phase III clinical trials for the treatment of acute heart failure. The 53 amino acid peptide hormone consists of two polypeptide chains (A and B) which are cross-linked by two inter-chains and one intra-A chain disulfide bridge. Although its cognate receptor is relaxin family peptide receptor (RXFP) 1, relaxin is also able to cross-react with RXFP2, for which the native ligand is INSL3. The “RXXXRXXI” motif in the B-chain of H2 relaxin is responsible for primary binding to LRR of the RXFP1 receptor (Bullesbach and Schwabe, J Biol Chem 280:14051–14056, 2005). Previous RXFP2 receptor mutation and molecular modelling studies strongly suggest that, in addition to this motif, the Trp-B28 residue in the B-chain is responsible for H2–RXFP2 interaction. To confirm this finding, here we have mutated H2 relaxin in which Trp-B28 was replaced with alanine. The synthetic relaxin analogue was then tested on cells expressing either RXFP1 or 2 to determine the affinity and potency for the respective receptors. Our results confirm that Trp-B28 in the B-chain is crucial for binding and activating RXFP2, but not for RXFP1.

Relaxin Regulates Hyaluronan Synthesis and Aquaporins in the Cervix of Late Pregnant Mice

Cervical ripening is associated with loss of structural integrity and tensile strength, thus enabling the cervix to dilate at term. It is characterized by changes in glycosaminoglycan composition, increased water content, and a progressive reorganization of the collagen network. The peptide hormone relaxin via interaction with its receptor, relaxin family peptide receptor 1 (RXFP1), promotes tissue hydration and increases cervical hyaluronan (HA) concentrations, but the mechanisms that regulate these effects are not known. This study in relaxin mutant (Rln(-/-)) mice tested the hypothesis that relaxin regulates HA synthase and aquaporin (AQP) expression in the cervix. We also assessed expression of the RXFP1 protein by immunohistochemistry. Pregnant Rln(-/-) mice had lower Has2 and Aqp3 expression on d 18.5 of pregnancy and decreased cervical HA compared with wild-type Rln(+/+) mice. Chronic infusion of relaxin for 4 or 6 d in pregnant Rln(-/-) mice reversed these phenotypes and increased Has2 and Aqp3 compared with placebo controls. Relaxin-treated mice also had lower Has1 and Aqp5. Changes in gene expression were paralleled by increases in cervical HA and variations in AQP3 and AQP5 protein localization in epithelial cells of Rln(-/-) cervices. Our findings demonstrate that relaxin alters AQP expression in the cervix and initiates changes in glycosaminoglycan composition through increased HA synthesis. These effects are likely mediated through RXFP1 localized to subepithelial stromal cells and epithelial cells. We suggest these actions of relaxin collectively promote water recruitment into the extracellular matrix to loosen the dense collagen fiber network.

Identification of Key Residues Essential for the Structural Fold and Receptor Selectivity within the A-chain of Human Gene-2 (H2) Relaxin

Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4-24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin.

New insights into ligand-receptor pairing and coevolution of relaxin family peptides and their receptors in teleosts.

Relaxin-like peptides (RLN/INSL) play diverse roles in reproductive and neuroendocrine processes in placental mammals and are functionally associated with two distinct types of receptors (RXFP) for each respective function. The diversification of RLN/INSL and RXFP gene families in vertebrates was predominantly driven by whole genome duplications (2R and 3R). Teleosts preferentially retained duplicates of genes putatively involved in neuroendocrine regulation, harboring a total of 10-11 receptors and 6 ligand genes, while most mammals have equal numbers of ligands and receptors. To date, the ligand-receptor relationships of teleost Rln/Insl peptides and their receptors have largely remained unexplored. Here, we use selection analyses based on sequence data from 5 teleosts and qPCR expression data from zebrafish to explore possible ligand-receptor pairings in teleosts. We find support for the hypothesis that, with the exception of RLN, which has undergone strong positive selection in mammalian lineages, the ligand and receptor genes shared between mammals and teleosts appear to have similar pairings. On the other hand, the teleost-specific receptors show evidence of subfunctionalization. Overall, this study underscores the complexity of RLN/INSL and RXFP ligand-receptor interactions in teleosts and establishes theoretical background for further experimental work in nonmammals.

The Different Ligand-Binding Modes of Relaxin Family Peptide Receptors RXFP1 and RXFP2

Relaxin and insulin-like peptide 3 (INSL3) are peptide hormones with a number of important physiological roles in reproduction, regulation of extracellular matrix turnover, and cardiovascular function. Relaxin and INSL3 mediate their actions through the closely related G-protein coupled receptors, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2), respectively. These receptors have large extracellular domains (ECD) that contain high-affinity ligand-binding sites within their 10 leucine-rich repeat (LRR)-containing modules. Although relaxin can bind and activate both RXFP1 and RXFP2, INSL3 can only bind and activate RXFP2. To investigate whether this difference is related to the nature of the high-affinity ECD binding site or to differences in secondary binding sites involving the receptor transmembrane (TM) domain, we created a suite of constructs with RXFP1/2 chimeric ECD attached to single TM helices. We show that by changing as little as one LRR, representing four amino acid substitutions, we were able to engineer a high-affinity INSL3-binding site into the ECD of RXFP1. Molecular modeling of the INSL3-RXFP2 interaction based on extensive experimental data highlights the differences in the binding mechanisms of relaxin and INSL3 to the ECD of their cognate receptors. Interestingly, when the engineered RXFP1/2 ECD were introduced into full-length RXFP1 constructs, INSL3 exhibited only low affinity and efficacy on these receptors. These results highlight critical differences both in the ECD binding and in the coordination of the ECD-binding site with the TM domain, and provide new mechanistic insights into the binding and activation events of RXFP1 and RXFP2 by their native hormone ligands.

Disturbed Relaxin Signaling Pathway and Testicular Dysfunction in Mouse Offspring upon Maternal Exposure to Simazine

Simazine is a triazine herbicide that is being widely applied worldwide and commonly detected in surface and groundwater. Despite its popular use in controlling weeds and algae, very limited information is available regarding its toxicity. In the present study, pregnant mice were orally exposed to low doses (0, 5, 50, or 500 µg/kg body weight per day) of simazine during gestation and lactation, during which no overt maternal toxic response was detected, and their offspring was assessed. Simazine-exposed male offspring showed decreased body, testicular, and epididymis weight, increased testicular apoptosis, and decreased sperm concentrations. Differentially-expressed genes in the testes of male offspring exposed to simazine were identified by DNA microarray, revealing 775 upregulated and 791 downregulated genes; among these, the relaxin-family peptide receptor 1 (Rxfp1), which is the receptor for relaxin hormone, was significantly downregulated. In addition, the expression of target genes in the relaxin pathway, including nitric oxide synthase 2 (Nos2) and Nos3, was significantly decreased in simazine-exposed F1 testes. Moreover, simazine inhibited NO release, and knockdown of Rxfp1 blocked the inhibitory action of simazine on NO production in testicular Leydig cells. Therefore, the present study provides a better understanding of the toxicities associated with the widely used herbicide simazine at environmentally relevant doses by demonstrating that maternal exposure interferes with the pleotropic relaxin-NO signaling pathway, impairing normal development and reproductive activity of male offspring.

Increased feeding and body weight gain in rats after acute and chronic activation of RXFP3 by relaxin-3 and receptor-selective peptides

This paper provides a review of the effects of relaxin-3 and structurally related analogues on food intake and related behaviours, in relation to hypothalamic neural networks and chemical messengers known to control feeding, metabolism and body weight, including other neuropeptides and hormones. Soon after relaxin-3 was discovered, pharmacological studies identified the ability of the native peptide to stimulate feeding acutely in adult rats. Although interpretation of these data was confounded by ligand cross-reactivity at relaxin-family peptide (RXFP) receptors, studies with relaxin-3 analogues selective for the native relaxin-3 receptor, RXFP3, confirmed that acute and chronic activation of RXFP3 increased feeding and weight gain, and produced changes in plasma leptin and insulin. These studies also identified the hypothalamus as a locus of action. Studies are now required to identify RXFP3-positive neuron populations involved in the effects of relaxin-3/RXFP3 signalling on metabolic and neuroendocrine homeostasis, and to determine whether peptide-based, nonpeptide-based or gene-based RXFP3 treatments can alter food intake and body weight in animal models of obesity and eating disorders, as a reflection of the therapeutic potential of this newly identified transmitter system.

Relaxin signals through a RXFP1-pERK-nNOS-NO-cGMP-dependent pathway to up-regulate matrix metalloproteinases: the additional involvement of iNOS.

The hormone, relaxin, inhibits aberrant myofibroblast differentiation and collagen deposition by disrupting the TGF-β1/Smad2 axis, via its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1), extracellular signal-regulated kinase (ERK)1/2 phosphorylation (pERK) and a neuronal nitric oxide (NO) synthase (nNOS)-NO-cyclic guanosine monophosphate (cGMP)-dependent pathway. However, the signalling pathways involved in its additional ability to increase matrix metalloproteinase (MMP) expression and activity remain unknown. This study investigated the extent to which the NO pathway was involved in human gene-2 (H2) relaxin's ability to positively regulate MMP-1 and its rodent orthologue, MMP-13, MMP-2 and MMP-9 (the main collagen-degrading MMPs) in TGF-β1-stimulated human dermal fibroblasts and primary renal myofibroblasts isolated from injured rats; by gelatin zymography (media) and Western blotting (cell layer). H2 relaxin (10–100 ng/ml) significantly increased MMP-1 (by ∼50%), MMP-2 (by ∼80%) and MMP-9 (by ∼80%) in TGF-β1-stimulated human dermal fibroblasts; and MMP-13 (by ∼90%), MMP-2 (by ∼130%) and MMP-9 (by ∼115%) in rat renal myofibroblasts (all p<0.01 vs untreated cells) over 72 hours. The relaxin-induced up-regulation of these MMPs, however, was significantly blocked by a non-selective NOS inhibitor (L-nitroarginine methyl ester (hydrochloride); L-NAME; 75–100 µM), and specific inhibitors to nNOS (N-propyl-L-arginine; NPLA; 0.2–2 µM), iNOS (1400W; 0.5–1 µM) and guanylyl cyclase (ODQ; 5 µM) (all p<0.05 vs H2 relaxin alone), but not eNOS (L-N-(1-iminoethyl)ornithine dihydrochloride; L-NIO; 0.5–5 µM). However, neither of these inhibitors affected basal MMP expression at the concentrations used. Furthermore, of the NOS isoforms expressed in renal myofibroblasts (nNOS and iNOS), H2 relaxin only stimulated nNOS expression, which in turn, was blocked by the ERK1/2 inhibitor (PD98059; 1 µM). These findings demonstrated that H2 relaxin signals through a RXFP1-pERK-nNOS-NO-cGMP-dependent pathway to mediate its anti-fibrotic actions, and additionally signals through iNOS to up-regulate MMPs; the latter being suppressed by TGF-β1 in myofibroblasts, but released upon H2 relaxin-induced inhibition of the TGF-β1/Smad2 axis.

Silencing Relaxin-3 in Nucleus Incertus of Adult Rodents: A Viral Vector-based Approach to Investigate Neuropeptide Function

Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.

Relaxin induces matrix-metalloproteinases-9 and -13 via RXFP1: Induction of MMP-9 involves the PI3K, ERK, Akt and PKC-ζ pathways

We determined the precise role of relaxin family peptide (RXFP) receptors-1 and -2 in the regulation of MMP-9 and -13 by relaxin, and delineated the signaling cascade that contributes to relaxin’s modulation of MMP-9 in fibrocartilaginous cells. Relaxin treatment of cells in which RXFP1 was silenced resulted in diminished induction of MMP-9 and -13 by relaxin, whereas overexpression of RXFP1 potentiated the relaxin-induced expression of these proteinases. Suppression or overexpression of RXFP2 resulted in no changes in the relaxin-induced MMP-9 and -13. Studies using chemical inhibitors and siRNAs to signaling molecules showed that PI3K, Akt, ERK and PKC-ζ and the transcription factors Elk-1, c-fos and, to a lesser extent, NF-κB are involved in relaxin’s induction of MMP-9. Our findings provide the first characterization of signaling cascade involved in the regulation of any MMP by relaxin and offer mechanistic insights on how relaxin likely mediates extracellular matrix turnover.

INSL5-Deficient Mice Display an Alteration in Glucose Homeostasis and an Impaired Fertility

Insulin-like factor 5 (INSL5), a member of the insulin superfamily, is expressed in the colorectum and hypothalamus. To facilitate studies into the role of INSL5, we generated Insl5(-/-) mice by gene targeting. Insl5(-/-) mice were born in the expected Mendelian ratio, reached normal body weight, but displayed impaired male and female fertility that are due to marked reduction in sperm motility and irregular length of the estrous cycle. Furthermore, Insl5(-/-) mice showed impairment in glucose homeostasis with characteristic elevation of serum glucose levels at an advanced age. Glucose and insulin tolerance tests revealed that the increased blood glucose in Insl5(-/-) mice was due to glucose intolerance resulting from reduced insulin secretion. Morphometric and immunohistological analyses revealed that the Insl5(-/-) mice had markedly reduced average islets area and β-cell numbers. Furthermore, immunohistochemistry showed the expression of INSL5 in enteroendocrine cells in the colorectal epithelium and the presence of its putative receptor relaxin family peptide receptor 4 in pancreatic islet cells. These results suggest the potential role of INSL5 signaling in the regulation of insulin secretion and β-cell homeostasis.

Effect of Relaxin on Human Sperm Functions

Relaxin is a circulating hormone with functions in pregnancy, parturition, and other aspects of female reproduction. It is also secreted from the prostate gland into the seminal fluid; however, the role of relaxin in male reproduction is debated. Studies conducted in the past have suggested possible actions on human spermatozoa, but the data were contrasting. Here, we show that the relaxin receptor RXFP1 (Relaxin Family Peptide Receptor 1) is expressed in human spermatozoa, and it mainly localizes in the astrodome. In vitro studies on human sperm demonstrated that this hormone attenuates the natural decline in sperm motility and maintains higher mitochondrial activity and lower apoptosis level. Furthermore, relaxin induced an increase in sperm hyperactivation, intracellular calcium and cAMP, and acrosome reaction. These effects were abolished by the use of the specific anti-RXFP1 antibody. Relaxin concentrations were low in the blood (x ± SD, 0.16 ± 0.03 nM) and very high in the seminal plasma (x ± SD, 10.3 ± 4.0 nM), confirming its secretion mainly by the prostate. Taken together, these data demonstrate that relaxin influences positively many sperm functions linked to fertilizing ability, and it preserves sperm functionality, with possible practical value in assisted reproduction techniques.

Distribution and targets of the relaxin‐3 innervation of the septal area in the rat

Neural tracing studies have revealed that the rat medial and lateral septum are targeted by ascending projections from the nucleus incertus, a population of tegmental GABA neurons. These neurons express the relaxin-family peptide, relaxin-3, and pharmacological modulation of relaxin-3 receptors in medial septum alters hippocampal theta rhythm and spatial memory. In an effort to better understand the basis of these interactions, we have characterized the distribution of relaxin-3 fibers/terminals in relation to different septal neuron populations identified using established protein markers. Dense relaxin-3 fiber plexuses were observed in regions of medial septum containing hippocampal-projecting choline acetyltransferase (ChAT)-, neuronal nitric oxide synthase (nNOS)-, and parvalbumin (PV)-positive neurons. In lateral septum (LS), relaxin-3 fibers were concentrated in the ventrolateral nucleus of rostral LS and the ventral nucleus of caudal LS, with sparse labeling in the dorsolateral and medial nuclei of rostral LS, dorsal nucleus of caudal LS, and ventral portion nuclei. Relaxin-3 fibers were also observed in the septofimbrial and triangular septal nuclei. In the medial septum, we observed relaxin-3-immunoreactive contacts with ChAT-, PV-, and glutamate decarboxylase-67-positive neurons that projected to hippocampus, and contacts between relaxin-3 terminals and calbindin- and calretinin-positive neurons. Relaxin-3 colocalized with synaptophysin in nerve terminals in all septal areas, and ultrastructural analysis revealed these terminals were symmetrical and contacted spines, somata, dendritic shafts, and occasionally other axonal terminals. These data predict that this GABA/peptidergic projection modulates septohippocampal activity and hippocampal theta rhythm related to exploratory navigation, defensive and ingestive behaviors, and responses to neurogenic stressors.

Using Paleogenomics to Study the Evolution of Gene Families: Origin and Duplication History of the Relaxin Family Hormones and Their Receptors

Recent progress in the analysis of whole genome sequencing data has resulted in the emergence of paleogenomics, a field devoted to the reconstruction of ancestral genomes. Ancestral karyotype reconstructions have been used primarily to illustrate the dynamic nature of genome evolution. In this paper, we demonstrate how they can also be used to study individual gene families by examining the evolutionary history of relaxin hormones (RLN/INSL) and relaxin family peptide receptors (RXFP). Relaxin family hormones are members of the insulin superfamily, and are implicated in the regulation of a variety of primarily reproductive and neuroendocrine processes. Their receptors are G-protein coupled receptors (GPCR's) and include members of two distinct evolutionary groups, an unusual characteristic. Although several studies have tried to elucidate the origins of the relaxin peptide family, the evolutionary origin of their receptors and the mechanisms driving the diversification of the RLN/INSL-RXFP signaling systems in non-placental vertebrates has remained elusive. Here we show that the numerous vertebrate RLN/INSL and RXFP genes are products of an ancestral receptor-ligand system that originally consisted of three genes, two of which apparently trace their origins to invertebrates. Subsequently, diversification of the system was driven primarily by whole genome duplications (WGD, 2R and 3R) followed by almost complete retention of the ligand duplicates in most vertebrates but massive loss of receptor genes in tetrapods. Interestingly, the majority of 3R duplicates retained in teleosts are potentially involved in neuroendocrine regulation. Furthermore, we infer that the ancestral AncRxfp3/4 receptor may have been syntenically linked to the AncRln-like ligand in the pre-2R genome, and show that syntenic linkages among ligands and receptors have changed dynamically in different lineages. This study ultimately shows the broad utility, with some caveats, of incorporating paleogenomics data into understanding the evolution of gene families.

Minimization of Human Relaxin-3 Leading to High-Affinity Analogues with Increased Selectivity for Relaxin-Family Peptide 3 Receptor (RXFP3) over RXFP1

Relaxin-3 is a neuropeptide that is implicated in the regulation of stress responses and memory. The elucidation of its precise physiological role(s) has, however, been hampered by cross-activation of the relaxin-2 receptor, RXFP1, in the brain. The current study undertook to develop analogues of human relaxin-3 (H3 relaxin) that can selectively bind and activate its receptor, RXFP3. We developed a high-affinity selective agonist (analogue 2) by removal of the intra-A chain disulfide bond and deletion of 10 residues from the N terminus of the A chain. Further truncation of this analogue from the C terminus of the B chain to Cys(B22) and addition of an Arg(B23) led to a high-affinity, RXFP3-selective, competitive antagonist (analogue 3). Central administration of analogue 2 in rats increased food intake, which was blocked by prior coadministration of analogue 3. These novel RXFP3-selective peptides represent valuable pharmacological tools to study the physiological roles of H3 relaxin/RXFP3 systems in the brain and important leads for the development of novel compounds for the treatment of affective and cognitive disorders.

The Structural Determinants of Insulin-Like Peptide 3 Activity

Insulin-like peptide 3 (INSL3) is a hormone and/or paracrine factor which is a member of the relaxin peptide family. It has key roles as a fertility regulator in both males and females. The receptor for INSL3 is the leucine rich repeat (LRR) containing G-protein coupled receptor 8 (LGR8) which is now known as relaxin family peptide receptor 2 (RXFP2). Receptor activation by INSL3 involves binding to the LRRs in the large ectodomain of RXFP2 by residues within the B-chain of INSL3 as well as an interaction with the transmembrane exoloops of the receptor. Although the binding to the LRRs is well characterized the features of the peptide and receptor involved in the exoloop interaction are currently unknown. This study was designed to determine the key INSL3 determinants for RXFP2 activation. A chimeric peptide approach was first utilized to demonstrate that the A-chain is critical for receptor activation. Replacement of the INSL3 A-chain with that from the related peptides INSL5 and INSL6 resulted in complete loss of activity despite only minor changes in binding affinity. Subsequent replacement of specific A-chain residues with those from the INSL5 peptide highlighted that the N-terminus of the A-chain of INSL3 is critical for its activity. Remarkably, replacement of the entire N-terminus with four or five alanine residues resulted in peptides with near native activity suggesting that specific residues are not necessary for activity. Additionally removal of two amino acids at the C-terminus of the A-chain and mutation of Lys-8 in the B-chain also resulted in minor decreases in peptide activity. Therefore we have demonstrated that the activity of the INSL3 peptide is driven predominantly by residues 5–9 in the A-chain, with minor additional contributions from the two C-terminal A-chain residues and Lys-8 in the B-chain. Using this new knowledge, we were able to produce a truncated INSL3 peptide structure which retained native activity, despite having 14 fewer residues than the parent peptide.

Profiling Insulin Like Factor 3 (INSL3) Signaling in Human Osteoblasts

BackgroundYoung men with mutations in the gene for the INSL3 receptor (Relaxin family peptide 2, RXFP2) are at risk of reduced bone mass and osteoporosis. Consistent with the human phenotype, bone analyses of Rxfp2 −/− mice showed decreased bone volume, alterations of the trabecular bone, reduced mineralizing surface, bone formation, and osteoclast surface. The aim of this study was to elucidate the INSL3/RXFP2 signaling pathways and targets in human osteoblasts.Methodology/Principal FindingsAlkaline phosphatase (ALP) production, protein phosphorylation, intracellular calcium, gene expression, and mineralization studies have been performed. INSL3 induced a significant increase in ALP production, and Western blot and ELISA analyses of multiple intracellular signaling pathway molecules and their phosphorylation status revealed that the MAPK was the major pathway influenced by INSL3, whereas it does not modify intracellular calcium concentration. Quantitative Real Time PCR and Western blotting showed that INSL3 regulates the expression of different osteoblast markers. Alizarin red-S staining confirmed that INSL3-stimulated osteoblasts are fully differentiated and able to mineralize the extracellular matrix.Conclusions/SignificanceTogether with previous findings, this study demonstrates that the INSL3/RXFP2 system is involved in bone metabolism by acting on the MAPK cascade and stimulating transcription of important genes of osteoblast maturation/differentiation and osteoclastogenesis.