ORCID Profile
0000-0001-6301-861X
Current Organisations
Teagasc Food Research Centre Moorepark
,
Florey Institute of Neuroscience and Mental Health
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Physiology | Animal Physiology - Systems | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Pharmacology and Pharmaceutical Sciences | Biochemistry and Cell Biology | Medicinal and Biomolecular Chemistry | Animal Physiology—Cell | Animal Physiology—Systems | Autonomic Nervous System | Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics) | Proteins and Peptides | Basic Pharmacology | Receptors and Membrane Biology | Analytical Biochemistry | Structural Biology (incl. Macromolecular Modelling)
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences | Diabetes | Expanding Knowledge in the Chemical Sciences | Digestive System Disorders | Health not elsewhere classified | Biological sciences |
Related Links
Publications
THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors.
Publisher: Wiley
Date: 16-09-2021
DOI: 10.1111/BPH.15538
Abstract: The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at oi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is ided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
Relaxin-like bioactivity of ovine insulin 3 (INSL3) analogues
Publisher: Wiley
Date: 12-2002
DOI: 10.1046/J.1432-1033.2002.03348.X
Abstract: Relaxin is an insulin-like peptide consisting of two separate chains (A and B) joined by two inter- and one intrachain disulfide bonds. Binding to its receptor requires an Arg-X-X-X-Arg-X-X-Ile motif in the B-chain. A related member of the insulin superfamily, INSL3, has a tertiary structure that is predicted to be similar to relaxin. It also possesses an Arg-X-X-X-Arg motif within its B-chain, although this is displaced by four amino acids towards the C-terminus from the corresponding position within relaxin. We have previously shown that synthetic INSL3 itself does not display relaxin-like activity although analogue (Analogue A) with an introduced arginine residue in the B-chain giving it an Arg cassette in the exact relaxin position does possess weak activity. In order to identify further the structural features that impart relaxin function, solid phase peptide synthesis was used to prepare three additional analogues for bioassay. Each of these contained point substitutions within the arginine cassette. Analogue D contained the full human relaxin binding cassette, Analogue G consisted of the native INSL3 sequence containing an Arg to Ala substitution, and Analogue E was a further modification of Analogue A, with the same substitution. Each analogue was fully chemically characterized by a number of criteria. Detailed circular dichroism spectroscopy analyses showed that the changes caused little alteration of secondary structure and, hence, overall conformation. However, each analogue displayed only weak relaxin-like activity. These results indicate that while the arginine cassette is vital for relaxin-like activity, there are additional, as yet unidentified structural requirements for relaxin binding.
The structural determinants of insulin-like peptide 3 activity
Publisher: Frontiers Media SA
Date: 2012
Structural insights into the function of relaxins
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03833.X
Abstract: The relaxin peptide hormones are members of the insulin superfamily and share a structural fold that is characterized by two peptide chains which are cross-braced by three disulfide bonds. On this framework, various amino acid side chains are presented, allowing specific interactions with different receptors. The relaxin receptors belong to two unrelated classes of G-protein-coupled receptors, but interestingly they are not selective for a single relaxin peptide. Relaxin-3, which is considered to be an extreme ex le of the relaxin family, can activate receptors from both classes and in fact interacts to some degree with all four receptors identified to date. To deduce how changes in the primary sequence can fine-tune the overall structure and thus the ability to interact with the various receptors, we have studied a range of relaxin-like peptides using solution nuclear magnetic resonance analysis. Three-dimensional structures of relaxin-3, insulin-like peptide 3 (INSL3), and INSL5 were determined and revealed a number of interesting features. All peptides showed a significant amount of line-broadening in certain regions, in particular around the intra-A-chain disulfide bond, suggesting that despite the disulfide bonds the fold is rather dynamic. Although the peptides share a common structural core there are significant differences, particularly around the termini. The structural data in combination with mutational studies provide valuable insights into the structure-activity relationships of relaxins.
Relaxin family peptide receptors - from orphans to therapeutic targets
Publisher: Elsevier BV
Date: 08-2008
DOI: 10.1016/J.DRUDIS.2008.04.002
Abstract: The relaxin family peptides have distinct expression profiles and physiological functions. Several of them are the cognate ligands for 4 G-protein-coupled relaxin family peptide receptors (RXFPs formerly LGR7, LGR8, GPCR135, GPCR142). The relaxin/RXFP1 system has roles in reproductive physiology but is also involved in fibrosis, wound healing and responses to infarction. Relaxin has a potential use in congestive heart failure where fibrosis plays an important role in organ failure. The INSL3/RXFP2 system has biological roles in reproductive biology that may have limited therapeutic potential. However, the recently characterized relaxin-3/RXFP3 system is important in stress/anxiety and body composition. RXFP3 receptor antagonists are potentially novel anti-anxiety and anti-obesity drugs.
Structure and function relationship of murine insulin-like peptide 5 (INSL5): Free C-terminus is essential for RXFP4 receptor binding and activation
Publisher: American Chemical Society (ACS)
Date: 08-09-2011
DOI: 10.1021/BI201093M
Abstract: Insulin-like peptide 5 (INSL5) is a member of insulin/relaxin superfamily of peptides. It has recently been identified as the cognate ligand for the G-protein-coupled receptor, RXFP4. Although the complete physiological role of this naturally occurring peptide is still under investigation, there is evidence that it acts to both stimulate appetite and activate colon motility. This suggests that both agonists and antagonists of the peptide may have potential therapeutic applications. To further investigate the physiological role of this peptide and because of the ready availability of the mouse as an experimental animal, the preparation of mouse INSL5 was undertaken. Because of its complex structure and the intractable nature of the two constituent chains, different solid phase synthesis strategies were investigated, including the use of a temporary B-chain solubilizing tag. Unfortunately, none provided significantly improved yield of purified mouse INSL5 which reflects the complexity of this peptide. In addition to the native peptide, two mouse INSL5 analogues were also prepared. One had its two chains as C-terminal amides, and the other contained a europium chelate monolabel for use in RXFP4 receptor assays. It was found that the INSL5 amide was substantially less potent than the native acid form. A similar observation was made for the human peptide acid and amide, highlighting the necessity for free C-terminal carboxylates for function. Two additional human INSL5 analogues were prepared to further investigate the necessity of a free C-terminal. The results together provide a first insight into the mechanism whereby INSL5 binds to and activates RXFP4.
Posterior pituitary of the newborn marsupial possum, Trichosurus vulpecula.
Publisher: Wiley
Date: 10-1993
Abstract: The fetal anterior pituitary-adrenal axis is thought to be involved in the initiation of birth in both eutherian and marsupial mammals. Little is known about the structure and function of the posterior pituitary at birth in the marsupial. Immunocytochemistry, high pressure liquid chromatography, and radioimmunoassay were used to identify vasopressin and mesotocin in the posterior pituitary of a newborn marsupial, the brushtail possum, Trichosurus vulpecula. The concentrations of vasopressin and mesotocin in the head of the newborn possum were 0.34 and 0.28 ng, respectively. The concentration of vasopressin was always greater than that of mesotocin, and the amounts of neuropeptides present in the head increased as the possum developed.
Evolution of the relaxin-like peptide family
Publisher: Springer Science and Business Media LLC
Date: 2005
Molecular Profiling of VGluT1 AND VGluT2 Ventral Subiculum to Nucleus Accumbens Shell Projections.
Publisher: Springer Science and Business Media LLC
Date: 05-04-2023
Arginine vasopressin- and oxytocin-like peptides in the testis of two Australian marsupials
Publisher: Elsevier BV
Date: 07-1993
DOI: 10.1016/0196-9781(93)90100-U
Abstract: High performance liquid chromatography (HPLC) and specific radioimmunoassay (RIA) for arginine vasopressin (AVP), mesotocin (MT), and oxytocin (OT) were used to identify and quantify these peptides in the testis of the brushtail possum (Trichosurus vulpecula) and the northern brown bandicoot (Isoodon macrourus). Arginine vasopressin (0.092 +/- 0.041 ng/g) and MT (0.198 +/- 0.089 ng/g), but not OT, were found in the possum testis, while the bandicoot testis contained AVP (0.061 ng/g), MT (0.108 +/- 0.024 ng/g), and OT (0.114 +/- 0.053 ng/g). The values correlate well with those reported for AVP- and OT-like peptides in the testis of eutherian mammals. It was concluded that there are neurohypophysial peptides present in the marsupial testis.
Probing the correlation between ligand efficacy and conformational diversity at the α1A-adrenoreceptor reveals allosteric coupling of its microswitches
Publisher: Elsevier BV
Date: 05-2020
Chemical synthesis and orexigenic activity of rat/mouse relaxin-3
Publisher: Springer Science and Business Media LLC
Date: 03-2013
DOI: 10.1007/S00726-013-1478-0
Abstract: The insulin-like peptide, relaxin-3 was first identified just a decade ago via a genomic database search and is now recognized to be a key neuropeptide with several roles including the regulation of arousal, stress responses and neuroendocrine homeostasis. It also has significant potential as a drug to treat stress and obesity. Its actions are mediated via its cognate G protein-coupled receptor, RXFP3, which is found in abundant numbers in the brain. However, much remains to be determined with respect to the mechanism of neurological action of this peptide. Consequently, the chemical synthesis of the rat and mouse (which share identical primary structures) two-chain, three disulfide peptide was undertaken and the resulting peptide subjected to detailed in vitro and in vivo assay. Use of efficient solid-phase synthesis methods provided the two regioselectively S-protected A- and B-chains which were readily combined via sequential disulfide bond formation. The synthetic rat/mouse relaxin-3 was obtained in high purity and good overall yield. It demonstrated potent orexigenic activity in rats in that central intracerebroventricular infusion led to significantly increased food intake and water drinking.
Relaxin family peptide receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database
Publisher: Edinburgh University Library
Date: 16-09-2019
DOI: 10.2218/GTOPDB/F60/2019.4
Abstract: Relaxin family peptide receptors (RXFP, nomenclature as agreed by the NC-IUPHAR Subcommittee on Relaxin family peptide receptors [18, 75]) may be ided into two pairs, RXFP1/2 and RXFP3/4. Endogenous agonists at these receptors are heterodimeric peptide hormones structurally related to insulin: relaxin-1, relaxin, relaxin-3 (also known as INSL7), insulin-like peptide 3 (INSL3) and INSL5. Species homologues of relaxin have distinct pharmacology and relaxin interacts with RXFP1, RXFP2 and RXFP3, whereas mouse and rat relaxin selectively bind to and activate RXFP1 [172]. relaxin-3 is the ligand for RXFP3 but it also binds to RXFP1 and RXFP4 and has differential affinity for RXFP2 between species [170]. INSL5 is the ligand for RXFP4 but is a weak antagonist of RXFP3. relaxin and INSL3 have multiple complex binding interactions with RXFP1 [176] and RXFP2 [84] which direct the N-terminal LDLa modules of the receptors together with a linker domain to act as a tethered ligand to direct receptor signaling [173]. INSL5 and relaxin-3 interact with their receptors using distinct residues in their B-chains for binding, and activation, respectively [211, 97].
Structure of the R3/I5 chimeric relaxin peptide, a selective GPCR135 and GPCR142 agonist
Publisher: Elsevier BV
Date: 08-2008
Prevention of Bleomycin-Induced Pulmonary Fibrosis by a Novel Antifibrotic Peptide with Relaxin-Like Activity
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 08-09-2010
Abstract: Pulmonary fibrosis is a progressive and lethal lung disease characterized by accumulation of extracellular matrix and loss of pulmonary function. No cure exists for this pathologic condition, and current treatments often fail to slow its progression or relieve its symptoms. Relaxin was previously shown to induce a matrix-degrading phenotype in human lung fibroblasts in vitro and to inhibit pulmonary fibrosis in vivo. A novel peptide that targets the relaxin RXFP1/LGR7 receptor was recently identified using our computational platform designed to predict novel G protein-coupled receptor peptide agonists. In this study, we examined the antifibrotic properties of this novel peptide, designated CGEN25009, in human cell-based assays and in a murine model of bleomycin-induced pulmonary fibrosis. Similar to relaxin, CGEN25009 was found to have an inhibitory effect on transforming growth factor-β1-induced collagen deposition in human dermal fibroblasts and to enhance MMP-2 expression. The peptide's biological activity was also similar to relaxin in generating cellular stimulation of cAMP, cGMP, and NO in the THP-1 human cell line. In vivo, 2-week administration of CGEN25009 in a preventive or therapeutic mode (i.e., concurrently with or 7 days after bleomycin treatment, respectively) caused a significant reduction in lung inflammation and injury and ameliorated adverse airway remodeling and peribronchial fibrosis. The results of this study indicate that CGEN25009 displays antifibrotic and anti-inflammatory properties and may offer a new therapeutic option for the treatment of pulmonary fibrosis.
The evolution of the relaxin peptide family and their receptors
Publisher: Springer New York
Date: 2007
DOI: 10.1007/978-0-387-74672-2_1
Abstract: The relaxin peptide family in humans consists of relaxin-1, 2 and 3 and the insulin-like peptides (INSL)-3, 4, 5 and 6. The evolution of this family has been controversial points of contention include the existence of an invertebrate relaxin and the absence of a ruminant relaxin. Over the past four years we have performed a comprehensive analysis of the relaxin peptide family using all available vertebrate and invertebrate genomes. Contrary to previous reports an invertebrate relaxin was not found sequence similarity searches indicate the family emerged during early vertebrate evolution. Phylogenetic analyses revealed the presence ofpotential relaxin-3, relaxin and INSL5 homologs in fish dating their emergence far earlier than previously believed. There are four known relaxin peptide family receptors the relaxin and INSL3 receptors, the leucine rich repeat containing G protein-coupled receptors (LGR), LGR7 and LGR8 respectively and the two relaxin-3 receptors, GPCR135 and GPCR142. Database searching identified several invertebrate ancestors of LGR7 and LGR8 the absence of an invertebrate relaxin suggests the presence of an unidentified invertebrate ligand for these receptors. No invertebrate ancestors of GPCR135 or GPCR142 were found. Based on the theory that interacting proteins co-evolve together, phylogenetic analyses of the relaxin peptide family receptors were performed to provide insight into interactions within the relaxin system. Co-evolution between INSL5 and GPCR142, as evidenced by the loss of both genes in the rat and dog and their similar expression profiles, predicted GPCR142 to be the endogeneous INSL5 receptor. This interaction has since been confirmed experimentally. The emergence and presence of multiple GPCR135 homologs in fish reflected similar findings for relaxin-3. It seems likely the ancestral relaxin system was relaxin-3 acting through GPCR135, before LGR7 was "acquired" as a relaxin receptor early in vertebrate development.
B7-33 replicates the vasoprotective functions of human relaxin-2 (serelaxin).
Publisher: Elsevier BV
Date: 07-2017
DOI: 10.1016/J.EJPHAR.2017.05.005
Abstract: Recombinant H2 relaxin (serelaxin) has gained considerable attention as a new vasoprotective drug, largely due to its potential therapeutic effects in heart failure and fibrosis. However, serelaxin is laborious and costly to produce. A single-chain peptidomimetic, B7-33, has been developed to overcome these problems but little is known about its biological actions in the vascular system. This study first compared the rapid vascular effects of an acute bolus injection of B7-33 compared with serelaxin. Male Wistar rats received a tail vein injection of placebo (20mM sodium acetate), B7-33 (13.3μg/kg) or serelaxin (26.6μg/kg). Three hours later vascular function in the mesenteric artery, small renal artery and abdominal aorta was assessed by wire myography. B7-33 and serelaxin selectively enhanced bradykinin-mediated endothelium-dependent relaxation in the rat mesenteric artery by increasing endothelium-derived hyperpolarization, but had no overall effects on relaxation in the small renal artery or aorta. We then compared the actions of B7-33 and serelaxin in an ex vivo model of vascular disease using virgin female mouse mesenteric arteries pre-incubated in placental trophoblast conditioned media to induce endothelial dysfunction characteristic of preecl sia. Co-incubation of these arteries in trophoblast conditioned media with B7-33 or serelaxin (15, 30nM) prevented the development of endothelial dysfunction. In conclusion, equimolar doses of B7-33 replicated the acute beneficial vascular effects of serelaxin in rat mesenteric arteries and also prevented endothelial dysfunction induced by placental trophoblast conditioned media in mouse mesenteric arteries. Therefore, B7-33 should be considered as a cost-effective vasoactive therapeutic in cardiovascular diseases, including preecl sia.
Structure and expression of the bovine oxytocin receptor gene.
Publisher: Mary Ann Liebert Inc
Date: 12-1995
Abstract: The gene for the bovine oxytocin receptor has been sequenced using a combination of clones derived from a bovine endometrial cDNA library from estrus and a bovine genomic DNA library, with confirmation of structure using reverse transcription PCR programmed by term myometrial RNA. The receptor belongs to the seven transmembrane domain family and predicts a protein of 391 amino acids. A comparison of the genomic sequence with the cDNA structure, as well as reverse transcription polymerase chain reaction (RT-PCR) analysis, shows there are two introns, one in the 5'noncoding region that appears to be differentially spliced in the bovine uterus and a conserved intron within the open reading frame between the regions encoding the transmembrane domains VI and VII. Northern blot analysis indicated three major transcripts in myometrium and endometrium in vivo at approximately 6.5 kb, 3.5 kb, and 2.0 kb. In situ hybridization analysis of uterine tissue at term showed highest mRNA concentrations in the endometrial epithelium, particularly in the deep glands, a pattern confirmed also at the immunohistochemical level by monoclonal antibodies raised against a human amino-terminal peptide. Further confirmation of the identity of the receptor was obtained by transient transfection of a reconstituted receptor construct into COS-7 cells. The expressed receptor was shown to have identical pharmacological properties in respect to various oxytocin analogs to the natural bovine endometrial receptor.
ML290 is a biased allosteric agonist at the relaxin receptor RXFP1.
Publisher: Springer Science and Business Media LLC
Date: 07-06-2017
DOI: 10.1038/S41598-017-02916-5
Abstract: Activation of the relaxin receptor RXFP1 has been associated with improved survival in acute heart failure. ML290 is a small molecule RXFP1 agonist with simple structure, long half-life and high stability. Here we demonstrate that ML290 is a biased agonist in human cells expressing RXFP1 with long-term beneficial actions on markers of fibrosis in human cardiac fibroblasts (HCFs). ML290 did not directly compete with orthosteric relaxin binding and did not affect binding kinetics, but did increase binding to RXFP1. In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and p38MAPK phosphorylation but not cGMP accumulation or ERK1/2 phosphorylation although prior addition of ML290 increased p-ERK1/2 responses to relaxin. In human primary vascular endothelial and smooth muscle cells that endogenously express RXFP1, ML290 increased both cAMP and cGMP accumulation but not p-ERK1/2. In HCFs, ML290 increased cGMP accumulation but did not affect p-ERK1/2 and given chronically activated MMP-2 expression and inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation. In vascular cells, ML290 was 10x more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290 caused strong coupling of RXFP1 to Gα s and Gα oB but weak coupling to Gα i3 . ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.
Relaxin requires the angiotensin II type 2 receptor to abrogate renal interstitial fibrosis
Publisher: Elsevier BV
Date: 07-2014
DOI: 10.1038/KI.2013.518
Abstract: Fibrosis is a hallmark of chronic kidney disease, for which there is currently no effective cure. The hormone relaxin is emerging as an effective antifibrotic therapy however, its mechanism of action is poorly understood. Recent studies have shown that relaxin disrupts the profibrotic actions of transforming growth factor-β1 (TGF-β1) by its cognate receptor, relaxin family peptide receptor 1 (RXFP1), extracellular signal-regulated kinase phosphorylation, and a neuronal nitric oxide synthase-dependent pathway to abrogate Smad2 phosphorylation. Since angiotensin II also inhibits TGF-β1 activity through its AT2 receptor (AT2R), we investigated the extent to which relaxin interacts with the AT2R. The effects of the AT2R antagonist, PD123319, on relaxin activity were examined in primary rat kidney myofibroblasts, and in kidney tissue from relaxin-treated male wild-type and AT2R-knockout mice subjected to unilateral ureteric obstruction. Relaxin's antifibrotic actions were significantly blocked by PD123319 in vitro and in vivo, or when relaxin was administered to AT2R-knockout mice. While heterodimer complexes were formed between RXFP1 and AT2Rs independent of ligand binding, relaxin did not directly bind to AT2Rs but signaled through RXFP1-AT2R heterodimers to induce its antifibrotic actions. These findings highlight a hitherto unrecognized interaction that may be targeted to control fibrosis progression.
The effects of relaxin and estrogen deficiency on collagen deposition and hypertrophy of nonreproductive organs
Publisher: The Endocrine Society
Date: 12-2006
DOI: 10.1210/EN.2006-0533
Abstract: In this study, we determined the effects of relaxin and estrogen deficiency and estrogen replacement therapy (ERT) on the cardiac, renal, and pulmonary phenotypes of female relaxin gene knockout (Rln1-/-) and age-matched wild-type (Rln1+/+) mice. One-month-old Rln1+/+ and Rln1-/- mice were bilaterally ovariectomized or sham-operated and aged until 9 or 12 months. A subgroup of ovariectomized mice received ERT from 9 to 12 months of age. At the appropriate time points, heart, kidney, and lung tissues from these mice were collected and analyzed for changes in organ fibrosis, hypertrophy, and airway thickening. Neither ovariectomy nor ERT had any effect on cardiac or renal collagen concentration in all groups studied. In contrast, total lung collagen concentration and airway subepithelial collagen deposition were significantly increased in ovariectomized Rln1+/+ mice (P<0.05 vs. sham) and to a greater extent in ovariectomized Rln1-/- mice (P<0.01 vs. sham). Ovariectomy of Rln1+/+ mice also led to a significant increase in airway smooth muscle (SM) (lung) thickening, which was further exaggerated in Rln1-/- mice. Cardiac hypertrophy, evidenced by increased heart weight and expression of hypertrophy-related genes (all P<0.05 vs. sham) was only observed in Rln1-/- mice. These findings demonstrated an increased pathology in mice that were deficient of both relaxin and estrogen. ERT significantly decreased airway fibrosis, airway SM thickening, and cardiac hypertrophy when administered to ovariectomized Rln1-/- mice (all P<0.05 vs. ovariectomy alone). These findings suggest that relaxin and estrogen appear to play protective roles against airway fibrosis, airway SM thickening, and cardiac hypertrophy in female mice.
Solution structure and novel insights into the determinants of the receptor specificity of human relaxin-3
Publisher: Elsevier BV
Date: 03-2006
Relaxin signals through a RXFP1-pERK-nNOS-NO-cGMP-dependent pathway to up-regulate matrix metalloproteinases: The additional involvement of iNOS
Publisher: Public Library of Science (PLoS)
Date: 22-08-2012
The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1.
Publisher: Springer Science and Business Media LLC
Date: 18-04-2016
DOI: 10.1038/NCOMMS11344
Abstract: H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1.
Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization.
Publisher: Frontiers Media SA
Date: 2013
Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3
Publisher: Elsevier BV
Date: 10-2018
Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.
Publisher: American Chemical Society (ACS)
Date: 08-08-2016
DOI: 10.1021/ACS.JMEDCHEM.6B00265
Abstract: Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.
Serelaxin-mediated signal transduction in human vascular cells: Bell-shaped concentration-response curves reflect differential coupling to G proteins
Publisher: Wiley
Date: 12-2015
DOI: 10.1111/BPH.12964
Chemically synthesized dicarba H2 relaxin analogues retain strong RXFP1 receptor activity but show an unexpected loss of in vitro serum stability.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5OB01539A
Abstract: Replacement of a disulfide bond with a non-reducible dicarba bond in an insulin-like peptide, relaxin, did not significantly alter functional activity but resulted in unexpected dramatic decrease in vitro serum stability.
Development and optimization of MicroRNA against relaxin-3
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2008.03816.X
Abstract: Recent in vivo studies suggest a role for relaxin-3 in feeding and stress. To further elucidate the function of relaxin-3 in the central nervous system, we have employed a complementary approach, based on RNA interference, to modulate relaxin-3 expression. We have designed, constructed, and characterized three microRNAs (miRNAs) targeting different regions of the relaxin-3 transcript. These were tested to determine the amount of miRNA required to achieve the greatest knockdown and for their ability to reduce the expression of relaxin-3 in transfected HEK293Tcells. All miRNA constructs significantly reduced relaxin-3-induced cAMP responses however, miR499 was most effective. This should be a useful tool for in vitro studies and central targeting of relaxin-3 in vivo using viral delivery systems.
Investigation of interactions at the extracellular loops of the relaxin family peptide receptor 1 (RXFP1)
Publisher: Elsevier BV
Date: 12-2014
Relaxin family peptide receptors in GtoPdb v.2021.3
Publisher: Edinburgh University Library
Date: 02-09-1970
DOI: 10.2218/GTOPDB/F60/2021.3
Abstract: Relaxin family peptide receptors (RXFP, nomenclature as agreed by the NC-IUPHAR Subcommittee on Relaxin family peptide receptors [18, 81]) may be ided into two pairs, RXFP1/2 and RXFP3/4. Endogenous agonists at these receptors are heterodimeric peptide hormones structurally related to insulin: relaxin-1, relaxin, relaxin-3 (also known as INSL7), insulin-like peptide 3 (INSL3) and INSL5. Species homologues of relaxin have distinct pharmacology and relaxin interacts with RXFP1, RXFP2 and RXFP3, whereas mouse and rat relaxin selectively bind to and activate RXFP1 [184]. relaxin-3 is the ligand for RXFP3 but it also binds to RXFP1 and RXFP4 and has differential affinity for RXFP2 between species [183]. INSL5 is the ligand for RXFP4 but is a weak antagonist of RXFP3. relaxin and INSL3 have multiple complex binding interactions with RXFP1 [189] and RXFP2 [91] which direct the N-terminal LDLa modules of the receptors together with a linker domain to act as a tethered ligand to direct receptor signaling [186]. INSL5 and relaxin-3 interact with their receptors using distinct residues in their B-chains for binding, and activation, respectively [225, 104].
Neohormone systems as exciting targets for drug development
Publisher: Elsevier BV
Date: 05-2006
Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein.
Publisher: Wiley
Date: 27-11-2015
Abstract: The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N(α) -deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca(2+) ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production.
The structure and regulation of the oxytocin receptor
Publisher: Wiley
Date: 03-2001
DOI: 10.1113/EPH8602185
Abstract: The oxytocin receptor (OTR) is part of an ancient hormone system expressed in erse phyla in relation to acute reproductive smooth muscle responses, such as egg-laying, birth, or milk letdown. The regulation of the OTR gene, while correlating with steroid levels in vivo, remains elusive. There appear to be both inhibitory and stimulatory influences acting upon a constitutive pattern of basal expression. We have found no evidence, however, for an effect of the sex steroids either directly on gene transcription, or on the receptor itself at the protein level. In the prostatic carcinoma cell line Du145, we have shown that up-regulation of the OTR gene transcription can be effected by cAMP. In an attempt to characterize the expression of the OTR protein in vivo, we have shown, using ligand-blotting, that the OTR can be expressed at different sizes in transfected cells and in myometrium. Also, in the myometrium at term, immunohistochemistry suggests that there is both an increase in OTR protein per cell, as well as in the number of smooth muscle cells expressing OTR, emphasizing that perinatal changes are the results of both in idual gene activation events and gross cellular differentiation. The OTR is a valuable model system reflecting molecular changes in the perinatal period. When we understand how this important molecule is regulated, we will also be a long way towards understanding the mechanisms controlling myometrial contractility at birth. Experimental Physiology (2001) 86.2, 289-296.
The importance of tryptophan B28 in H2 relaxin for RXFP2 binding and activation
Publisher: Springer Science and Business Media LLC
Date: 26-10-2013
The role of insulin 3, testosterone, Müllerian inhibiting substance and relaxin in rat gubernacular growth.
Publisher: Oxford University Press (OUP)
Date: 10-2002
Abstract: Transabdominal testicular descent is influenced by various anatomical and hormonal factors and is mediated by gubernacular enlargement and regression of the cranial suspensory ligament, but its mechanism remains controversial. The aim of this study was to determine which hormones have a direct effect on the proliferation of cells in the day 17 fetal rat gubernaculum in vitro, using an organ culture system. The effects of synthetic rat insulin 3 (INSL3), inactive INSL3, dihydrotestosterone (DHT), DHT+INSL3, human Müllerian inhibiting substance (hMIS), hMIS+INSL3 and human gene 2 relaxin were tested, together with co-culture with fetal rat testis. Cell proliferation was assessed using a bromodeoxyuridine labelling index. The results showed that MIS and relaxin have a mild effect on gubernacular growth, whilst INSL3 and DHT have a more marked effect. The combination of MIS+INSL3 showed an effect close to that of co-culture with testis. However, the most pronounced effect was caused by DHT+INSL3. RT-PCR analysis indicated that the fetal rat gubernaculum strongly expresses putative INSL3 receptors, weakly expresses MIS type II receptors and does not express relaxin receptors. In conclusion, a number of different hormones directly influence growth of the gubernaculum in vitro, including the recently reported hormone INSL3. INSL3 shows a direct stimulatory effect on the swelling reaction, while DHT and MIS may have roles in augmenting this growth.
Development of Novel High-Affinity Antagonists for the Relaxin Family Peptide Receptor 1
Publisher: American Chemical Society (ACS)
Date: 03-05-2023
Promise and Limitations of Relaxin-based Therapies in Chronic Fibrotic Lung Diseases.
Publisher: American Thoracic Society
Date: 12-2016
Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis
Publisher: Wiley
Date: 30-07-2019
DOI: 10.1002/PRP2.513
Relaxin therapy reverses large artery remodeling and improves arterial compliance in senescent spontaneously hypertensive rats
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 05-2010
DOI: 10.1161/HYPERTENSIONAHA.109.149369
Abstract: Hypertension and aging are associated with large artery structural remodeling and stiffening, which are known to increase cardiovascular risk. Relaxin is a peptide hormone with potent antifibrotic action in multiple organs. Although relaxin is able to reduce peripheral vascular resistance and improve arterial compliance in rats, it remains unclear whether the improvement in compliance is indirectly attributed to a vasodilatory action or whether relaxin is able to reverse arterial remodeling and stiffening directly in aged hypertensive animals. Senescent spontaneously hypertensive rats (17 months old) were treated with relaxin for 2 weeks (0.5 mg/kg per day) followed by a 1-week washout period. We determined large artery compliance using in vivo and in vitro techniques and quantified arterial remodeling by morphological and chemical means. Relaxin therapy significantly reversed aortic remodeling (ie, increases in vessel size, wall thickness, and collagen content) and improved arterial compliance, effects independent of its vasodilatory action. In relaxin-treated spontaneously hypertensive rats, arterial collagen content showed a greater reduction (−31% P .05) than that of elastin (−8%), resulting in an increased elastin:collagen ratio (0.63±0.03 versus 0.47±0.02 P .05). In conclusion, our results demonstrated that relaxin is potent in mediating reversal of arterial remodeling and improving arterial structural compliance in aged hypertensive rats.
Roles of the receptor, the ligand, and the cell in the signal transduction pathways utilized by the relaxin family peptide receptors 1-3
Publisher: Wiley
Date: 04-2009
A Lipidated Single-B-Chain Derivative of Relaxin Exhibits Improved In Vitro Serum Stability without Altering Activity
Publisher: MDPI AG
Date: 04-2023
DOI: 10.3390/IJMS24076616
Abstract: Human relaxin-2 (H2 relaxin) is therapeutically very important due to its strong anti-fibrotic, vasodilatory, and cardioprotective effects. Therefore, relaxin’s receptor, relaxin family peptide receptor 1 (RXFP1), is a potential target for the treatment of fibrosis and related disorders, including heart failure. H2 relaxin has a complex two-chain structure (A and B) and three disulfide bridges. Our laboratory has recently developed B7-33 peptide, a single-chain agonist based on the B-chain of H2 relaxin. However, the peptide B7-33 has a short circulation time in vitro in serum (t1/2 = ~6 min). In this study, we report structure-activity relationship studies on B7-33 utilizing different fatty-acid conjugations at different positions. We have shown that by fatty-acid conjugation with an appropriate spacer length, the in vitro half-life of B7-33 can be increased from 6 min to 60 min. In the future, the lead lipidated molecule will be studied in animal models to measure its PK/PD properties, which will lead to their pre-clinical applications.
Relaxin family peptide receptors - Former orphans reunite with their parent ligands to activate multiple signalling pathways
Publisher: Wiley
Date: 03-2007
Relaxin family peptides
Publisher: Elsevier
Date: 2011
Design, synthesis, and characterization of a single-chain peptide antagonist for the relaxin-3 receptor RXFP3
Publisher: American Chemical Society (ACS)
Date: 08-03-2011
DOI: 10.1021/JA110567J
Abstract: Relaxin-3 is a two-chain disulfide-rich peptide that is the ancestral member of the relaxin peptide family and, together with its G protein-coupled receptor RXFP3, is highly expressed in the brain. Strong evolutionary conservation of relaxin-3 suggests a critical biological function and recent studies have demonstrated modulation of sensory, neuroendocrine, metabolic, and cognitive systems. However, detailed studies of central relaxin-3-RXFP3 signaling have until now been severely h ered by the lack of a readily available high-affinity antagonist for RXFP3. Previous studies have utilized a complex two-chain chimeric relaxin peptide, R3(BΔ23-27)R/I5, in which a truncated relaxin-3 B-chain carrying an additional C-terminal Arg residue was combined with the insulin-like peptide 5 (INSL5) A-chain. In this study we demonstrate that, by replacing the native Cys in this truncated relaxin-3 B-chain with Ser, a single-chain linear peptide of 23 amino acids that retains high-affinity antagonism for RXFP3 can be achieved. In vivo studies demonstrate that this peptide, R3 B1-22R, antagonized relaxin-3/RXFP3 induced increases in feeding in rats after intracerebroventricular injection. Thus, R3 B1-22R represents an excellent tool for biological studies probing relaxin pharmacology and a lead molecule for the development of synthetically tractable, single-chain RXFP3 modulators for clinical use.
The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.
Publisher: Springer Science and Business Media LLC
Date: 11-12-2015
DOI: 10.1007/S00726-015-2144-5
Abstract: Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops.
Evidence that RXFP4 is located in enterochromaffin cells and can regulate production and release of serotonin.
Publisher: Portland Press Ltd.
Date: 04-2023
DOI: 10.1042/BSR20221956
Abstract: RXFP4 is a G protein–coupled receptor (GPCR) in the relaxin family. It has recently been recognised that this receptor and its cognate ligand INSL5 may have a role in the regulation of food intake, gut motility, and other functions relevant to metabolic health and disease. Recent data from reporter-mice showed co-location of Rxfp4 and serotonin (5-HT) in the lower gut. We used human single-cell RNA sequence data (scRNASeq) to show that RXFP4 is in a subset of gut enterochromaffin cells that produce 5-HT in humans. We also used RNAScope to show co-location of Rxfp4 mRNA and 5-HT in mouse colon, confirming prior findings. To understand whether RXFP4 might regulate serotonin production, we developed a cell model using Colo320, a human gut-derived immortalised cell line that produces and releases serotonin. Overexpression of RXFP4 in these cells resulted in a constitutive decrease in cAMP levels in both the basal state and in cells treated with forskolin. Treatment of cells with two RXFP4 agonists, INSL5 derived peptide INSL5-A13 and small molecule compound-4, further reduced cAMP levels. This was paralleled by a reduction in expression of mRNA for TPH1, the enzyme controlling the rate limiting step in the production of serotonin. Overexpression of RXFP4 also attenuated the cAMP-induced release of serotonin from Colo320 cells. Together this demonstrates that serotonin producing enterochromaffin cells are the major site of RXFP4 expression in the gut and that RXFP4 can have inhibitory functional impacts on cAMP production as well as TPH1 expression and serotonin release.
Synthetic short-chain peptide analogues of H1 relaxin lack affinity for the RXFP1 receptor and relaxin-like bioactivity. Clues to a better understanding of relaxin agonist design
Publisher: Frontiers Media SA
Date: 11-08-2022
DOI: 10.3389/FPHAR.2022.942178
Abstract: The peptide hormone relaxin (RLX), also available as clinical-grade recombinant protein (serelaxin), holds great promise as a cardiovascular and anti-fibrotic agent but is limited by the pharmacokinetic issues common to all peptide drugs. In this study, by a computational modelling chemistry approach, we have synthesized and tested a set of low molecular weight peptides based on the putative receptor-binding domain of the B chain of human H1 RLX isoform, with the objective to obtain RLX analogues with improved pharmacokinetic features. Some of them were stabilized to induce the appropriate 3-D conformation by intra-chain tri-azolic staples, which should theoretically enhance their resistance to digestive enzymes making them suited for oral administration. Despite these favourable premises, none of these H1 peptides, either linear or stapled, revealed a sufficient affinity to the specific RLX receptor RXFP1. Moreover, none of them was endowed with any RLX-like biological effects in RXFP1-expressing THP-1 human monocytic cells and mouse NIH-3T3-derived myofibroblasts in in vitro culture, in terms of significantly relevant cAMP elevation and ERK1/2 phosphorylation, which represent two major signal transduction events downstream RXFP1 activation. This was at variance with authentic serelaxin, which induced a clear-cut, significant activation of both these classical RLX signaling pathways. Albeit negative, the results of this study offer additional information about the structural requirements that new peptide therapeutics shall possess to effectively behave as RXFP1 agonists and RLX analogues.
Optimization and 13CH3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies.
Publisher: Elsevier BV
Date: 06-2018
DOI: 10.1016/J.BBAMEM.2018.03.020
Abstract: Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS
Synthetic human insulin 4 does not activate the G-protein-coupled receptors LGR7 or LGR8
Publisher: Wiley
Date: 26-04-2004
DOI: 10.1002/PSC.521
Abstract: In contrast to the cellular receptors for insulin and insulin-like growth factors that are known to be protein tyrosine kinases, those of both insulin 3 and relaxin have recently been identified as being members of the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, LGR8 and LGR7, respectively. This has prompted an examination into the possibility that they might also be specific for another member of the insulin superfamily, namely, insulin 4. Towards this aim, a two-chain peptide corresponding to the predicted primary structure of insulin 4 was prepared by solid phase synthesis. As conventional aeration and combination of the two S-reduced chains in solution at high pH failed to produce target product, selectively S-protected A- and B-chains were prepared followed by stepwise, in idual formation of each of the three disulfides, one intramolecular within the A-chain and two intermolecular. Chemical characterization confirmed the purity and identity of the synthetic insulin 4 analogue. However, secondary structural analysis indicated that the peptide was devoid of tertiary conformation suggesting that the native peptide may well be either significantly longer in length or is similar to insulin-like growth factor I or II in that it is a single chain product. Screening of the synthetic analogue for activation of transfected cells bearing LGR7, and LGR7 splice variant or LGR8 failed to identify a specific interaction. Thus, the in vivo structural identity of insulin 4 and its receptor (if any) as well as its potential function remains unknown.
Resolving the unconventional mechanisms underlying RXFP1 and RXFP2 receptor function
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03949.X
Abstract: 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.
Gram scale preparation of clozapine N-oxide (CNO), a synthetic small molecule actuator for muscarinic acetylcholine DREADDs.
Publisher: Elsevier BV
Date: 2018
Endogenous relaxin does not affect chronic pressure overload-induced cardiac hypertrophy and fibrosis
Publisher: The Endocrine Society
Date: 08-11-2008
DOI: 10.1210/EN.2007-1220
Abstract: The effect of endogenous relaxin on the development of cardiac hypertrophy, dysfunction, and fibrosis remains completely unknown. We addressed this question by subjecting relaxin-1 deficient (Rln1−/−) and littermate control (Rln1+/+) mice of both genders to chronic transverse aortic constriction (TAC). The extent of left ventricular (LV) remodeling and dysfunction were studied by serial echocardiography over an 8-wk period and by micromanometry. The degree of hypertrophy was estimated by LV weight, cardiomyocyte size, and expression of relevant genes. Cardiac fibrosis was determined by hydroxyproline assay and quantitative histology. Expression of endogenous relaxin during the course of TAC was also examined. In response to an 8-wk period of pressure overload, TAC mice of both genotypes developed significant LV hypertrophy, fibrosis, hypertrophy related gene profile, and signs indicating congestive heart failure when compared with respective sham controls. The severity of these alterations was not statistically different between the two genotypes of either gender. Relaxin mRNA expression was up-regulated, whereas that of its receptor was unchanged in the hypertrophic myocardium of wild-type mice. Collectively, the extent of pressure overload-induced LV hypertrophy, fibrosis, and dysfunction were comparable between Rln1+/+ and Rln1−/− mice. Thus, although up-regulated in its expression, endogenous relaxin had no significant effect on the progression of cardiac maladaptation and dysfunction in the setting of chronic pressure overload.
Enhanced serelaxin signalling in co-cultures of human primary endothelial and smooth muscle cells.
Publisher: Wiley
Date: 15-01-2016
DOI: 10.1111/BPH.13371
Complete Genome Sequence of Listeria monocytogenes Strain DPC6895, a Serotype 1/2b Isolate from Bovine Raw Milk
Publisher: American Society for Microbiology
Date: 25-06-2015
Abstract: Listeria monocytogenes is a foodborne pathogen and is the causative agent of listeriosis among humans and animals. The draft genome sequence of L. monocytogenes DPC6895, a serotype 1/2b strain isolated from the raw milk of a cow with subclinical bovine mastitis, is reported.
Relaxin family peptide receptors
Publisher: Elsevier
Date: 2011
Identification of the N-linked glycosylation sites of the human relaxin receptor and effect of glycosylation on receptor function
Publisher: American Chemical Society (ACS)
Date: 06-06-2008
DOI: 10.1021/BI800535B
Abstract: 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 in idual 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.
Relaxin-2 Does Not Ameliorate Nephropathy in an Experimental Model of Type-1 Diabetes
Publisher: S. Karger AG
Date: 2015
DOI: 10.1159/000368484
Abstract: b i Background/Aims: /i /b In diabetic nephropathy (DN), the current angiotensin-II-blocking pharmacotherapy is frequently failing. For diabetic cardiomyopathy (DC), there is no specific remedy available. Relaxin-2 (Rlx) - an anti-fibrotic, anti-inflammatory, and vasoprotecting peptide - is a candidate drug for both. b i Methods: /i /b Low-dose (32 µg/kg/day) and high-dose (320 µg/kg/day) Rlx were tested against vehicle (n = 20 each) and non-diabetic controls (n = 14) for 12 weeks in a model of type-1 diabetes induced in endothelial nitric oxide synthase knock-out (eNOS-KO) mice by intraperitoneal injection of streptozotocin. b i Results: /i /b Diabetic animals showed normal plasma creatinine, markedly increased albuminuria and urinary malonyldialdehyde, elevated relative kidney weight, glomerulosclerosis, and increased glomerular size, but no relevant interstitial fibrosis. Neither dose of Rlx affected these changes although the drug was active and targeted plasma levels were achieved. Of note, we found no activation of the renal TGF-β pathway in this model. In the hearts of diabetic animals, no fibrotic alterations indicative of DC could be determined which precluded testing of the initial hypothesis. b i Conclusions: /i /b We investigated a model showing early DN without overt tubulo-interstitial fibrosis and activation of the TGF-β-Smad-2/3 pathway. In this model, Rlx proved ineffective however, the same may not apply to other models and types of diabetes.
Relaxin family peptide systems and the central nervous system
Publisher: Springer Science and Business Media LLC
Date: 07-03-2010
DOI: 10.1007/S00018-010-0304-Z
Abstract: Since its discovery in the 1920s, relaxin has enjoyed a reputation as a peptide hormone of pregnancy. However, relaxin and other relaxin family peptides are now associated with numerous non-reproductive physiologies and disease states. The new millennium bought with it the sequence of the human genome and subsequently new directions for relaxin research. In 2002, the ancestral relaxin gene RLN3 was identified from genome databases. The relaxin-3 peptide is highly expressed in a small region of the brain and in species from teleost to primates and has both conserved sequence and sites of expression. Combined with the discovery of the relaxin family peptide receptors, interest in the role of the relaxin family peptides in the central nervous system has been reignited. This review explores the relaxin family peptides that are expressed in or act upon the brain, the receptors that mediate their actions, and what is currently known of their functions.
Leucine-rich repeat-containing G-protein-coupled receptor 8 in mature glomeruli of developing and adult rat kidney and inhibition by insulin-like peptide-3 of glomerular cell proliferation
Publisher: Bioscientifica
Date: 05-2006
DOI: 10.1677/JOE.1.06697
Abstract: Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the precise roles of this receptor in many of these tissues are unknown. In an effort to better understand INSL3–LGR8 systems in the rat, we cloned the full-length Lgr8 cDNA and investigated the presence and cellular localization of Lgr8 mRNA expression in adult and developing rat kidney. On the basis of these findings, we investigated the presence and distribution of renal 125 I-labelled human INSL3-binding sites and the nature of INSL3–LGR8 signalling in cultured renal cells. Thus, using in situ hybridization histochemistry, cells expressing Lgr8 mRNA were observed in glomeruli of renal cortex from adult rats and were tentatively identified as mesangial cells. Quantitative, real-time PCR analysis of the developmental profile of Lgr8 mRNA expression in kidney revealed highest relative levels at late stage gestation (embryonic day 18), with a sharp decrease after birth and lowest levels in the adult. During development, silver grains associated with Lgr8 mRNA hybridization were observed overlying putative mesangial cells in mature glomeruli, with little or no signal associated with less-mature glomeruli. In adult and developing kidney, specific 125 I-INSL3-binding sites were associated with glomeruli throughout the renal cortex. In primary cultures of glomerular cells, synthetic human INSL3 specifically and dose-dependently inhibited cell proliferation over a 48 h period, further suggesting the presence of functional LGR8 (receptors) on these cells (mesangial and others). These findings suggest INSL3–LGR8 signalling may be involved in the genesis and/or developmental maturation of renal glomeruli and possibly in regulating mesangial cell density in adult rat kidney.
Evolution of the relaxin-like peptide family from neuropeptide to reproduction
Publisher: Wiley
Date: 05-2005
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 relaxin's original function was likely in the brain, its reproductive role was acquired just prior to the ergence of hibians. This phylogeny also illuminates inconsistencies in relaxin evolution in invertebrates, chickens, and cows.
Development of lanthanide-labeled human INSL3 as an alternative probe to radioactively labeled INSL3 for use in bioassays
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03839.X
Abstract: The use of radioisotope-labeled peptides and proteins in bioassays is diminishing as a consequence of the emergence of fluorescent lanthanide-labeled probes. We describe a simple, single-step labeling of synthetic human insulin-like peptide 3 (INSL3) with a commercially available activated europium chelator. The labeled peptide was then used for the development of a robust and highly reproducible receptor-binding assay which is amenable to automated high-throughput screening of ligands. Europium-labeled INSL3 showed a similar binding profile to radioactively labeled INSL3.
Detection, localization, and action of the INSL3 receptor, LGR8, in rat kidney
Publisher: Wiley
Date: 05-2005
Abstract: Recent molecular and pharmacologic studies have identified LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family, as a cognate receptor for insulin-like peptide-3 (INSL3). LGR8 mRNA has been detected in various tissues, but the precise roles of these INSL3-LGR8 systems are unknown. In this study we first investigated the presence and cellular localization of LGR8 mRNA in both adult and developing rat kidney and subsequently examined the possible role of INSL3-LGR8 signaling in cultured mesangial cells. LGR8 mRNA was detected in the kidney by polymerase chain reaction and localized by in situ hybridization in mature glomerular mesangial cells within the renal cortex, with highest levels detected at embryonic day 18 and lowest levels in adult kidney. Synthetic INSL3 inhibited the proliferation of mesangial cells in primary culture, indicating the presence of functional LGR8 on these cells. These findings suggest that INSL3/LGR8 signaling may be involved in the genesis and/or developmental maturation of renal glomeruli and in the regulation of mesangial cell density in the adult kidney.
Structural insights into the modes of relaxin-binding and tethered-agonist activation of RXFP1 and RXFP2
Publisher: Cold Spring Harbor Laboratory
Date: 07-06-2021
DOI: 10.1101/2021.06.06.446989
Abstract: Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signalling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors.
Restricted expression of LGR8 in intralaminar thalamic nuclei of rat brain suggests a role in sensorimotor systems
Publisher: Wiley
Date: 05-2005
Abstract: LGR8 was recently identified as a cognate receptor for insulin-like peptide-3 (INSL3), and INSL3-LGR8 signaling is best known for its role in testis descent during development. LGR8 mRNA has been detected in various human tissues including brain, but the regional and cellular distribution of LGR8 expression in the mammalian central nervous system is unknown. Therefore, in this study we investigated the presence and localization of LGR8 mRNA in rat brain using reverse transcription-polymerase chain reaction and in situ hybridization histochemistry. Results revealed a distinct distribution of LGR8 in forebrain, with transcripts principally restricted to the posterior thalamus and highest densities detected in the parafascicular nucleus of both adult and developing rats. Unexpectedly, INSL3 mRNA was not detected in brain by similar methods, but preliminary electrophysiologic studies of parafascicular neurons revealed that INSL3 altered their activity. These findings suggest that LGR8 signaling may be involved in sensorimotor control in the rat and perhaps other species, particularly via actions on parafascicular neurons that project to basal ganglia and are depleted in Parkinson's disease.
The chemically synthesized human relaxin-2 analog, B-R13/17K H2, is an RXFP1 antagonist
Publisher: Springer Science and Business Media LLC
Date: 31-12-2010
DOI: 10.1007/S00726-009-0454-1
Abstract: Relaxin is a pleiotropic hormone which exerts its biological functions through its G-protein coupled receptor, RXFP1. While relaxin is well known for its reproductive and antifibrotic roles, recent studies suggest that it is produced by cancer cells and acts on RXFP1 to induce growth and metastasis. Furthermore, more recently Silvertown et al. demonstrated that lentiviral production of a human gene-2 (H2) relaxin analog reduced the growth of prostate xenograft tumors. The authors proposed that the lentivirally produced peptide was an RXFP1 antagonist however, the processed form of the peptide produced was not demonstrated. In this study, we have chemically synthesized the H2 relaxin analog, B-R13/17K H2 relaxin, and subjected it to detailed chemical characterization by HPLC, MALDI-TOF mass spectrometry, and amino acid analysis. The biological activity of the synthetic peptide was then tested in three different cell lines. It was found to bind with 500-fold lower affinity than H2 relaxin to RXFP1 receptors over-expressed in HEK-293T cells where it acted as a partial agonist. However, in cells which natively express the RXFP1 receptor, rat renal myofibroblasts and MCF-7 cancer cells, it acted as a full antagonist. Importantly, it was able to significantly inhibit cell invasion induced by H2 relaxin in MCF-7 cells consistent with the results of the lentiviral-driven expression in prostate cancer cells. The relaxin analog, B-R13/17K H2, can now be used as a tool to further understand RXFP1 function, and serve as a template for drug design for a therapeutic to treat prostate and other cancers.
Swim stress excitation of nucleus incertus and rapid induction of relaxin-3 expression via CRF1 activation
Publisher: Elsevier BV
Date: 2010
DOI: 10.1016/J.NEUROPHARM.2009.06.019
Abstract: Relaxin-3 (RLX3), a newly identified member of the relaxin peptide family, is distinguished by its enriched expression in GABA projection neurons of the pontine nucleus incertus (NI), which are postulated to participate in forebrain neural circuits involved in behavioural activation and stress responses. In this regard, corticotrophin-releasing factor-1 receptor (CRF(1)) is abundantly expressed by NI neurons central CRF administration activates c-fos expression in NI and various stressors have been reported to increase NI neuron activity. In studies to determine whether a specific neurogenic stressor would activate RLX3 expression, we assessed the effect of a repeated forced swim (RFS) on levels of RLX3 mRNA and heteronuclear (hn) RNA in rat NI by in situ hybridization histochemistry of exon- and intron-directed oligonucleotide probes, respectively. Exposure of rats to an RFS (10 min at 23 degrees C, 24 h apart), markedly increased RLX3 mRNA levels in NI at 30-60 min after the second swim, before a gradual return to basal levels over 2-4 h, while RLX3 hnRNA levels were significantly up-regulated at 60-120 min post-RFS, following a transient decrease at 30 min. Systemic treatment of rats with a CRF(1) antagonist, antalarmin (20 mg/kg, i.p.) 30 min prior to the second swim, blunted the stress-induced effects on RLX3 transcripts. Relative levels of RLX3-immunostaining in NI neurons appeared elevated at 3 h post-swim, but not at earlier time points (30-60 min). These results suggest that acute stress-induced CRF secretion can rapidly alter RLX3 gene transcription by activation of CRF(1) present on NI neurons. More generally, these studies support a role for RLX3 neural networks in the normal neural and physiological response to neurogenic stressors in the rat.
An apically located hybrid guanylate cyclase–ATPase is critical for the initiation of Ca2+ signaling and motility in Toxoplasma gondii
Publisher: Elsevier BV
Date: 05-2019
Relaxin-3: Improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo
Publisher: American Chemical Society (ACS)
Date: 22-12-2006
DOI: 10.1021/BI052233E
Abstract: Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.
Mesotocin and oxytocin in the brain and plasma of an australian marsupial, the northern brown bandicoot, isoodon macrourus
Publisher: Elsevier BV
Date: 05-1992
DOI: 10.1016/0300-9629(92)90009-F
Abstract: 1. Mesotocin (MT) and oxytocin (OT) were measured in the brain and plasma of bandicoots using reverse phase high performance liquid chromatography and specific radioimmunoassays. 2. MT and OT were found in the pituitary (1.25 +/- 0.10 micrograms/MT 0.725 +/- 0.077 micrograms/OT) and hypothalamus (38.37 +/- 6.46 ng/MT 19.1 +/- 4.61 ng/OT). Smaller amounts were present in the cerebral cortex. 3. Basal plasma concentrations ranged from 1.5 to 8.1 pg/ml for both peptides (N = 14) and were elevated by stress. 4. It was concluded that both MT and OT are secreted by the bandicoot brain and that stress stimulates secretion.
Differential expression of mesotocin receptors in the uterus and ovary of the pregnant tammar wallaby
Publisher: Bioscientifica
Date: 05-2005
DOI: 10.1530/REP.1.00505
Abstract: Mesotocin, an oxytocin-like peptide, is released in highest concentrations during parturition in macropodid marsupials. In late pregnant wallabies, uterine sensitivity to mesotocin increases markedly in the myometrium of the gravid uterus. This coincides with a significant increase in myometrial mesotocin receptor concentrations 3–4 days before term. To date, there is no information on mesotocin receptor gene expression in female wallaby reproductive tissues. This study aimed to examine mesotocin receptor gene expression in the uterus and ovaries of pregnant tammar wallabies, and to localise mesotocin receptors within the uterus. An RT-PCR strategy produced a consensus nucleotide sequence of 834 bp, which encoded 278 amino acids of transmembrane domains I to VI. This protein sequence has approximately 80% homology with the bovine and rat oxytocin receptor exon 2 region. Only one mesotocin receptor was detected in the tammar genome. The myometrium and mammary gland both expressed a 4.1 kb mesotocin receptor gene transcript. Myometrial mesotocin receptor gene expression increased on day 22 of the 26-day gestation and was significantly higher in the gravid than the non-gravid uterus in late pregnancy. This pattern of mesotocin receptor gene expression paralleled mesotocin receptor concentrations. Mesotocin binding sites were localised only to the myometrium, the highest densities being observed in the gravid uterus. Finally, this study showed high expression of mesotocin receptors in the corpus luteum. The pattern of luteal mesotocin receptor expression differed from the myometrium, with a decrease in mesotocin receptors occurring on the day of expected births.
Bi-allelic variants in INSL3 and RXFP2 cause bilateral cryptorchidism and male infertility.
Publisher: Oxford University Press (OUP)
Date: 19-05-2023
Abstract: What is the impact of variants in the genes INSL3 (Insulin Like 3) and RXFP2 (Relaxin Family Peptide Receptor 2), respectively, on cryptorchidism and male infertility? Bi-allelic loss-of-function (LoF) variants in INSL3 and RXFP2 result in bilateral cryptorchidism and male infertility, whereas heterozygous variant carriers are phenotypically unaffected. The small heterodimeric peptide INSL3 and its G protein-coupled receptor RXFP2 play a major role in the first step of the biphasic descent of the testes, and variants in the INSL3 and RXFP2 genes have long been implicated in inherited cryptorchidism. However, only one single homozygous missense variant in RXFP2 has clearly been linked to familial bilateral cryptorchidism, so the effects of bi-allelic variants in INSL3 and heterozygous variants in both genes on cryptorchidism and male infertility remain unclear. Exome data of 2412 men from the MERGE (Male Reproductive Genomics) study cohort including 1902 infertile men with crypto-/azoospermia, of whom 450 men had a history of cryptorchidism, were screened for high-impact variants in INSL3 and RXFP2. For patients with rare, high-impact variants in INSL3 and RXFP2, detailed clinical data were collected and the testicular phenotype was determined. Genotyping of family members was performed to analyse the co-segregation of candidate variants with the condition. Immunohistochemical staining for INSL3 in patient testicular tissue and measuring serum INSL3 concentration was performed to analyse the functional impact of a homozygous loss-of-function variant in INSL3. For a homozygous missense variant in RXFP2, its impact on the protein’s cell surface expression and ability to respond to INSL3 in CRE reporter gene assay was determined. This study presents homozygous high-impact variants in INSL3 and RXFP2 and clearly correlates these to bilateral cryptorchidism. Functional impact of the identified INSL3 variant was demonstrated by absence of INSL3-specific staining in patients’ testicular Leydig cells as well as undetectable blood serum levels. The identified missense variant in RXFP2 was demonstrated to lead to reduced RXFP2 surface expression and INSL3 mediated receptor activation. Further investigations are needed to explore a potential direct impact of bi-allelic INSL3 and RXFP2 variants on spermatogenesis. With our data, we cannot determine whether the infertility observed in our patients is a direct consequence of the disruption of a possible function of these genes on spermatogenesis or whether it occurs secondarily due to cryptorchidism. In contrast to previous assumptions, this study supports an autosomal recessive inheritance of INSL3- and RXFP2-related bilateral cryptorchidism while heterozygous LoF variants in either gene can at most be regarded as a risk factor for developing cryptorchidism. Our findings have diagnostic value for patients with familial/bilateral cryptorchidism and additionally shed light on the importance of INSL3 and RXFP2 in testicular descent and fertility. This study was carried out within the frame of the German Research Foundation (DFG) funded by Clinical Research Unit ‘Male Germ Cells: from Genes to Function’ (DFG, CRU326). Research at the Florey was supported by an NHMRC grant (2001027) and the Victorian Government Operational Infrastructure Support Program. A.S.B. is funded by the DFG (‘Emmy Noether Programme’ project number 464240267). The authors declare no conflict of interest. N/A.
Relaxin-3 inputs target hippocampal interneurons and deletion of hilar relaxin-3 receptors in floxed-RXFP3 mice impairs spatial memory.
Publisher: Wiley
Date: 31-01-2017
DOI: 10.1002/HIPO.22709
Abstract: Hippoc us is innervated by γ-aminobutyric acid (GABA) "projection" neurons of the nucleus incertus (NI), including a population expressing the neuropeptide, relaxin-3 (RLN3). In studies aimed at gaining an understanding of the role of RLN3 signaling in hippoc us via its G
The different ligand-binding modes of relaxin family peptide receptors RXFP1 and RXFP2
Publisher: The Endocrine Society
Date: 11-2012
DOI: 10.1210/ME.2012-1188
The actions of relaxin family peptides on signal transduction pathways activated by the relaxin family peptide receptor RXFP4.
Publisher: Springer Science and Business Media LLC
Date: 26-11-2016
DOI: 10.1007/S00210-016-1321-8
Abstract: The relaxin family peptide receptor 4 (RXFP4) is a G protein-coupled receptor (GPCR) expressed in the colorectum with emerging roles in metabolism and appetite regulation. It is activated by its cognate ligand insulin-like peptide 5 (INSL5) that is expressed in enteroendocrine L cells in the gut. Whether other evolutionarily related peptides such as relaxin-2, relaxin-3, or INSL3 activate RXFP4 signal transduction mechanisms with a pattern similar to or distinct from INSL5 is still unclear. In this study, we compare the signaling pathways activated by various relaxin family peptides to INSL5. We found that, like INSL5, relaxin-3 activated ERK1/2, p38MAPK, Akt, and S6RP phosphorylations leading to increased cell proliferation and also caused GRK and β-arrestin-mediated receptor internalization. Interestingly, relaxin-3 was slightly more potent than INSL5 in ERK1/2 and Akt phosphorylations, but both peptides were almost equipotent in adenylyl cyclase inhibition, S6RP phosphorylation, and cell proliferation. In addition, relaxin-3 showed greater efficacy only in Akt phosphorylation but not in the other pathways investigated. In contrast, no signaling activity or receptor internalization mechanisms were observed following relaxin-2 and INSL3. In conclusion, relaxin-3 is a high-efficacy agonist at RXFP4 with a comparable signal transduction profile to INSL5.
Discovery and characterization of a thermostable two-domain GH6 endoglucanase from a compost metagenome
Publisher: Public Library of Science (PLoS)
Date: 24-05-2018
Paracrine regulation of mammalian oocyte maturation and male germ cell survival
Publisher: Proceedings of the National Academy of Sciences
Date: 03-05-2004
Abstract: Mammalian oocytes are arrested at the prophase of meiosis before induction of maturation by the preovulatory luteinizing hormone (LH) surge. LH also promotes the survival of meiotic male germ cells in the testis. Because LH binds somatic cells, the mechanism underlying its regulation of germ cell function is unclear. We found that LH stimulates Leydig insulin-like 3 (INSL3) transcripts in ovarian theca and testicular Leydig cells. INSL3, in turn, binds a G protein-coupled receptor, LGR8 (leucine-rich repeat-containing G protein-coupled receptor 8), expressed in germ cells to activate the inhibitory G protein, thus leading to decreases in cAMP production. Treatment with INSL3 initiates meiotic progression of arrested oocytes in preovulatory follicles in vitro and in vivo and suppresses male germ cell apoptosis in vivo , thus demonstrating the importance of the INSL3-LGR8 paracrine system in mediating gonadotropin actions.
Coevolution of the relaxin-like peptides and their receptors
Publisher: Wiley
Date: 05-2005
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.
Dynamic changes in the expression of relaxin-like factor (INSL3), cholesterol side-chain cleavage cytochrome p450, and 3beta-hydroxysteroid dehydrogenase in bovine ovarian follicles during growth and atresia.
Publisher: Oxford University Press (OUP)
Date: 04-2002
DOI: 10.1095/BIOLREPROD66.4.934
Abstract: Relaxin-like factor (RLF) is a new member of the insulin-relaxin gene family known to be expressed in the ovarian follicular thecal cells of ruminants. To investigate the pattern of RLF expression in development and atresia of bovine follicles, antisera were raised in rats and rabbits to recombinantly expressed bovine pro-RLF and to chemically synthesized ovine RLF B chain, respectively. On dot blotting analysis, the rat antiserum bound to pro-RLF and less strongly to a synthetic mature ovine RLF lacking the C-domain, whereas the rabbit antiserum bound the mature form of ovine RLF. These antisera were used to immunostain bovine ovarian follicles of differing sizes and stages of health and atresia. 3beta-Hydroxysteroid dehydrogenase was colocalized with pro-RLF (n = 86 follicles), and cholesterol side-chain cleavage cytochrome P450 was localized in another section of many of the same follicles (n = 66). Not all follicles expressed pro-RLF in the theca interna, so the results are presented as the proportion of follicles expressing pro-RLF. Both mature and pro-RLF were immunolocalized to steroidogenic thecal cells of healthy follicles. As follicles enlarged to >5 mm, the proportion expressing pro-RLF declined (19/19 for 6 mm). Atresia was ided into antral (antral granulosa cells dying first) or basal (basal cells dying first) and further ided into early, middle, and late. For antral atresia of small follicles (2-5 mm), no decline in the proportion expressing pro-RLF was observed (early 6/6, middle 2/2) until the late stages (1/4). For basal atresia, which only occurs in small follicles (2-5 mm), the proportion expressing pro-RLF declined in the middle (2/5) and late (0/8) stages. In larger follicles (>6 to <10 mm), the proportion expressing pro-RLF also declined with atresia (1/13). These declines in RLF expression with atresia or increasing size were not accompanied by a decline in the expression of steroidogenic enzymes in the theca interna. A significant (P < 0.001) inverse relationship in the expression of pro-RLF and 3beta-hydroxysteroid dehydrogenase in the membrana granulosa was observed. We conclude that the expression of pro-RLF in the theca interna is switched off as follicles enlarge or enter atresia, whereas the expression of steroidogenic enzymes is maintained in the theca interna.
Activation of relaxin-related receptors by short, linear peptides derived from a collagen-containing precursor
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03827.X
Abstract: In a screening effort based on algorithmic predictions for novel G-protein-coupled receptor (GPCR) peptide activators, we were able to identify and examine two novel peptides (P59 and P74) which are short, linear, and derived from a natural, previously unidentified precursor protein containing a collagen-like repeat. Both peptides seemed to show an apparent cAMP-related effect on CHO-K1 cells transiently transfected with either LGR7 or LGR8, usually after treatment with cAMP-generating forskolin, compared to the same cells treated with forskolin plus relaxin. This activation was not found for the relaxin-3 receptor (GPR135). In a set of follow-up experiments, both peptides were found to stimulate cAMP production, mostly upon initial stimulation of cAMP production by 5 micro M forskolin in cells transfected with either LGR7 or LGR8. In a dye-free cell impedance GPCR activation assay, we were able to show that these peptides were also able to activate a cellular response mediated by these receptors. Although untransfected CHO-K1 cells showed some cellular activation by both relaxin and at least one of our newly discovered peptides, both LGR7- and LGR8-transfected cells showed a stronger response, indicating stimulation of a cellular pathway through activation of these receptors. In conclusion, we were able to show that these newly discovered peptides, which have no similarity to any member of the relaxin-insulin-like peptide family, are potential ligands for the relaxin-related family of receptors and as such might serve as novel candidates for relaxin-related therapeutic indications. Both peptides are linear and were found to be active after being chemically synthesized.
Relaxin family peptide receptor (RXFP1) coupling to Gαi3, involves the C-terminal Arg752 and localization within membrane raft microdomains
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 24-11-2009
Abstract: The relaxin family peptide receptors (RXFP) 1 and 2 are targets for the relaxin family peptides relaxin and insulin-like peptide 3 (INSL3), respectively. Although both receptors and peptides share a high degree of sequence identity, the cAMP signaling pathways activated by the two systems are quite distinct. Relaxin activation of RXFP1 initially results in accumulation of cAMP via G(alpha)(s), but this is modulated by inhibition of cAMP through G(alpha)(oB). Over time, RXFP1 recruits coupling to G(alpha)(i3), causing additional cAMP accumulation via a G(alpha)(i3)-Gbetagamma-phosphoinositide 3-kinase (PI3K)-protein kinase C (PKC)zeta pathway. In contrast, INSL3 activation of RXFP2 results in accumulation of cAMP only via G(alpha)(s), modulated by cAMP inhibition through G(alpha)(oB). Thus, the aim of this study was to identify the cause of differential G-protein coupling between these highly similar receptors. Construction of chimeric receptors revealed that G(alpha)(i3) coupling is dependent upon the transmembrane region of RXFP1 and independent of the receptor ectodomain or ligand bound. Generation of C-terminal truncated receptors identified the terminal 10 amino acids of the RXFP1 C terminus as essential for G(alpha)(i3) signaling, and point mutations revealed an obligatory role for Arg(752). RXFP1-mediated G(alpha)(i3), but not G(alpha)(s) or G(alpha)(oB), signaling was also found to be dependent upon membrane rafts, and RXFP1 coupled to G(alpha)(i3) after only 3 min of receptor stimulation. Therefore, RXFP1 coupling to the G(alpha)(i3)-Gbetagamma-PI3K-PKCzeta pathway requires the terminal 10 amino acids of the RXFP1 C terminus and membrane raft localization, and the observed delay in this pathway occurs downstream of G(alpha)(i3).
The relaxin receptor as a therapeutic target - perspectives from evolution and drug targeting.
Publisher: Elsevier BV
Date: 07-2018
DOI: 10.1016/J.PHARMTHERA.2018.02.008
Abstract: The peptide relaxin was first identified as an important circulating hormone during pregnancy over 90 years ago. Research over many years defined the numerous biological roles that relaxin plays throughout pregnancy in many mammalian species. These important biological actions have led to the testing of relaxin as a therapeutic agent for a number of indications. The discovery of the relaxin receptor, RXFP1, in 2002 facilitated the better understanding of the cellular targets of relaxin, its mechanism of action and enabled the development of relaxin mimetics and screening for small molecule agonists. Additionally, the rapid expansion of the genome databases and bioinformatics tools has significantly advanced our understanding of the evolution of the relaxin/RXFP1 signaling system. It is now clear that the relaxin-RXFP1 signaling axis is far more ancient than previously appreciated with important roles for invertebrate relaxin-like peptides in reproductive and non-reproductive functions. This review summarizes these advances as well as developments in drug targeting of RXFP1. Hence the complex mode of activation of RXFP1 is discussed as is the discovery and development of a peptide mimetic and small molecule agonist. Detailed signaling studies are summarized which highlight the cell specific signaling of a peptide mimetic and biased signaling of a small molecule agonist. These studies highlight the complexities of targeting peptide GPCRs such as RXFP1.
Structural properties of relaxin chimeras: NMR characterization of the R3/I5 relaxin peptide
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2008.03805.X
Abstract: Relaxin-3 interacts with high potency with three relaxin family peptide receptors (RXFP1, RXFP3, and RXFP4). Therefore, the development of selective agonist and antagonist analogs is important for in vivo studies characterizing the biological significance of the different receptor-ligand systems and for future pharmaceutical applications. Recent reports demonstrated that a peptide selective for RXFP3 and RXFP4 over RXFP1 can be generated by the combination of the relaxin-3 B chain with the A chain from insulin-like peptide 5 (INSL5), creating an R3/I5 chimera. We have used NMR spectroscopy to determine the three-dimensional structure of this peptide to gain structural insights into the consequences of combining chains from two different relaxins. The R3/I5 structure reveals a similar backbone conformation for the relaxin-3 B chain compared to native relaxin-3, and the INSL5 A chain displays a relaxin/insulin-like fold with two parallel helices. The findings indicate that binding and activation of RXFP3 and RXFP4 mainly require the B chain and that the A chain functions as structural support. RXFP1, however, demonstrates a more complex binding mechanism, involving both the A chain and the B chain. The creation of chimeras is a promising strategy for generating new structure-activity data on relaxins.
Regulation of the oxytocin receptor in bovine reproductive tissues and the role of steroids
Publisher: Wiley
Date: 06-2000
The single-chain relaxin mimetic, B7-33, maintains the cardioprotective effects of relaxin and more rapidly reduces left ventricular fibrosis compared to perindopril in an experimental model of cardiomyopathy
Publisher: Elsevier BV
Date: 04-2023
Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS1)
Publisher: Elsevier BV
Date: 2023
Expression of the insulin-like peptide 3 (INSL3) hormone-receptor (LGR8) system in the testis
Publisher: Oxford University Press (OUP)
Date: 05-2006
DOI: 10.1095/BIOLREPROD.105.048165
Abstract: The new peptide hormone insulin-like peptide 3 (INSL3) is a member of the insulin-relaxin family, yet, unlike insulin, it signals through a new G-protein coupled receptor, LGR8, distantly related to the receptors for LH and FSH. INSL3 is produced in large amounts by the Leydig cells of the testis in both fetal and adult mammals. Using a combination of mRNA analysis by RT-PCR, immunohistochemistry, ligand-binding, and/or bioactivity assays, the distribution of LGR8 expression was assessed in testicular tissues and cells and in the epididymis. There was consistent agreement that LGR8 was expressed in meiotic and particularly postmeiotic germ cells and in Leydig cells, though not in Sertoli or peritubular cells. Leydig cells appear to express only a low level of the LGR8 gene product other transcripts may be present, representing nonfunctional products. Messenger RNA analysis suggested that LGR8 transcripts in germ cells represented mostly full-length forms. LGR8 mRNA was also expressed in the epididymis, though no function can yet be ascribed to this expression. Therefore, the INSL3/LGR8 system represents a further paracrine hormone-receptor system in the testis, which conveys information about Leydig cell status to germ cells, and possibly as part of an autocrine feedback loop.
Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5)
Publisher: Wiley
Date: 11-07-2008
Modulation of feeding by chronic rAAV expression of a relaxin-3 peptide agonist in rat hypothalamus
Publisher: Springer Science and Business Media LLC
Date: 08-11-2013
DOI: 10.1038/GT.2012.83
Abstract: Relaxin-3 is a neuropeptide that is abundantly expressed by discrete brainstem neuron populations that broadly innervate forebrain areas rich in the relaxin-3 G-protein-coupled-receptor, RXFP3. Acute and subchronic central administration of synthetic relaxin-3 or an RXFP3-selective agonist peptide, R3/I5, increase feeding and body weight in rats. Intrahypothalamic injection of relaxin-3 also increases feeding. In this study, we developed a recombinant adeno-associated virus 1/2 (rAAV1/2) vector that drives expression and constitutive secretion of bioactive R3/I5 and assessed the effect of intrahypothalamic injections on daily food intake and body weight gain in adult male rats over 8 weeks. In vitro testing revealed that the vector rAAV1/2-fibronectin (FIB)-R3/I5 directs the constitutive secretion of bioactive R3/I5 peptide. Bilateral injection of rAAV1/2-FIB-R3/I5 vector into the paraventricular nucleus produced an increase in daily food intake and body weight gain (P<0.01, ~23%, respectively), relative to control treatment. In a separate cohort of rats, quantitative polymerase chain reaction analysis of hypothalamic mRNA revealed strong expression of R3/I5 transgene at 3 months post-rAAV1/2-FIB-R3/I5 infusion. Levels of mRNA transcripts for the relaxin-3 receptor RXFP3, the hypothalamic 'feeding' peptides neuropeptide Y, AgRP and POMC, and the reproductive hormone, GnRH, were all similar to control, whereas vasopressin and oxytocin (OT) mRNA levels were reduced by ~25% (P=0.051) and ~50% (P<0.005), respectively, in rAAV1/2-FIB-R3/I5-treated rats (at 12 weeks, n=9/8 rats per group). These data demonstrate for the first time that R3/I5 is effective in modulating feeding in the rat by chronic hypothalamic RXFP3 activation and suggest a potential underlying mechanism involving altered OT signalling. Importantly, there was no desensitization of the feeding response over the treatment period and no apparent deleterious health effects, indicating that targeting the relaxin-3-RXFP3 system may be an effective long-term therapy for eating disorders.
Minimum active structure of insulin-like peptide 5
Publisher: American Chemical Society (ACS)
Date: 21-11-2013
DOI: 10.1021/JM400924P
Abstract: Insulin-like peptide 5 (INSL5) is a complex two-chain peptide hormone constrained by three disulfide bonds in a pattern identical to insulin. High expression of INSL5 in the colon suggests roles in activation of colon motility and appetite control. A more recent study indicates it may have significant roles in the regulation of insulin secretion and β-cell homeostasis. This peptide thus has considerable potential for the treatment of eating disorders, obesity, and/or diabetes. However, the synthesis of INSL5 is extremely challenging either by chemical or recombinant means. The A-chain is very poorly soluble and the B-chain is highly aggregating in nature which, together, makes their postsynthesis handling and purification very difficult. Given these difficulties, we have developed a highly active INSL5 analogue that has a much simpler structure with two disulfide bonds and is thus easier to assemble compared to native INSL5. This minimized peptide represents an attractive new mimetic for investigating the functional role of INSL5.
Comparison of signaling pathways activated by the relaxin family peptide receptors, RXFP1 and RXFP2, using reporter genes
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 25-10-2007
Abstract: The receptors for H2 relaxin and insulin-like peptide 3, relaxin family peptide receptor (RXFP) 1 and RXFP2, respectively, were recently identified, but their signaling pathways are not yet well characterized. Although previous work has suggested that cAMP is a major signaling pathway activated by these receptors, RXFP1 has also been shown to activate a number of other signaling proteins. To this end, we examined the effect of stimulation of RXFP1 and RXFP2 receptors [expressed in human embryonic kidney (HEK) 293T cells] with human relaxin family peptides on a number of transcription factor-response elements coupled to reporter genes. Hence, reporter gene activity measured by enzyme activity in the cell media is a measure of the activation of a particular signaling pathway. Eight reporter genes were tested at both receptors as a screen to identify other signaling pathways activated by RXFP1 and RXFP2. The cAMP-response element reporter was strongly activated by both receptors. This effect was enhanced by preincubation with pertussis toxin (PTX), suggesting that Gs and inhibitory Gi/Go proteins mediate this response. Only activation of RXFP1 inhibited nuclear factor kappaB transcription, and this was reversed by PTX and the phosphoinositide-3-kinase inhibitor wortmannin. In addition, the glucocorticoid-response element was activated by RXFP1 but not by RXFP2 and was not activated in the parent HEK293T cells. Thus, the use of reporter genes enabled differences in signaling between these two receptors to be revealed and also threw light on the wide range of effects attributed to relaxin.
Challenges in the design of insulin and relaxin/insulin-like peptide mimetics.
Publisher: Elsevier BV
Date: 06-2017
DOI: 10.1016/J.BMC.2017.09.030
Abstract: Peptidomimetics are designed to overcome the poor pharmacokinetics and pharmacodynamics associated with the native peptide or protein on which they are based. The design of peptidomimetics starts from developing structure-activity relationships of the native ligand-target pair that identify the key residues that are responsible for the biological effect of the native peptide or protein. Then minimization of the structure and introduction of constraints are applied to create the core active site that can interact with the target with high affinity and selectivity. Developing peptidomimetics is not trivial and often challenging, particularly when peptides' interaction mechanism with their target is complex. This review will discuss the challenges of developing peptidomimetics of therapeutically important insulin superfamily peptides, particularly those which have two chains (A and B) and three disulfide bonds and whose receptors are known, namely insulin, H2 relaxin, H3 relaxin, INSL3 and INSL5.
A Chemogenetic Tool that Enables Functional Neural Circuit Analysis.
Publisher: Elsevier BV
Date: 09-2020
Negative cooperativity in H2 relaxin binding to a dimeric relaxin family peptide receptor 1
Publisher: Elsevier BV
Date: 12-2008
DOI: 10.1016/J.MCE.2008.07.014
Abstract: H2 relaxin, a member of the insulin superfamily, binds to the G-protein-coupled receptor RXFP1 (relaxin family peptide 1), a receptor that belongs to the leucine-rich repeat (LRR)-containing subgroup (LGRs) of class A GPCRs. We recently demonstrated negative cooperativity in INSL3 binding to RXFP2 and showed that this subgroup of GPCRs functions as constitutive dimers. In this work, we investigated whether the binding of H2 relaxin to RXFP1 also shows negative cooperativity, and whether this receptor functions as a dimer using BRET(2). Both binding and dissociation were temperature dependent, and the pH optimum for binding was pH 7.0. Our results showed that RXFP1 is a constitutive dimer with negative cooperativity in ligand binding, that dimerization occurs through the 7TM domain, and that the ectodomain has a stabilizing effect on this interaction. Dimerization and negative cooperativity appear to be general properties of LGRs involved in reproduction as well as other GPCRs.
Identification and characterization of the mouse and rat relaxin receptors as the novel orthologues of human leucine-rich repeat-containing G-protein-coupled receptor 7
Publisher: Wiley
Date: 11-2004
DOI: 10.1111/J.1440-1681.2004.04075.X
Abstract: 1. Relaxin is an extracellular matrix (ECM)-remodelling hormone that is functionally important in reproductive tissues, brain, lung and heart. 2. Recently, the human relaxin receptor was identified as leucine-rich repeat-containing G-protein-coupled receptor 7 (LGR7). 3. Using human LGR7 as a template, we identified mouse and rat LGR7 orthologues in the Celera and National Centre for Biotechnology Information databases. 4. At the protein level, mouse and rat LGR7 share 85.2 and 85.7% identity with human LGR7, respectively. 5. Mouse LGR7 mRNA was detected in all tissues where relaxin binding is observed. 6. Mouse and rat LGR7 bound [33P]-relaxin with high affinity and, upon relaxin treatment, both receptors stimulated cAMP production in transfected HEK 293T cells. 7. These results indicate that mouse and rat LGR7 are the relaxin receptors in these species. 8. The actions of relaxin in rodents are well characterized, providing an established platform for research into the molecular pharmacology of the highly conserved relaxin receptor.
Design and recombinant expression of insulin-like peptide 5 precursors and the preparation of mature human INSL5
Publisher: Springer Science and Business Media LLC
Date: 11-04-2010
DOI: 10.1007/S00726-010-0586-3
Abstract: Insulin-like peptide 5 (INSL5) is a recently identified insulin superfamily member. Although it binds to and activates the G-protein coupled receptor, RXFP4, its precise biological function remains unknown. To help determine its function, significant quantities of INSL5 are required. In the present work, three single-chain INSL5 precursors were designed, two of which were successfully expressed in E. coli cells. The expressed precursors were solubilized from inclusion bodies, purified almost to homogeneity by immobilized metal-ion affinity chromatography, and then refolded in vitro. One precursor could be converted to two-chain human INSL5 bearing an extended N-terminus of the A-chain (designated long-INSL5) by sequential Lys-C endoproteinase and carboxypeptidase B treatment. The 6 residue A-chain N-terminal extension of long-INSL5 was subsequently removed by Aeromonas aminopeptidase to yield native INSL5 that was designated short-INSL5. Circular dichroism spectroscopic analysis and peptide mapping showed that the recombinant INSL5s adopted an insulin-like conformation and possessed the expected characteristic insulin-like disulfide linkages. Activity assay showed that both long- and short-INSL5 had full RXFP4 receptor activity compared with chemically synthesized human INSL5. This suggested that extension of the N-terminus of the A-chain of long-INSL5 did not adversely impact upon the binding to or activation of the RXFP4 receptor. However, the single-chain INSL5 precursor was inactive which indicated that a free C-terminus of the B-chain is critical for the activity of INSL5. Our present work thus provides an efficient approach for preparation of INSL5 and its analogs through recombinant expression in E. coli cells.
Regulation of neurohypophysial hormone secretion in an Australian marsupial.
Publisher: American Physiological Society
Date: 05-1995
DOI: 10.1152/AJPREGU.1995.268.5.R1319
Abstract: The brushtail possum secretes the typically reptilian mesotocin (MT) and arginine vasopressin (AVP) as its neurohypophysial hormones. In this study we have looked at the regulation of MT and AVP secretion in conscious possums by studying the effects of surgical stress and handling of animals, hypertonic saline infusion, hemorrhage, and angiotensin II (ANG II) infusion on the plasma concentrations of MT and AVP. Surgical insertion of a jugular catheter and handling stress increased MT secretion for 3 days after surgery without affecting plasma AVP concentrations. Hypertonic saline infusion induced a gradual increase in plasma osmolarity and Na+ concentration throughout the infusion, which steadily increased plasma AVP without affecting plasma K+ or hematocrit. The relationship between plasma osmolarity and AVP was exponential. Plasma MT was stimulated only by supraphysiological plasma osmolarities. ANG II increased plasma MT and AVP concentrations equipotently throughout the infusion. Hemorrhage was a relatively specific stimulus for AVP secretion, but MT secretion was highly stimulated during severe hypovolemia. It was concluded that MT and AVP secretion is differentially regulated in the possum.
Mesotocin gene expression and evidence of gene duplication in the tammar wallaby
Publisher: Wiley
Date: 05-1998
DOI: 10.1111/J.1749-6632.1998.TB10829.X
Abstract: Adolescent mental health (AMH) is a critical driver of HIV outcomes, but is often overlooked in HIV research and programming. The implementation science Exploration, Preparation, Implementation, Sustainment (EPIS) framework informed development of a questionnaire that was sent to a global alliance of adolescent HIV researchers, providers, and implementors working in sub-Saharan Africa with the aim to (1) describe current AMH outcomes incorporated into HIV research within the alliance (2) identify determinants (barriers/gaps) of integrating AMH into HIV research and care and (3) describe current AMH screening and referral systems in adolescent HIV programs in sub-Saharan Africa. Respondents reported on fourteen named studies that included AMH outcomes in HIV research. Barriers to AMH integration in HIV research and care programs were explored with suggested implementation science strategies to achieve the goal of integrated and sustained mental health services within adolescent HIV programs.
Identification of key residues essential for the structural fold and receptor selectivity within the A-chain of human gene-2 (H2) relaxin
Publisher: Elsevier BV
Date: 11-2012
Preliminary structure-function relationship studies on insulin-like peptide 5 (INSL5)
Publisher: Springer Science and Business Media LLC
Date: 17-01-2013
Examination of relaxin and its receptors expression in pig gametes and embryos
Publisher: Springer Science and Business Media LLC
Date: 2011
Determinants of Ligand Subtype-Selectivity at α1A-Adrenoceptor Revealed Using Saturation Transfer Difference (STD) NMR
Publisher: American Chemical Society (ACS)
Date: 14-03-2202
DOI: 10.1021/ACSCHEMBIO.8B00191
Abstract: α
A missense mutation in LRR8 of RXFP2 is associated with cryptorchidism
Publisher: Springer Science and Business Media LLC
Date: 10-2010
DOI: 10.1007/S00335-010-9291-5
Abstract: Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2 ( DG/DG ) mice compared to Rxfp2 (+/DG ) and Rxfp2 (+/+) mice. Testicular histology of the Rxfp2 ( DG/DG ) mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2 (-)) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2 ( DG/-)) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2 (+/-) littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7-9 indicated that aspartic acid 294 is located within the β-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.
Understanding relaxin signalling at the cellular level.
Publisher: Elsevier BV
Date: 05-2018
DOI: 10.1016/J.MCE.2018.12.017
Abstract: The peptide hormone relaxin mediates many biological actions including anti-fibrotic, vasodilatory, angiogenic, anti-inflammatory, anti-apoptotic, and organ protective effects across a range of tissues. At the cellular level, relaxin binds to the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1) to activate a variety of downstream signal transduction pathways. This signalling cascade is complex and also varies in erse cellular backgrounds. Moreover, RXFP1 signalling shows crosstalk with other receptors to mediate some of its physiological functions. This review summarises known signalling pathways induced by acute versus chronic treatment with relaxin across a range of cell types, it describes RXFP1 crosstalk with other receptors, signalling pathways activated by other ligands targeting RXFP1, and it also outlines physiological relevance of RXFP1 signalling outputs. Comprehensive understanding of the mechanism of relaxin actions in fibrosis, vasodilation, as well as organ protection, will further support relaxin's clinical potential.
H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2
Publisher: Elsevier BV
Date: 03-2003
Characterisation of a cell-free synthesised G-protein coupled receptor
Publisher: Springer Science and Business Media LLC
Date: 24-04-2017
DOI: 10.1038/S41598-017-01227-Z
Abstract: G-protein coupled receptors are the largest family of integral membrane proteins found within the human genome. They function as receptors and modulators to a wide range of ligands and responses which are crucial for human health. GPCR study, specifically the investigation of structure and interaction to cognate ligands, is of high priority. Limitations for structural study can be traced in part, to obtaining suitable quantities of recombinant protein. We sought to address the limitations of traditional recombinant technologies by utilising an Escherichia coli based cell-free protein synthesis (CFPS) approach for production of a thermostable neurotensin receptor 1 (en2NTS 1 ). Initial results were promising, with a high amount (up to 2 mg/mL) of en2NTS 1 produced, that had attained correct secondary structure. Meanwhile, concurrent experiments indicated that CFPS produced en2NTS 1 showed non-competitive binding to the peptide ligand neurotensin8–13 when compared to E. coli produced en2NTS 1 . 1 H- 13 C HMQC SOFAST NMR spectra were indicative of disrupted tertiary structure for CFPS produced 13 CH 3 -methionine labelled en2NTS 1 . The results obtained, indicate CFPS produced en2NTS 1 is not forming a discrete tertiary structure and that further development of the CFPS technique needs to be carried out.
Distinct but overlapping binding sites of agonist and antagonist at the relaxin family peptide 3 (RXFP3) receptor
Publisher: Elsevier BV
Date: 10-2018
Minimization of human relaxin-3 leading to high-affinity analogues with increased selectivity for relaxin-family peptide 3 receptor (RXFP3) over RXFP1
Publisher: American Chemical Society (ACS)
Date: 08-02-2012
DOI: 10.1021/JM201505P
Abstract: 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 h ered 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.
Mesotocin gene expression in the diencephalon of domestic fowl: cloning and sequencing of the MT cDNA and distribution of MT gene expressing neurons in the chicken hypothalamus.
Publisher: Wiley
Date: 10-1997
DOI: 10.1046/J.1365-2826.1997.00643.X
Abstract: This study focuses on the structure and expression of the mesotocin (MT) gene in the chicken hypothalamus. Using an anchored and nested RT-PCR strategy, combined with circular RACE-PCR, the full length sequence of the chicken MT cDNA was obtained. The cDNA and derived amino acid sequences conformed to the structure of the oxytocin-like gene family. However, unlike most mammalian species, there does not appear to be frequent gene conversion between the MT and AVT cDNA sequences. A single specific hybridization signal of 1.2 kb was detected by Southern analysis of chicken genomic DNA, indicating only a single gene copy in the chicken genome. Northern analysis of hypothalamic RNA revealed a single band at approximately 0.6 kb. Using the same probe for in situ hybridization histochemistry, MT-mRNA was demonstrated to be predominantly localized in the parvocellular, magnocellular and periventricular subgroups of the paraventricular nucleus and, when compared to the distribution of neurons containing arginine-vasotocin (AVT)-mRNA in the same region, with far fewer neurons expressing the MT gene in the lateral subgroups. Only few and scattered neurons expressing the MT gene were found in the ventral and external subgroups of the supraoptic nucleus in which many neurons contain AVT transcripts, as demonstrated in consecutive sections. In all nuclei investigated, the intensity of AVT and MT hybridization signals per cell was approximately equal. No specific labelling for MT-mRNA was found in the bed nucleus of the stria terminalis, nor the nucleus accumbens. Using immunocytochemical detection of AVT and in situ hybridization for neurons expressing MT-mRNA, some neurons were found to contain both AVT and MT gene products in the paraventricular nucleus but not in the supraoptic nucleus.
Relaxin signaling in reproductive tissues
Publisher: Elsevier BV
Date: 04-2003
DOI: 10.1016/S0303-7207(03)00078-9
Abstract: The insulin/relaxin peptide family includes insulin, IGFs, relaxin1-3, INSL3/RLF, INSL4, INSL5/RIF2 and INSL6/RIF1, many without functional characterization. Based on analysis of transgenic phenotypes and phylogenetic profiling, we have discovered that two orphan leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8, are cognate receptors for relaxin whereas INSL3 is a specific ligand for LGR8. With the identification of the relaxin receptors, it is now possible to investigate specific cells and tissues that are responsive to relaxin in erse physiological and pathological conditions as well as to develop agonists and antagonists for LGR7 and LGR8 as therapeutics to treat different labor disorders. Furthermore, future functional characterization of the specificity of these pluripoentent receptors with peptide ligands could lead to the understanding of related orphan ligands and receptors.
Role of the intra-A-chain disulfide bond of insulin-like peptide 3 in binding and activation of its receptor, RXFP2
Publisher: Elsevier BV
Date: 09-2010
DOI: 10.1016/J.PEPTIDES.2010.05.021
Abstract: INSL3 is a member of the insulin-IGF-relaxin superfamily and plays a key role in male fetal development and in adult germ cell maturation. It is the cognate ligand for RXFP2, a leucine-rich repeat containing G-protein coupled receptor. To date, and in contrast to our current knowledge of the key structural features that are required for the binding of INSL3 to RXFP2, comparatively little is known about the key residues that are required to elicit receptor activation and downstream cell signaling. Early evidence suggests that these are contained principally within the A-chain. To further explore this hypothesis, we have undertaken an examination of the functional role of the intra-A-chain disulfide bond. Using solid-phase peptide synthesis together with regioselective disulfide bond formation, two analogs of human INSL3 were prepared in which the intra-chain disulfide bond was replaced, one in which the corresponding Cys residues were substituted with the isosteric Ser and the other in which the Cys were removed altogether. Both of these peptides retained nearly full RXFP2 receptor binding but were devoid of cAMP activity (receptor activation), indicating that the intra-A-chain disulfide bond makes a significant contribution to the ability of INSL3 to act as an RXFP2 agonist. Replacement of the disulfide bond with a metabolically stable dicarba bond yielded two isomers of INSL3 that each exhibited bioactivity similar to native INSL3. This study highlights the critical structural role played by the intra-A-chain disulfide bond of INSL3 in mediating agonist actions through the RXFP2 receptor.
Mesotocin receptor gene and protein expression in the prostate gland, but not testis, of the tammar wallaby, Macropus eugenii
Publisher: Oxford University Press (OUP)
Date: 11-1998
DOI: 10.1095/BIOLREPROD59.5.1101
Abstract: Evidence suggests that systemic oxytocin (OT) causes contractions of the prostate gland during ejaculation in eutherians, although functional OT receptors in this tissue have not been identified. Male marsupials secrete mesotocin (MT) from the pituitary and have relatively large, muscular prostate glands, so we examined MT receptors (MTRs) in the reproductive tract of the male tammar wallaby at the mRNA and protein level. We first obtained a partial (588 base pair) sequence of the tammar MTR cDNA that showed high homology to eutherian OT receptors (74-77%) and low homology to vasopressin receptors (38-52%). Analysis by reverse transcription-polymerase chain reaction demonstrated MTR mRNA in the adult, juvenile, and pouch young prostate and epididymis, but not testis. MTR transcripts were observed in the smooth muscle layers surrounding the urethral lumen and in the fibromuscular capsule. There was a single high-affinity 125I-D(CH2)5[Tyr(Me)2, Tyr4, Orn8, Tyr-NH29]-vasotocin (125I-OTA) binding site in the adult prostate. Competitive binding assays revealed identical ligand-binding profiles to the myometrium MTR (OTA > OT = MT > arginine vasopressin [AVP] antagonist > AVP). A lower-affinity 125I-OTA-binding site was present in the testis, with ligand-binding profiles indicating binding to vasopressin receptors. MTR concentrations in the prostate were 8-fold lower than concentrations in the myometrium. Our data demonstrate the presence of an MTR gene and functional receptor protein in the prostate gland, but not the testis, of the tammar. Localization of MTRs to the smooth muscle fibers in the capsule and surrounding the urethral lumen suggests a contractile function for MT during ejaculation.
Characterization of a new potent and long-lasting single chain peptide agonist of RXFP1 in cells and in vivo translational models.
Publisher: Springer Science and Business Media LLC
Date: 28-11-2022
DOI: 10.1038/S41598-022-24716-2
Abstract: Despite beneficial effects in acute heart failure, the full therapeutic potential of recombinant relaxin-2 has been h ered by its short half-life and the need for intravenous administration limiting its use to intensive care units. A multiparametric optimization of the relaxin B-chain led to the identification of single chain lipidated peptide agonists of RXFP1 like SA10SC-RLX with subcutaneous bioavailability and extended half-life. SA10SC-RLX has sub nanomolar activity on cells expressing human RXFP1 and molecular modeling associated with the study of different RXFP1 mutants was used to decipher the mechanism of SA10SC-RLX interaction with RXFP1. Telemetry was performed in rat where SA10SC-RLX was able to engage RXFP1 after subcutaneous administration without tachyphylaxis after repeated dosing. Renal blood flow was then used as a translational model to evaluate RXFP1 activation. SA10SC-RLX increased renal blood flow and decreased renal vascular resistance in rats as reported for relaxin in humans. In conclusion, SA10SC-RLX mimics relaxin activity in in vitro and in vivo models of acute RXFP1 engagement. SA10SC-RLX represents a new class of long-lasting RXFP1 agonist, suitable for once daily subcutaneous administration in patients and potentially paving the way to new treatments for chronic fibrotic and cardiovascular diseases.
A Novel Antagonist Peptide Reveals a Physiological Role of Insulin-Like Peptide 5 in Control of Colorectal Function
Publisher: American Chemical Society (ACS)
Date: 30-08-2021
Relaxin antagonizes hypertrophy and apoptosis in neonatal rat cardiomyocytes
Publisher: The Endocrine Society
Date: 04-2007
DOI: 10.1210/EN.2006-1324
Abstract: The pregnancy hormone relaxin has recently been shown to be cardio-protective. Despite its well-established antifibrotic actions in the heart, the effects of relaxin on cardiomyocytes (CM) remain to be determined. We investigated effects of isoform 2 of the human relaxin (H2-relaxin) on CM hypertrophy and apoptosis. In cultured neonatal rat CM, phenylephrine (50 μm) and cardiac fibroblast-conditioned medium were used respectively to induce CM hypertrophy. The degree of hypertrophy was indicated by increased cell size, protein synthesis and gene expression of atrial natriuretic peptide. Although H2-relaxin (16.7 nm) alone failed to suppress hypertrophy induced by phenylephrine, it repressed the cardiac fibroblast-conditioned medium-induced increase in protein synthesis by 24% (P & 0.05) and reversed the increase in cell size (P & 0.001) and atrial natriuretic peptide expression (P& .01). We further studied the effect of H2-relaxin on CM apoptosis induced by H2O2 (200 μm). Studies of DNA laddering and nuclear staining demonstrated that H2-relaxin treatment reduced H2O2-induced DNA fragmentation. Real-time PCR and Western blot analysis revealed a significant increase in the Bcl2/Bax ratio in H2-relaxin-treated CM. Further analysis showed that activation of Akt (1.8-fold, P& 0.001) and ERK (2.0-fold, P& .01) were involved in the antiapoptotic action of H2-relaxin in CM, and that Gi/o coupling of relaxin receptors was associated with the H2-relaxin-induced Akt activation in CM. In conclusion, these results extend our current knowledge of the cardiac actions of relaxin by demonstrating that H2-relaxin indirectly inhibits CM hypertrophy and directly protects CM from apoptosis.
INSL3/RXFP2 Signaling in Testicular Descent
Publisher: Wiley
Date: 04-2009
Engineering of a Novel Simplified Human Insulin-Like Peptide 5 Agonist.
Publisher: American Chemical Society (ACS)
Date: 12-02-2016
DOI: 10.1021/ACS.JMEDCHEM.5B01786
Abstract: Insulin-like peptide 5 (INSL5) has recently been discovered as only the second orexigenic gut hormone after ghrelin. As we have previously reported, INSL5 is extremely difficult to assemble and oxidize into its two-chain three-disulfide structure. The focus of this study was to generate structure-activity relationships (SARs) of INSL5 and use it to develop a potent and simpler INSL5 mimetic with RXFP4 agonist activity. A series of human and mouse INSL5 (hINSL5/mINSL5) analogues were designed and chemically synthesized, resulting in a chimeric INSL5 analogue exhibiting more than 10-fold higher potency (0.35 nM) at human RXFP4 compared with native hINSL5 (4.57 nM). The SAR study also identified a key residue (K(A15)) in the A-chain of mINSL5 that contributes to improved RXFP4 affinity and potency of mINSL5 compared with hINSL5. This knowledge ultimately led us to engineer a minimized hINSL5 mimetic agonist that retains native hINSL5-like RXFP4 affinity and potency at human RXFP4. This minimized analogue was synthesized in 17.5-fold higher yield and in less time compared with hINSL5.
Searching the human genome database for novel relaxin- and insulin-like peptides
Publisher: Springer Science and Business Media LLC
Date: 2002
Coatings Releasing the Relaxin Peptide Analogue B7-33 Reduce Fibrotic Encapsulation
Publisher: American Chemical Society (ACS)
Date: 12-11-2019
Abstract: The development of antifibrotic materials and coatings that can resist the foreign body response (FBR) continues to present a major hurdle in the advancement of current and next-generation implantable medical devices, biosensors, and cell therapies. From an implant perspective, the most important issue associated with the FBR is the prolonged inflammatory response leading to a collagenous capsule that ultimately blocks mass transport and communication between the implant and the surrounding tissue. Up to now, most attempts to reduce the capsule thickness have focused on providing surface coatings that reduce protein fouling and cell attachment. Here, we present an approach that is based on the sustained release of a peptide drug interfering with the FBR. In this study, the biodegradable polymer poly(lactic-
INSL5 is a high affinity specific agonist for GPCR142 (GPR100)
Publisher: Elsevier BV
Date: 2005
The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors
Publisher: Elsevier BV
Date: 06-2008
Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions
Publisher: American Chemical Society (ACS)
Date: 05-06-2008
DOI: 10.1021/BC800127P
Abstract: An efficient solid-phase synthesis protocol has been developed which, together with regioselective sequential formation of the three disulfide bonds, enabled the preparation of specifically monolanthanide (europium)-labeled human insulin-like peptide 3 (INSL3) for the study of its interaction with its G-protein-coupled receptor, RXFP2, via time-resolved fluorometry. A commercially available chelator, diethylene triamine pentaacetic acid (DTPA), was coupled to the N-terminus of the INSL3 A-chain on the solid phase, and then a coordination complex between europium ion and DTPA was formed using EuCl 3 to protect the chelator from production of an unidentified adduct during subsequent combination of the A- and B-chains. The labeled peptide was purified in high yield using high-performance liquid chromatography with nearly neutral pH buffers to prevent the liberation of Eu (3+) from the chelator. Using time-resolved fluorometry, saturation binding assays were undertaken to determine the binding affinity (p K d) of labeled INSL3 for RXFP2 in HEK-293T cells stably expressing RXFP2. The dissociation constant of DTPA-labeled INSL3 (9.05 +/- 0.03, n = 3) that was obtained from saturation binding experiments was comparable to that of (125)I-labeled INSL3 (9.59 +/- 0.09, n = 3). The receptor binding affinity (p K i) of human INSL3 was determined to be 9.27 +/- 0.06, n = 3, using Eu-DTPA-INSL3 as a labeled ligand, which again is similar to that obtained when (125)I-INSL3 was used as labeled ligand (9.34 +/- 0.02, n = 4). This novel lanthanide-coordinated, DTPA-labeled INSL3 has excellent sensitivity, stability, and high specific activity, properties that will be particularly beneficial in high-throughput screening of INSL3 analogues in structure-activity studies.
Spatial Learning Requires mGlu5 Signalling in the Dorsal Hippocampus.
Publisher: Springer Science and Business Media LLC
Date: 10-05-2015
DOI: 10.1007/S11064-015-1595-0
Abstract: We examined the role of hippoc al metabotropic glutamate receptor 5 (mGlu5) in spatial learning and memory. Although it has been shown that mGlu5 signalling is required for certain forms of learning and memory, its role in spatial learning is unclear since studies using pharmacological or knockout mice models provide inconsistent findings. Additionally, the location in the brain where mGlu5 signalling may modulate such learning is yet to be precisely delineated. We stereotaxically injected rAAV-Cre into the dorsal hippoc us of mGlu5(loxP/loxP) mice to knockdown mGlu5 in that region. We show for the first time that knockdown of mGlu5 in the dorsal hippoc us is sufficient to impair spatial learning in Morris Water Maze. Locomotor activity and memory retrieval were unaffected by the mGlu5 knockdown. Taken together, these findings support a key role for dorsal hippoc al mGlu5 signalling in spatial learning.
Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3.
Publisher: Elsevier BV
Date: 09-2006
High-Throughput Screening Campaign Identified a Potential Small Molecule RXFP3/4 Agonist
Publisher: MDPI AG
Date: 11-12-2021
DOI: 10.3390/MOLECULES26247511
Abstract: Relaxin/insulin-like family peptide receptor 3 (RXFP3) belongs to class A G protein-coupled receptor family. RXFP3 and its endogenous ligand relaxin-3 are mainly expressed in the brain with important roles in the regulation of appetite, energy metabolism, endocrine homeostasis and emotional processing. It is therefore implicated as a potential target for treatment of various central nervous system diseases. Since selective agonists of RXFP3 are restricted to relaxin-3 and its analogs, we conducted a high-throughput screening c aign against 32,021 synthetic and natural product-derived compounds using a cyclic adenosine monophosphate (cAMP) measurement-based method. Only one compound, WNN0109-C011, was identified following primary screening, secondary screening and dose-response studies. Although displayed agonistic effect in cells overexpressing the human RXFP3, it also showed cross-reactivity with the human RXFP4. This hit compound may provide not only a chemical probe to investigate the function of RXFP3/4, but also a novel scaffold for the development of RXFP3/4 agonists.
Increased feeding and body weight gain in rats after acute and chronic activation of RXFP3 by relaxin-3 and receptor-selective peptides
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 09-2012
Expression and regulation of relaxin-like factor gene transcripts in the bovine ovary: Differentiation-dependent expression in theca cell cultures
Publisher: Oxford University Press (OUP)
Date: 10-1999
DOI: 10.1095/BIOLREPROD61.4.1090
Abstract: The relaxin-like factor (RLF) was recently discovered as a new member of the insulin-insulin-like growth factor-relaxin family of growth factors and hormones predominantly in the Leydig cells of the testis. In cattle, in contrast to other species, the RLF gene is also expressed to a high level in the ovary, where its expression pattern in the corpus luteum of the late cycle and pregnancy is similar to that of relaxin in the pig. The RLF gene was also transcribed to a high level in the theca cells of estrogen-rich, large antral follicles. Long-term primary cultures of bovine theca cells showed that expression was insulin dependent. After an initial decline in specific mRNA concentrations, there was a switch to a transcript with a longer poly(A) tail at about Day 6 of culture, which continued to increase to very high levels by Day 15 of culture. Addition of fetal calf serum to cultures caused a rapid and irreversible down-regulation of the RLF gene. Also, LH caused a decline in specific gene expression in long-term primary theca cell cultures. As in the Leydig cells of the testis, the pattern of RLF gene expression appears to reflect the differentiation state of the ovarian theca-luteal cell lineage, and should prove useful for mapping the fate of these cells under differing stimulation regimes.
Insulin-relaxin family peptide signaling and receptors in mouse brain membranes and neuronal cells
Publisher: Wiley
Date: 05-2005
Abstract: Several orphan G-protein-coupled receptors (GPCRs), LGR7 and LGR8, GPCR135 and GPCR142, were recently identified as putative, native receptors for different relaxin-family peptides, and their cell signaling mechanisms were elucidated in stably transfected cell lines. Anatomic studies have demonstrated that discrete populations of neurons in rat brain express relaxin and relaxin-3 mRNA eptide, relaxin and relaxin-3 binding sites, and LGR7 and GPCR135 mRNAs. Thus, we began to assess the ability of relaxin-family peptides to alter cAMP production in brain and the involvement of the different native receptors. In mouse cortical membranes, a fixed concentration of relaxin peptides (100 nM) inhibited forskolin-induced cAMP production, but further studies in normal and receptor knockout mouse strains are required to assess the specificity of these effects. In addition, whole-cell signaling mechanisms are being investigated in a mouse hypothalamic cell line, GT1-7. Such studies will help to establish the actions of relaxin-family peptides via their different GPCRs in different brain pathways.
Physiology and molecular biology of the relaxin peptide family
Publisher: Elsevier
Date: 2006
Relaxin deficiency in mice is associated with an age-related progression of pulmonary fibrosis.
Publisher: Wiley
Date: 11-2002
Exploring the Use of Helicogenic Amino Acids for Optimising Single Chain Relaxin-3 Peptide Agonists
Publisher: MDPI AG
Date: 14-10-2020
DOI: 10.3390/BIOMEDICINES8100415
Abstract: Relaxin-3 is a highly conserved two-chain neuropeptide that acts through its endogenous receptor the Relaxin Family Peptide-3 (RXFP3) receptor. The ligand/receptor system is known to modulate several physiological processes, with changes in food intake and anxiety-levels the most well studied in rodent models. Agonist and antagonist analogues based on the native two-chain peptide are costly to synthesise and not ideal drug leads. Since RXFP3 interacting residues are found in the relaxin B-chain only, this has been the focus of analogue development. The B-chain is unstructured without the A-chain support, but in single-chain variants structure can be induced by dicarba-based helical stapling strategies. Here we investigated whether alternative helical inducing strategies also can enhance structure and activity at RXFP3. Combinations of the helix inducing α-aminoisobutyric acid (Aib) were incorporated into the sequence of the relaxin-3 B-chain. Aib residues at positions 13, 17 and 18 partially reintroduce helicity and activity of the relaxin-3 B-chain, but other positions are generally not suited for modifications. We identify Thr21 as a putative new receptor contact residue important for RXFP3 binding. Cysteine residues were also incorporated into the sequence and cross-linked with dichloroacetone or α, α’-dibromo-m-xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.
Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat
Publisher: Elsevier BV
Date: 2007
DOI: 10.1016/J.NEUROSCIENCE.2006.08.072
Abstract: Relaxin-3 (RLX3) is a newly identified member of the relaxin/insulin peptide family that is highly conserved across a range of species from fish to mammals and is highly expressed in rat, mouse and human brain. Extensive pharmacological studies have demonstrated that RLX3 is a high affinity, selective ligand for G-protein-coupled receptor-135 (GPCR135, now classified as relaxin family peptide-3 receptor RXFP3). In ongoing studies to understand the physiological functions of RLX3, the distribution of RLX3-containing neuronal elements in rat brain was determined by immunohistochemistry, using an affinity-purified polyclonal antiserum raised against a conserved segment of the RLX3 C-peptide (AS-R3(85-101)). Consistent with the distribution of RLX3 mRNA, neurons containing RLX3-like immunoreactivity (LI) were observed in the pontine nucleus incertus and the majority of these cells, which are known to express corticotropin-releasing factor receptor-1, were shown to express glutamic acid decarboxylase-65-immunoreactivity, suggesting a GABA phenotype. Nerve fibers and terminals containing RLX3-LI were observed adjacent to cells in the nucleus incertus and in various forebrain regions known to receive afferents from the nucleus incertus, including cortex, septum, hippoc us, thalamus, hypothalamus and midbrain. Regions that contained highest densities of RLX3-positive fibers included the medial septum, lateral preoptic area, lateral hypothalamus/medial forebrain bundle and ventral hippoc us and additional fibers were observed in olfactory bulb and olfactory and frontal/cingulate cortices, bed nucleus of the stria terminalis, dorsal endopiriform, intergeniculate, and supramammillary nuclei, and the periaqueductal gray and dorsal raphe. The RLX3-positive network overlapped the regional distribution of GPCR135 mRNA and specific binding sites for an [125I]-GPCR135-selective, chimeric peptide. These anatomical findings further support the proposition that RLX3 is the endogenous ligand for GPCR135 in rat brain and provide evidence for broad modulatory activity of RLX3 in behavioral activation relating to autonomic and neuroendocrine control of metabolism and reproduction and higher-order processes such as stress and cognition.
Bovine endometrial epithelial cells as a model system to study oxytocin receptor regulation
Publisher: Oxford University Press (OUP)
Date: 09-1998
Abstract: Endometrial epithelial cell cultures were established from bovine uterine tissue collected during the oestrous cycle from commercially slaughtered animals. These cells were shown to express moderately high levels of oxytocin receptors (OTR) (up to 30000 per cell) after about one week in culture. These receptors have been characterized at the molecular, pharmacological and functional level and shown to be identical to those expressed in the bovine endometrium in vivo. Preliminary experiments to investigate the regulation of the OTR and its gene using this system, have shown that expression is to a large degree constitutive, the receptors being spontaneously upregulated during culture. Sex steroids at concentrations close to or above the serum limits observed in vivo appeared to have no effect, although the cells were shown to express mRNA for the specific steroid receptors throughout culture. Only the blastocyst product, interferon-tau, showed a significant effect, downregulating both OTR and their gene transcripts in the cultured endometrial epithelial cells. Although more extensive studies are necessary, these results support the view that the OTR gene is controlled in part at least by a combination of constitutive and inhibitory elements.
Targeted viral vector transduction of relaxin-3 neurons in the rat nucleus incertus using a novel cell-type specific promoter
Publisher: Elsevier BV
Date: 06-2020
Purification and characterization of relaxin from the tammar wallaby (Macropus eugenii): bioactivity and expression in the corpus luteum.
Publisher: Oxford University Press (OUP)
Date: 07-2002
DOI: 10.1095/BIOLREPROD67.1.293
Abstract: The objective of this study was to isolate and purify prorelaxin or mature relaxin from the tammar wallaby corpus luteum (CL), determine their structure and bioactivity, and test the hypothesis that enzymatic cleavage of prorelaxin occurs in late gestation. Tammar relaxin peptides were extracted from pooled corpora lutea of late pregnant tammars using a combination of HPLC methods, and they were identified using Western blotting with a human (H2) relaxin antisera and matrix-assisted laser desorption ionization time of flight mass spectrometry. Although no prorelaxin was identified, multiple 6-kDa peptides were detected, which corresponded to the predicted mature tammar relaxin amino acid sequence, with an A chain of 24 amino acids, and different B chain lengths of 28, 29, 30, and 32 amino acids. Tammar relaxin bound with high affinity to rat cortical relaxin receptors and stimulated cAMP production in the human monocytic cell line, THP-1, which expresses the relaxin receptor. Analysis of in idual CL indicated that equivalent amounts of mature relaxin peptides were present throughout gestation and also in unmated tammars at equivalent stages of the luteal phase in the nonpregnant cycle. Immunoreactive relaxin was localized specifically to the luteal cells of the CL and the intensity of immunostaining did not vary between gestational stages. These data show that the CL of both pregnant and unmated tammar wallabies produces mature relaxin and suggests that relaxin expression in this species is not influenced by the conceptus. Moreover, the presence of mature relaxin throughout gestation implies that prohormone cleavage is not limited to the later stages of pregnancy
Silencing relaxin-3 in nucleus incertus of adult rodents: A viral vector-based approach to investigate neuropeptide function
Publisher: Public Library of Science (PLoS)
Date: 02-08-2012
Effect of graded adrenaline infusion on arterial adrenaline clearance in normotensive and hypertensive man.
Publisher: Wiley
Date: 04-1990
DOI: 10.1111/J.1440-1681.1990.TB01317.X
Abstract: 1. Arterial adrenaline (ADR) levels were measured during graded ADR infusions in normotensives (NT) and in mild hypertensives (HT), in order to test the hypothesis that ADR enhances its own clearance in NT. 2. Although infusion of ADR resulted in similar arterial ADR levels in NT and HT, increases in systolic blood pressure (SBP) and heart rate (HR) and decreases in diastolic blood pressure (DBP) were greater during ADR infusion in HT than in NT. 3. Twenty minutes after cessation of infusion, SBP and HR remained elevated in HT but not in NT. 4. No evidence was found supporting enhanced ADR clearance during increasing ADR levels in NT or HT.
Solid phase synthesis and structural analysis of novel A-chain dicarba analogs of human relaxin-3 (INSL7) that exhibit full biological activity
Publisher: Royal Society of Chemistry (RSC)
Date: 2009
DOI: 10.1039/B821882J
Abstract: Replacement of disulfide bonds with non-reducible isosteres can be a useful means of increasing the in vivo stability of a protein. We describe the replacement of the A-chain intramolecular disulfide bond of human relaxin-3 (H3 relaxin, INSL7), an insulin-like peptide that has potential applications in the treatment of stress and obesity, with the physiologically stable dicarba bond. Solid phase peptide synthesis was used to prepare an A-chain analogue in which the two cysteine residues that form the intramolecular bond were replaced with allylglycine. On-resin microwave-mediated ring closing metathesis was then employed to generate the dicarba bridge. Subsequent cleavage of the peptide from the solid support, purification of two isomers and their combination with the B-chain via two intermolecular disulfide bonds, then furnished two isomers of dicarba-H3 relaxin. These were characterized by CD spectroscopy, which suggested a structural similarity to the native peptide. Additional analysis by solution NMR spectroscopy also identified the likely cis/trans form of the analogs. Both peptides demonstrated binding affinities that were equivalent to native H3 relaxin on RXFP1 and RXFP3 expressing cells. However, although the cAMP activity of the analogs on RXFP3 expressing cells was similar to the native peptide, the potency on RXFP1 expressing cells was slightly lower. The data confirmed the use of a dicarba bond as a useful isosteric replacement of the disulfide bond.
Modeling the primary hormone-binding site of RXFP1 and RXFP2.
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03950.X
Abstract: The primary binding sites of the relaxin and insulin-like peptide 3 (INSL3) receptors, RXFP1 and RXFP2, are found within the leucine-rich repeats (LRRs) of the ectodomains. Specific B-chain residues in the peptides interact with residues in the inner beta-sheets of the LRRs of the receptors. Relaxin binds to RXFP2 with high affinity, although INSL3 has a very poor affinity for RXFP1. In this paper we present evidence that relaxin binds to the LRRs of RXFP2 in a manner similar to INSL3 binding to its receptor. Additionally, we introduce a model of this binding interaction and compare it to an alternate model for relaxin-RXFP1 binding.
C-terminus of the B-chain of relaxin-3 is important for receptor activity
Publisher: Public Library of Science (PLoS)
Date: 11-12-2013
Signal switching after stimulation of LGR7 receptors by human relaxin 2
Publisher: Wiley
Date: 05-2005
Abstract: Previous studies have described a biphasic cAMP response after stimulation of LGR7 by human gene 2 (H2) relaxin, involving both adenylate cyclase and PI3-kinase activity. The current study identifies the upstream involvement of Gi in the PI3-kinase-mediated response, likely the result of receptor signal switching. Amino acid sequence analysis of the LGR7 C-terminal tail and intracellular loops revealed multiple putative phosphorylation sites, suggesting that signal switching from Gs to Gi may occur after receptor phosphorylation. This study supports a time-dependent biphasic cAMP response: an initial short Gs-adenylate cyclase-mediated cAMP response is followed by receptor signal switching to a Gi-PI3-kinase-mediated response.
Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor.
Publisher: Wiley
Date: 13-07-2016
DOI: 10.1111/BPH.13522
Effects of C-Terminal B-Chain Modifications in a Relaxin 3 Agonist Analogue.
Publisher: American Chemical Society (ACS)
Date: 22-10-2020
In a Class of Their Own - RXFP1 and RXFP2 are Unique Members of the LGR Family.
Publisher: Frontiers Media SA
Date: 07-09-2015
The structural and functional role of the B-chain C-terminal arginine in the relaxin-3 peptide antagonist, R3(BΔ23-27)R/I5
Publisher: Wiley
Date: 18-12-2009
DOI: 10.1111/J.1747-0285.2008.00756.X
Abstract: Relaxin-3, a member of the insulin superfamily, is involved in regulating stress and feeding behavior. It is highly expressed in the brain and is the endogenous ligand for the receptor RXFP3. As relaxin-3 also interacts with the relaxin receptor RXFP1, selective agonists and antagonists are crucial for studying the physiological function(s) of the relaxin-3/RXFP3 pair. The analog R3(BDelta23-27)R/I5, in which a C-terminally truncated human relaxin-3 (H3) B-chain is combined with the INSL5 A-chain, is a potent selective RXFP3 antagonist and has an Arg residue remaining on the B-chain C-terminus as a consequence of the recombinant protein production process. To investigate the role of this residue in the RXFP3 receptor binding and activation, the analogs R3(BDelta23-27)R/I5 and R3(BDelta23-27)R containing the B-chain C-terminal Arg as well as R3(BDelta23-27)/I5 and R3(BDelta23-27), both lacking the Arg, were chemically assembled and their secondary structure and receptor activity assessed. The peptides generally had a similar conformation but those with the extra Arg residue displayed a significantly increased affinity for the RXFP3. Interestingly, in contrast to R3(BDelta23-27)R and R3(BDelta23-27)R/I5, the peptide R3(BDelta23-27) is a weak agonist. This suggests that the C-terminal Arg, although increasing the affinity, alters the manner in which the peptide binds to the receptor and thereby prevents activation, giving R3(BDelta23-27)R/I5 its potent antagonistic activity.
Relaxin: New peptides, receptors and novel actions
Publisher: Elsevier BV
Date: 07-2003
DOI: 10.1016/S1043-2760(03)00081-X
Abstract: Relaxin has long been known as a hormone of pregnancy. Until recently, little was known of potential roles for relaxin in non-pregnant females and males. The identification of a new gene encoding relaxin-3 (RLN3), the discovery of the elusive relaxin receptor and a novel role for relaxin-1 in regulating the normal turnover of collagen has provided us with unique insights into potential new roles for this peptide family. The Rln3 gene appears to be predominantly expressed in the brain, and mapping studies indicate a highly developed network of Rln3, Rln1 and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides might have important roles in the central nervous system. Rln1-knockout mice show progressive tissue fibrosis as they age, and this fibrosis leads to functional changes in both the heart and lungs. Hence, the biological significance of this enigmatic peptide family is expanding, as are its potential clinical uses.
The role of oxytocin and regulation of uterine oxytocin receptors in pregnant marsupials.
Publisher: Wiley
Date: 03-2000
DOI: 10.1111/J.1469-445X.2000.TB00012.X
Abstract: The oxytocin-like peptide of most Australian marsupials is mesotocin, which differs from oxytocin by a single amino acid. This substitution has no functional significance as both peptides have equivalent affinity for and biological activity on the marsupial oxytocin-like receptor. A role for mesotocin in marsupial parturition has been demonstrated in the tammar wallaby where plasma mesotocin concentrations increase less than one minute before birth. Infusion of an oxytocin receptor antagonist at the end of gestation disrupts normal parturition, probably by preventing mesotocin from stimulating uterine contractions. In the absence of mesotocin receptor activation, a peripartum surge in prostaglandins is delayed which suggests a functional relationship between mesotocin, prostaglandin release and luteolysis. Female marsupials have anatomically separate uteri and in monovular species, such as the tammar wallaby, only one uterus is gravid with a single fetus whereas the contralateral uterus remains non-gravid. We have used this unique animal model to differentiate systemic and fetal-specific factors in the regulation of uterine function during pregnancy. The gravid uterus in the tammar wallaby becomes increasingly sensitive to mesotocin as gestation proceeds, with the maximum contractile response observed at term. This is reflected in a large increase in mesotocin receptor concentrations in the gravid uterus, and a downregulation in the non-gravid uterus in late pregnancy. The upregulation in myometrial mesotocin receptors is pregnancy-specific and independent of systemic steroids. One factor that may influence mesotocin receptor upregulation in the gravid uterus in late pregnancy is mechanical stretch of the uterus caused by the growing fetus. Our data highlight that a local fetal influence is more important than systemic factors in the regulation of mesotocin receptors in the tammar wallaby.
Signaling pathways of the LGR7 and LGR8 receptors determined by reporter genes
Publisher: Wiley
Date: 05-2005
Abstract: Although much is known about the pleiotropic effects mediated by relaxin, the exact signaling pathways involved remain relatively elusive. This study examines LGR7 and LGR8 signaling using reporter gene technology. The greatest response was observed at the CRE reporter (indicates activation of cAMP-PKA and p38/JNK pathways), although INSL3 treatment of LGR8 produced a lower response than H2 relaxin treatment of LGR7. AP1 (which indicates activation of JNK pathways) was stimulated to a lesser degree. Three other reporters produced no response. The reporter gene studies suggest that ligand stimulation of LGR7 and LGR8 involves cAMP-PKA and p38/JNK signaling.
Diazepam is not a direct allosteric modulator of α1-adrenoceptors, but modulates receptor signaling by inhibiting phosphodiesterase-4.
Publisher: Wiley
Date: 26-12-2018
DOI: 10.1002/PRP2.455
Design and development of analogues of dimers of insulin-like peptide 3 B-chain as high-affinity antagonists of the RXFP2 receptor.
Publisher: Wiley
Date: 2011
DOI: 10.1002/BIP.21484
Abstract: Insulin-like peptide 3 (INSL3) is one of 10 members of the human relaxin-insulin superfamily of peptides. It is a peptide hormone that is expressed by fetal and postnatal testicular Leydig cells and postnatal ovarian thecal cells. It mediates testicular descent during fetal life and suppresses sperm apoptosis in adult males, whereas, in females, it causes oocyte maturation. INSL3 has also been shown to promote thyroid tumor growth and angiogenesis in human. These actions of INSL3 are mediated through its G protein-coupled receptor, RXFP2. INSL3, a two-chained peptide, binds to its receptor primarily via its B-chain, whereas elements of the A-chain are essential for receptor activation. In an attempt to design a high-affinity antagonist with potential clinical application as an anticancer agent as well as a contraceptive, we have previously prepared a synthetic parallel dimer of INSL3 B-chain and demonstrated that it binds to RXFP2 with high affinity. In this work, we undertook full pharmacological characterization of this peptide and show that it can antaogonize INSL3-mediated cAMP signaling through RXFP2. Further refinement by truncation of 18 residues yielded a minimized analogue that retained full binding affinity and INSL3 antagonism. It is an attractive lead peptide for in vivo evaluation as an inhibitor of male and female fertility and of INSL3-mediated carcinogenesis.
Chimeric RXFP1 and RXFP2 receptors highlight the similar mechanism of activation utilizing their N-terminal low-density lipoprotein class A modules
Publisher: Frontiers Media SA
Date: 2013
Characterization of novel splice variants of LGR7 and LGR8 reveals that receptor signaling is mediated by their unique low density lipoprotein class A modules
Publisher: Elsevier BV
Date: 11-2006
Relaxin reverses cardiac and renal fibrosis in spontaneously hypertensive rats
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 08-2005
DOI: 10.1161/01.HYP.0000171930.00697.2F
Abstract: The antifibrotic effects of the peptide hormone relaxin on cardiac and renal fibrosis were studied in 9- to 10-month-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Rats (n=8 to 9 per group) were allocated into 3 groups: WKY controls, vehicle-treated SHR (SHR-V), and relaxin-treated SHR (SHR-R). Relaxin (0.5 mg/kg per day) was administered via subcutaneously implanted osmotic mini-pumps over 2 weeks before hearts and kidneys were harvested for analysis. Collagen content was analyzed by hydroxyproline assay, gel electrophoresis, and quantitative histology. Zymography was used to determine matrix metalloproteinase (MMP) expression and Western blotting to determine proliferating cell nuclear antigen (PCNA) expression and α-smooth muscle actin (α-SMA)/myofibroblast expression, whereas cardiac hypertrophy was assessed by myocyte size and real-time polymerase chain reaction of associated genes. The left ventricular (LV) myocardium of SHR-V contained increased collagen levels (by 25±1%, P .01 using biochemical analysis and 3-fold P .01 using quantitative histology), enhanced expression of PCNA (by 70±8% P .01), α-SMA (by 32±2% P .05), and the collagen-degrading enzyme MMP-9 (by 70±6% P .05) versus respective levels measured in WKY controls. The kidneys of SHR-V also contained increased collagen (25±2%, P .05 using biochemical analysis and 2.4-fold P .01 using quantitative histology). Relaxin treatment significantly normalized collagen content in the LV ( P .01) and kidney ( P .05), completely inhibited cell proliferation ( P .01) and fibroblast differentiation ( P .05) in the LV, and increased MMP-2 expression (by 25±1% P .05) without affecting MMP-9 in the LV compared with that measured in SHR-V. Thus, relaxin is a potent antifibrotic hormone with a rapid-occurring efficacy that may have therapeutic potential for hypertensive disease.
De novo design and synthesis of cyclic and linear peptides to mimic the binding cassette of human relaxin
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03840.X
Abstract: A synthetic cyclic regioselectively addressable functionalized template was used to prepare six analogs that presented the side chains of the key receptor-binding residues of each of H2 and H3 relaxins and insulin-like peptide 3. None showed any binding affinity for RXFP1, RXFP2, or RXFP3, indicating that the key residues were either incorrectly oriented or that additional residues are required for receptor binding.
Engineering of chimeric peptides as antagonists for the G protein-coupled receptor, RXFP4
Publisher: Springer Science and Business Media LLC
Date: 28-11-2019
DOI: 10.1038/S41598-019-53707-Z
Abstract: Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes. However, INSL5 with two chains and three disulfide bridges is an extremely difficult peptide to assemble by chemical or recombinant means. In a recent study, we were able to engineer a simplified INSL5 analogue 13 which is a relaxin family peptide receptor 4 (RXFP4)-specific agonist. To date, however, no RXFP4-specific antagonist (peptide or small molecule) has been reported in the literature. The focus of this study was to utilize the non-specific RXFP3/RXFP4 antagonist ΔR3/I5 as a template to rationally design an RXFP4 specific antagonist. Unexpectedly, we demonstrated that ΔR3/I5 exhibited partial agonism at RXFP4 when expressed in CHO cells which is associated with only partial antagonism of INSL5 analogue activation. In an attempt to improve RXFP4 specificity and antagonist activity we designed and chemically synthesized a series of analogues of ΔR3/I5. While all the chimeric analogues still demonstrated partial agonism at RXFP4, one peptide (Analogue 17) exhibited significantly improved RXFP4 specificity. Importantly, analogue 17 has a simplified structure which is more amenable to chemical synthesis. Therefore, analogue 17 is an ideal template for further development into a specific high affinity RXFP4 antagonist which will be an important tool to probe the physiological role of RXFP4/INSL5 axis.
Stabilization of pre-existing neurotensin receptor conformational states by β-arrestin-1 and the biased allosteric modulator ML314
Publisher: Springer Science and Business Media LLC
Date: 07-06-2023
DOI: 10.1038/S41467-023-38894-8
Abstract: The neurotensin receptor 1 (NTS 1 ) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS 1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13 C ε H 3 -methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor’s intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs – without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS 1 :G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.
Relaxin family peptides and their receptors
Publisher: American Physiological Society
Date: 2013
DOI: 10.1152/PHYSREV.00001.2012
Abstract: There are seven relaxin family peptides that are all structurally related to insulin. Relaxin has many roles in female and male reproduction, as a neuropeptide in the central nervous system, as a vasodilator and cardiac stimulant in the cardiovascular system, and as an antifibrotic agent. Insulin-like peptide-3 (INSL3) has clearly defined specialist roles in male and female reproduction, relaxin-3 is primarily a neuropeptide involved in stress and metabolic control, and INSL5 is widely distributed particularly in the gastrointestinal tract. Although they are structurally related to insulin, the relaxin family peptides produce their physiological effects by activating a group of four G protein-coupled receptors (GPCRs), relaxin family peptide receptors 1–4 (RXFP1–4). Relaxin and INSL3 are the cognate ligands for RXFP1 and RXFP2, respectively, that are leucine-rich repeat containing GPCRs. RXFP1 activates a wide spectrum of signaling pathways to generate second messengers that include cAMP and nitric oxide, whereas RXFP2 activates a subset of these pathways. Relaxin-3 and INSL5 are the cognate ligands for RXFP3 and RXFP4 that are closely related to small peptide receptors that when activated inhibit cAMP production and activate MAP kinases. Although there are still many unanswered questions regarding the mode of action of relaxin family peptides, it is clear that they have important physiological roles that could be exploited for therapeutic benefit.
Responses of GPCR135 to human gene 3 (H3) relaxin in CHO-K1 cells determined by microphysiometry
Publisher: Wiley
Date: 05-2005
Abstract: This study examined the functional response to human relaxin 2 (H2 relaxin), human relaxin 3 (H3 relaxin), porcine relaxin, and human INSL3 in the cytosensor microphysiometer, using CHO-K1 cells stably expressing human GPCR135. CHO-K1 cells stably expressing GPCR135 were generated by the serial dilution method and receptor properties were assessed. Saturation studies of [125I] H3 relaxin binding to GPCR135 in these cells gave a Bmax of 32.61 +/- 6.5 fmol/mg protein and Kd of 0.12 +/- 0.08 nM. The functional response to H3 relaxin and other relaxin/insulin peptides of GPCR135 expressed in CHO-K1 cells was measured in the cytosensor microphysiometer and analyzed using inhibitors of signal transduction proteins.
Relaxin inhibits renal myofibroblast differentiation via RXFP1, the nitric oxide pathway, and Smad2
Publisher: Wiley
Date: 10-12-2009
DOI: 10.1096/FJ.08-120857
Abstract: The hormone relaxin inhibits renal myofibroblast differentiation by interfering with TGF-beta1/Smad2 signaling. However, the pathways involved in the relaxin-TGF-beta1/Smad2 interaction remain unknown. This study investigated the signaling mechanisms by which human gene-2 (H2) relaxin regulates myofibroblast differentiation in vitro by examining its effects on mixed populations of fibroblasts and myofibroblasts propagated from injured rat kidneys. Cultures containing approximately 60-70% myofibroblasts were used to determine which relaxin receptors, G-proteins, and signaling pathways were involved in the H2 relaxin-mediated regulation of alpha-smooth muscle actin (alpha-SMA a marker of myofibroblast differentiation). H2 relaxin only inhibited alpha-SMA immunostaining and collagen concentration in the presence of relaxin family peptide receptor 1 (RXFP1). H2 relaxin also induced a transient rise in cAMP in the presence of G(i/o) inhibition, and a sustained increase in extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. Furthermore, inhibition of neuronal nitric oxide synthase (nNOS), NO, and cGMP significantly blocked the inhibitory effects of relaxin on alpha-SMA and Smad2 phosphorylation, while the NO inhibitor, L-nitroarginine methyl ester (hydrochloride) (L-NAME) significantly blocked the inhibitory actions of relaxin on collagen concentration in vivo. These findings suggest that relaxin signals through RXFP1, and a nNOS-NO-cGMP-dependent pathway to inhibit Smad2 phosphorylation and interfere with TGF-beta1-mediated renal myofibroblast differentiation and collagen production.
Molecular biology of the oxytocin receptor: a comparative approach.
Publisher: Portland Press Ltd.
Date: 08-1997
DOI: 10.1042/BST0251058
Abstract: Left ventricular assist device (LVAD) evaluation includes a psychosocial assessment, conducted by social workers (SWs) on the advanced heart failure multidisciplinary team. Postdischarge caregiving plans are central to psychosocial evaluation. Caregiving's relationship with LVAD outcomes is mixed, and testing patients' social resources may disadvantage those from historically undertreated groups. We describe variation in policies defining adequate caregiving plans post-LVAD implant and possible impacts on patients from marginalized groups. This was a 2-phase sequential mixed-methods study: (1) phase 1, survey of US-based LVAD SWs, describing assessment structure and policies guiding candidacy outcomes and (2) phase 2, in idual interviews with SWs to further describe how caregiving plan adequacy impacts LVAD candidacy. Sixty-seven SWs returned surveys (rr=47%) from unique programs. Caregiving plan inadequacy (n=30) was the most common psychosocial dealbreaker. When asked what duration of caregiving is required, 23% indicated ≥3 months, 27% 4 to 12 weeks, and 30% <4 weeks. Two reported no duration requirement, 6 stated an indefinite 24/7 commitment was necessary. Across 22 interviews, SWs mirrored that caregiving plans were the most common psychosocial contraindication. How caregiving is operationalized varied. Participants voiced a tension between extended caregiving improving outcomes and the sense that some people of color, women, or low socioeconomic status patients struggle to meet stringent requirements. Policies regarding adequate duration of 24/7 caregiving vary, but inadequate caregiving plans are the most common psychosocial contraindication. Participants worry about patients' ability to meet restrictive requirements, particularly from historically undertreated groups. This highlights a need to operationalize quality caregiving, standardize assessment, and support medically appropriate patients with strained social resources.
The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class a module to mediate receptor activation
Publisher: Elsevier BV
Date: 09-2013
Brain content and plasma concentrations of arginine vasopressin in an Australian marsupial, the brushtail possum Trichosurus vulpecula
Publisher: Elsevier BV
Date: 11-1992
DOI: 10.1016/0016-6480(92)90253-G
Abstract: Arginine vasopressin (AVP) has been identified and quantified in the brain and plasma of the possum using a highly specific radioimmunoassay and high-performance liquid chromatography. Large amounts of AVP were found in the pituitary (16.3 +/- 0.56 micrograms ituitary, n = 5) and hypothalamus (398 +/- 82.5 ng/hypothalamus), and significant amounts of AVP were also present in the cerebral cortex (26.8 +/- 11.5 ng/cortex). Plasma AVP concentrations were significantly lower (2.2 +/- 0.45 pg/ml, n = 10) during anesthesia compared to concentrations while conscious (4.5 +/- 1.19 pg/ml). Severe hemorrhage markedly increased plasma concentrations to 1091 +/- 225 pg/ml (n = 8). It was concluded that AVP is present in the possum brain, pituitary, and plasma, and that its secretion is stimulated by hypovolemia and inhibited by surgical stress.
Functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production
Publisher: Proceedings of the National Academy of Sciences
Date: 25-03-2013
Abstract: Ovarian androgen synthesis is essential for normal ovarian follicle development and female fertility in animals and humans. However, ovarian androgen excess, a feature of the widespread polycystic ovarian syndrome in women, is detrimental to fertility and has other pathophysiological consequences. Our findings reveal the importance of the intraovarian growth factor insulin-like peptide 3 signaling for maintaining androgen production by ovarian theca cells and show that the suppressive action of bone morphogenetic proteins on androgen production is linked to their inhibitory effect on insulin-like peptide 3 signaling, likely mediated via down-regulation of the nuclear transcription factor steroidogenic factor-1.
Regioselective disulfide solid phase synthesis, chemical characterization and in vitro receptor binding activity of equine relaxin
Publisher: Springer Science and Business Media LLC
Date: 19-05-2006
Listeria monocytogenes in Irish Farmhouse cheese processing environments
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.IJFOODMICRO.2010.10.012
Abstract: Sixteen cheesemaking facilities were s led during the production season at monthly intervals over a two-year period. Thirteen facilities were found to have s les positive for Listeria monocytogenes. S les were ided into 4 categories cheese, raw milk, processing environment and external to the processing environment (s les from the farm such as silage, bedding, and pooled water). In order to attempt to identify the source, persistence and putative transfer routes of contamination with the L. monocytogenes isolates, they were differentiated using PFGE and serotyping. Of the 250 isolates, there were 52 different pulsotypes. No pulsotype was found at more than one facility. Two facilities had persistent pulsotypes that were isolated on s ling occasions at least 6 months apart. Of the s les tested, 6.3% of milk, 13.1% of processing environment and 12.3% of s les external to the processing environment, respectively, were positive for L. monocytogenes. Pulsotypes found in raw milk were also found in the processing environment, however, one of the pulsotypes from raw milk was found in cheese on only one occasion. One of the pulsotypes isolated from the environment external to the processing facility was found on the surface of cheese, however, a number of them were found in the processing environment. The results suggest that the farm environment external to the processing environment may in some cases be the source of processing environment contamination with L. monocytogenes.
Membrane receptors: Structure and function of the relaxin family peptide receptors
Publisher: Elsevier BV
Date: 05-2010
DOI: 10.1016/J.MCE.2010.02.003
Abstract: The receptors for members of the relaxin peptide family have only recently been discovered and are G-protein-coupled receptors (GPCRs). Relaxin and insulin-like peptide 3 (INSL3) interact with the leucine-rich-repeat-containing GPCRs (LGRs) LGR7 and LGR8, respectively. These receptors show closest similarity to the glycoprotein hormone receptors and contain large ectodomains with 10 leucine-rich repeats (LRRs) but are unique members of the LGR family (class C) as they have an LDL class A (LDLa) module at their N-terminus. In contrast, relaxin-3 and INSL5 interact with another class of type I GPCRs which lack a large ectodomain, the peptide receptors GPCR135 and GPCR142, respectively. These receptors are now classified as relaxin family peptide (RXFP) receptors, RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135) and RXFP4 (GPCR142). This review outlines the identification of the peptides and receptors, their expression profiles and physiological roles and the functional interactions of the peptides with their unique receptors.
Knockdown of corticotropin-releasing factor 1 receptors in the ventral tegmental area enhances conditioned fear.
Publisher: Elsevier BV
Date: 09-2016
DOI: 10.1016/J.EURONEURO.2016.06.002
Abstract: The neuropeptide corticotropin-releasing factor (CRF) coordinates the physiological and behavioural responses to stress. CRF receptors are highly expressed in the ventral tegmental area (VTA), an important region for motivated behaviour. Therefore, we examined the role of CRF receptor type 1 (CRFR1) in the VTA in conditioned fear, using a viral-mediated RNA interference approach. Following stereotaxic injection of a lentivirus that contained either shCRF-R1 or a control sequence, mice received tone-footshock pairings. Intra-VTA shCRF-R1 did not affect tone-elicited freezing during conditioning. Once conditioned fear was acquired, however, shCRF-R1 mice consistently showed stronger freezing to the tone even after extinction and reinstatement. These results implicate a novel role of VTA CRF-R1 in conditioned fear, and suggest how stress may modulate aversive learning and memory.
Investigations into the inhibitory effects of relaxin on renal myofibroblast differentiation
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2008.03823.X
Abstract: Derived from fibroblasts, myofibroblasts are the principal cells that are responsible for the synthesis and reorganization of excess matrix in renal interstitial fibrosis. Recognized from their de novo expression of alpha-smooth muscle actin, myofibroblast differentiation and activity can be influenced by several factors, including a combination of growth factors and other soluble mediators, extracellular matrix components, and mechanical stress. Relaxin has previously been shown to inhibit renal myofibroblast differentiation in vitro, an effect partly mediated through its ability to interfere with the transforming growth factor-beta1 (TGF-beta1) pathway via inhibition of Smad2 phosphorylation and translocation. Furthermore, endogenous relaxin has been shown to protect the kidney from a myofibroblast-mediated model of injury in vivo. However, the pathways involved in the interaction between relaxin and TGF-beta1 remain unknown. In this report, the inhibitory actions of relaxin on TGF-beta1-induced renal myofibroblast differentiation are summarized to date, and the potential signaling pathways that are implicated in relaxin's inhibitory actions are discussed.
Solution structure, aggregation behavior, and flexibility of human relaxin-2
Publisher: American Chemical Society (ACS)
Date: 15-01-2015
DOI: 10.1021/CB500918V
Abstract: Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function.
Relaxin remodels fibrotic healing following myocardial infarction
Publisher: Elsevier BV
Date: 05-2011
DOI: 10.1038/LABINVEST.2010.198
Abstract: In the setting of myocardial infarction (MI), implanted stem cell viability is low and scar formation limits stem cell homing, viability, and integration. Thus, interventions that favorably remodel fibrotic healing may benefit stem cell therapies. However, it remains unclear whether it is feasible and safe to remodel fibrotic healing post-MI without compromising ventricular remodeling and dysfunction. This study, therefore, determined the anti-fibrotic and other effects of the hormone, relaxin in a mouse model of MI. Adult male mice underwent left coronary artery ligation-induced MI and were immediately treated with recombinant human relaxin (MI+RLX) or vehicle (MI+VEH) over 7 or 30 days, representing time points of early and mature fibrotic healing. Cardiac function was assessed by echocardiography and catheterization, while comprehensive immunohistochemistry, morphometry, and western blotting were performed to explore the relaxin-induced mechanisms of action post-MI. RLX significantly inhibited the MI-induced progression of cardiac fibrosis over 7 and 30 days, which was associated with a reduction in TGF-β1 expression, myofibroblast differentiation, and cardiomyocyte apoptosis in addition to a promotion of matrix metalloproteinase-13 levels and de novo blood vessel growth (all P<0.05 vs respective measurements from MI+VEH mice). Despite the evident fibrotic healing post-MI, relaxin did not adversely affect the incidence of ventricular free-wall rupture or the extent of LV remodeling and dysfunction. These combined findings demonstrate that RLX favorably remodels the process of fibrotic healing post-infarction by lowering the density of mature scar tissue in the infarcted myocardium, border zone, and non-infarcted myocardium, and may, therefore, facilitate cell-based therapies in the setting of ischemic heart disease.
Evidence for a local fetal influence on myometrial oxytocin receptors during pregnancy in the tammar wallaby (Macropus eugenii)
Publisher: Oxford University Press (OUP)
Date: 1997
DOI: 10.1095/BIOLREPROD56.1.200
Abstract: Mesotocin (MT), the oxytocin-like peptide of the tammar wallaby (Macropus eugenii) is important for delivery of live young. The tammar mesotocin receptor (MTR) was first characterized using the iodinated oxytocin receptor antagonist [125I]d(CH2)5 [Tyr(Me)2, Tyr4, Orn8, Tyr-NH(2)9]-vasotocin. MTR concentrations were then measured in matched s les of gravid and nongravid myometrium and median vagina at different stages of the 26-day pregnancy. MTR concentrations in both the gravid and nongravid myometrium changed significantly (ANOVA, p < 0.01) during pregnancy. There was no difference in MTR concentrations between uteri on Days 8-22. From Day 23 of pregnancy, MTR concentrations in the gravid myometrium increased (615.8 +/- 144.0 fmol/mg protein), whereas in the nongravid myometrium, they remained unchanged (248.6 +/- 65.5 fmol/mg protein). Receptor concentrations were high in the gravid myometrium during the last 3 days of pregnancy but decreased significantly in the nongravid myometrium. In the median vagina, MTR concentrations were low compared with myometrial tissues and did not increase at term. Changes in MTR concentrations paralleled changes in uterine responsiveness to exogenous MT in vitro. Our data show that MTR concentrations and the responsiveness to MT differ between the gravid and nongravid myometrium during pregnancy. The increase in MTRs in the gravid myometrium and the decrease in the nongravid suggest that different factors influence these receptors in the separate uteri, independent of systemic influence.
'Relaxin' the stiffened heart and arteries: The therapeutic potential for relaxin in the treatment of cardiovascular disease
Publisher: Elsevier BV
Date: 11-2006
DOI: 10.1016/J.PHARMTHERA.2005.05.012
Abstract: Although originally characterised as a reproductive hormone, relaxin has emerged as a multi-functional endocrine and paracrine factor that plays a number of important roles in several organs, including the normal and diseased cardiovascular system. The recent discovery of the H3/relaxin-3 gene, and the elusive receptors for relaxin (Relaxin family peptide receptor RXFP1) and relaxin-3 (RXFP3/RXFP4) have led to the re-classification of a distinct relaxin peptide/receptor family. Additionally, the identification of relaxin and RXFP1 mRNA and/or relaxin binding sites in the heart and blood vessels has confirmed that the cardiovascular system is a target for relaxin peptides. While evidence for the production of relaxins within the cardiovascular system is limited, several studies have established that the relaxin genes are upregulated in the diseased human and rodent heart where they likely act as cardioprotective agents. The ability of relaxin to protect the heart is most likely mediated via its antifibrotic, anti-hypertrophic, anti-inflammatory and vasodilatory actions, but it may also directly stimulate myocardial regeneration and repair. This review describes relaxin and its primary receptor (RXFP1) in relation to the roles and effects of relaxin in the normal and pathological cardiovascular system. It is becoming increasingly clear that relaxin has a number of erse physiological and pathological roles in the cardiovascular system that may have important therapeutic and clinical implications.
Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis.
Publisher: Elsevier BV
Date: 2018
Expression of LGR7 and LGR8 by neonatal porcine uterine tissues and transmission of milk-borne relaxin into the neonatal circulation by suckling
Publisher: The Endocrine Society
Date: 09-2006
DOI: 10.1210/EN.2006-0397
Abstract: Estrogen receptor-dependent organizational events between birth [postnatal day (PND) 0] and PND 14 affect development and function of porcine uterine tissues. Observations that uterotrophic effects of relaxin (RLX) in neonatal gilts were inhibited by the antiestrogen ICI 182,780 suggested that a RLX signaling system, capable of cross-talk with the estrogen receptor, evolves during a critical period for uterine programming (PND 0-14). Objectives were to determine 1) effects of age and estrogen exposure from birth on porcine uterine RLX/insulin-like 3 receptor (LGR7/LGR8) expression and 2) whether milk serves as a natural source of RLX in neonatal pigs. Uterine LGR7/LGR8 expression, detected by RT-PCR and in situ hybridization on PND 0, 7, and 14, was predominantly stromal for LGR7, myometrial for LGR8, and increased with age and after treatment with estradiol valerate (50 microg/kg body weight x d) from birth. Stromal expression of LGR7 was also detected immunohistochemically. Milk RLX concentrations declined (P < 0.001) from 17.3 +/- 1.4 ng/ml (lactation d 0) to 1.7 +/- 0.3 ng/ml (lactation d 14). RLX, present in the serum of nursing pigs on PND 0 and 1, was undetectable before nursing and in neonates fed RLX-free milk replacer for 12 h. Thus, a developmentally regulated, estrogen-sensitive LGR7 and LGR8 receptor system is present in the porcine uterus at birth and may be activated by milk-borne RLX delivered into the circulation during the first 48 h of postnatal life. Maternal lactocrine contributions to the neonatal hormonal milieu could affect the developmental programming of uterine and other somatic tissues.
Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain
Publisher: Wiley
Date: 2007
DOI: 10.1002/PSC.807
Abstract: Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin-insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G-protein coupled receptor, LGR8. Previous structure-activity studies have shown that the key binding site of INSL3 is situated within its B-chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B-chain, which we have shown to compete with the binding of [33P]-relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP-mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure-activity relationships of cyclic analogues of the INSL3 B-chain, we used a structure-based approach to design a series of cyclic, disulfide-constrained INSL3 B-chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide-constrained mimetics of the INSL3 B-chain. The peptides were synthesized by solid-phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B-chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]-relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000-fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their alpha-helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B-chain mimetics that might better present key receptor binding residues in the native INSL3-like conformation.
Improved chemical synthesis and demonstration of the relaxin receptor binding affinity and biological activity of mouse relaxin
Publisher: American Chemical Society (ACS)
Date: 11-04-2007
DOI: 10.1021/BI700238H
Abstract: The primary stored and circulating form of relaxin in humans, human gene-2 (H2) relaxin, has potent antifibrotic properties with rapidly occurring efficacy. However, when administered to experimental models of fibrosis, H2 relaxin can only be applied over short-term (2-4 week) periods, due to rodents mounting an antibody response to the exogenous human relaxin, resulting in delayed clearance and, hence, increased and variable circulating levels. To overcome this problem, the current study investigated the therapeutic potential of mouse relaxin over long-term exposure in vivo. Mouse relaxin is unique among the known relaxins in that it possesses an extra residue within the C-terminal region of its A-chain. To enable a detailed assessment of its receptor interaction and biological properties, it was chemically synthesized in good overall yield by the separate preparation of each of its A- and B-chains followed by regioselective formation of each of the intramolecular and two intermolecular disulfide bonds. Murine relaxin was shown to bind with high affinity to the human, mouse, and rat RXFP1 (primary relaxin) receptor but with a slightly lower affinity to that of H2 relaxin. When administered to relaxin-deficient mice (which undergo an age-dependent progression of organ fibrosis) over a 4 month treatment period, mouse relaxin was able to significantly inhibit the progression of collagen accumulation in several organs including the lung, kidney, testis, and skin (all p < 0.05 vs untreated group), consistent with the actions of H2 relaxin. These combined data demonstrate that mouse relaxin can effectively inhibit collagen deposition and accumulation (fibrosis) over long-term treatment periods.
Mesotocin in the brain and plasma of an Australian marsupial, the brushtail possum (Trichosurus vulpecula)
Publisher: Elsevier BV
Date: 07-1990
DOI: 10.1016/0143-4179(90)90123-G
Abstract: Oxytocic peptides extracted from the brain and plasma of the brushtail possum, Trichosurus vulpecula, were separated by reverse-phase high pressure lipid chromatography (HPLC) and quantified by specific radioimmunoassays for oxytocin (OT) and mesotocin (MT). The pituitary, hypothalamus and cerebral cortex were found to contain MT only in quantities of 3.9 +/- 0.2 (SE) ug, 17.6 +/- 0.6 ng and 21.0 +/- 2.6 ng respectively. The plasma concentration of MT varied according to the degree of stress of the possum. In anaesthetized animals values of 39.7 +/- 9.7 pg/ml (11 males) and 31.5 +/- 12.9 pg/ml (6 females) were obtained in four conscious catheterized animals, 9.4 +/- 6.3 pg/ml. S les taken from three anaesthetized animals during exsanguination contained 271 +/- 102 (SD) pg MT/ml. It was concluded that hypothalamic and extra-hypothalamic MT is present in the marsupial brain and that as in placental mammals, stress stimulates the secretion of mesotocin.
The anti-fibrotic actions of relaxin are mediated through AT2 R-associated protein phosphatases via RXFP1-AT2 R functional crosstalk in human cardiac myofibroblasts.
Publisher: Wiley
Date: 16-04-2020
International union of pharmacology LVII: Recommendations for the nomenclature of receptors for relaxin family peptides
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 28-02-2006
DOI: 10.1124/PR.58.1.9
Abstract: Although the hormone relaxin was discovered 80 years ago, only in the past 5 years have the receptors for relaxin and three other receptors that respond to related peptides been identified with all four receptors being G-protein-coupled receptors. In this review it is suggested that the receptors for relaxin (LGR7) and those for the related peptides insulin-like peptide 3 (LGR8), relaxin-3 (GPCR135), and insulin-like peptide 5 (LGPCR142) be named the relaxin family peptide receptors 1 through 4 (RXFP1-4). RXFP1 and RXFP2 are leucine-rich repeat-containing G-protein-coupled receptors with complex binding characteristics involving both the large ectodomain and the transmembrane loops. RXFP1 activates adenylate cyclase, protein kinase A, protein kinase C, phosphatidylinositol 3-kinase, and extracellular signaling regulated kinase (Erk1/2) and also interacts with nitric oxide signaling. RXFP2 activates adenylate cyclase in recombinant systems, but physiological responses are sensitive to pertussis toxin. RXFP3 and RXFP4 resemble more conventional peptide liganded receptors and both inhibit adenylate cyclase, and in addition RXFP3 activates Erk1/2 signaling. Physiological studies and examination of the phenotypes of transgenic mice have established that relaxin has roles as a reproductive hormone involved in uterine relaxation (some species), reproductive tissue growth, and collagen remodeling but also in the cardiovascular and renal systems and in the brain. The connective tissue remodeling properties of relaxin acting at RXFP1 receptors have potential for the development of agents effective for the treatment of cardiac and renal fibrosis, asthma, and scleroderma and for orthodontic remodelling. Agents acting at RXFP2 receptors may be useful for the treatment of cryptorchidism and infertility, whereas antagonists may be used as contraceptives. The brain distribution of RXFP3 receptors suggests that actions at these receptors have the potential for the development of antianxiety and antiobesity drugs.
INSL5 activates multiple signalling pathways and regulates GLP-1 secretion in NCI-H716 cells.
Publisher: Bioscientifica
Date: 04-2018
DOI: 10.1530/JME-17-0152
Abstract: Insulin-like peptide 5 (INSL5) is a newly discovered gut hormone expressed in colonic enteroendocrine L-cells but little is known about its biological function. Here, we show using RT-qPCR and in situ hybridisation that Insl5 mRNA is highly expressed in the mouse colonic mucosa, colocalised with proglucagon immunoreactivity. In comparison, mRNA for RXFP4 (the cognate receptor for INSL5) is expressed in various mouse tissues, including the intestinal tract. We show that the human enteroendocrine L-cell model NCI-H716 cell line, and goblet-like colorectal cell lines SW1463 and LS513 endogenously express RXFP4. Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr 202 /Tyr 204 ), AKT (Thr 308 and Ser 473 ) and S6RP (Ser 235/236 ) and inhibited cAMP production but did not stimulate Ca 2+ release. Acute INSL5 treatment had no effect on GLP-1 secretion mediated by carbachol or insulin, but modestly inhibited forskolin-stimulated GLP-1 secretion in NCI-H716 cells. However, chronic INSL5 pre-treatment (18 h) increased basal GLP-1 secretion and prevented the inhibitory effect of acute INSL5 administration. LS513 cells were found to be unresponsive to INSL5 despite expressing RXFP4 . Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of Rxfp4 and were unresponsive to INSL5. This study provides novel insights into possible autocrine aracrine roles of INSL5 in the intestinal tract.
Mapping key regions of the RXFP2 low-density lipoprotein class-A module that are involved in signal activation
Publisher: American Chemical Society (ACS)
Date: 10-07-2014
DOI: 10.1021/BI500797D
Abstract: The peptide hormone INSL3 and its receptor, RXFP2, have co-evolved alongside relaxin and its receptor, RXFP1. Both RXFP1 and RXFP2 are G protein-coupled receptors (GPCRs) containing the hallmark seven transmembrane helices in addition to a distinct ectodomain of leucine-rich repeats (LRRs) and a single low-density lipoprotein class-A (LDLa) module at the N-terminus. RXFP1 and RXFP2 are the only mammalian GPCRs known to contain an LDLa, and its removal does not perturb primary ligand binding to the LRRs however, signaling is abolished. This presents a general mechanism whereby ligand binding induces a conformational change in the receptor to position the LDLa to elicit a signal response. Although the LDLa interaction site has not been identified, the residues important to the action have been mapped within the RXFP1 LDLa module. In this study, we comprehensively study the RXFP2 LDLa module. We determine its structure using nuclear magnetic resonance (NMR) and concurrently investigate the signaling of an RXFP2 with the LDLa removed (RXFP2-short), confirming that the LDLa is essential to signaling. We then replaced the LDLa with the second ligand binding module from the LDL receptor, LB2, creating the RXFP2-LB2 chimera. Unlike that in the equivalent RXFP1-LB2 chimera, signaling is rescued albeit modestly. Guided by the NMR structure, we dissected regions of the RXFP2 LDLa to identify specific residues that are important to signal activation. We determine that although the module is important to the activation of RXFP2, unlike the RXFP1 receptor, specific residues in the N-terminus of the domain are not involved in signal activation.
Reproductive biology of the relaxin-like factor (RLF/INSL3)
Publisher: Oxford University Press (OUP)
Date: 09-2002
DOI: 10.1095/BIOLREPROD.102.005199
Abstract: The relaxin-like factor (RLF), which is the product of the insulin-like factor 3 (INSL3) gene, is a new circulating peptide hormone of the relaxin-insulin family. In male mammals, it is a major secretory product of the testicular Leydig cells, where it appears to be expressed constitutively but in a differentiation-dependent manner. In the adult testis, RLF expression is a good marker for fully differentiated adult-type Leydig cells, but it is only weakly expressed in prepubertal immature Leydig cells or in Leydig cells that have become hypertrophic or transformed. It is also an important product of the fetal Leydig cell population, where it has been demonstrated using knockout mice to be responsible for the second phase of testicular descent acting on the gubernaculum. INSL3 knockout mice are cryptorchid, and in estrogen-induced cryptorchidism, RLF levels in the testis are significantly reduced. RLF is also made in female tissues, particularly in the follicular theca cells of small antral follicles and in the corpus luteum of the cycle and pregnancy. The ruminant ovary has a very high level of RLF expression, and analysis of primary cultures of ovarian theca-lutein cells indicated that, as in the testis, expression is probably constitutive but differentiation dependent. Female INSL3 knockout mice have altered estrous cycles, where RLF may be involved in follicle selection, an idea strongly supported by observations on bovine secondary follicles. Recently, a novel 7-transmembrane domain receptor (LGR8 or Great) has been tentatively identified as the RLF receptor, and its deletion in mice leads also to cryptorchidism.
Familial bilateral cryptorchidism is caused by recessive variants in RXFP2
Publisher: BMJ
Date: 05-06-2019
DOI: 10.1136/JMEDGENET-2019-106203
Abstract: Cryptorchidism or failure of testicular descent is the most common genitourinary birth defect in males. While both the insulin-like peptide 3 (INSL3) and its receptor, relaxin family peptide receptor 2 (RXFP2), have been demonstrated to control testicular descent in mice, their link to human cryptorchidism is weak, with no clear cause–effect demonstrated. To identify the genetic cause of a case of familial cryptorchidism. We recruited a family in which four boys had isolated bilateral cryptorchidism. A fourth-degree consanguineous union in the family was reported. Whole exome sequencing was carried out for the four affected boys and their parents, and variants that segregated with the disorder and had a link to testis development/descent were analysed. Functional analysis of a RXFP2 variant in cell culture included receptor localisation, ligand binding and cyclic AMP (cAMP) pathway activation. Genomic analysis revealed a homozygous missense variant in the RXFP2 gene (c.1496G A .p.Gly499Glu) in all four affected boys and heterozygous in both parents. No other variant with a link to testis biology was found. The RXFP2 variant is rare in genomic databases and predicted to be damaging. It has not been previously reported. Functional analysis demonstrated that the variant protein had poor cell surface expression and failed to bind INSL3 or respond to the ligand with cAMP signalling. This is the first reported genomic analysis of a family with multiple in iduals affected with cryptorchidism. It demonstrates that recessive variants in the RXFP2 gene underlie familial cryptorchidism and solidifies the link between this gene and testicular descent in humans.
A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells
Publisher: MDPI AG
Date: 08-02-2022
DOI: 10.3390/IJMS23031908
Abstract: Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.
Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290.
Publisher: Frontiers Media SA
Date: 17-08-2015
Cooperative binding of insulin-like peptide 3 to a dimeric relaxin family peptide receptor 2
Publisher: The Endocrine Society
Date: 06-12-2008
DOI: 10.1210/EN.2007-0412
Abstract: Insulin-like peptide 3 (INSL3) binds to a G protein-coupled receptor (GPCR) called relaxin family peptide receptor 2 (RXFP2). RXFP2 belongs to the leucine-rich repeat-containing subgroup (LGR) of class A GPCRs. Negative cooperativity has recently been demonstrated in other members of the LGR subgroup. In this work, the kinetics of INSL3 binding to HEK293 cells stably transfected with RXFP2 (HEK293-RXFP2) have been investigated in detail to study whether negative cooperativity occurs and whether this receptor functions as a dimer. Our results show that negative cooperativity is present and that INSL3-RXFP2 binding shows both similarities and differences with insulin binding to the insulin receptor. A dose-response curve for the negative cooperativity of INSL3 binding had a reverse bell shape reminiscent of that seen for the negative cooperativity of insulin binding to its receptor. This suggests that binding of INSL3 may happen in a trans rather than in a cis way in a receptor dimer. Bioluminescence resonance energy transfer (BRET2) experiments confirmed that RXFP2 forms constitutive homodimers. Heterodimerization between RXFP2 and RXFP1 was also observed.
Mesotocin and arginine-vasopressin in the corpus luteum of an Australian marsupial, the brushtail possum (Trichosurus vulpecula)
Publisher: Elsevier BV
Date: 02-1994
Abstract: The neuropeptide hormones arginine-vasopressin (AVP) and oxytocin (OT) have been found in the ovarian follicles and corpora lutea (CL) of many eutherian mammals. In ruminants, there is persuasive evidence that luteal OT is involved in luteolysis via stimulation of uterine prostaglandins. However, based on scant evidence, the marsupial ovary has been viewed as being devoid of OT-like and AVP-like peptides. In this study, corpora lutea from the brushtail possum were examined for OT, AVP, and mesotocin (MT) by a combination of reverse phase HPLC, radioimmunoassay, and immunohistochemistry (IHC). Peptides extracted from each of five CL were separated by HPLC and each fraction was assayed for AVP, MT, and OT. Two immunoreactive peaks were found, corresponding to AVP and MT standards. The amount of each peptide was 8.7 +/- 2.22 pmol MT/g (mean +/- SEM) and 5.7 +/- 1.0 pmol AVP/g, respectively. The mean MT/AVP ratio was 1.55 compared to 0.26 for the pituitary. IHC (streptavidin-peroxidase method) of Bouin's-fixed CL showed staining for MT in the cytoplasm of luteal cells which was absent in stromal tissue and nonluteal ovarian tissue. Not all luteal cells were immunopositive and no topographical distribution of stained cells was observed. IHC localization of AVP was not attempted. It was concluded that the CL of the brushtail possum contains low quantities of MT and AVP, which in the case of MT is probably synthesized by the immunochemically staining cells of the CL.
Prolonged RXFP1 and RXFP2 signaling can be explained by poor internalization and a lack of β-arrestin recruitment
Publisher: American Physiological Society
Date: 05-2009
DOI: 10.1152/AJPCELL.00581.2008
Abstract: Relaxin induces sustained physiological responses, which brings into question the deactivation processes typical of most G protein-coupled receptors (GPCR) for its receptor, relaxin family peptide receptor 1 (RXFP1). Here, we examined relaxin-dependent phosphorylation of RXFP1 and the related insulin-like peptide 3 (INSL3) receptor, RXFP2, as well as the capacity of these receptors to recruit β-arrestins and internalize in response to ligand stimulation. We confirmed in human embryonic kidney (HEK)-293T cells, expressing RXFP1 or RXFP2, that both receptors elicit prolonged cAMP responses up to 6 h after stimulation. Receptors immunoprecipitated from 32 P metabolically labeled cells were used to investigate the agonist-specific phosphorylation. Rapid and robust receptor phosphorylation was not observed for either RXFP1 or RXFP2, although some 32 P-incorporation was observed at 30 min however, this was not statistically significant. In accord with this result, RXFP1 and RXFP2 demonstrated poor internalization in response to relaxin or INSL3, as compared with the angiotensin II type 1 receptor (AT 1 R), which undergoes rapid and robust phosphorylation and internalization in response to angiotensin II. Additionally, coexpression of GPCR kinases has no effect on the rate of internalization for either RXFP1 or RXFP2. Confocal microscopy was used to follow the trafficking of green fluorescent protein-labeled β-arrestins after receptor activation. Neither RXFP1 nor RXFP2 activation results in recruitment of β-arrestins to the cell surface, whereas AT 1 R rapidly recruits both β-arrestins-1 and -2. The apparent lack of classical regulation for RXFP1 and RXFP2 provides the molecular basis for the prolonged signaling and physiological actions of relaxin and related peptides.
RXFP1 couples to the Gαi3-Gβγ-PI3K-PKCζ pathway via the final 10 amino acids of the receptor C-terminal tail
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2008.03813.X
Abstract: The relaxin family peptide receptors RXFP1 and RXFP2 are highly similar receptors that share approximately 80% amino acid sequence homology. Constitutively active receptors couple to increased cAMP accumulation, which is important for relaxin-mediated decidualization and myometrial inhibition. Despite the high homology, the receptors couple to different G-proteins to affect cAMP accumulation. This study aimed to determine the region of RXFP1 that directs coupling to the delayed Galpha(i3) pathway by using receptor mutagenesis. Receptor chimeras suggested that activation of this pathway by RXFP1 was dependent upon the membrane-anchored domain of the receptor. Further receptor mutagenesis showed that activation of the Galpha(i3)-Gbetagamma-PI3K-PKCzeta cAMP pathway by RXFP1 is dependent upon the C-terminal 10 amino acids of the receptor and absolutely requires Arg(752).
Dimerization and negative cooperativity in the relaxin family peptide receptors
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03835.X
Abstract: Peptides of the relaxin family bind to the relaxin family peptide receptors or RXFPs, members of the G-protein-coupled receptor (GPCR) superfamily. For many years, ligand binding to GPCRs was thought to take place as monomeric complexes, ignoring early evidence of negative cooperativity. However, recent research has shown that most GPCRs form constitutive dimers or larger oligomers. The connection between dimerization and negative cooperativity has now been shown for several GPCRs, including the thyroid-stimulating hormone, luteinizing hormone, and follicle-stimulating hormone receptors, which like RXFP1 and -2 belong to the leucine-rich repeat-containing subgroup of class A GPCRs. We recently demonstrated homodimerization and negative cooperativity for RXFP1 and RXFP2 as well as their heterodimerization. Another study showed that RXFP1 has to homodimerize in order to be transported from the endoplasmic reticulum to the cell membrane.
Analogs of insulin-like peptide 3 (INSL3) B-chain are LGR8 antagonists in vitro and in vivo
Publisher: Elsevier BV
Date: 05-2006
Recombinant expression of an insulin-like peptide 3 (INSL3) precursor and its enzymatic conversion to mature human INSL3
Publisher: Wiley
Date: 27-08-2009
DOI: 10.1111/J.1742-4658.2009.07216.X
Abstract: Insulin-like peptide 3 (INSL3), which is primarily expressed in the Leydig cells of the testes, is a member of the insulin superfamily of peptide hormones. One of its primary functions is to initiate and mediate descent of the testes of the male fetus via interaction with its G protein-coupled receptor, RXFP2. Study of the peptide has relied upon chemical synthesis of the separate A- and B-chains and subsequent chain recombination. To establish an alternative approach to the preparation of human INSL3, we designed and recombinantly expressed a single-chain INSL3 precursor in Escherichia coli cells. The precursor was solubilized from the inclusion body, purified almost to homogeneity by immobilized metal-ion affinity chromatography and refolded efficiently in vitro. The refolded precursor was subsequently converted to mature human INSL3 by sequential endoproteinase Lys-C and carboxypeptidase B treatment. CD spectroscopic analysis and peptide mapping showed that the refolded INSL3 possessed an insulin-like fold with the expected disulfide linkages. Recombinant human INSL3 demonstrated full activity in stimulating cAMP activity in RXFP2-expressing cells. Interestingly, the activity of the single-chain precursor was comparable with that of the mature two-chain INSL3, suggesting that the receptor-binding region within the mid- to C-terminal of B-chain is maintained in an active conformation in the precursor. This study not only provides an efficient approach for mature INSL3 preparation, but also resulted in the acquisition of a useful single-chain template for additional structural and functional studies of the peptide.
Distribution of relaxin-3 and RXFP3 within arousal, stress, affective, and cognitive circuits of mouse brain
Publisher: Wiley
Date: 04-08-2010
DOI: 10.1002/CNE.22442
Abstract: Relaxin-3 (RLN3) and its native receptor, relaxin family peptide 3 receptor (RXFP3), constitute a newly identified neuropeptide system enriched in mammalian brain. The distribution of RLN3/RXFP3 networks in rat brain and recent experimental studies suggest a role for this system in modulation of arousal, stress, metabolism, and cognition. In order to facilitate exploration of the biology of RLN3/RXFP3 in complementary murine models, this study mapped the neuroanatomical distribution of the RLN3/RXFP3 system in mouse brain. Adult, male wildtype and RLN3 knock-out (KO)/LacZ knock-in (KI) mice were used to map the central distribution of RLN3 gene expression and RLN3-like immunoreactivity (-LI). The distribution of RXFP3 mRNA and protein was determined using [(35)S]-oligonucleotide probes and a radiolabeled RXFP3-selective agonist ([(125)I]-R3/I5), respectively. High densities of neurons expressing RLN3 mRNA, RLN3-associated beta-galactosidase activity and RLN3-LI were detected in the nucleus incertus (or nucleus O), while smaller populations of positive neurons were observed in the pontine raphé, the periaqueductal gray and a region adjacent to the lateral substantia nigra. RLN3-LI was observed in nerve fibers/terminals in nucleus incertus and broadly throughout the pons, midbrain, hypothalamus, thalamus, septum, hippoc us, and neocortex, but was absent in RLN3 KO/LacZ KI mice. This RLN3 neural network overlapped the regional distribution of RXFP3 mRNA and [(125)I]-R3/I5 binding sites in wildtype and RLN3 KO/LacZ KI mice. These findings provide further evidence for the conserved nature of RLN3/RXFP3 systems in mammalian brain and the ability of RLN3/RXFP3 signaling to modulate "behavioral state" and an array of circuits involved in arousal, stress responses, affective state, and cognition.
Characterization and localization of oxytocin receptors in the rat testis.
Publisher: Bioscientifica
Date: 05-1994
Abstract: In this study oxytocin (OT) receptors have been characterized and localized in the testis of the rat using the radioiodinated OT receptor antagonist 125 I-labelled d(CH 2 ) 5 [Tyr(Me) 2 ,Thr 4 ,Tyr 9 -NH 2 ]-vasotocin (OTA). Receptor density and localization have been compared with the rat testis arginine vasopressin (AVP) receptor using the radioiodinated AVP V 1a receptor antagonist 125 I-labelled d(CH 2 ) 5 Sar 7 -AVP and the radioiodinated linear AVP V 1a antagonist 125 I-labelled [(C 6 H 5 -CH 2 CO)-O-methyl- d -Tyr-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH 2 ]. 125 I-labelled OTA bound with high affinity to membrane fractions of the rat testis ( K a = 13·8 ± 1·25 litres/nmol), mammary tissue ( K a =20·3± 4·36 litres/nmol) and uterus ( K a =27·8±0·74 litres/nmol). Competition studies with various OT and AVP receptor agonists and antagonists confirmed that the binding was to OT receptors. AVP receptors in the testis were found to be identical to AVP V 1a receptors in the liver. The AVP receptor density in the testis was much higher than the OT receptor density (109 ±12·3 vs 5·2 ±0·79 (mean ± s.e.m. ) fmol/mg protein). Autoradiographical localization showed that both OT and AVP receptors were present in the interstitial spaces in the testis consistent with binding to Leydig cells. AVP receptors were also localized on the epithelial surfaces of the seminiferous tubules and on testicular blood vessels. This study has, for the first time, found OT receptors in the testis of the rat which have similar ligand-binding characteristics to mammary and uterine OT receptors. The receptor localizations are consistent with binding to Leydig cells. Journal of Endocrinology (1994) 141, 343–352
Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity
Publisher: Oxford University Press (OUP)
Date: 28-07-2005
Abstract: LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.
Relaxin family peptides and receptors in mammalian brain: Anatomical insights and diverse functional possibilities
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03956.X
Abstract: As a foundation for regulatory and functional studies of central relaxin family peptide receptor systems, we are mapping the distribution of the different receptors in the brain of rat, mouse, and nonhuman primates, attempting to identify the nature of the receptor-positive neurons in key circuits and establish the complementary distribution of the respective ligands in these species. Here we review progress in mapping RXFP1, RXFP2, and RXFP3 (mRNAs and proteins) and their respective ligands and discuss some of the putative functions for these peptides and receptors that are being explored using receptor-selective agonist and antagonist peptides and receptor and peptide gene deletion mouse strains. Comparative studies reveal an association of RXFP1 and RXFP2 with excitatory neurons but a differential regional or cellular distribution, in contrast to the association of RXFP3 with inhibitory neurons. These studies also reveal differences in the distribution of RXFP1 and RXFP2 in rat and mouse brain, whereas the distribution of RXFP3 is more conserved across these species. Enrichment of RXFP1/2/3 in olfactory, cortical, thalamic, limbic, hypothalamic, midbrain, and pontine circuits suggests a erse range of modulatory actions for these receptors. For ex le, experimental evidence in the rat reveals that RXFP1 activation in the amygdala inhibits memory consolidation, RXFP2 activation in striatum produces sniffing behavior, and RXFP3 modulation has effects on feeding and metabolism, the activity of the septohippoc al pathway, and spatial memory. Further studies are now required to reveal additional details of these and other functions linked to relaxin family peptide receptor signaling in mammalian brain and the precise mechanisms involved.
Relaxin family peptides: structure-activity relationship studies.
Publisher: Wiley
Date: 19-01-2017
DOI: 10.1111/BPH.13684
Structural Insights into the Unique Modes of Relaxin-Binding and Tethered-Agonist Mediated Activation of RXFP1 and RXFP2.
Publisher: Elsevier BV
Date: 10-2021
Characterization of the Mouse and Rat Relaxin Receptors
Publisher: Wiley
Date: 05-2005
Abstract: Rodent models have been used for many years to probe the actions of relaxin. Identification of the orthologs of human leucine-rich repeat-containing g-protein-coupled receptor 7 (LGR7), the relaxin receptor, in mouse and rat will enable characterization of the response of LGR7 to relaxin in these species. Partial LGR7 homologous sequences from mouse and rat were discovered in the Celera and NCBI gene databases, lified, cloned, and sequenced. At the protein level, mouse and rat LGR7 are 85.2% and 85.7% identical to human LGR7. Mouse and rat LGR7 were able to bind to and be activated by relaxin ligands.
Elucidation of relaxin-3 binding interactions in the extracellular loops of RXFP3
Publisher: Frontiers Media SA
Date: 2013
Relaxin research in the postgenomic era
Publisher: Wiley
Date: 05-2005
Abstract: Because of the coevolution of ligands and their cognate receptors, analysis of human genomic sequences allows prediction of the pairing of these elements. Initially, we identified a group of five human leucine-rich repeat-containing G-protein-coupled receptor (LGR) genes homologous to LH, FSH, and TSH receptors. Based on common phenotypes of INSL3 null mice and transgenic mice with LGR8 gene deletion, we hypothesized that INSL3, relaxin, and related genes are likely ligands for the paralogous LGR7 and LGR8 genes. Matching the relaxin family peptides with these two orphan LGRs led to the finding that relaxin is capable of activating LGR7 and LGR8 through the Gs pathway. In addition, INSL3 and relaxin 3 were found to be specific ligands for LGR8 and LGR7, respectively. Based on the known production of INLS3 by testicular Leydig cells and ovarian theca cells, we demonstrated the expression of the INSL3 receptor LGR8 in oocytes in ovary and in male germ cells in the testis. Furthermore, we found that LH stimulates INSL3 transcripts in ovarian theca and testicular Leydig cells. INSL3, in turn, binds LGR8 expressed in germ cells to initiate the meiotic progression of arrested oocytes in preovulatory follicles in vitro and in vivo and to suppress male germ cell apoptosis in vivo. INSL3 interacts with germ cells to activate the inhibitory G protein, thus leading to decreases in cAMP production. Our data demonstrate the importance of the INSL3-LGR8 paracrine system in mediating gonadotropic actions in both ovary and testis.
Reversal of cardiac fibrosis and related dysfunction by relaxin experimental findings
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2008.03780.X
Abstract: As a hallmark of heart disease, cardiac fibrosis contributes to the development of heart failure and arrhythmias and forms a key therapeutic target. There is a major unmet need for selective, potent, and safe antifibrotic drugs. Earlier studies revealed a cardiac fibrosis phenotype in relaxin-1-deficient mice. Recent studies in several rodent models of cardiac fibrosis have documented reversal of fibrosis by treatment with relaxin peptide or virally mediated relaxin gene delivery. In mice with surgically induced transmural myocardial infarction, relaxin therapy inhibited scar density. In these studies, however, functional benefits achieved by relaxin therapy were limited or less explored. Collectively, there is good experimental evidence that relaxin is able to reverse cardiac fibrosis due to distinct mechanisms. Future research needs to explore functional improvement following fibrosis reversal by relaxin and the usefulness of relaxin in antiarrhythmic or stem cell-based therapy.
The human LGR7 low-density lipoprotein class A module requires calcium for structure
Publisher: Wiley
Date: 05-2005
Abstract: The relaxin and INSL3 receptors, LGR7 and LGR8, are the only human G-protein-coupled receptors to contain a low-density lipoprotein class-A (LDL-A) module. LDL-A modules are well characterized in a variety of erse biological functions that involve ligand binding to elicit a response. Common features of the LDL-A modules characterized to date are the conservation of three disulfide bonds and the structural arrangement around a bound calcium ion. In this study we recombinantly produce the human LGR7 LDL-A module for NMR studies and demonstrate that calicum is required for the module to form a stable and correctly folded structure.
Synthesis, conformation, receptor binding and biological activities of monobiotinylated human insulin-like peptide 3
Publisher: Wiley
Date: 02-2004
DOI: 10.1111/J.1399-3011.2003.00118.X
Abstract: Biotin-avidin immobilization has been routinely used as a tool to study peptide-receptor and peptide-antibody interactions. Biotinylated peptides can also be employed to localize cells that express the peptides' receptor, and to analyse ligand-receptor binding. Insulin-like peptide 3 (INSL3) is a peptide hormone which contains A- and B-chains connected by two disulphide bonds and plays a role in testicular descent during sexual development. In order to study the interaction of INSL3 with its receptor LGR8, a G protein-coupled receptor, we chemically synthesized Nalpha-mono-biotinylated human INSL3 (B-hINSL3) and compared it structurally and biologically with hINSL3. Both peptides exhibited similar, but high, receptor binding affinities on human foetal kidney fibroblast 293T cells transfected human LGR8 based on a competition radioreceptor assay with 33P-labelled relaxin H2 (B33). The modified B-hINSL3 showed full biological activity as determined by the stimulation of gubernacular cell proliferation. The labelled B-hINSL3 contains a higher alpha-helix content, and this increased helical structure is accompanied by an increase in ability to stimulate cAMP accumulation in 293T cells expressing LGR8. Our results suggest that the N-terminal region of the A-chain is not involved in the interaction of INSL3 with its receptor. However, the introduction of biotin onto the N-terminus of the A-chain promoted conformational stability which, in turn, permitted better receptor activation.
INSL3/Leydig insulin-like peptide activates the LGR8 receptor important in testis descent
Publisher: Elsevier BV
Date: 08-2002
LGR7-truncate is a splice variant of the relaxin receptor LGR7 and is a relaxin antagonist in vitro
Publisher: Wiley
Date: 05-2005
Abstract: The relaxin receptor (LGR7) and the insulin-like peptide 3 (INSL3) receptor (LGR8) are unique LGR family members in possessing a single, functionally important amino terminal LDL-A module.1 Mouse and rat cDNA was screened for LGR7 and LGR7 splice variant expression. A uterus-specific exon 4 deleted variant was identified and named LGR7-Truncate. Deletion of exon 4 results in a premature stop codon and a transcript that putatively encodes a secreted protein containing LGR7's LDL-A module. Expression of LGR7-Truncate with LGR7 in HEK-293T cells resulted in decreased relaxin-induced signaling of LGR7. LGR7-Truncate is potentially an endogenous regulator of LGR7 signaling.
Identification of binding sites with differing affinity and potency for relaxin analogues on LGR7 and LGR8 Receptors
Publisher: Wiley
Date: 05-2005
Abstract: This study defines the pharmacologic characteristics of LGR7 and LGR8, the receptors for H2 relaxin and INSL3 respectively, and determines the relative activity of relaxin-related peptides. We show, for the first time, the availability of two binding sites at LGR8 and confirm the presence of two sites at LGR7. Relaxin-related peptides had differing rank orders of affinity and potency at LGR7 and LGR8, but chimeric receptors were highly similar to their ectodomain-origin native receptors. The high-affinity site on the ectodomain coupled efficiently to cAMP production, whereas the low-affinity site in the transmembrane region coupled with decreased efficiency.
Characterization of the rat INSL3 receptor
Publisher: Wiley
Date: 05-2005
Abstract: Human LGR8, initially discovered as a low-affinity relaxin receptor, has now been characterized as the INSL3 receptor. To investigate LGR8 function in the rat, an LGR8 ortholog was identified in the rat genome, and the full-length sequence was cloned and expressed. Rat LGR8 bound INSL3 with high affinity, clearly demonstrating that it is the rat INSL3 receptor. Interestingly, native rat relaxin did not activate rat LGR8, indicating that relaxin is not an endogenous ligand for rat LGR8. LGR8 mRNA expression was demonstrated in the gubernaculum at the time of testis descent and in the testis associated with germ cells.
The chemistry and biology of human relaxin-3
Publisher: Wiley
Date: 05-2005
Abstract: A novel member of the human relaxin subclass of the insulin superfamily was recently discovered during a genomics database search and named relaxin-3. Like human relaxin-1 and relaxin-2, relaxin-3 is predicted to consist of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken. In contrast to human relaxin-1 and relaxin-2, however, relaxin-3 could not be successfully prepared by simple combination of the in idual chains, thus necessitating recourse to the use of a regioselective disulfide bond formation strategy. Solid phase synthesis of the separate, selectively S-protected A and B chains followed by their purification and the subsequent stepwise formation of each of the three disulfides led to the successful acquisition of human relaxin-3. Comprehensive chemical characterization confirmed both the correct chain orientation and the integrity of the synthetic product. Relaxin-3 was found to bind to and activate native relaxin receptors in vitro and stimulate water drinking through central relaxin receptors in vivo. Recent studies have demonstrated that relaxin-3 will bind to and activate human LGR7, but not LGR8, in vitro. Secondary structural analysis showed it to adopt a less ordered confirmation than either relaxin-1 or relaxin-2, reflecting the presence in the former of a greater percentage of nonhelical forming amino acids. NMR spectroscopy and simulated annealing calculations were used to determine the three-dimensional structure of relaxin-3 and to identify key structural differences between the human relaxins.
Single chain peptide agonists of relaxin receptors
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.MCE.2019.01.008
Abstract: There are seven human relaxin family peptides that have two chains (A and B) and three disulfide bonds. The target receptors for four of these peptides are known as relaxin family peptide receptors, RXFP1-RXFP4. Detailed structure-activity relationship (SAR) studies of relaxin family peptides have been reported over the years and have led to the design of new analogs with agonistic and antagonistic properties. This review briefly summarizes the SAR of human relaxin 2 (H2 relaxin) and human relaxin 3 (H3 relaxin) leading to the design and development of single-B-chain only agonists, B7-33 and peptide 5. The physiological functions of these new peptides agonists in cellular and animal models are also described.
Studies on soluble ectodomain proteins of relaxin (LGR7) and insulin 3 (LGR8) receptors
Publisher: Wiley
Date: 05-2005
Abstract: The ectodomains of both the relaxin (LGR7) and the INSL3 (LGR8) receptors can be expressed on the cell surface using only a single transmembrane domain. These membrane-anchored proteins retain the ability to bind relaxin and can be cleaved from the cell surface. The subsequent LGR7 protein, 7BP, binds relaxin and can act as a functional relaxin antagonist. By contrast, the equivalent LGR8 protein 8BP does not bind relaxin or antagonize LGR8 activity. The 7BP protein has been successfully immobilized onto chemically derivatized surfaces for the capture of relaxin peptides and subsequent identification via SELDI-MS analysis.
Relaxin-like factor gene is highly expressed in the bovine ovary of the cycle and pregnancy: sequence and messenger ribonucleic acid analysis.
Publisher: Oxford University Press (OUP)
Date: 12-1996
DOI: 10.1095/BIOLREPROD55.6.1452
Abstract: Relaxin-like factor (RLF) is a new member of the insulin/insulin-like growth factor family of hormones and growth factors, which appears to be predominantly expressed in the Leydig cells of the testis. An analysis of male and female bovine tissues indicated that in the cow the RLF gene is also highly expressed in the female, mainly in the follicular theca interna and in the corpus luteum, with a pattern of gene expression very similar to that for the related peptide relaxin in other species. Sequence analysis of bovine testicular and luteal RLF and cDNA shows that the same gene is expressed in both male and female gonads. Because the bovine RLF sequence, like those from pigs, humans, and mice, retains, the putative receptor binding motif described for relaxin, it seems plausible that RLF might functionally substitute for relaxin in the cow, in which the latter peptide appears not to be significantly expressed.
Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies.
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.BBAMEM.2014.07.015
Abstract: The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening c aigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS1) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS1 using robotic ligand binding assays. NTS1 is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery.
International union of basic and clinical pharmacology. XCV. Recent advances in the understanding of the pharmacology and biological roles of relaxin family peptide receptors 1–4, the receptors for relaxin family peptides
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 11-03-2015
Chemical synthesis and biological activity of rat INSL3
Publisher: Wiley
Date: 2001
DOI: 10.1002/PSC.344
Abstract: The recently identified protein, insulin 3 (INSL3), has structural features that make it a bona fide member of the insulin superfamily. Its predicted amino acid sequence contains the classic two-peptide chain (A- and B-) structure with conserved cysteine residues that results in a disulphide bond disposition identical to that of insulin. Recently, the generation of insl3 knockout mice has demonstrated that testicular descent is blocked due to the failure of a specific ligament, the gubernaculum, to develop. The mechanism by which INSL3 exerts its action on the gubernaculum is currently unknown. The purpose of this study was to, for the first time, synthesize rat INSL3 and test its action on organ cultures of foetal rat gubernaculum. INSL3 also contains a cassette of residues Arg-X-X-X-Arg within the B-chain, a motif that is essential for characteristic activity of another related member of the superfamily, relaxin. Hence, the relaxin activity of rat INSL3 was also tested in two different relaxin bioassays. The primary structure of rat INSL3 was determined by deduction from its cDNA sequence and successfully prepared by solid phase peptide synthesis of the two constituent chains followed by their combination in solution. Following confirmation of its chemical integrity by a variety of analytical techniques, circular dichroism spectroscopy confirmed the presence of high beta-turn and alpha-helical content, with a remarkable spectral similarity to the synthetic ovine INSL3 peptide and to synthetic rat relaxin. The synthetic rat INSL3 bound with very low affinity to rat relaxin receptors and had no activity in a relaxin bioassay. Furthermore, it did not augment or antagonize relaxin activity. The rat INSL3 did however induce growth of foetal rat gubernaculum in whole organ cultures demonstrating that INSL3 has a direct action on this structure.
Nucleus incertus promotes cortical desynchronization and behavioral arousal.
Publisher: Springer Science and Business Media LLC
Date: 20-05-2016
DOI: 10.1007/S00429-016-1230-0
Abstract: Arousal and vigilance are essential for survival and relevant regulatory neural circuits lie within the brainstem, hypothalamus and forebrain. The nucleus incertus (NI) is a distinct site within the pontine periventricular gray, containing a substantial population of GABAergic neurons with long-range, ascending projections. Existing neuroanatomical data and functional studies in anesthetized rats, suggest the NI is a central component of a midline behavioral control network well positioned to modulate arousal, vigilance and exploratory navigation, yet none of these roles have been established experimentally. We used a chemogenetic approach-clozapine-N-oxide (CNO) activation of virally delivered excitatory hM3Dq-DREADDs-to activate the NI in rats and examined the behavioral and physiological effects, relative to effects in naïve rats and appropriate viral-treated controls. hM3Dq activation by CNO resulted in long-lasting depolarization of NI neurons with action potentials, in vitro. Peripheral injection of CNO significantly increased c-Fos immunoreactivity in the NI and promoted cortical electroencephalograph (EEG) desynchronization. These brain changes were associated with heightened arousal, and increased locomotor activity in the homecage and in a novel environment. Furthermore, NI activation altered responses in a fear conditioning paradigm, reflected by increased head-scanning, vigilant behaviors during conditioned fear recall. These findings provide direct evidence that the NI promotes general arousal via a broad behavioral activation circuit and support early hypotheses, based on its connectivity, that the NI is a modulator of cognition and attention, and emotional and motivated behaviors.
Relaxins enhance growth of spontaneous murine breast cancers as well as metastatic colonization of the brain
Publisher: Springer Science and Business Media LLC
Date: 21-08-2014
Up-regulation of mesotocin receptors in the tammar wallaby myometrium is pregnancy-specific and independent of estrogen.
Publisher: Oxford University Press (OUP)
Date: 05-2002
DOI: 10.1095/BIOLREPROD66.5.1237
Abstract: The oxytocin-like peptide of most Australian marsupials is mesotocin, which stimulates uterine contractions and is important for normal birth in the tammar wallaby. Female marsupials have two uteri and, in monovular species such as the tammar, one uterus is gravid with a single fetus, whereas the contralateral uterus is nongravid. A significant increase in myometrial mesotocin receptor concentrations occurs only in the gravid uterus on Day 23 of the 26-day gestation. This study examined whether or not mesotocin receptors are present in the myometrium and are up-regulated at the equivalent stage of the luteal phase in unmated tammars. In contrast to the marked increase in mesotocin receptor mRNA and protein concentrations in the myometrium of the gravid uterus during pregnancy, receptors did not increase in the unmated animals. There were also no significant differences between the two uteri, except on Day 27. Plasma profiles of peripheral estradiol-17beta and progesterone did not differ significantly between pregnant and nonpregnant cycles. However, progesterone concentrations were significantly lower on Day 1 postpartum compared with Day 27 of the nonpregnant cycle. In pregnant tammars, the molar ratio of circulating estradiol-17beta to progesterone increased significantly between Day 25 of gestation and 1 day postpartum, but was not correlated with an increase in mesotocin receptor concentrations in either uterus. The data confirm that a local fetal influence is more important than systemic factors, such as estrogen, in the regulation of uterine mesotocin receptors in the tammar wallaby.
Design, synthesis and pharmacological evaluation of tricyclic derivatives as selective RXFP4 agonists.
Publisher: Elsevier BV
Date: 05-2021
Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.PEPTIDES.2012.02.021
Abstract: Human gene-2 (H2) relaxin is a member of the insulin-relaxin peptide superfamily. Because of the potential clinical applications of H2 relaxin, there is a need for novel analogs that have improved biological activity and receptor specificity. In this respect, we have chemically assembled chimeric peptides consisting of the B-chain of H2 relaxin in combination with A-chains from other insulin/relaxin family members. The peptides were prepared using solid phase peptide synthesis together with regioselective disulfide bond formation and characterized by RP-HPLC, MALDI-TOF MS and amino acid analysis. Their in vitro activity was assessed in RXFP1 or RXFP2 expressing cells. Replacement of the H2 relaxin A-chain resulted in parallel losses of binding affinity and activity on RXFP1. Not surprisingly H1A-H2B demonstrated the highest activity as the H1 A-chain shares high homology with H2 relaxin whereas INSLA-H2B, which shows low homology, had very poor activity. Importantly A-chain replacements had a dramatic effect on RXFP2 activity similar to previous results demonstrating different modes of activation of A-chain variants on RXFP1 and RXFP2. H3A-H2B is particularly interesting as it displays moderate activity at RXFP1 but poor activity at RXFP2 indicating that it may be a template for specific RXFP1 agonist development. Our study confirms that the activity of H2 relaxin at both RXFP1 and RXFP2 relies on interactions with both the B- and A-chains, and also provide new biochemical insights into the mechanism of relaxin action that the A-chain needs to be in native or near-native form for strong RXFP1 or RXFP2 agonist activity.
Transcriptional regulation of the bovine oxytocin receptor gene
Publisher: Oxford University Press (OUP)
Date: 03-2003
DOI: 10.1095/BIOLREPROD.102.008961
Abstract: The oxytocin receptor (OTR) is expressed in the cow uterus at high levels at estrus and at term of pregnancy. This expression appears to be controlled mostly at the transcriptional level and correlates with increasing estrogen concentration and progesterone withdrawal. Approximately 3200 base pairs of the upstream region of the bovine OTR gene were cloned and analyzed using a combination of bioinformatic, electrophoretic mobility shift (EMSA), and transfection analyses. Using nuclear proteins from high- and low-expressing tissues, EMSA indicated no significant quantitative or qualitative changes in specific DNA-protein binding, suggesting that transcription is probably controlled by signalling systems targeting constitutive factors. Using various cell types, including primary and immortalized ruminant endometrial epithelial cells, as hosts for transfection of promoter-reporter constructs showed that endogenous activity resided only in the longest, i.e., 3.2-kb, construct but not in those shorter than 1.0 kb. While estrogen appears to be important in vivo, no effect of estradiol was found on any construct directly only when the longest 3.2-kb construct was used in combination with some cotransfected steroid receptor cofactors, e.g., SRC1e, was an estradiol-dependent effect observed. A putative interferon-responsive element (IRE) was found at approximately -2,400 from the transcription start site. This element was shown to bind mouse IRF1 and IRF2 as well as similar proteins from bovine endometrial and myometrial nuclear extracts. This element also responded to these factors when cotransfected into various cell types. The bovine equivalents to IRF1 and IRF2 were molecularly cloned from endometrial tissue and shown to be expressed in a temporal fashion, supporting the role of interferon-tau in maternal recognition of pregnancy. Of many factors tested or analyzed, these components of the IFN system are the only ones found to significantly influence the transcription of the bovine OTR gene.
Erratum: Relaxin signaling in reproductive tissues (Molecular and Cellular Endocrinology (2003) 202 (165-175) DOI: 10.1016/S0303-7207(03)00078-9)
Publisher: Elsevier BV
Date: 09-2005
Modern Perspectives on the Structure, Function and Evolution of the Relaxin-Like Peptides and their Receptors
Publisher: Bentham Science Publishers Ltd.
Date: 10-2005
A Novel Ultra-Stable, Monomeric Green Fluorescent Protein For Direct Volumetric Imaging of Whole Organs Using CLARITY.
Publisher: Springer Science and Business Media LLC
Date: 12-01-2018
DOI: 10.1038/S41598-017-18045-Y
Abstract: Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D, generated monomeric usGFP (muGFP). Neurons in whole mouse brains were virally transduced with either EGFP or muGFP and subjected to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/ In situ hybridization-compatible Tissue-hYdrogel (CLARITY) clearing. muGFP fluorescence was retained after CLARITY whereas EGFP fluorescence was highly attenuated, thus demonstrating muGFP is a novel FP suitable for applications where high fluorescence stability and minimal self-association are required.
Probing the functional domains of relaxin-3 and the creation of a selective antagonist for RXFP3/GPCR135 over relaxin receptor RXFP1/LGR7
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2008.03790.X
Abstract: Both relaxin-3 and its receptor (RXFP3, also known as GPCR135) are predominantly expressed in brain regions known to play important roles in processing sensory signals. Recent studies have shown that relaxin-3 is involved in the regulation of stress and feeding behaviors. The mechanisms underlying the involvement of relaxin-3/RXFP3 in the regulation of stress, feeding, and other potential functions remain to be studied. Since relaxin-3 also activates the relaxin receptor (RXFP1, also known as LGR7), which is also expressed in the brain, selective RXFP3 agonists and antagonists are crucial for study of the physiological functions of relaxin-3 and RXFP3 in vivo. The finding that the B chain of relaxin-3 is an agonist for RXFP3 (albeit at low potency) but not RXFP1 suggests that the B chain of relaxin-3 plays a dominant role for RXFP3 binding and activation. Chimeric peptide studies using the B chain from relaxin-3 and the A chains from different members of the insulin and relaxin family have confirmed this hypothesis and led to the generation of R3/I5 (a chimeric peptide with relaxin-3 B chain and INSL5 A chain) as a selective agonist for RXFP3 over RXFP1. Truncation of the C-terminus of the B chain of R3/I5 results in a high-affinity antagonist, R3(BDelta23-27)R/I5, for RXFP3 over RXFP1. R3(BDelta23-27)R/I5 has pA2 values of 9.15 and 9.6 for human and rat RXFP3, respectively, but has no affinity or agonistic activity for the human and rat RXFP1. Ongoing and future in vivo studies using the selective agonist and antagonist for RXFP3 will shed light on the physiological role of the relaxin-3 system.
Relaxin activates multiple cAMP signaling pathway profiles in different target cells
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2008.03814.X
Abstract: Although RXFP1-cAMP signaling in HEK293T cell systems is now relatively well-defined, the signaling pathways activated by relaxin in its target cells and tissues are still unclear. This study aimed to examine the cAMP signaling of RXFP1 in cells that endogenously express the receptor. Seven cell types derived from various backgrounds were screened for receptor expression. Only in THP-1 cells and rat cardiac fibroblasts was there activation of the Galpha(i3)-Gbetagamma-phosphatidylinositol 3-kinase-protein kinase Czeta pathway, leading to cAMP accumulation. In all other cells there was activation of a combination of the initial pathways to affect cAMP. T-47D cells could activate only Galpha(s), whereas Colo 16 and rat renal fibroblasts from obstructed kidney could activate both Galpha(s) and Galpha(oB) pathways. Thus, the signaling pathways activated by relaxin are highly dependent upon the cell type under investigation, and this may help to explain the varied physiological responses exerted by relaxin in its different target tissues.
Receptors for relaxin family peptides
Publisher: Wiley
Date: 05-2005
Abstract: Recent studies have identified four receptors that are the physiological targets for relaxin family peptides. All are class I (rhodopsin like) G-protein-coupled receptors with LGR7 (RXFP1) and LGR8 (RXFP2) being type C leucine-rich repeat-containing receptors, whereas GPCR135 (RXFP3) and GPCR142 (RXFP4) resemble receptors that respond to small peptides such as somatostatin and angiotensin II. The cognate ligands for the receptors have been identified: relaxin for RXFP1 INSL3 for RXFP2 relaxin 3 for RXFP3 and INSL5 for RXFP4. RXFP1 and RXFP2 receptors produce increases in intracellular cAMP levels upon stimulation, although the response is complex and contains a component sensitive to PI-3-kinase inhibitors. There is also evidence that RXFP1 can activate Erk1/2 and nitric oxide synthase, and relaxin has been reported to enter cells and activate glucocorticoid receptors. In contrast, RXFP3 and RXFP4 couple to Gi by a pertussis toxin-sensitive mechanism to cause inhibition of cAMP production. Now that the receptors for relaxin family peptides and their cognate ligands have been identified, we suggest a nomenclature for both the peptides and the receptors that we hope will be helpful to researchers in this rapidly advancing field.
Relaxin family peptide receptors in GtoPdb v.2023.1
Publisher: Edinburgh University Library
Date: 26-04-2023
DOI: 10.2218/GTOPDB/F60/2023.1
Abstract: Relaxin family peptide receptors (RXFP, nomenclature as agreed by the NC-IUPHAR Subcommittee on Relaxin family peptide receptors [23, 119]) may be ided into two pairs, RXFP1/2 and RXFP3/4. Endogenous agonists at these receptors are heterodimeric peptide hormones structurally related to insulin: relaxin-1, relaxin, relaxin-3 (also known as INSL7), insulin-like peptide 3 (INSL3) and INSL5. Species homologues of relaxin have distinct pharmacology and relaxin interacts with RXFP1, RXFP2 and RXFP3, whereas mouse and rat relaxin selectively bind to and activate RXFP1 [260]. relaxin-3 is the ligand for RXFP3 but it also binds to RXFP1 and RXFP4 and has differential affinity for RXFP2 between species [259]. INSL5 is the ligand for RXFP4 but is a weak antagonist of RXFP3. relaxin and INSL3 have multiple complex binding interactions with RXFP1 [267] and RXFP2 [132] which direct the N-terminal LDLa modules of the receptors together with a linker domain to act as a tethered ligand to direct receptor signaling [262]. INSL5 and relaxin-3 interact with their receptors using distinct residues in their B-chains for binding, and activation, respectively [321, 152].
Relaxin modulates cardiac fibroblast proliferation, differentiation, and collagen production and reverses cardiac fibrosis in vivo
Publisher: The Endocrine Society
Date: 09-2004
DOI: 10.1210/EN.2004-0209
Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone
Publisher: Elsevier BV
Date: 04-2012
DOI: 10.1016/J.BBRC.2012.02.141
Abstract: Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide.
Defining the LGR8 residues involved in binding insulin-like peptide 3.
Publisher: The Endocrine Society
Date: 07-2007
DOI: 10.1210/ME.2007-0097
Abstract: The peptide hormone insulin-like peptide 3 (INSL3) is essential for testicular descent and has been implicated in the control of adult fertility in both sexes. The human INSL3 receptor leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8) binds INSL3 and relaxin with high affinity, whereas the relaxin receptor LGR7 only binds relaxin. LGR7 and LGR8 bind their ligands within the 10 leucine-rich repeats (LRRs) that comprise the majority of their ectodomains. To define the primary INSL3 binding site in LGR8, its LRRs were first modeled on the crystal structure of the Nogo receptor (NgR) and the most likely binding surface identified. Multiple sequence alignment of this surface revealed the presence of seven of the nine residues implicated in relaxin binding to LGR7. Replacement of these residues with alanine caused reduced [125I]INSL3 binding, and a specific peptide/receptor interaction point was revealed using competition binding assays with mutant INSL3 peptides. This point was used to crudely dock the solution structure of INSL3 onto the LRR model of LGR8, allowing the prediction of the INSL3 Trp-B27 binding site. This prediction was then validated using mutant INSL3 peptide competition binding assays on LGR8 mutants. Our results indicated that LGR8 Asp-227 was crucial for binding INSL3 Arg-B16, whereas LGR8 Phe-131 and Gln-133 were involved in INSL3 Trp-B27 binding. From these two defined interactions, we predicted the complete INSL3/LGR8 primary binding site, including interactions between INSL3 His-B12 and LGR8 Trp-177, INSL3 Val-B19 and LGR8 Ile-179, and INSL3 Arg-B20 with LGR8 Asp-181 and Glu-229.
The relaxin peptide family and their novel G-protein coupled receptors
Publisher: Springer Science and Business Media LLC
Date: 09-2003
DOI: 10.1007/BF02442569
Insulin 3: From chemical synthesis to biological function
Publisher: Springer Science and Business Media LLC
Date: 09-2003
DOI: 10.1007/BF02442568
Synthesis and pharmacological characterization of a europium-labelled single-chain antagonist for binding studies of the relaxin-3 receptor RXFP3
Publisher: Springer Science and Business Media LLC
Date: 20-03-2015
DOI: 10.1007/S00726-015-1961-X
Abstract: Relaxin-3 and its endogenous receptor RXFP3 are involved in fundamental neurological signalling pathways, such as learning and memory, stress, feeding and addictive behaviour. Consequently, this signalling system has emerged as an attractive drug target. Development of leads targeting RXFP3 relies on assays for screening and ligand optimization. Here, we present the synthesis and in vitro characterization of a fluorescent europium-labelled antagonist of RXFP3. This ligand represents a cheap and safe but powerful tool for future mechanistic and cell-based receptor-ligand interaction studies of the RXFP3 receptor.
Multiple binding sites revealed by interaction of relaxin family peptides with native and chimeric relaxin family peptide receptors 1 and 2 (LGR7 and LGR8)
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 12-01-2005
Abstract: Relaxin family peptide 1 (RXFP1) receptor (LGR7) and RXFP2 receptor (LGR8) were recently identified as the receptor targets for H2 relaxin and insulin-like peptide 3 (INSL3), respectively. In this study, we define the pharmacology of these two receptors by using a number of receptor chimeras and relaxin family peptides. We have identified two binding sites on these receptors: one primary, high-affinity site within the ectodomain and a secondary, lower affinity site within the transmembrane region. The primary site was found to dictate receptor binding characteristics, although the lower affinity site also exerts some influence and modulates ligand affinity for the primary site in a manner dependent upon the peptide in question. Not all relaxin peptides were able to bind to the RXFP2 receptor, indicating that the relaxin-RXFP2 receptor interaction is species-specific. INSL3 was found to exhibit characteristics of a partial agonist at the RXFP2 and chimeric RXFP1/2 receptors, with low maximal cAMP responses but high potency in coupling to this pathway. cAMP accumulation studies also revealed that the binding sites couple to cAMP signaling pathways with differing efficiency: the high-affinity site signals with high efficiency, whereas the lower affinity site signals with little to no efficiency. Comparisons between RXFP1, RXFP2, the chimeric receptors, and the truncated receptors revealed that the interaction between receptor sites is critical for optimal ligand binding and signal transduction and that the ectodomain is essential for signaling. Evidence obtained in this study supports a two-stage binding model of receptor activation: binding to the primary site allows a conformational change and interaction with the low-affinity transmembrane site.
Ligand recognition mechanism of the human relaxin family peptide receptor 4 (RXFP4)
Publisher: Springer Science and Business Media LLC
Date: 30-01-2023
DOI: 10.1038/S41467-023-36182-Z
Abstract: Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs). Here, we report three cryo-electron microscopy structures of RXFP4–G i protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.
Further Developments towards a Minimal Potent Derivative of Human Relaxin-2
Publisher: MDPI AG
Date: 11-08-2023
Abstract: Human relaxin-2 (H2 relaxin) is a peptide hormone with potent vasodilatory and anti-fibrotic effects, which is of interest for the treatment of heart failure and fibrosis. H2 relaxin binds to the Relaxin Family Peptide Receptor 1 (RXFP1). Native H2 relaxin is a two-chain, three-disulfide-bond-containing peptide, which is unstable in human serum and difficult to synthesize efficiently. In 2016, our group developed B7-33, a single-chain peptide derived from the B-chain of H2 relaxin. B7-33 demonstrated poor affinity and potency in HEK cells overexpressing RXFP1 however, it displayed equivalent potency to H2 relaxin in fibroblasts natively expressing RXFP1, where it also demonstrated the anti-fibrotic effects of the native hormone. B7-33 reversed organ fibrosis in numerous pre-clinical animal studies. Here, we detail our efforts towards a minimal H2 relaxin scaffold and attempts to improve scaffold activity through Aib substitution and hydrocarbon stapling to re-create the peptide helicity present in the native H2 relaxin.
Human Insulin-like Peptide 5 (INSL5). Identification of a Simplified Version of Two-Chain Analog A13
Publisher: American Chemical Society (ACS)
Date: 19-10-2020
Relaxin down-regulates renal fibroblast function and promotes matrix remodelling in vitro
Publisher: Oxford University Press (OUP)
Date: 06-02-2004
DOI: 10.1093/NDT/GFG598
Abstract: Renal fibroblasts are important effector cells in tubulointerstitial fibrosis, with experimental antifibrotic strategies focusing on the functional down-regulation of these cells. Several experimental models of fibrosis have provided evidence for the effectiveness of the polypeptide hormone relaxin as a potential antifibrotic agent. This study was conducted to further elucidate the antifibrotic mechanisms of relaxin on renal fibroblasts in vitro. Rat cortical fibroblasts were obtained from outgrowth culture of renal tissue isolated from kidneys 3 days post-unilateral ureteric obstruction and constituted 100% of cells studied. A relaxin radio-receptor assay was used to establish binding of relaxin to renal fibroblasts in vitro. Functional studies then examined the effects of H2 relaxin (0, 1, 10 and 100 ng/ml) on fibroblast kinetics, expression of alpha-smooth muscle actin (alpha-SMA), total collagen synthesis, collagenase production and collagen-I lattice contraction. CTGF mRNA expression was also measured by northern analysis. H2 relaxin bound with high affinity to rat renal fibroblasts, but receptor numbers were low. Consistent with its previously reported bimodal effect, transforming growth factor (TGF-beta 1) reduced fibroblast proliferation, an effect abrogated by H2 relaxin. Fibroblasts exposed to H2 relaxin (100 ng/ml) for 24 h demonstrated decreased immunostaining for alpha-SMA and reduced alpha-SMA protein expression compared with controls. There was a trend for a relaxin-mediated reduction in total collagen synthesis and alpha 1(I) mRNA expression with large dose-related increases in collagenase protein expression being observed. TGF-beta 1-stimulated collagen-I lattice contraction was significantly inhibited following co-incubation with 100 ng/ml relaxin. Incremental doses of H2 relaxin had no significant effect on CTGF mRNA expression. The findings of this study suggest that the antifibrotic effects of relaxin involve down-regulation of fibroblast activity, increase in collagenase synthesis and restructuring of collagen-I lattices, which are consistent with its known physiological role of matrix remodelling. Although there appears to be an interaction between TGF-beta 1 and H2 relaxin, this does not appear to involve a reduction in CTGF mRNA expression.
The NMR solution structure of the relaxin (RXFP1) receptor lipoprotein receptor class A module and identification of key residues in the N-terminal region of the module that mediate receptor activation
Publisher: Elsevier BV
Date: 02-2007
Relaxin receptors - New drug targets for multiple disease states
Publisher: Bentham Science Publishers Ltd.
Date: 2007
DOI: 10.2174/138945007779315650
Abstract: Relaxin was discovered more than 75 years prior to the identification of the receptors that mediate its actions. There has been a slow emergence in understanding the role of relaxin, with it being denoted initially as a hormone of pregnancy due to its observed effects to relax pubic ligaments and soften the cervix of guinea pigs to facilitate parturition. However, many other physiological roles have been identified for relaxin, including cardiovascular and neuropeptide functions and an ability to induce the matrix metalloproteinases, so it is clear that relaxin is not exclusively a hormone of pregnancy but has a much wider role in vivo. The recent de-orphanisation of four receptors LGR7, LGR8, GPCR135 (SALPR) and GPCR142 (GPR100) that respond to and bind at least one of the three forms of relaxin identified to date, allows dissection of this system to determine the precise role of each receptor and enable the identification of new targets for treatment of numerous disease states. Relaxin has the potential to be useful for the treatment of scleroderma, fibrosis, in orthodontics and to facilitate embryo implantation in humans. Relaxin antagonists may act as contraceptives or prevent the development of breast cancer metastases. Recent research has added considerable knowledge to the signalling pathways activated by relaxin, which will aid our understanding of how relaxin produces its effects. The focus of this review is to bring together recent developments in the relaxin receptor field and to highlight their potential as drug targets.
Structure and activity in the relaxin family of peptides.
Publisher: Wiley
Date: 04-2009
DOI: 10.1111/J.1749-6632.2009.03955.X
Abstract: The availability of improved peptide synthesis procedures, convenient and sensitive assays for receptor binding and activation, together with advances in methods for structural characterization, has enabled the key structural features of the relaxin family of peptides responsible for biological activity to be defined. Not surprisingly, despite the similarities in primary amino acid sequences, different structural domains and residues are involved in the binding and activation at the four known relaxin family peptide receptors (RXFP1 to -4). Most of our knowledge on structure and function relates to the relaxin-RXFP1, insulin-like peptide 3 (INSL3)-RXFP2, and relaxin-3-RXFP3 systems, with information accumulating not only on the critical ligand structures but also the domains and residues on the receptor itself that are required for specificity and activation. These studies provide the framework for the design of small-molecule mimetics. While the B-chain cassette R-X-X-X-R-X-X-I, defined by Büllesbach and Schwabe, is essential for binding and activation of RXFP1, it is now recognized that the A chain, particularly the N-terminal domain, is also critical for receptor specificity. Studies of the various endogenous ligand-receptor pairs have led to the design of potent and specific agonists and antagonists. The relaxin-3 A chain-INSL5 B chain chimeric peptide and analogs with C-terminal truncations of the B chain, developed by Liu and colleagues at Johnson & Johnson, have provided selective agonist and antagonist peptides that are proving invaluable for in vivo studies of the relaxin-3-RXFP3 system.
Cardiovascular effects of relaxin: From basic science to clinical therapy
Publisher: Springer Science and Business Media LLC
Date: 24-11-2010
DOI: 10.1038/NRCARDIO.2009.198
Abstract: Although substantial advances have been achieved in recent decades in the clinical management of heart diseases, new therapies that provide better or additional efficacy with minimal adverse effects are urgently required. Evidence that has accumulated since the 1990s indicates that the peptide hormone relaxin has multiple beneficial actions in the cardiovascular system under pathological conditions and, therefore, holds promise as a novel therapeutic intervention. Clinical trials for heart failure therapy using relaxin revealed several beneficial actions. Here we review findings from mechanistic and applied research in this field, comment on the outcomes of recent phase I/II clinical trails on patients with heart failure, and highlight settings of cardiovascular diseases where relaxin might be effective.
Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene: Novel members of the relaxin peptide family
Publisher: Elsevier BV
Date: 2002
Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET‐based biosensor
Publisher: Wiley
Date: 24-09-2018
DOI: 10.1002/PRP2.432
The relaxin gene-knockout mouse: A model of progressive fibrosis
Publisher: Wiley
Date: 05-2005
Abstract: Relaxin is well known for its actions on collagen remodeling. To improve our understanding of the physiologic role(s) of relaxin, the relaxin gene-knockout (RLX-KO) mouse was established by our group and subsequently phenotyped. Pregnant RLX-KO mice underwent inadequate development of the pubic symphysis as well as the mammary glands and nipples compared to wild-type mice, thus preventing lactation. Later studies showed that these deficiencies were associated with increased collagen, primarily in the nipple and vagina. Analysis of male RLX-KO mice also demonstrated inadequate reproductive tract development. The testis, epididymis, and prostate of RLX-KO mice showed delayed tissue maturation and growth associated with increased collagen deposition. In nonreproductive tissues, an age-related increase in interstitial collagen (fibrosis) was also detected in the lung, heart, and kidneys of RLX-KO mice and was associated with organ dysfunction. From 6-9 months of age and onwards, all organs of RLX-KO mice, particularly male mice, underwent progressive increases in tissue weight and collagen content (all P < .05) compared with wild-type animals. The increased fibrosis contributed to bronchiole epithelium thickening and alveolar congestion (lung), atrial hypertrophy and increased ventricular chamber stiffness (heart) in addition to glomerulosclerosis (kidney). Treatment of RLX-KO mice with recombinant human relaxin in early and developed stages of fibrosis caused the reversal of collagen deposition in the lung, heart, and kidneys. Together, these findings suggest that relaxin is a naturally occurring inhibitor of collagen deposition during normal development, aging, and pregnancy and can be used to prevent the progression of fibrosis.
Mammalian mesotocin: cDNA sequence and expression of an oxytocin-like gene in a macropodid marsupial, the tammar wallaby
Publisher: Elsevier BV
Date: 05-2000
Abstract: The oxytocin (OT)-like peptide of most Australian marsupials is mesotocin (MT), which differs from OT by substitution of isoleucine for leucine at position 8. To date, the only information on the evolution of the OT peptide in marsupials is based on the sequence of the 9-amino acid peptide itself. The main objective of this study was to obtain the nucleotide and derived amino acid sequences of a marsupial MT precursor for comparison with known OT and MT precursors of eutherians and nonmammalian vertebrates. The structural organization and sequence of the MT gene and its specific transcript were established in a macropodid marsupial, the tammar wallaby, using PCR strategies with a combination of genomic DNA and reverse-transcribed hypothalamic RNA. A consensus genomic sequence of 1221 bp was produced which, by comparison with the expressed cDNA sequence, included two intron sequences of 480 and 188 bp. The tammar MT precursor molecule consists of a 32-amino acid signal peptide, followed by the MT-encoding region and the Gly-Lys-Arg carboxy-terminal cleavage and amidation signal which separates the nonapeptide from the 92-amino acid neurophysin. At the amino acid level, the MT precursor is more similar to eutherian OT precursors than to nonmammalian MT, isotocin, or vasotocin precursors. Northern analysis demonstrated a single transcript of approximately 0.6 kB in the hypothalamus. Mesotocin mRNA is also present in several tissues of the reproductive tract, including the corpus luteum, follicle, uterus, and placenta. Within the ovary, MT transcripts are localized predominantly in the granulosa cells of antral follicles with some positive hybridization signals in cells of the theca interna. This pattern of MT gene expression in marsupials is very similar to that of OT in eutherians and suggests a conserved physiology in the mammalian ovary.
Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain
Publisher: Wiley
Date: 09-2002
DOI: 10.1046/J.1471-4159.2002.01114.X
Abstract: Relaxin is a peptide hormone with known actions associated with female reproductive physiology, but it has also been identified in the brain. Only one relaxin gene had been characterized in rodents until recently when a novel human relaxin gene, human gene-3 (H3) and its mouse equivalent (M3) were identified. The current study reports the identification of a rat homologue, rat gene-3 (R3) relaxin that is highly expressed in a discrete region of the adult brain. The full R3 relaxin cDNA was generated using RT-PCR and 3' and 5' RACE protocols. The derived amino acid sequence of R3 relaxin retains all the characteristic features of a relaxin peptide and has a high degree of homology with H3 and M3 relaxin. The distribution of R3 relaxin mRNA in adult rat brain was determined and highly abundant expression was only detected in neurons of the ventromedial dorsal tegmental nucleus (vmDTg) in the pons, whereas all other brain areas were unlabelled or contained much lower mRNA levels. Relaxin binding sites and relaxin immunoreactivity were also detected in the vmDTg. These together with earlier findings provide strong evidence for a role(s) for multiple relaxin peptides as neurotransmitters and/or modulators in the rat CNS.
Effect of helix-promoting strategies on the biological activity of novel analogues of the B-chain of INSL3
Publisher: Springer Science and Business Media LLC
Date: 07-12-2010
DOI: 10.1007/S00726-008-0219-2
Abstract: Insulin-like 3 (INSL3) is a novel circulating peptide hormone that is produced by testicular Leydig cells and ovarian thecal and luteal cells. In males, INSL3 is responsible for testicular descent during foetal life and suppresses germ cell apoptosis in adult males, whereas in females, it causes oocyte maturation. Antagonists of INSL3 thus have significant potential clinical application as contraceptives in both males and females. Previous work has shown that the INSL3 receptor binding region is largely confined to the B-chain central alpha-helix of the hormone and a conformationally constrained analogue of this has modest receptor binding and INSL3 antagonist activity. In the present study, we have employed and evaluated several approaches for increasing the alpha-helicity of this peptide in order to better present the key receptor binding residues and increase its affinity for the receptor. Analogues of INSL3 with higher alpha-helicity generally had higher receptor binding affinity although other structural considerations limit their effectiveness.
Relaxin family peptide receptors RXFP1 and RXFP2 modulate cAMP signaling by distinct mechanisms
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 28-03-2006
Abstract: Two orphan leucine-rich repeat-containing G protein-coupled receptors were recently identified as targets for the relaxin family peptides relaxin and insulin-like peptide (INSL) 3. Human gene 2 relaxin is the cognate ligand for relaxin family peptide receptor (RXFP) 1, whereas INSL3 is the ligand for RXFP2. Constitutively active mutants of both receptors when expressed in human embryonic kidney (HEK) 293T cells signal through Galphas to increase cAMP. However, recent studies using cells that endogenously express the receptors revealed greater complexity: cAMP accumulation after activation of RXFP1 involves a time-dependent biphasic pathway with a delayed phase involving phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) zeta, whereas the RXFP2 response involves inhibition of adenylate cyclase via pertussis toxin-sensitive G proteins. The aim of this study was to compare and contrast the cAMP signaling pathways used by these two related receptors. In HEK293T cells stably transfected with RXFP1, preliminary studies confirmed the biphasic cAMP response, with an initial Galphas component and a delayed response involving PI3K and PKCzeta. This delayed pathway was dependent upon G-betagamma subunits derived from Galphai3. An additional inhibitory pathway involving GalphaoB affecting cAMP accumulation was also identified. In HEK293T cells stably transfected with RXFP2, the cAMP response involved Galphas and was modulated by inhibition mediated by GalphaoB and release of inhibitory G-betagamma subunits. Thus, initially both RXFP1 and RXFP2 couple to Galphas and an inhibitory GalphaoB pathway. Differences in cAMP accumulation stem from the ability of RXFP1 to recruit coupling to Galphai3, release G-betagamma subunits and thus activate a delayed PI3K-PKCzeta pathway to further increase cAMP accumulation.
Synthetic covalently linked dimeric form of h2 relaxin retains native RXFP1 activity and has improved in vitro serum stability
Publisher: Hindawi Limited
Date: 2015
DOI: 10.1155/2015/731852
Abstract: Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG 7 linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin.
Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin.
Publisher: Springer Science and Business Media LLC
Date: 12-06-2017
DOI: 10.1038/S41598-017-03638-4
Abstract: Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker’s highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate the RXFP2 transmembrane domains in conjunction with the INSL3 A-chain. In contrast, relaxin activation of RXFP2 involves a more RXFP1-like mechanism involving binding to the LDLa-linker, reorientation of the LDLa module and activation of the transmembrane domains by the LDLa alone.
A single-chain derivative of the relaxin hormone is a functionally selective agonist of the G protein-coupled receptor, RXFP1.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C5SC04754D
Abstract: A single-chain derivative of the relaxin hormone ameliorates fibrosis without side-effects.
The minimal active structure of human relaxin-2
Publisher: Elsevier BV
Date: 10-2011
R3(BΔ23-27)R/I5 chimeric peptide, a selective antagonist for GPCR135 and GPCR142 over relaxin receptor LGR7: In vitro and in vivo characterization
Publisher: Elsevier BV
Date: 08-2007
Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies
Publisher: Springer Science and Business Media LLC
Date: 21-12-2012
DOI: 10.1007/S00726-011-1164-Z
Abstract: Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various natural or designed ligands with these receptors. Using this site-specific labeling strategy, other functional probes, such as fluorescent dyes, biotin, or nanoparticles could also be introduced to the A-chain N-terminal of the recombinant human relaxin-3. Additionally, we improved the time-resolved fluorescence assay for the DOTA-bound europium ion which paves the way for the use of DOTA as a lanthanide chelator for protein and peptide labeling in future studies.
A simple approach for the preparation of mature human relaxin-3
Publisher: Elsevier BV
Date: 11-2010
DOI: 10.1016/J.PEPTIDES.2010.07.022
Abstract: Relaxin-3 (also known as INSL7) is the most recently identified member of the insulin-like family. It is predominantly expressed in the nucleus incertus of the brain and involved in the control of stress response, food intake, and reproduction. In the present work, we have established a simple approach for the preparation of the mature human relaxin-3 peptide. We first designed and recombinantly expressed a single-chain relaxin-3 precursor in E. coli cells. After purification by immobilized metal ion affinity chromatography, refolding in vitro through disulfide reshuffling, and digestion by endoproteinase Asp-N, mature human relaxin-3 was obtained in high yield and at low cost. Peptide mapping and circular dichroism spectroscopy studies suggested that the recombinant relaxin-3 adopted an insulin-like fold with the expected disulfide linkages. The recombinant mature relaxin-3 was fully active in both RXFP3 binding and activation assays. The activity of the single-chain precursor was very low, suggesting that a free C-terminus of the B-chain is necessary for receptor-binding and activation of relaxin-3. Our present work provides a highly efficient approach for the preparation of relaxin-3 as well as its analogues for functional and structural analyses.
Localization of LGR7 (relaxin receptor) mRNA and protein in rat forebrain: Correlation with relaxin binding site distribution
Publisher: Wiley
Date: 05-2005
Abstract: Discrete neuronal populations in brain express relaxin and relaxin-3, and molecular studies have identified former-orphan, G-protein-coupled receptors LGR7 and GPCR135 as their native receptors. To better understand the role of central relaxin systems, we began to assess the anatomic distribution of these receptors and ligands in brain. This study documents the widespread distribution of LGR7 mRNA and LGR7-like immunoreactivity (LI) throughout adult rat forebrain areas shown to contain specific [33P]-relaxin binding sites. High densities of LGR7 mRNA hybridization were detected in deep layers of neocortex, hypothalamic paraventricular and supraoptic nuclei and within hippoc al subiculum and CA3, the basolateral amygdala and subfornical organ. Low to moderate hybridization was detected in septum, midline thalamic nuclei, arcuate and supramammillary nuclei, and regions of the midbrain pons. Complementary expression of LGR7-LI was observed in cortical pyramidal neurons, hypothalamic magnocellular neurons, and hippoc al pyramidal and interneurons. These findings provide further evidence for actions of relaxin as a modulator in somatosensory, autonomic, and neuroendocrine pathways.
Localization of LGR7 gene expression in adult mouse brain using LGR7 knock-out/LacZ knock-in mice: Correlation with LGR7 mRNA distribution
Publisher: Wiley
Date: 05-2005
Abstract: Knowledge of the distribution of the relaxin receptor, LGR7, in the brain provides a basis for studies of the physiologic actions of relaxin. LGR7 knock-out (KO) mice were produced by the in-frame replacement of LGR7 exon 10 and 11 with a LacZ-reporter cassette (knock-in [KI]), and in this study we used LGR7-KO/LacZ-KI mice to determine the regional/cellular distribution of LGR7 gene expression in adult mouse brain by assessing beta-galactosidase activity in perfusion-fixed sections. High densities of beta-galactosidase-positive neurons were detected in anterior olfactory and claustrum/endopiriform nuclei, deep layers of cortex (particularly somatosensory), and the subiculum. Low to moderate densities were detected in olfactory bulb (periglomerular layer), cingulate cortex, subfornical organ, hippoc al CA2/dentate hilus, amygdala, hypothalamus, and thalamus. This LGR7/LacZ expression appears to recapitulate that of native LGR7 in wild-type mice and provides a model to further investigate the phenotype of LGR7-responsive neurons in the brain and to help reveal functions associated with central relaxin signaling.
The role of N-terminal glycosylation in the human oxytocin receptor.
Publisher: Oxford University Press (OUP)
Date: 11-1997
Abstract: The human oxytocin receptor includes three N-glycosylation sites in its extracellular N-terminal domain. We have established permanent cell-lines in which the gene for the human oxytocin receptor (OTR) has been introduced into HeLa cells. These cells differ by the disruption of one or more of the N-terminal N-glycosylation sites by site-directed mutagenesis of the transfected OTR constructs. The binding capacity of each transfectant, calculated per mg membrane protein, was 5-17 times higher than that of human term myometrium. The pharmacological characteristics of the transfected wild-type OTR are very similar to those of native myometrial OTR. The mutation of N-glycosylation sites (Asn-X-Ser/Thr), namely OTR-D8N15N26 (Asn8-->Asp8), -N8D15N26(Asn15-->Asp15), -N8D15D26(Asn15-->Asp15, Asn26-->Asp26) and -D8N15D26 (Asn8-->Asp8, Asn26-->Asp26) appear to affect neither their dissociation constant (Kd), nor the affinities for various oxytocin related ligands. As a high level of cell surface binding was retained for each clone, receptor trafficking appears to be normal. This suggests that the full glycosylation of OTR observed in vivo is not essential for its activity. These results indicate also that these cell lines may prove very useful for pharmacological screening of oxytocin related products.
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