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Roger J. Summers - One of the best experts on this subject based on the ideXlab platform.
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signalling profiles of h3 Relaxin h2 Relaxin and r3 bδ23 27 r i5 acting at the Relaxin family peptide receptor 3 rxfp3
British Journal of Pharmacology, 2014Co-Authors: Martina Kocan, John D. Wade, Mohammed Akhter Hossain, Mohsin Sarwar, Roger J. SummersAbstract:Background and Purpose Relaxin family peptide receptor 3 (RXFP3) is expressed in brain areas important for processing sensory information and feeding, suggesting that it may be a target for anti-anxiety and anti-obesity drugs. We examined the effects of H3 Relaxin, the biased agonist H2 Relaxin and the antagonist, R3(BΔ23–27)R/I5, on RXFP3 signalling to establish their suitability as tools to assess the physiological roles of RXFP3. Experimental Approach The signalling profile of the RXFP3 ligands was determined using reporter gene assays, multiplexed signalling assays and direct examination of receptor–G protein and receptor–β-arrestin interactions using BRET. Key Results H2 Relaxin activated p38MAPK and ERK1/2 with lower efficacy than H3 Relaxin, but had similar efficacy for JNK1/2 phosphorylation. H2 or H3 Relaxin activation of p38MAPK, JNK1/2 or ERK1/2 involved Pertussis toxin-sensitive G-proteins. R3(BΔ23–27)R/I5 blocked H3 Relaxin AP-1 reporter gene activation, but not H2 Relaxin AP-1 activation or H3 Relaxin NF-κB activation. R3(BΔ23–27)R/I5 activated the SRE reporter, but did not inhibit either H2 or H3 Relaxin SRE activation. R3(BΔ23–27)R/I5 blocked H3 Relaxin-stimulated p38MAPK and ERK1/2 phosphorylation, but was a weak partial agonist for p38MAPK and ERK1/2 signalling. p38MAPK activation by R3(BΔ23–27)R/I5 was G protein-independent. H3 Relaxin-activated RXFP3 interacts with Gαi2, Gαi3, GαoA and GαoB whereas H2 Relaxin or R3(BΔ23–27)R/I5 induce interactions only with Gαi2 or GαoB. Only H3 Relaxin promoted RXFP3/β-arrestin interactions that were blocked by R3(BΔ23–27)R/I5. Conclusion and Implications Understanding signalling profile of drugs acting at RXFP3 is essential for development of therapies targeting this receptor.
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h2 Relaxin is a biased ligand relative to h3 Relaxin at the Relaxin family peptide receptor 3 rxfp3
Molecular Pharmacology, 2010Co-Authors: Emma T Van Der Westhuizen, John D. Wade, Arthur Christopoulos, Patrick M Sexton, Roger J. SummersAbstract:Relaxin family peptide 3 receptors (RXFP3) are activated by H3-Relaxin to inhibit forskolin-stimulated cAMP accumulation and stimulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In this study, we sought to identify novel signaling pathways coupled to RXFP3 and to investigate whether other members of the Relaxin peptide family activated these pathways. Two patterns of signaling were observed in RXFP3-expressing Chinese hamster ovary (CHO)-K1 and human embryonic kidney (HEK)-293 cells (CHO-RXFP3 and HEK-RXFP3) and murine septal neuron SN56 cell lines: 1) strong inhibition of forskolin-stimulated cAMP accumulation, ERK1/2 activation and nuclear factor (NF)-κB reporter gene activation in cells stimulated with H3 Relaxin, with weaker activity observed for H2 Relaxin, porcine Relaxin, or insulin-like peptide (INSL) 3 and 2) strong stimulation of activator protein (AP)-1 reporter genes by H2 Relaxin, with weaker activation observed with H3 or porcine Relaxin. Two distinct ligand binding sites were identified on RXFP3-expressing cells using two different radioligands. 125 I-INSL5 A-chain/Relaxin-3 B-chain chimera bound with high affinity to the RXFP3-expressing cells with competition by H3 Relaxin or a H3 Relaxin B-chain dimeric peptide, consistent with previous reports. Binding studies with 125 I-H2 Relaxin revealed a distinct binding site with potent competition observed with H2 Relaxin, H3 Relaxin, or INSL3 and weaker competition with porcine Relaxin. Thus H3 Relaxin potently activates all signaling pathways coupled to RXFP3, whereas H2 Relaxin is an AP-1-biased ligand relative to H3 Relaxin.
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Relaxin inhibits renal myofibroblast differentiation via rxfp1 the nitric oxide pathway and smad2
The FASEB Journal, 2009Co-Authors: Ishanee Mookerjee, Geoffrey W. Tregear, Roger J. Summers, Ross A D Bathgate, Tim D Hewitson, Michelle L Halls, Michael L Mathai, Chrishan S SamuelAbstract: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.
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H2 Relaxin Is a Biased Ligand Relative to H3 Relaxin at the Relaxin Family Peptide Receptor 3 (RXFP3)□S
2009Co-Authors: Emma T Van Der Westhuizen, John D. Wade, Arthur Christopoulos, Patrick M Sexton, Roger J. SummersAbstract:Relaxin family peptide 3 receptors (RXFP3) are activated by H3-Relaxin to inhibit forskolin-stimulated cAMP accumulation and stimulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In this study, we sought to identify novel signaling pathways coupled to RXFP3 and to investigate whether other members of the Relaxin peptide family activated these pathways. Two patterns of signaling were observed in RXFP3-expressing Chinese hamster ovary (CHO)-K1 and hu-man embryonic kidney (HEK)-293 cells (CHO-RXFP3 and HEK-RXFP3) and murine septal neuron SN56 cell lines: 1) strong inhibition of forskolin-stimulated cAMP accumulation, ERK1/2 activation and nuclear factor (NF)-B reporter gene activation in cells stimulated with H3 Relaxin, with weaker activity observed for H2 Relaxin, porcine Relaxin, or insulin-like peptide (INSL)
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responses of gpcr135 to human gene 3 h3 Relaxin in cho k1 cells determined by microphysiometry
Annals of the New York Academy of Sciences, 2005Co-Authors: Emma T Van Der Westhuizen, Ross A D Bathgate, Patrick M Sexton, Roger J. SummersAbstract: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 [ 1 2 5 I] H3 Relaxin binding to GPCR135 in these cells gave a B m a x of 32.61 ′ 6.5 fmol/mg protein and K d 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.
Ross A D Bathgate - One of the best experts on this subject based on the ideXlab platform.
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Relaxin family peptides structure activity relationship studies
British Journal of Pharmacology, 2017Co-Authors: John D. Wade, Mohammed Akhter Hossain, Ross A D Bathgate, Johan K Rosengren, Nitin A Patil, Frances SeparovicAbstract:The human Relaxin peptide family consists of seven cystine-rich peptides, four of which are known to signal through Relaxin family peptide receptors, RXFP1–4. As these peptides play a vital role physiologically and in various diseases, they are of considerable importance for drug discovery and development. Detailed structure–activity relationship (SAR) studies towards understanding the role of important residues in each of these peptides have been reported over the years and utilized for the design of antagonists and minimized agonist variants. This review summarizes the current knowledge of the SAR of human Relaxin 2 (H2 Relaxin), human Relaxin 3 (H3 Relaxin), human insulin-like peptide 3 (INSL3) and human insulin-like peptide 5 (INSL5). Linked Articles This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc
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activation of Relaxin family receptor 1 from different mammalian species by Relaxin peptide and small molecule agonist ml290
Frontiers in Endocrinology, 2015Co-Authors: Zaohua Huang, Ross A D Bathgate, Courtney Myhr, Amaya Bueno, Jingbo Xiao, Noel Southall, Elena Barnaeva, Irina U Agoulnik, Juan Marugan, Marc FerrerAbstract:Relaxin peptide (RLN), which signals through the Relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to Relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to Relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the 7-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the forth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit Relaxins. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled Relaxin to rabbit RXFP1 was detected, suggesting that in this species RXFP1 might be non-functional. We used chimeric rabbit-human and guinea pig-human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing.
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probing the functional domains of Relaxin 3 and the creation of a selective antagonist for rxfp3 gpcr135 over Relaxin receptor rxfp1 lgr7
Annals of the New York Academy of Sciences, 2009Co-Authors: Changlu Liu, John D. Wade, Mohammed Akhter Hossain, Chester Kuei, Steven W. Sutton, Diane Nepomuceno, Jonathan Shelton, Jessica Zhu, Ross A D Bathgate, Pascal BonaventureAbstract: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.
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Relaxin inhibits renal myofibroblast differentiation via rxfp1 the nitric oxide pathway and smad2
The FASEB Journal, 2009Co-Authors: Ishanee Mookerjee, Geoffrey W. Tregear, Roger J. Summers, Ross A D Bathgate, Tim D Hewitson, Michelle L Halls, Michael L Mathai, Chrishan S SamuelAbstract: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.
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the a chain of human Relaxin family peptides has distinct roles in the binding and activation of the different Relaxin family peptide receptors
Journal of Biological Chemistry, 2008Co-Authors: Mohammed Akhter Hossain, John D. Wade, Geoffrey W. Tregear, Sharon Layfield, Tania Ferraro, Norelle L Daly, Johan K Rosengren, Linda M Haugaardjonsson, Soude Zhang, Ross A D BathgateAbstract:The Relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the Relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the Relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human Relaxin-2 and -3 (H2 and H3) Relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 Relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 Relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 Relaxin. In contrast, A-chain-truncated H3 Relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 Relaxin-RXFP3 interaction. Our results provide new insights into the action of Relaxins and demonstrate that the role of the A-chain for Relaxin activity is both peptide- and receptor-dependent.
John D. Wade - One of the best experts on this subject based on the ideXlab platform.
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Relaxin family peptides structure activity relationship studies
British Journal of Pharmacology, 2017Co-Authors: John D. Wade, Mohammed Akhter Hossain, Ross A D Bathgate, Johan K Rosengren, Nitin A Patil, Frances SeparovicAbstract:The human Relaxin peptide family consists of seven cystine-rich peptides, four of which are known to signal through Relaxin family peptide receptors, RXFP1–4. As these peptides play a vital role physiologically and in various diseases, they are of considerable importance for drug discovery and development. Detailed structure–activity relationship (SAR) studies towards understanding the role of important residues in each of these peptides have been reported over the years and utilized for the design of antagonists and minimized agonist variants. This review summarizes the current knowledge of the SAR of human Relaxin 2 (H2 Relaxin), human Relaxin 3 (H3 Relaxin), human insulin-like peptide 3 (INSL3) and human insulin-like peptide 5 (INSL5). Linked Articles This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc
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signalling profiles of h3 Relaxin h2 Relaxin and r3 bδ23 27 r i5 acting at the Relaxin family peptide receptor 3 rxfp3
British Journal of Pharmacology, 2014Co-Authors: Martina Kocan, John D. Wade, Mohammed Akhter Hossain, Mohsin Sarwar, Roger J. SummersAbstract:Background and Purpose Relaxin family peptide receptor 3 (RXFP3) is expressed in brain areas important for processing sensory information and feeding, suggesting that it may be a target for anti-anxiety and anti-obesity drugs. We examined the effects of H3 Relaxin, the biased agonist H2 Relaxin and the antagonist, R3(BΔ23–27)R/I5, on RXFP3 signalling to establish their suitability as tools to assess the physiological roles of RXFP3. Experimental Approach The signalling profile of the RXFP3 ligands was determined using reporter gene assays, multiplexed signalling assays and direct examination of receptor–G protein and receptor–β-arrestin interactions using BRET. Key Results H2 Relaxin activated p38MAPK and ERK1/2 with lower efficacy than H3 Relaxin, but had similar efficacy for JNK1/2 phosphorylation. H2 or H3 Relaxin activation of p38MAPK, JNK1/2 or ERK1/2 involved Pertussis toxin-sensitive G-proteins. R3(BΔ23–27)R/I5 blocked H3 Relaxin AP-1 reporter gene activation, but not H2 Relaxin AP-1 activation or H3 Relaxin NF-κB activation. R3(BΔ23–27)R/I5 activated the SRE reporter, but did not inhibit either H2 or H3 Relaxin SRE activation. R3(BΔ23–27)R/I5 blocked H3 Relaxin-stimulated p38MAPK and ERK1/2 phosphorylation, but was a weak partial agonist for p38MAPK and ERK1/2 signalling. p38MAPK activation by R3(BΔ23–27)R/I5 was G protein-independent. H3 Relaxin-activated RXFP3 interacts with Gαi2, Gαi3, GαoA and GαoB whereas H2 Relaxin or R3(BΔ23–27)R/I5 induce interactions only with Gαi2 or GαoB. Only H3 Relaxin promoted RXFP3/β-arrestin interactions that were blocked by R3(BΔ23–27)R/I5. Conclusion and Implications Understanding signalling profile of drugs acting at RXFP3 is essential for development of therapies targeting this receptor.
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Design, Synthesis, and Characterization of a Single-Chain Peptide Antagonist for the Relaxin-3 Receptor RXFP3
Journal of the American Chemical Society, 2011Co-Authors: Linda M. Haugaard-kedström, John D. Wade, Andrew L Gundlach, Mohammed Akhter Hossain, David J Craik, Fazel Shabanpoor, Richard J. Clark, Philip Ryan, K Johan RosengrenAbstract: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 hampered 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.
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the roles of the a and b chains of human Relaxin 2 and 3 on their biological activity
Current Protein & Peptide Science, 2010Co-Authors: Mohammed Akhter Hossain, John D. WadeAbstract:Two members of the human insulin/Relaxin superfamily, Relaxins-2 and 3 (H2 and H3 respectively), are separated by nearly 75 years in terms of chronological identification but are both the subject of intense recent biological study. The physiological effects of H2 Relaxin include vasodilatory, anti-inflammatory, extracellular matrix remodeling, and angiogenic and anti-ischemic. Because of its potent systemic and renal vasodilatory effects, it is currently undergoing phase III clinical trial for the treatment of acute heart failure. In contrast, H3 Relaxin is a highly conserved neuropeptide that has rapidly emerged as an important regulator of homeostatic physiology and complex behaviors. Because of their immense clinical potential, an understanding of the structural features that control their functions is critical for rational drug design and development. The native receptor for H2 Relaxin is RXFP1. It also strongly binds to the related receptor, RXFP2. The native receptor for H3 Relaxin is the unrelated receptor, RXFP3; however, it also has high affinity for another related receptor, RXFP4. Interestingly, H3 Relaxin also has a high affinity for RXFP1 and can interact with RXFP2 with a significantly lower affinity. H3 Relaxin thus interacts with all four of the Relaxin family receptors. Previous studies have shown that H2 and H3 Relaxins interact with their receptors primarily using their B-chain specific residues. However, more recent studies suggest that the role of the respective A and B chains for their activity is both peptide- and receptor-dependent. This mini-review summarizes these recent findings on the structure-activity relationships of H2 and H3 Relaxins.
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h2 Relaxin is a biased ligand relative to h3 Relaxin at the Relaxin family peptide receptor 3 rxfp3
Molecular Pharmacology, 2010Co-Authors: Emma T Van Der Westhuizen, John D. Wade, Arthur Christopoulos, Patrick M Sexton, Roger J. SummersAbstract:Relaxin family peptide 3 receptors (RXFP3) are activated by H3-Relaxin to inhibit forskolin-stimulated cAMP accumulation and stimulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In this study, we sought to identify novel signaling pathways coupled to RXFP3 and to investigate whether other members of the Relaxin peptide family activated these pathways. Two patterns of signaling were observed in RXFP3-expressing Chinese hamster ovary (CHO)-K1 and human embryonic kidney (HEK)-293 cells (CHO-RXFP3 and HEK-RXFP3) and murine septal neuron SN56 cell lines: 1) strong inhibition of forskolin-stimulated cAMP accumulation, ERK1/2 activation and nuclear factor (NF)-κB reporter gene activation in cells stimulated with H3 Relaxin, with weaker activity observed for H2 Relaxin, porcine Relaxin, or insulin-like peptide (INSL) 3 and 2) strong stimulation of activator protein (AP)-1 reporter genes by H2 Relaxin, with weaker activation observed with H3 or porcine Relaxin. Two distinct ligand binding sites were identified on RXFP3-expressing cells using two different radioligands. 125 I-INSL5 A-chain/Relaxin-3 B-chain chimera bound with high affinity to the RXFP3-expressing cells with competition by H3 Relaxin or a H3 Relaxin B-chain dimeric peptide, consistent with previous reports. Binding studies with 125 I-H2 Relaxin revealed a distinct binding site with potent competition observed with H2 Relaxin, H3 Relaxin, or INSL3 and weaker competition with porcine Relaxin. Thus H3 Relaxin potently activates all signaling pathways coupled to RXFP3, whereas H2 Relaxin is an AP-1-biased ligand relative to H3 Relaxin.
Sharon Layfield - One of the best experts on this subject based on the ideXlab platform.
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the a chain of human Relaxin family peptides has distinct roles in the binding and activation of the different Relaxin family peptide receptors
Journal of Biological Chemistry, 2008Co-Authors: Mohammed Akhter Hossain, John D. Wade, Geoffrey W. Tregear, Sharon Layfield, Tania Ferraro, Norelle L Daly, Johan K Rosengren, Linda M Haugaardjonsson, Soude Zhang, Ross A D BathgateAbstract:The Relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the Relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the Relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human Relaxin-2 and -3 (H2 and H3) Relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 Relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 Relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 Relaxin. In contrast, A-chain-truncated H3 Relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 Relaxin-RXFP3 interaction. Our results provide new insights into the action of Relaxins and demonstrate that the role of the A-chain for Relaxin activity is both peptide- and receptor-dependent.
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Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the Relaxin receptor LGR7 both in vitro and in vivo.
Biochemistry, 2006Co-Authors: Feng Lin, Sharon Layfield, Nicola F. Hanson, Laszlo Otvos, Angelo Guidolin, Chris Giannakis, Stan Bastiras, Tania FerraroAbstract: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.
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Relaxin 3 improved synthesis strategy and demonstration of its high affinity interaction with the Relaxin receptor lgr7 both in vitro and in vivo
Biochemistry, 2006Co-Authors: Ross A D Bathgate, Sharon Layfield, Tania Ferraro, Nicola F. Hanson, Laszlo Otvos, Angelo Guidolin, Chris Giannakis, Stan Bastiras, Chongxin Zhao, Andrew L GundlachAbstract: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 (LGR...
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studies on soluble ectodomain proteins of Relaxin lgr7 and insulin 3 lgr8 receptors
Annals of the New York Academy of Sciences, 2005Co-Authors: Sharon Layfield, Tania Ferraro, Satoko Sudo, Jin Kumagai, Yan Yan, Jin Cai, Jianguo Tang, Eleni GiannakisAbstract: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.
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The chemistry and biology of human Relaxin-3.
Annals of the New York Academy of Sciences, 2005Co-Authors: Geoffrey W. Tregear, Andrew L Gundlach, Sharon Layfield, Tania Ferraro, Feng Lin, Nicola F. Hanson, Roger J. Summers, Johan RosengrenAbstract: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 individual 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.
Geoffrey W. Tregear - One of the best experts on this subject based on the ideXlab platform.
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Relaxin inhibits renal myofibroblast differentiation via rxfp1 the nitric oxide pathway and smad2
The FASEB Journal, 2009Co-Authors: Ishanee Mookerjee, Geoffrey W. Tregear, Roger J. Summers, Ross A D Bathgate, Tim D Hewitson, Michelle L Halls, Michael L Mathai, Chrishan S SamuelAbstract: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.
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the a chain of human Relaxin family peptides has distinct roles in the binding and activation of the different Relaxin family peptide receptors
Journal of Biological Chemistry, 2008Co-Authors: Mohammed Akhter Hossain, John D. Wade, Geoffrey W. Tregear, Sharon Layfield, Tania Ferraro, Norelle L Daly, Johan K Rosengren, Linda M Haugaardjonsson, Soude Zhang, Ross A D BathgateAbstract:The Relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the Relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the Relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human Relaxin-2 and -3 (H2 and H3) Relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 Relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 Relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 Relaxin. In contrast, A-chain-truncated H3 Relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 Relaxin-RXFP3 interaction. Our results provide new insights into the action of Relaxins and demonstrate that the role of the A-chain for Relaxin activity is both peptide- and receptor-dependent.
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improved chemical synthesis and demonstration of the Relaxin receptor binding affinity and biological activity of mouse Relaxin
Biochemistry, 2007Co-Authors: Chrishan S Samuel, Mohammed Akhter Hossain, Geoffrey W. Tregear, Tania Ferraro, Feng Lin, Ross A D Bathgate, Chongxin Zhao, John D. WadeAbstract: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.
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Solution Structure and Novel Insights into the Determinants of the Receptor Specificity of Human Relaxin-3
The Journal of biological chemistry, 2005Co-Authors: K Johan Rosengren, John D. Wade, Geoffrey W. Tregear, Feng Lin, Norelle L Daly, David J CraikAbstract:Relaxin-3 is the most recently discovered member of the Relaxin family of peptide hormones. In contrast to Relaxin-1 and -2, whose main functions are associated with pregnancy, Relaxin-3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human Relaxin-3, the first structure of a Relaxin family member to be solved by NMR methods. Overall, Relaxin-3 adopts an insulin-like fold, but the structure differs crucially from the crystal structure of human Relaxin-2 near the B-chain terminus. In particular, the B-chain C terminus folds back, allowing Trp(B27) to interact with the hydrophobic core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the Relaxin receptor LGR7 and may account for the lower affinity of Relaxin-3 relative to Relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135.
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evolution of the Relaxin like peptide family
BMC Evolutionary Biology, 2005Co-Authors: Tracey N Wilkinson, Geoffrey W. Tregear, Terence P Speed, Ross A D BathgateAbstract:The Relaxin-like peptide family belongs in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; Relaxin-1, 2 and 3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. The functions of Relaxin-3, INSL4, INSL5, INSL6 remain uncharacterised. The evolution of this family has been contentious; high sequence variability is seen between closely related species, while distantly related species show high similarity; an invertebrate Relaxin sequence has been reported, while a Relaxin gene has not been found in the avian and ruminant lineages. Sequence similarity searches of genomic and EST data identified homologs of Relaxin-like peptides in mammals, and non-mammalian vertebrates such as fish. Phylogenetic analysis was used to resolve the evolution of the family. Searches were unable to identify an invertebrate Relaxin-like peptide. The published Relaxin cDNA sequence in the tunicate, Ciona intestinalis was not present in the completed C. intestinalis genome. The newly discovered Relaxin-3 is likely to be the ancestral Relaxin. Multiple Relaxin-3-like sequences are present in fugu fish (Takifugu rubripes) and zebrafish (Danio rerio), but these appear to be specific to the fish lineage. Possible Relaxin-1 and INSL5 homologs were also identified in fish and frog species, placing their emergence prior to mammalia, earlier than previously believed. Furthermore, estimates of synonymous and nonsynonymous substitution rates (dN/dS) suggest that the emergence of Relaxin-1, INSL4 and INSL6 during mammalia was driven by positive Darwinian selection, hence these peptides are likely to have novel and in the case of Relaxin-1, which is still under positive selection in humans and the great apes, possibly still evolving functions. In contrast, Relaxin-3 is constrained by strong purifying selection, demonstrating it must have a highly conserved function, supporting its hypothesized important neuropeptide role. We present a phylogeny describing the evolutionary history of the Relaxin-like peptide family and show that positive selection has driven the evolution of the most recent members of the family.
