title: Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo.
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.
authors.affiliation: Howard Florey Institute, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Ross A D
authors.initials: RA
authors.lastname: Bathgate
authors.forename: Feng
authors.initials: F
authors.lastname: Lin
authors.forename: Nicola F
authors.initials: NF
authors.lastname: Hanson
authors.forename: Laszlo
authors.initials: L
authors.lastname: Otvos
authors.forename: Angelo
authors.initials: A
authors.lastname: Guidolin
authors.forename: Chris
authors.initials: C
authors.lastname: Giannakis
authors.forename: Stan
authors.initials: S
authors.lastname: Bastiras
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Layfield
authors.forename: Tania
authors.initials: T
authors.lastname: Ferraro
authors.forename: Sherie
authors.initials: S
authors.lastname: Ma
authors.forename: Chongxin
authors.initials: C
authors.lastname: Zhao
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Gundlach
authors.forename: Chrishan S
authors.initials: CS
authors.lastname: Samuel
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Tregear
authors.forename: John D
authors.initials: JD
authors.lastname: Wade
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D007527:Isoenzymes; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011498:Protein Precursors; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012065:Relaxin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D008565:Membrane Proteins; D011498:Protein Precursors; C480238:RLN3 protein, human; C417543:RXFP1 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C038656:prorelaxin; D012065:Relaxin; D020778:Matrix Metalloproteinase 2