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

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Abstract

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

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References

  • Bathgate RA, Ivell R, Sanborn BM, Sherwood OD, Summers RJ (2006) International Union of Pharmacology LVII: recommendations for the nomenclature of receptors for relaxin family peptides. Pharmacol Rev 58:7–31

    Article  PubMed  CAS  Google Scholar 

  • Büllesbach EE, Schwabe C (1999) Tryptophan B27 in the relaxin-like factor (RLF) is crucial for RLF receptor-binding. Biochemistry 38:3073–3078

    Article  PubMed  Google Scholar 

  • Büllesbach EE, Schwabe C (2000) The relaxin receptor-binding site geometry suggests a novel gripping mode of interaction. J Biol Chem 275:35276–35280

    Article  PubMed  Google Scholar 

  • Büllesbach EE, Schwabe C (2005) The trap-like relaxin-binding site of the leucine-rich G-protein-coupled receptor 7. J Biol Chem 280:14051–14056

    Article  PubMed  Google Scholar 

  • Büllesbach EE, Yang S, Schwabe C (1992) The receptor-binding site of human relaxin II. A dual prong-binding mechanism. J Biol Chem 267:22957–22960

    PubMed  Google Scholar 

  • Chan LJ, Hossain MA, Samuel CS, Separovic F, Wade JD (2011) The relaxin peptide family: structure, function and clinical applications. Protein Pept Lett 18:220–229

    Article  PubMed  CAS  Google Scholar 

  • Eigenbrot C, Randal M, Quan C et al (1991) X-ray structure of human relaxin at 1.5 A. Comparison to insulin and implications for receptor binding determinants. J Mol Biol 221:15–21

    PubMed  CAS  Google Scholar 

  • Halls ML, Bathgate RA, Sudo S et al (2005a) Identification of binding sites with differing affinity and potency for relaxin analogues on LGR7 and LGR8 receptors. Ann N Y Acad Sci 1041:17–21

    Article  PubMed  CAS  Google Scholar 

  • Halls ML, Bond CP, Sudo S et al (2005b) Multiple binding sites revealed by interaction of relaxin family peptides with native and chimeric relaxin family peptide receptors 1 and 2 (LGR7 and LGR8). J Pharmacol Exp Ther 313:677–687

    Article  PubMed  CAS  Google Scholar 

  • Hossain MA, Zhang S, Lin F et al (2006) Regioselective disulfide solid phase synthesis, chemical characterization and in vitro receptor binding activity of equine relaxin. Int J Pept Res Ther 12:211–215

    Article  CAS  Google Scholar 

  • Hossain MA, Bathgate RA, Kong CK et al (2008) Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5). ChemBioChem 9:1816–1822

    Article  CAS  Google Scholar 

  • Hossain MA, Rosengren KJ, Samuel CS et al (2011) The minimal active structure of human relaxin-2. J Biol Chem 286:37555–37565

    Article  PubMed  CAS  Google Scholar 

  • Hsu SY, Nakabayashi K, Nishi S et al (2002) Activation of orphan receptors by the hormone relaxin. Science 295:671–674

    Article  PubMed  CAS  Google Scholar 

  • Kumagai J, Hsu SY, Matsumi H et al (2002) INSL3/Leydig insulin-like peptide activates the LGR8 receptor important in testis descent. J Biol Chem 277:31283–31286

    Article  PubMed  CAS  Google Scholar 

  • Rosengren KJ, Zhang S, Lin F et al (2006) Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3. J Biol Chem 281:28287–28295

    Article  PubMed  CAS  Google Scholar 

  • Scott DJ, Fu P, Shen PJ et al (2005) Characterization of the rat INSL3 receptor. Ann N Y Acad Sci 1041:13–16

    Article  PubMed  CAS  Google Scholar 

  • Scott DJ, Layfield S, Yan Y et al (2006) Characterization of novel splice variants of LGR7 and LGR8 reveals that receptor signaling is mediated by their unique low density lipoprotein class A modules. J Biol Chem 281:34942–34954

    Article  PubMed  CAS  Google Scholar 

  • Scott DJ, Wilkinson TN, Zhang S et al (2007) Defining the LGR8 residues involved in binding insulin-like peptide 3. Mol Endocrinol 21:1699–1712

    Article  PubMed  CAS  Google Scholar 

  • Scott DJ, Tregear GW, Bathgate RAD (2009) Modeling the primary hormone-binding site of RXFP1 and RXFP2. Ann N Y Acad Sci 1160:74–77

    Article  PubMed  CAS  Google Scholar 

  • Scott DJ, Rosengren KJ, Bathgate RAD (2012) The different ligand-binding modes of relaxin family peptide receptors RXFP1 and RXFP2. Mol Endocrinol 26 (in press)

  • Shabanpoor F, Bathgate RA, Belgi A et al (2012) Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone. Biochem Biophys Res Commun 420:253–256

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Tania Ferraro and Sharon Layfield for their assistance with the biochemical assays. This work was supported by an NHMRC Grant (APP #1023078) to JDW, RADB and MAH, and (APP #1023321) to MAH. JDW and RADB are recipients of NHMRC Principal and Senior Research Fellowships, respectively. Studies at the FNI were supported by the Victorian Government’s Operational Infrastructure Support Program.

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Correspondence to John D. Wade or Mohammed Akhter Hossain.

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Chan, L.J., Wade, J.D., Separovic, F. et al. The Importance of Tryptophan B28 in H2 Relaxin for RXFP2 Binding and Activation. Int J Pept Res Ther 19, 55–60 (2013). https://doi.org/10.1007/s10989-012-9332-x

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  • DOI: https://doi.org/10.1007/s10989-012-9332-x

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