title: Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin.
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.
authors.affiliation: Florey Neuroscience Institutes, The University of Melbourne, VIC 3010, Australia. akhter.hossain@florey.edu.au
authors.forename: Mohammed Akhter
authors.initials: MA
authors.lastname: Hossain
authors.forename: John D
authors.initials: JD
authors.lastname: Wade
authors.forename: Ross A D
authors.initials: RA
authors.lastname: Bathgate
mesh_terms: D000242:Cyclic AMP / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008024:Ligands; D010446:Peptide Fragments / Q000494:pharmacology*; D011485:Protein Binding; D011506:Proteins / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism; D018000:Receptors, Peptide / Q000819:agonists / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D012065:Relaxin / Q000494:pharmacology*
chemical_list: D007328:Insulin; C084797:Leydig insulin-like protein; D008024:Ligands; D010446:Peptide Fragments; D011506:Proteins; C095068:RLN2 protein, human; C417543:RXFP1 protein, human; C472867:RXFP2 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D012065:Relaxin; D000242:Cyclic AMP