The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors.
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.
Hossain
Mohammed Akhter
MA
Howard Florey Institute and Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria, Australia.
Rosengren
K Johan
KJ
Haugaard-Jönsson
Linda M
LM
Zhang
Soude
S
Layfield
Sharon
S
Ferraro
Tania
T
Daly
Norelle L
NL
Tregear
Geoffrey W
GW
Wade
John D
JD
Bathgate
Ross A D
RA
D000595:Amino Acid Sequence; D006801:Humans; D007328:Insulin / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry; D018000:Receptors, Peptide / Q000737:chemistry; D012065:Relaxin / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D007328:Insulin; C084797:Leydig insulin-like protein; D010455:Peptides; D011506:Proteins; C480238:RLN3 protein, human; C417543:RXFP1 protein, human; C410092:RXFP3 protein, human; C480239:RXFP4 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D012065:Relaxin