Regulation of Receptor Signaling by Relaxin A Chain Motifs

Abstract

Relaxin peptides are important hormones for the regulation of reproductive tissue remodeling and the renal cardiovascular system during pregnancy. Recent studies demonstrated that two of the seven human relaxin family peptides, relaxin H2 (RLN2) and INSL3, signal exclusively through leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8. Although it was well characterized that an RXXXRXXI motif at the RLN2 B chain confers receptor activation activity, it is not clear what roles RLN2 A chain plays in receptor interaction. Analyses of relaxin family genes on syntenic regions of model tetrapods showed that the A chain of RLN2 orthologs exhibited a greater sequence divergence as compared with the receptorbinding domain-containing B chain, foreshadowing a potential role in receptor interactions; hence, defining receptor selectivity in this fast evolving peptide hormone. To test our hypothesis that select residues in the human RLN2 A chain play key roles in receptor interaction, we studied mutant peptides with residue substitution(s) in the A chain. Here, we showed that alanine substitution at the A16 and A17 positions enhances LGR8-activation activity of RLN2, whereas mutation at the A22-23 region (RLN2 A22-23 ) ablates LGR8, but not LGR7, activation activity. In addition, we demonstrated that the functional characteristics of the RLN2 A22-23 mutant are mainly attributed to modifications at the Phe A23 position. Taken together, our studies indicated that Thr A16 , Lys A17 , and Phe A23 constitute part of the receptorbinding interface of human RLN2, and that modification of these residues has led to the generation of novel human RLN2 analogs that would allow selective activation of human LGR7, but not LGR8, in vivo.In humans, the relaxin gene family contains a total of seven members: relaxin H1 (RLN1), 3 relaxin H2 (RLN2), relaxin 3/INSL7 (RLN3), INSL3/RLF, INSL4/EPIL, INSL5/RIF2, and INSL6/RIF1 (1-5). Among these family members, RLN2 and INSL3 signal through two leucine-rich repeat-containing GPCRs, LGR7 (RFXR1) and/or LGR8 (RFXR2). Whereas RLN2 is capable of activating both LGR7 and LGR8, INSL3 is a selective ligand for LGR8. In addition to the better characterized RLN2 and INSL3, RLN3 was shown to activate LGR7, GPCR135, and GPCR142, but not LGR8 (6 -10); therefore, the relaxin family peptides exhibit overlapping specificity on the activation of LGR7 and LGR8. Although the physiological roles of RLN3 and other family peptides remain to be studied, analysis of null mice models showed that the ablation of relaxin and INSL3 genes caused phenotypes similar to that of LGR7-and LGR8-deficient mice, suggesting that LGR7 and LGR8 are the main receptors for the single relaxin and INSL3 peptides in rodents, respectively (11-15).In addition to the role in remodeling of reproductive tissues, relaxin has been shown to effect endometrial differentiation during embryo implantation, nipple and mammary gland development, angiogenesis, wound healing, and renal cardiovascular responses (12, 13, 16 -19). Furthermore...

The Minimal Active Structure of Human Relaxin-2

“…This finding is consistent with studies by Park et al (18), which suggest that Tyr A16 and Lys A17 are important for the H2 relaxin interaction with RXFP1. However, the nature of such an interaction is unclear as no drop in affinity for RXFP1 was seen when either of these residues were mutated by Ala but only when replaced by Gly (18), which may suggest a secondary effect related to structural changes. Clearly, further mutational studies targeting the midto C-terminal part of H2 relaxin A-chain are required to shed light on its role in receptor activation, in particular for the development of a highly sought after high affinity RXFP1 antagonist.…”

“…1). These included the following: four B-chain N-terminal truncated analogues (H2-(B3-29), H2-(B5-29), H2-(B7-29), and H2-(B9 -29)), six B-chain C-terminal truncated analogues (H2-(B1-28), H2-(B1-27), H2-(B1-26), H2-(B1-25), H2-(B1-24), and H2-(B1-23)), four B-chain Nand C-terminal truncated analogues (H2-(B7-25), H2-(B7-24), H2-(B8 -25), and H2-(B8 -24)), and six A-and B-chain truncated analogues (H2-(A2-24)(B7-24), H2-(A3-24)(B7-24), H2-(A4 -24)(B7-24), H2-(A5-24)(B7-24), H2-(A7-24)(B7-24), and H2-(A9 -24) (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)). All of these H2 analogues were amidated at the C termini of both the A-and B-chains.…”

“…Thus, H2 relaxin is binding to and activating both RXFP1 and RXFP2 receptors in a similar manner but via a mechanism that is different compared with INSL3 (21). This structure activity relationship study indicates that the active site of H2 relaxin most likely consists of the mid-region of the B-chain helix and the C-terminal region of the A-chain (18,23). In this study, we have carried out truncations at other regions within the A-and/or B-chains and evaluated the resulting structure and function of these minimized peptides.…”

“…Combinations of A-and B-chain Truncations of H2 RelaxinIn our previous work, we investigated the role of the H2 relaxin A-chain in the interaction with RXFP1 (18). We showed that analogues with up to five residues truncated retained close to full activity.…”

RXFP1 Receptor Activation by Relaxin-2 Induces Vascular Relaxation in Mice via a Gαi2-Protein/PI3Kß/γ/Nitric Oxide-Coupled Pathway

“…Binding of relaxins to RXFP1 is mediated via high-affinity binding to extracellular domain of RXFP1 and an additional binding site in the transmembrane (TM) exoloops ( Sherwood, 2004 ). A recent study has shown that specific residues in the center of the H2 relaxin A-chain are necessary for ligand activity at RXFP1 ( Park et al, 2008 ). Importantly, modeling of the ligand–receptor interaction for different RXFP receptors suggests that once the B-chains of the ligands are bound to the primary binding site in a large ectodomain with 10 leucine-rich repeats (LRRs) that the A-chain is presented in a favorable orientation for interaction with the TM exoloops ( Hartley et al, 2009 ).…”