Structural characterization of a H3-INSL5 relaxin peptide chimera

Introduction Human relaxin-3 (H3 relaxin) and insulin-like peptide 5 (INSL5) belong to the relaxin peptide hormone family, a subfamily of the insulin super family. Structurally the members are characterized by two peptide chains (A and B) connected with two disulfide bonds, with the A-chain comprising a third intra chain bond [1]. H3 relaxin is primarily expressed in the brain and is suggested to play an important role as a central nervous system signalling substance involved in regulating food intake and stress response [2,3]. The biological function of INSL5 is not yet well understood, but INSL5 is expressed in various peripheral tissues, particularly in the colon [4]. The relaxins bind to G-protein coupled receptors (GPCR). To date four receptors have been identified namely GPCR135 (RXFP3), GPCR142 (RXFP4), leucine rich-repeat containing G-protein coupled receptor 7 (LGR7; RXFP1) and LGR8 (RXFP2). The GPCR135 and GPCR142 are of the classic peptide ligand GPCR type, whereas the LGRs belong to a different subfamily and are characterized by a large leucine rich repeat containing ectodomain, which contains the major ligand binding site. Despite these differences among the relaxin receptors, H3 relaxin has the ability to bind and activate both types of receptors, namely GPCR135, GPCR142 and LGR7, with GPCR135 being established as the endogenous receptor for H3 relaxin based on expression profiles. In contrast, the rest of the relaxin like-peptide members with known receptors, H2 relaxin, INSL3 and INSL5, can only activate receptors from the same subgroup (Figure 1) [5-7]. Since the full biological significance of many relaxins is poorly understood, selective ligands for the different relaxin receptors are highly desirable to enable studies of each receptor-signalling pathway individually in vivo. Excitingly it was recently shown that by combining the B-chain from relaxin-3 with the A-chain from INSL5 (H3-INSL5), a chimera peptide found to be selective for GPCR135 and GPCR142 over LGR7 is achieved [8,9]. Since GPCR142 is a pseudo gene in rats, the H3-INSL5 chimeric peptide is a good tool to try to unravel the in vivo function of GPCR135 and try to get closer to the answer about what properties in H3 relaxin enables the binding and activation of both types of GPCRs. Results and Discussion To gain further knowledge about what structural features within the relaxin peptide hormone family are responsible for the binding and activation of the four different receptors, the chimera peptide H3-INSL5 was subjected to nuclear magnetic resonance (NMR) studies for structure determination. The two-dimensional NMR spectra recorded at 600 MHz were of good quality and a sequential assignment strategy was used to identify nearly all protons in the structure (Figure 2). The overall structure is well defined and displays a typical insulin/relaxin fold, with a central α-helix in the B-chain and two α-helices located in the A-chain. The B-chain α-helix, which contains the receptor binding motif, RXXXRXXI is retained (Figure 3).