Structure of the R3/I5 Chimeric Relaxin Peptide, a Selective GPCR135 and GPCR142 Agonist

Abstract

The human relaxin family comprises seven peptide hormones with various biological functions mediated through interactions with G-protein-coupled receptors. Interestingly, among the hitherto characterized receptors there is no absolute selectivity toward their primary ligand. The most striking example of this is the relaxin family ancestor, relaxin-3, which is an agonist for three of the four currently known relaxin receptors: GPCR135, GPCR142, and LGR7. Relaxin-3 and its endogenous receptor GPCR135 are both expressed predominantly in the brain and have been linked to regulation of stress and feeding. However, to fully understand the role of relaxin-3 in neurological signaling, the development of selective GPCR135 agonists and antagonists for in vivo studies is crucial. Recent reports have demonstrated that such selective ligands can be achieved by making chimeric peptides comprising the relaxin-3 B-chain combined with the INSL5 A-chain. To obtain structural insights into the consequences of combining A-and B-chains from different relaxins we have determined the NMR solution structure of a human relaxin-3/INSL5 chimeric peptide. The structure reveals that the INSL5 A-chain adopts a conformation similar to the relaxin-3 A-chain, and thus has the ability to structurally support a native-like conformation of the relaxin-3 B-chain. These findings suggest that the decrease in activity at the LGR7 receptor seen for this peptide is a result of the removal of a secondary LGR7 binding site present in the relaxin-3 A-chain, rather than conformational changes in the primary B-chain receptor binding site.The human relaxin family of peptide hormones comprises seven members in humans, including relaxins 1-3 (1-3), and the insulin-like peptides INSLs 3-6 (4 -7). Relaxin-1 is the result of a gene duplication only present in higher primates and relaxin-2 is the human equivalent of "relaxin" in all other mammals. The relaxins are structurally related to insulin and, together with insulin and the insulin-like growth factors I and II, they make up the insulin/relaxin superfamily (Fig. 1). These peptides share the structural characteristics of two peptide chains, A and B, which comprise around 24 and 30 amino acids, respectively, and are cross-braced by three disulfide bonds (8). Despite being structural relatives of insulin and the insulin-like growth factors, which activate tyrosine kinase receptors, it was recently established that relaxins interact with G-protein-coupled receptors (GPCRs).3 To date four so-called relaxin family peptide receptors (RXFPs) have been characterized and identified as the endogenous receptors of relaxin, INSL3, relaxin-3, and INSL5, namely LGR7 (RXFP1) (9), LGR8 (RXFP2) (10), GPCR135 (RXFP3) (11), and GPCR142 (RXFP4) (12), respectively. Interestingly, despite these receptors interacting with similar ligands, GPCR135 and GPCR142 are of the classic peptide ligand receptor types, whereas LGR7 and LGR8 belong to an unrelated class, which is characterized by a large extracellular leucine-rich repeat containi...

The Minimal Active Structure of Human Relaxin-2

“…Furthermore, a chimeric peptide comprising the H3 relaxin B-chain and the A-chain of INSL5 loses the ability of native H3 relaxin to interact with RXFP1 (49). Structural studies on this peptide revealed that this effect is not related to structural changes as the H3 relaxin B-chain adopts an identical conformation in the chimera as when associated with its native A-chain (50). Thus, at least in part, the domain responsible for activation of the RXFP1 and RXFP2 receptors must be located in the A-chain.…”

“…Data points were measured in triplicate, and each experiment was repeated at least three times. Statistical differences in pEC 50 values were analyzed using one-way analysis of variance coupled to a Newman Keul's multiple comparison test for multiple group comparisons in Graphpad Prism 4.…”

“…of the percentage of the total specific binding of triplicate wells, repeated in at least three separate experiments, and curves were fitted using onesite binding curves in Graphpad Prism 4 (Graphpad Software). Statistical differences in pIC 50 values were analyzed using oneway analysis of variance coupled to a Newman Keul's multiple comparison test for multiple group comparisons in Graphpad Prism 4.…”

Increased feeding and body weight gain in rats after acute and chronic activation of RXFP3 by relaxin-3 and receptor-selective peptides

“…Soon after relaxin-3 was discovered, central injection studies identified the ability of synthetic native peptide to stimulate feeding markedly in satiated adult rats (McGowan et al, 2005). Although the interpretation of these and subsequent data was confounded by ligand cross-reactivity at RXFP1 (Sudo et al, 2003;Liu et al, 2005), the development and use of relaxin-3 analogues selective for the native relaxin-3 receptor, RXFP3 518 Behavioural Pharmacology 2012, Vol 23 No 5&6 (Liu et al, 2005;Kuei et al, 2007;Haugaard-Jonsson et al, 2008;Haugaard-Kedstrom et al, 2011) (Table 1; Fig. 1), subsequently confirmed that the activation of RXFP3 increased feeding and these effects were blocked by RXFP3 antagonists (Kuei et al, 2007;Haugaard-Kedstrom et al, 2011;Shabanpoor et al, 2012) (Fig.…”

Relaxin-3/RXFP3 system regulates alcohol-seeking

“…The RXFP3 antagonists R3(B1-22)R and R3(BΔ23-27)R/I5 were synthesized using solid-phase peptide synthesis and purified using reverse-phase HPLC (38,48,49). The identity and purity of the peptide was confirmed by reverse-phase HPLC and MALDI-TOF mass spectrometry.…”