Proceedings of the 4th International Peptide Symposium in conjunction with the 7th Australian Peptide Conference and the 2nd Asia-Pacific International Peptide Symposium, 2007 1 Jackie Wilce (Editor) on behalf of the Australian Peptide Association Fig. 1. Sequence alignment of the seven members of the human relaxin family. The conserved cysteines are shown in yellow and their connectivities are indicated by black lines. Structural Insights into the Action of Relaxin Peptide Hormones K. Johan Rosengren 1 *, Ross A.D. Bathgate 2 , David J. Craik 3 , Norelle L. Daly 3 , Linda M. Haugaard-Jönsson 1 , M. Akther Hossain 2 , Feng Lin 2 , and John D. Wade 2 1 School of Pure and Applied Natural Sciences, University of Kalmar, Kalmar, SE-391 82, Sweden; 2 Howard Florey Institute, University of Melbourne, Melbourne, VIC 3010, Australia; 3 Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia. E-mail: johan.rosengren@hik.se Introduction The human relaxin family of peptide hormones comprises seven members: H1, H2 and H3 relaxin and the insulin-like peptides INSL3-6. These hormones play a number of distinct physiological roles, many of which are yet to be characterized, but they all have the common structural characteristics of two peptide chains that are folded tightly together into a compact globular fold that is stabilized by three disulfide bonds (Fig. 1). H2 relaxin is the human ortholog of the mammalian relaxin hormone that was first isolated over 80 years ago and which has long been regarded as a hormone associated with pregnancy [1]. Its actions include the remodeling of the connective tissue of the reproductive tract during pregnancy, but it does have a number of other physiological roles not related to pregnancy, including regulation of collagen and vasodilatation of various tissues. INSL3 similarly plays a role in reproduction, the highest production being found in the testis and ovaries, and a key role is the initiation of testes decent during fetal development. In contrast, the most recently discovered member of the relaxin family, H3 relaxin [2], is primarily expressed in the brain and has been suggested to play a role in the neurological signaling of stress responses in the central nervous system [3]. For the remaining members of the family the physiological roles are largely unknown. Relaxins, unlike their structural relatives insulin and the insulin-like growth factors, which interact with tyrosine kinase receptors, activate two unrelated classes of G-protein coupled receptors (GPCRs) [4,5,6]. The H2 relaxin and INSL3 receptors LGR7 and LGR8 belong to the Leucine-rich repeat containing GPCRs, which have a large N-terminal ligand binding domain, while the H3 relaxin and INSL5 receptors belong to the classic peptide ligand GPCR family. Despite these differences, interesting cross-reactivity is observed between hormones and receptors, with H3 relaxin being able to active three of the four currently identified receptors (Fig. 2). These observations raise the questions: What features are needed for interacting with the various receptors and what is the nature of these interactions? Here we have employed nuclear magnetic resonance (NMR) spectroscopy to characterize the structural differences of the relaxin hormones in order to gain new insights into how differences in the primary sequence can alter the three-dimensional structure and, as a result, the ability of the peptides to interact with the various receptors. Results and Discussion The overall aim of this work is to develop a structural understanding of how relaxins differ in structure and how these differences affect the ability to interact with their various receptors. Such an understanding will facilitate the design of relaxin analogues that are selective agonists or antagonist for each of the individual receptors. These analogues may be invaluable for pharmaceutical applications, as relaxins have great potential as drugs and as pharmacological probes to help deduce the in vivo functions of the various relaxins. Peptide Synthesis – The A- and B-chains of the various relaxins used for structural studies were produced by FMOC solid-phase peptide synthesis. In most cases a Fig. 2. Relaxin hormone-receptor pairs. Block arrows represent the interaction between the relaxins and their endogenous receptors, with additional cross-reactivity indicated by thin arrows.