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Structure and function of relaxins
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The relaxin/insulin superfamily is a group of peptide hormones that consists of ten members in human, namely relaxins 1-3, insulin-like peptides (INSL) 3-6, insulin and insulin-like growth factors (IGF) I-II. These peptides have various functions in the body, such as regulating growth, blood glucose levels,  collagen metabolism, germ cell maturation and appetite. Misregulation of these mechanisms is associated with disease and accordingly they are of interest as potential pharmaceutical targets. Structurally the hormones are characterised by two peptide chains, A and B, which are held together by one intra A-chain and two inter chain disulfide bonds. Four different G-protein coupled receptors (GPCR) called relaxin family peptide receptor (RXFP) 1-4 have been found to respond to stimuli by different relaxin peptides. RXFP3 and RXFP4 are classic peptide ligand GPCRs, whereas RXFP1 and RXFP2 are characterised by a large extracellular leucine rich-repeat domain. Relaxin-3, which is the relaxin family ancestor, is the only relaxin peptide known to be able to bind and activate both subtypes of GPCRs, namely RXFP1, RXFP3 and RXFP4.

The aim of this thesis was to analyse the structure-function relationship of the relaxin ligands and receptors, and to use this information to develop selective ligands for the relaxin receptors, which can be used as drug leads or pharmacological tools for investigating the physiological roles of the RXFPs.

The 3D structures of native INSL5 and relaxin-2 were determined by solution NMR spectroscopy. The peptides showed an insulin/relaxin-like overall fold. A relaxin chimera peptide, consisting of the A-chain from INSL5 and the B-chain from relaxin-3, R3/I5, which has been shown to be selective for RXFP3 and RXFP4 over RXFP1, was also subjected to NMR studies. The R3/I5 peptide maintained an insulin/relaxin-like overall fold, and the relaxin-3 B-chain adopted a conformation identical to that in native relaxin-3, confirming that the activity of R3/I5 can be directly related to its primary sequence. Furthermore, a truncation study was undertaken to ascertain the importance of the termini for structure and function. By using the knowledge generated from the structure-function relationship, a single-chain high affinity RXFP3 selective antagonist was developed.

In conclusion, this thesis has contributed to broaden the knowledge of the structure-function relationship of the relaxin ligands and the development of a selective RXFP3 antagonist, which is currently a drug lead for treatment of neurological disorders including stress and obesity.

Place, publisher, year, edition, pages
Växjö, Kalmar: Linneaus University Press , 2011.
Series
Linnaeus University Dissertations, 31/2011
Keyword [en]
relaxin, insulin-like peptide, peptide hormone, peptide synthesis and NMR
National Category
Organic Chemistry
Research subject
Chemistry, Organic Chemistry
Identifiers
URN: urn:nbn:se:lnu:diva-9793ISBN: 978-91-86491-55-0 (print)OAI: oai:DiVA.org:lnu-9793DiVA: diva2:380522
Public defence
2011-01-28, N2007, Smålandsgatan 26B, Kalmar, 09:30 (English)
Opponent
Supervisors
Available from: 2010-12-21 Created: 2010-12-21 Last updated: 2017-04-18Bibliographically approved
List of papers
1. Structure of the human insulin-like peptide 5 and characterization of conserved hydrogen bonds and electrostatic interactions within the relaxin framework
Open this publication in new window or tab >>Structure of the human insulin-like peptide 5 and characterization of conserved hydrogen bonds and electrostatic interactions within the relaxin framework
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2009 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 419, 619-627 p.Article in journal (Refereed) Published
Abstract [en]

INSL5 (insulin-like peptide 5) is a two-chain peptide hormone related to insulin and relaxin. It was recently discovered through searches of expressed sequence tag databases and, although the fulfil biological significance of INSL5 is still being elucidated, high expression in peripheral tissues such as the colon, as well as in the brain and hypothalamus, suggests roles in gut contractility and neuroendocrine signalling. INSL5 activates the relaxin family peptide receptor 4 with high potency and appears to be the endogenous ligand for this receptor, on the basis of overlapping expression profiles and their apparent co-evolution. In the present Study, we have used solution-state NMR to characterize the three-dimensional structure of synthetic human INSL5. The structure reveals an insulin/relaxin-like fold with three helical segments that are braced by three disulfide bonds and enclose a hydrophobic core. Furthermore, we characterized in detail the hydrogen-bond network and electrostatic interactions between charged groups in INSL5 by NMR-monitored temperature and pH titrations and Undertook a comprehensive structural comparison with other members of the relaxin family, thus identifying the conserved structural features of the relaxin fold. The B-chain helix, which is the primary receptor-binding site of the relaxins, is longer in INSL5 than in its close relative relaxin-3. As this feature results in a different positioning of the receptor-activation domain Arg(B23) and Trp(B24), it may be an important contributor to the difference in biological activity observed for these two peptides. Overall, the structural Studies provide mechanistic insights into the receptor selectivity of this important family of hormones. 

National Category
Organic Chemistry
Research subject
Chemistry, Organic Chemistry
Identifiers
urn:nbn:se:lnu:diva-1953 (URN)10.1042/BJ20082353 (DOI)
Available from: 2010-04-06 Created: 2010-04-06 Last updated: 2017-04-18Bibliographically approved
2. Solution structure, aggregration behaviour and flexibility of human relaxin-2 - implications for biological function
Open this publication in new window or tab >>Solution structure, aggregration behaviour and flexibility of human relaxin-2 - implications for biological function
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:lnu:diva-9788 (URN)
Available from: 2010-12-21 Created: 2010-12-21 Last updated: 2017-04-18Bibliographically approved
3. Structure of the R3/I5 chimeric relaxin peptide, a selective GPCR135 and GPCR142 agonist
Open this publication in new window or tab >>Structure of the R3/I5 chimeric relaxin peptide, a selective GPCR135 and GPCR142 agonist
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 35, 23811-23818 p.Article in journal (Refereed) Published
Abstract [en]

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. 

National Category
Medical and Health Sciences Organic Chemistry
Research subject
Natural Science, Biomedical Sciences; Chemistry, Organic Chemistry
Identifiers
urn:nbn:se:lnu:diva-1877 (URN)10.1074/jbc.M800489200 (DOI)
Available from: 2010-04-06 Created: 2010-04-06 Last updated: 2017-04-18Bibliographically approved
4. The A-chain of the human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors
Open this publication in new window or tab >>The A-chain of the human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 25, 17287-17297 p.Article in journal (Refereed) Published
Abstract [en]

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. 

National Category
Organic Chemistry
Research subject
Chemistry, Organic Chemistry
Identifiers
urn:nbn:se:lnu:diva-1812 (URN)10.1074/jbc.M801911200 (DOI)
Available from: 2010-04-06 Created: 2010-04-06 Last updated: 2017-04-18Bibliographically approved
5. Design, synthesis, and characterization of a single-chain peptide antagonist for the relaxin-3 receptor RXFP3
Open this publication in new window or tab >>Design, synthesis, and characterization of a single-chain peptide antagonist for the relaxin-3 receptor RXFP3
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2011 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 133, no 13, 4965-4974 p.Article in journal (Refereed) Published
National Category
Chemical Sciences
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-9785 (URN)10.1021/ja110567j (DOI)
Available from: 2010-12-21 Created: 2010-12-20 Last updated: 2017-04-19Bibliographically approved

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