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J. Biol. Chem., Vol. 281, Issue 38, 28287-28295, September 22, 2006

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Solution Structure and Characterization of the LGR8 Receptor Binding Surface of Insulin-like Peptide 3^*

K. Johan Rosengren, Suode Zhang^¶, Feng Lin^¶, Norelle L. Daly¹, Daniel J. Scott^¶, Richard A. Hughes^||, Ross A. D. Bathgate^¶, David J. Craik², and John D. Wade^¶3

From the Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-392 81 Kalmar, Sweden, the Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia, and the ^¶Howard Florey Institute and ^||Department of Pharmacology, University of Melbourne, Parkville, Victoria 3010, Australia

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg^B16 and Val^B19, with His^B12 and Arg^B20 playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp^B27, to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Received for publication, April 21, 2006 , and in revised form, July 21, 2006.

The atomic coordinates and structure factors (code 2H8B) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from Åke Wiberg's Foundation and the University of Kalmar (to K. J. R.) and NHMRC Project Grants 350245 (to J. D. W., R. A. H., and R. A. D. B.) and 300012 (to R. A. D. B. and J. D. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Table S1.

¹ An NHMRC Industry Fellow.

² An ARC Professorial Fellow.

³ To whom correspondence should be addressed: Howard Florey Institute, University of Melbourne, Victoria 3010, Australia. Tel.: 61-3-8344-7285; Fax: 61-3-9348-1707; E-mail: j.wade{at}hfi.unimelb.edu.au.

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