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J. Biol. Chem., Vol. 283, Issue 9, 5355-5363, February 29, 2008

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An Investigation of the Ligand Binding Properties and Negative Cooperativity of Soluble Insulin-like Growth Factor Receptors^*

Katharina Helen Surinya, Briony E. Forbes, Filomena Occhiodoro, Grant W. Booker, Geoffrey Leonard Francis^¶, Kenneth Siddle^||, John C. Wallace, and Leah J. Cosgrove¹

From the School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia, Commonwealth Scientific and Industrial Research Organisation, Molecular and Health Technologies, P.O. Box 10041 BC, Adelaide 5000, Australia, ^¶Novozymes-GroPep Ltd., P.O. Box 10065 BC, Adelaide 5000, Australia, and the ^||Department of Clinical Biochemistry, University of Cambridge, Cambridge CB2 2QR, United Kingdom

To investigate the interaction of the insulin-like growth factor (IGF) ligands with the insulin-like growth factor type 1 receptor (IGF-1R), we have generated two soluble variants of the IGF-1R. We have recombinantly expressed the ectodomain of IGF-1R or fused this domain to the constant domain from the Fc fragment of mouse immunoglobulin. The ligand binding properties of these soluble IGF-1Rs for IGF-I and IGF-II were investigated using conventional ligand competition assays and BIAcore biosensor technology. In ligand competition assays, the soluble IGF-1Rs both bound IGF-I with similar affinities and a 5-fold lower affinity than that seen for the wild type receptor. In addition, both soluble receptors bound IGF-II with similar affinities to the wild type receptor. BIAcore analyses showed that both soluble IGF-1Rs exhibited similar ligand-specific association and dissociation rates for IGF-I and for IGF-II. The soluble IGF-1R proteins both exhibited negative cooperativity for IGF-I, IGF-II, and the 24-60 antibody, which binds to the IGF-1R cysteine-rich domain. We conclude that the addition of the self-associating Fc domain to the IGF-1R ectodomain does not affect ligand binding affinity, which is in contrast to the soluble ectodomain of the IR. This study highlights some significant differences in ligand binding modes between the IGF-1R and the insulin receptor, which may ultimately contribute to the different biological activities conferred by the two receptors.

Received for publication, August 22, 2007 , and in revised form, December 3, 2007.

^* This work was supported by an Australian Government Cooperative Research Centre grant. 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. 1 and 2.

¹ To whom correspondence should be addressed: CSIRO Molecular and Health Technologies, P.O. Box 10041 BC, Adelaide 5000, Australia. Tel.: 61-8-8303-8833; Fax: 61-8-8303-8899; E-mail: leah.cosgrove{at}csiro.au.

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