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J. Biol. Chem., Vol. 283, Issue 30, 20821-20829, July 25, 2008

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Alanine Scanning of a Putative Receptor Binding Surface of Insulin-like Growth Factor-I^*

Lisbeth Gauguin¹, Carlie Delaine, Clair L. Alvino, Kerrie A. McNeil, John C. Wallace, Briony E. Forbes, and Pierre De Meyts

From the Receptor Systems Biology Laboratory, Hagedorn Research Institute, 2820 Gentofte, Denmark and School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia

Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor. The interaction of these two surfaces with the insulin receptor results in a high affinity cross-linking of the two receptor subunits and leads to receptor activation. Evidence suggests that insulin-like growth factor-I (IGF-I) may activate the IGF-I receptor in a similar mode. So far IGF-I residues structurally corresponding to the residues of the insulin site 1 together with residues in the C-domain of IGF-I have been found to be important for binding of IGF-I to the IGF-I receptor (e.g. Phe²³, Tyr²⁴, Tyr³¹, Arg³⁶, Arg³⁷, Val⁴⁴, Tyr⁶⁰, and Ala⁶²). However, an IGF-I second binding surface similar to site 2 of insulin has not been identified yet. In this study, we have analyzed whether IGF-I residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor. Six single-substituted IGF-I analogues were produced, each containing an alanine substitution in one of the following positions (corresponding insulin residues in parentheses): Glu⁹ (His^B10), Asp¹² (Glu^B13), Phe¹⁶ (Leu^B17), Asp⁵³ (Ser^A12), Leu⁵⁴ (Leu^A13), and Glu⁵⁸ (Glu^A17). In addition, two analogues with 2 and 3 combined alanine substitutions were also produced (E9A,D12A IGF-I and E9A,D12A,E58A IGF-I). The results show that introducing alanine in positions Glu⁹, Asp¹², Phe¹⁶, Leu⁵⁴, and Glu⁵⁸ results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding. The multiple substitutions resulted in a 33–100-fold reduction in IGF-I receptor binding affinity. These data suggest that IGF-I, in addition to the C-domain, uses surfaces similar to those of insulin in contacting its cognate receptor, although the relative contribution of the side chains of homologous residues varies.

Received for publication, April 4, 2008 , and in revised form, May 8, 2008.

Note Added in Proof—After submission of this paper, Glendorf et al. (55) published an elegant systematic amino acid scanning of residues in the insulin B-chain central helix showing major effects of substitutions at B12 and B16 (in our site 1) and B10 and B13 (in our site 2), confirming the importance of the B-chain central helix as a receptor binding motif as previously shown by photoaffinity cross-linking (56).

^* 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.

¹ Supported in part by an Industrial Ph.D. scholarship from the Danish Ministry of Science, Technology, and Innovation. To whom correspondence should be addressed. Tel.: 45-44439317; Fax: 45-44438000; E-mail: lgau{at}hagedorn.dk.

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