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J. Biol. Chem., Vol. 279, Issue 51, 53232-53240, December 17, 2004

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Role of N- and C-terminal Residues of Insulin-like Growth Factor (IGF)-binding Protein-3 in Regulating IGF Complex Formation and Receptor Activation^*

Xiaolang Yan, Briony E. Forbes, Kerrie A. McNeil, Robert C. Baxter, and Sue M. Firth¶

From the Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia and the School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia 5005, Australia

Insulin-like growth factor-binding protein-3 (IGFBP-3), the major IGFBP in the circulation, sequesters IGF in a stable ternary complex with the acid-labile subunit. The high affinity IGF-binding site is proposed to reside within an N-terminal hydrophobic domain in IGFBP-3, but C-terminal residues have also been implicated in the homologous protein IGFBP-5. We have mutated in various combinations Leu⁷⁷, Leu⁸⁰, and Leu⁸¹ in the N terminus and Gly²¹⁷ and Gln²²³ in the C terminus of IGF-BP-3. All mutants retained immunoreactivity toward a polyclonal IGFBP-3 antibody, whereas IGF ligand blotting showed that all of the mutants had reduced binding to IGFs. Both solution IGF binding assays and BIAcore analysis indicated that mutations to the N-terminal region caused greater reduction in IGF binding activity than C-terminal mutations. The combined N- and C-terminal mutants showed undetectable binding to IGF-I but retained <10% IGF-II binding activity. Reduced ternary complex formation was seen only in mutants that had considerably reduced IGF-I binding, consistent with previous studies indicating that the binary IGF·IGFBP-3 complex is required for acid-labile subunit binding. Decreased IGF binding was also reflected in the inability of the mutants to inhibit IGF-I signaling in IGF receptor overexpressing cells. However, when present in excess, IGFBP-3 analogs defined as non-IGF-binding by biochemical assays could still inhibit IGF signaling. This suggests that residual binding activity of IGFBP-3 mutants may still be sufficient to inhibit IGF biological activity and questions the use of such analogs to study IGF-independent effects of IGFBP-3.

Received for publication, August 16, 2004 , and in revised form, September 23, 2004.

^* This work was supported by Project Grant 211257 (to S. M. F. and R. C. B.) from the National Health and Medical Research Council of Australia. 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.

^¶ To whom correspondence should be addressed: Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia. Tel.: 61-2-9926-8486; Fax: 61-2-9926-8484; E-mail: sfirth{at}med.usyd.edu.au.

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