Total Alanine-Scanning Mutagenesis of Insulin-like Growth Factor I (IGF-I) Identifies Differential Binding Epitopes for IGFBP-1 and IGFBP-3

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• 作者：Yves Dubaquié ; and Henry B. Lowman
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：May 18, 1999
• 年：1999
• 卷：38
• 期：20
• 页码：6386 - 6396
• 全文大小：180K
• 年卷期：v.38,no.20(May 18, 1999)
• ISSN：1520-4995

文摘

The bioavailability of insulin-like growth factor I (IGF-I) in the serum and tissues is controlledby members of the IGF binding protein family (IGFBP). These proteins form high-affinity complexeswith IGF-I and thereby either inhibit or potentiate its mitogenic and metabolic effects. Thus, understandingthe IGF-IGFBP interaction at the molecular level is crucial for attempts to modulate IGF-I activity invivo. We have systematically investigated the binding contribution of each IGF-I amino acid side chaintoward IGFBP-1 and IGFBP-3, combining alanine-scanning mutagenesis and monovalent phage display.Surprisingly, most IGF-I residues could be substituted by alanines, resulting in less than 5-fold affinitylosses for IGFBP-3. In contrast, binding of IGFBP-1 was more sensitive to alanine substitutions in IGF-I.The glutamate and phenylalanine at positions 3 and 49 were identified as major specificity determinantsfor IGFBP-1: the corresponding alanine mutations, E3A and F49A, selectively decreased IGFBP-1 bindingby 34- and 100-fold, whereas IGFBP-3 affinity was not affected or reduced maximally 4-fold. No sidechain specificity determinant was found for IGFBP-3. Instead, our results suggest that the N-terminalbackbone region of IGF-I is important for binding to IGFBP-3. The fact that the functional binding epitopeson IGF-I are overlapping but distinct for both binding proteins may be exploited to design binding protein-specific IGF variants.