Total alanine-scanning mutagenesis of insulin-like growth factor I (IGF-I)identifies differential binding epitopes for IGFBP-1 and IGFBP-3

Citation
Y. Dubaquie et Hb. Lowman, Total alanine-scanning mutagenesis of insulin-like growth factor I (IGF-I)identifies differential binding epitopes for IGFBP-1 and IGFBP-3, BIOCHEM, 38(20), 1999, pp. 6386-6396
Citations number
54
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
38
Issue
20
Year of publication
1999
Pages
6386 - 6396
Database
ISI
SICI code
0006-2960(19990518)38:20<6386:TAMOIG>2.0.ZU;2-Q
Abstract
The bioavailability of insulin-like growth factor I (IGF-I) in the serum an d tissues is controlled by members of the IGF binding protein family (IGFBP ). These proteins form high-affinity complexes with IGF-I and thereby eithe r inhibit or potentiate its mitogenic and metabolic effects. Thus, understa nding the IGF-IGFBP interaction at the molecular level is crucial for attem pts to modulate IGF-I activity in vivo. We have systematically investigated the binding contribution of each IGF-I amino acid side chain toward 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 affinity losses for IGFBP-3. In contrast, b inding 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 determinants for IGFBP-I: the corresponding alanine mutations, E3A and F49A, selectively decreased IGFBP-1 binding by 34- and 100-fold, w hereas IGFBP-3 affinity was not affected or reduced maximally 4-fold. No si de chain specificity determinant was found for IGFBP-3. Instead, our result s suggest that the N-terminal backbone region of IGF-I is important for bin ding to IGFBP-3. The fact that the functional binding epitopes on IGF-I are overlapping but distinct for both binding proteins may be exploited to des ign binding protein-specific IGF variants.