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
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.