ALANINE SCREENING MUTAGENESIS ESTABLISHES TYROSINE-60 OF BOVINE INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-2 AS A DETERMINANT OF INSULIN-LIKE-GROWTH-FACTOR BINDING

The determinants of insulin-like growth factor (IGF) binding to its bi nding proteins (IGFBPs) are poorly characterized in terms of important residues in the IGFBP molecule. We have previously used tyrosine iodi nation to implicate Tyr-60 in the IGF-binding site of bovine IGFBP-2 ( Hobba, G. D., Forbes, B. E., Parkinson, E. J., Francis, G;. L., and Wa llace, J. C, (1996) J. Biol. Chem. 271, 30529-30536). In this report, we show that the mutagenic replacement of Tyr-60 with either Ala or Ph e reduced the affinity of bIGFBP-2 for IGF-I (4.0- and 8.4-fold, respe ctively) and for IGF-II (3.5- and 4.0-fold, respectively). Although ad jacent residues Val-59, Thr-61, Pro-62, and Arg-63 are well conserved in IGFBP family members, Ala substitution for these residues did not r educe the IGF affinity of bIGFBP-2. Kinetic analysis of the bIGFBP-2 m utants on IGF biosensor chips in the BIAcore instrument revealed that Tyr-60 --> Phe bIG-FBP-2 bound to the IGF-I surface 3.0-fold more slow ly than bIGFBP-2 and was released 2.6-fold more rapidly than bIGFBP-2. We therefore propose that the hydroxyl group of Tyr-60 participates i n a hydrogen bond that is important for the initial complex formation with IGF-I and the stabilization of this complex. In contrast, Tyr-60 --> Ala bIGFBP-2 associated with the IGF-I surface 5.0-fold more rapid ly than bIGFBP-2 but exhibited an 18.4-fold more rapid release from th is surface compared with bIGFBP-2. Thus both the aromatic nature and t he hydrogen bonding potential of the tyrosyl side chain of Tyr-60 are important structural determinants of the IGF-binding site of bIGFBP-2.