ALANINE SCREENING MUTAGENESIS ESTABLISHES TYROSINE-60 OF BOVINE INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-2 AS A DETERMINANT OF INSULIN-LIKE-GROWTH-FACTOR BINDING
Gd. Hobba et al., 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 Journal of biological chemistry, 273(31), 1998, pp. 19691-19698
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.