Mutation of three critical amino acids of the N-terminal domain of IGF-binding protein-3 essential for high affinity IGF binding

The N-terminal domain is conserved in all members of the IGF-binding protei n superfamily. Most recently, studies have demonstrated the importance of a n IGF-binding protein N-terminal hydrophobic pocket for IGF binding. To exa mine more critically the amino acids important for IGF binding within the f ull-length IGF-binding protein-3 protein while minimizing changes in the te rtiary structure, we targeted residues I56, L80, and L81 within the propose d hydrophobic pocket for mutation. With a single change at these sites to t he nonconserved glycine there was a notable decrease in binding. A greater reduction was seen when both L80 and L81 were substituted with glycine, and complete loss of affinity for lGF-I and IGF-II occurred when all three tar geted amino acids were changed to glycine. Furthermore, the ability of the IGF-binding protein-3 mutants to inhibit IGF-I-stimulated phosphorylation o f its receptor was a reflection of their affinity for IGF, with the lowest affinity mutants having the least inhibitory effect. These studies, thus, support the hypothesis that an N-terminal hydrophobic pocket is the primary site of high affinity binding of IGF to IGF-binding p rotein-3. The mutants provide a tool for future studies directed at IGF-dep endent and IGF-independent actions of IGF-binding protein-3.