Heparin binding to insulin-like growth factor (IGF)-binding protein 5 (IGFBP-5) leads to a 17-fold decrease in its affinity for IGF-I, and a region that contains several basic amino acids (Arg(201)-Arg(218)) may be involved in this affinity shift. In the present study, mutagenesis was used to analyze the effect of substitutions for basic amino acids in the Arg(201)-Arg(218) region of IGFBP-5 on heparin-binding and the heparin-induced affinity shift. Nine mutant forms were prepared, Their association constants (K-alpha) for IGF-I were similar to native IGFBP-5, When 10 mu g/ml of heparin was added, the K-alpha of native IGFBP-5 decreased 17-fold, and the K-alpha of the K134A/R136A mutant decreased 16-fold. In contrast, substitutions for specific basic amino acids in the Arg(201)-Arg(218) region decrease the affinity shift to 1.1-3.2-fold, Lys(211) was especially important. When a mutant containing that single substitution was tested, heparin caused only a 2.5-fold reduction in IGF-I affinity. Affinity cross-linking studies showed that heparin was equipotent in inhibiting the formation of I-125-IGF-I . K134A/R136A mutant complexes compared to native IGFBP-5. In contrast, heparin had minimal effects on the formation of complexes between I-125-IGF-I and the other mutants. The heparin-binding activity of each mutant was determined. Four mutants, R201A/K202N, K202A/K206A/R207A, R201A/K202N/K206N/K208N, and K211N/R214A/K217A/R218A, had reduced heparin binding compared to native IGFBP-5. The other five mutants, including the K211N mutant, showed no change in heparin binding. The four mutants with reduced heparin binding could be dissociated from heparin-Sepharose with much lower NaCl concentrations, indicating that they had reduced affinity. These findings suggest that Arg(201), Lys(202), Lys(206), and Arg(214) are important for heparin binding. In contrast, Lys(211) , not important for the binding of IGFBP-5 to heparin, but substitution for it reduced the heparin-induced affinity shift.
