Substitution of specific amino acids in insulin-like growth factor (IGF) binding protein 5 alters heparin binding and its change in affinity for IGF-I response to heparin.
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 (Arg201-Arg218) 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 Arg201-Arg218 region of IGFBP-5 on heparin-binding and the heparin-induced affinity shift. Nine mutant forms were prepared. Their association constants (Ka) for IGF-I were similar to native IGFBP-5. When 10 microg/ml of heparin was added, the Ka of native IGFBP-5 decreased 17-fold, and the Ka of the K134A/R136A mutant decreased 16-fold. In contrast, substitutions for specific basic amino acids in the Arg2O1-Arg218 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 125I-IGF-I-K134A/Rl36A mutant complexes compared to native IGFBP-5. In contrast, heparin had minimal effects on the formation of complexes between 125I-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 K21IN 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 Arg201 Lys202, LysS206, and Arg214 are important for heparin binding. In contrast, LyS211 is not important for the binding of IGFBP-5 to heparin, but substitution for it reduced the heparin-induced affinity shift.
Arai
T
T
Department of Medicine, University of North Carolina, School of Medicine, Chapel Hill 27599, USA.
Clarke
J
J
Parker
A
A
Busby
W
W
Nam
T
T
Clemmons
D R
DR
D000345:Affinity Labels; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000235:genetics; D006025:Glycosaminoglycans / Q000378:metabolism; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D006025:Glycosaminoglycans; D018975:Insulin-Like Growth Factor Binding Protein 5; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D006493:Heparin