Structural determinants of ligand and cell surface binding of insulin-like growth factor-binding protein-3.
Among the well defined insulin-like growth factor (IGF)-binding proteins (IGFBPs), IGFBP-3 is characterized by its interaction with an acid-labile glycoprotein (ALS) in the presence of IGFs. To identify the structural determinants on IGFBP-3 required for ligand binding and cell association, five recombinant human IGFBP-3 variants were expressed in Chinese hamster ovary cells: deletions of amino acids 89-264, 89-184, and 185-264, and site-specific mutations 228KGRKR --> MDGEA and 253KED --> RGD. The basic carboxyl-terminal region of IGFBP-3 was required for binding to heparin. The deletion variants had greatly decreased IGF binding ability as assessed by ligand blotting and solution binding assays; affinity cross-linking indicated at least a 20-fold decrease in IGF affinity. The RGD mutant had a 4-6-fold reduced affinity for both IGFs, but the MDGEA mutant bound IGF-I with near normal affinity and IGF-II with a 3-fold reduction in affinity. The three deletion variants were incapable of binding ALS; but of the site-specific variants, the MDGEA mutant bound ALS with 90% lower affinity (Ka = 2.5 +/- 0.9 liters/nmol) than seen for rhIGFBP-3 (Ka = 24.3 +/- 5.2 liters/nmol), whereas the RGD mutation had no effect on ALS affinity (Ka = 21.7 +/- 4.5 liters/nmol). The ability of IGFBP-3 to associate with the cell surface was lost in variants lacking residues 185-264 and in the 228KGRKR --> MDGEA mutant. We conclude that residues 228-232 of IGFBP-3 are essential for cell association and are required for normal ALS binding affinity.
Firth
S M
SM
Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. sfirth@med.usyd.edu.au
Ganeshprasad
U
U
Baxter
R C
RC
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008024:Ligands; D016296:Mutagenesis; D011485:Protein Binding; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D018076:DNA, Complementary; D018972:Insulin-Like Growth Factor Binding Protein 3; D008024:Ligands; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II