Structural determinants of ligand and cell surface binding of insulin-like growth factor-binding protein-3.

The Journal of Biological Chemistry
S M FirthR C Baxter

Abstract

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 =...Continue Reading

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