Modulation of human IGF binding protein-3 activity by structural modification

To delinate regions of IGFBP-3 involved in ligand and cell-surface binding, DNAs encoding human IGFBP-3[1–264] and several variants were transfected into CHO cells. Of three deletion (Δ) mutants, IGFBP-3[1–88], [1–184], and [Δ89–184], none bound IGF-I tracer by ligand blotting, although all were detectable by immunoblotting. No ALS binding was detectable, as predicted by the lack of IGF binding. Normal-sequence IGFBP-3 associated with the CHO cells and was partly displaceable by IGF-I. Whereas IGFBP-3[1–88] and [1–184] failed to cell-associate, the non-IGF-binding central deletion variant [Δ89–184] did associate with CHO cells but was not displaced by IGF-I. To further examine the role of the carboxy-terminal domain in cell-association, the basic sequence IGFBP-3[228–232] (KGRKR) was altered to the corresponding IGFBP-1 residues MDGEA, a major charge reversal. This variant showed reduced IGF-I binding, and bound ALS with decreased affinity as determined by Scatchard analysis. It showed no cell binding, implicating the basic domain in cell-association. We conclude that, whereas the central and carboxy-terminal domain deletions fail to bind IGF-I, the ability to cell associate requires the carboxy-terminal but not the central domain. Specifically, the basic region [228–232] is essential for cell binding, and also affects IGF-I binding, and independently, ALS affinity.