STRUCTURE-FUNCTION ANALYSIS OF THE HUMAN INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-4

To identify the molecular mechanism by which insulin-like growth facto r binding protein-4 (IGFBP-4) exerts its inhibitory effects on insulin -like growth factor (IGF) actions, we localized and determined the rol e of the IGF binding domain in modulating IGF actions in human osteobl asts. Deletion analysis using IGFBP-4 expressed in bacteria revealed t hat the N-terminal sequence Leu(72)-Ser(91) was essential for IGF bind ing. The C-terminal fragments (His(121)-Glu(237) or Arg(142)-Glu(237)) did not bind to IGF but loss of these regions decreased IGF binding a ctivity. Detailed deletion analysis identified the residues Cys(205)-V al(214) as the motif to facilitate IGF binding. Mitogenic studies reve aled that an IGFBP-4 mutant (His(74) replaced by Pro(74)) and an N-ter minal peptide (N terminus to Thr(71)) with little IGF binding activity failed to inhibit IGF-II-induced human osteoblast proliferation. An N -terminal peptide (N terminus to Asn(182)) with reduced IGF binding ac tivity inhibited IGF action but with lower potency. In contrast, an IG FBP-4 mutant (His(74) replaced with Ala(74)) exhibited similar IGF bin ding activity and potency in inhibiting the activity of IGF-II compare d with the wild type. Therefore, the N-terminal sequence (Leu(72)-Ser( 91)) and the C-terminal sequence (Cys(205)-Val(214)) are necessary to form the high affinity IGF binding domain, which is the major structur al determinant of the IGFBP-4 function.