The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain

The IGF-binding protein (IGFBP)-5 protein contains consensus heparin bindin g motifs in both its carboxy (C)-terminal and central domains, although onl y the C-terminal site has previously been shown to be functional. We have m ade two chimeric IGFBP proteins by switching domains between rat IGFBP-5 an d -2, named BP552 and BP522 to reflect the domains present, and a truncated rat IGFBP-5 mutant (1-168), named BP550. The ability of these proteins and wild-type (wt) IGFBPs-5 and -2 to bind to either IGFs or heparin was deter mined using biosensor real-time analysis and heparin ligand blotting respec tively. We report that the chimeric molecules have IGF binding affinities c omparable to those of the native IGFBPs from which they were derived and, a s expected, the binding of BP550 to IGFs was greatly compromised. More surp rising was the finding that the ability of BP552 and BP550 to bind to hepar in was equivalent to that of wtIGFBP-5, whereas wtIGFBP-2 and BP522 failed to bind. These results demonstrate that the active heparin binding site in BP552 and BP550 is contained within the central domain of IGFBP-5, and that this site is active only in the absence of the C-terminal domain. We subse quently mutated two basic amino acids (R136A:R137A) in the central consensu s binding sites between residues 132-140. This resulted in the loss of hepa rin binding for BP550, confirming the importance of these two basic amino a cids in the central domain heparin binding activity. In light of these find ings, we suggest that C-terminally truncated fragments of IGFBP-5 generated in vivo by proteolysis could retain heparin/extracellular matrix binding p roperties.