Interaction of Insulin-Like Growth Factor-Binding Protein 3 With Hyaluronan and Its Regulation by Humanin and CD44

Insulin-like growth factor-binding protein-3 (IGFBP-3) belongs to a family of IGF-binding proteins. Humanin is a peptide known to bind residues 215-232 of mature IGFBP-3 in the C-terminal region of the protein. This region of IGFBP-3 was shown earlier to bind certain glycosaminoglycans including hyaluronan (HA). Here, we characterized the binding affinities of the IGFBP-3 protein and peptide (²¹⁵-KKGFYKKKQCRPSKGRKR-²³²) to HA and to humanin and found that HA binds with a weaker affinity to this region than does humanin. Either HA or humanin could bind to this IGFBP-3 segment, but not simultaneously. The HA receptor, CD44, blocked HA binding to IGFBP-3 but had no effect on binding of humanin to either IGFBP-3 or its peptide. Upon incubation of HA with CD44 and either IGFBP-3 protein or peptide, humanin was effective at binding and sequestering IGFBP-3 or peptide, thereby enabling access of CD44 to HA. We show that IGFBP-3 and humanin in the medium of A549 lung cancer cells can immunoprecipitate in a complex. However, the fraction of IGFBP-3 in the medium that is able to bind HA was not complexed with humanin suggesting that HA binding to the 215-232 segment renders it inaccessible for binding to humanin. Moreover, while the cytotoxic effects of IGFBP-3 on cell viability were reversed by humanin, blocking HA-CD44 interaction with an anti-CD44 antibody in combination with IGFBP-3 did not have an additive negative effect on cell viability suggesting that IGFBP-3 exerts its cytotoxic effects on cell survival through a mechanism that depends on HA-CD44 interactions.