A PROTEOLYTIC FRAGMENT OF INSULIN-LIKE GROWTH-FACTOR (IGF) BINDING PROTEIN-3 THAT FAILS TO BIND IGFS INHIBITS THE MITOGENIC EFFECTS OF IGF-I AND INSULIN

Limited proteolysis of insulin-like growth factor binding protein-3 (I GFBP-3) is increasingly becoming recognized as an essential mechanism in the regulation of insulin-like growth factor (IGF) bioavailability, both in the bloodstream and at cellular level. Plasmin generated on c ontact with various cell types provokes proteolytic cleavages that ape similar to those induced in vivo by (as yet unidentified) IGFBP-3 pro teases. Experimental conditions were determined to achieve plasmin-ind uced limited proteolysis of recombinant human nonglycosylated IGFBP-9. Two major fragments of 22/25 kilodaltons (kDa) and one of 16 kDa were identified by Western immunoblotting and isolated by reverse-phase ch romatography, The 22/25-kDa fragments correspond to the major approxim ate to 30-kDa glycosylated fragment of IGFBP-3 in serum and the 16-kDa fragment, to one of the same size, that is nonglycosylated. Western L igand blot analysis, affinity cross-linking, and competitive binding e xperiments using radiolabeled IGF and unlabeled IGF-I or -II showed th at in the high performance liquid chromatography eluate containing the 16-kDa fragment, all affinity for IGFs had been lost, whereas the aff inity of the 22/25-kDa fragment was considerably reduced. Scatchard an alysis of the data indicated a 20-fold loss of affinity for IGF-II and an 50-fold loss for IGF-I compared with that of recombinant human IGF BP-3. In a chick embryo fibroblast assay in which DNA synthesis was st imulated both by IGF-I and by insulin (at 100-fold concentrations, so that interaction with the Type 1 IGF receptor would occur), IGFBP-3 wa s found to inhibit IGF-I-induced stimulation almost totally. It had no effect on stimulation by insulin, which has no affinity for the IGFBP s. With the 22/25-kDa fragments, barely 50% inhibition of IGF-I stimul ation was achieved and no inhibition of insulin stimulation. Unexpecte dly, with the fraction containing the 16-kDa fragment (despite the tot al lack of affinity for IGF-I), IGF-I-induced stimulation was inhibite d to nearly the same extent as with intact IGFBP-3. In addition, insul in-induced stimulation was inhibited with similar potency. IGFBP-3 pro teolysis therefore generates two types of fragment with different acti vities. One has weak affinity for IGF-I and is only a weak antagonist of IGF action. The other lacks affinity for the IGFs, but nevertheless inhibits IGF-stimulated mitogenesis, thus acting by a mechanism that is independent of the IGFs.