IDENTIFICATION AND CHARACTERIZATION OF CELLULAR-BINDING PROTEINS (RECEPTORS) FOR RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2B, AN INITIATOR OF BONE DIFFERENTIATION CASCADE

Bone morphogenetic protein 2B (BMP 2B), is a heparin-binding bone differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously. The molecular mechanism of action of this differentiation factor is not known, and as a first step we have examined BMP 2B-responsive cells for the presence of specific cellular binding proteins. Using I-125-labeled BMP 2B, specific high-affinity binding sites for recombinant human BMP 2B on MC3T3 E1 osteoblast-like cells as well as on NIH 3T3 fibroblasts were identified. Platelet-derived growth factor, insulin-like growth factor 1, basic fibroblast growth factor, epidermal growth factor, and transforming growth factor-beta did not compete for the binding of radiolabeled BMP 2B. The binding of BMP 2B is a time- and temperature-dependent process. Chemical crosslinking of radiolabeled BMP showed two components (apparent size, 200 and 70 kDa in MC3T3 E1 cells and 200 and 90 kDa in NIH 3T3 cells). A minor component at 60 kDa was also detected in both cell lines. Scatchard analysis of the binding data showed a high-affinity receptor with an apparent dissociation constant of 128 +/- 40 pM in MC3T3 E1 cells. These data demonstrate specific, high-affinity cell-surface binding proteins for BMP 2B.