Osteoblast-like cells secrete insulin-like growth factor (IGF) binding protein-5 (IGFBP-5), which may act to enhance IGF-stimulated osteoblast function. We recently demonstrated that carboxyl-truncated IGFBP-5 (IGFBP-5) binds to the osteoblast surface and stimulates mitogenesis by a pathway that is independent of IGF action. The present study was conducted to determine the mechanism of osteoblast binding of IGFBP-5, beginning with the assumption that cell surface glycosaminoglycans may mediate the binding of this heparin binding protein. Intact I-IGFBP-5 and I-IGFBP-5 exhibited one-site binding to mouse osteoblast monolayers with dissociation constants of 28 and 6 nM for intact I-IGFBP-5 and I-IGFBP-5, respectively. Osteoblast binding of intact I-IGFBP-5 was inhibited by low heparin concentrations, while I-IGFBP-5 binding was stimulated by heparin. Treatment of cells with heparinase or chlorate to decrease surface glycosaminoglycan density failed to reduce the binding of either form of IGFBP-5. In contrast, pretreatment of cells with IGFBP-5 caused down-regulation of I-IGFBP-5 binding. Cross-linking studies revealed that both intact I-IGFBP-5 and I-IGFBP-5 bind to proteins in Triton extracts of osteoblast membranes, which were absent in osteoblast-derived matrix. Purification of membrane extracts by IGFBP-5 affinity chromatography revealed a 420-kDa band on reduced SDS-polyacrylamide gels. While the membrane protein internalized both forms of IGFBP-5, heparin treatment inhibited the internalization of intact I-IGFBP-5 but stimulated I-IGFBP-5 internalization. These data indicate that IGFBP-5 binds to and is internalized by an osteoblast membrane protein, which does not appear to be a proteoglycan. Glycosaminoglycans, however, modulate the binding and internalization of IGFBP-5 in a way that may preferentially favor the intracellular accumulation of the carboxyl-truncated form.

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