Identification of Dual α₄β₁ Integrin Binding Sites within a 38 Amino Acid Domain in the N-terminal Thrombin Fragment of Human Osteopontin*

Previous work from our laboratory demonstrates that the α₄β₁ integrin is an adhesion receptor for OPN and that α₄β₁ binding site(s) are present in the N-terminal thrombin fragment of osteopontin (OPN) (Bayless, K. J., Meininger, G. A., Scholtz, J. M., and Davis, G. E. (1998) J. Cell Sci. 111, 1165–1174). The work presented here identifies two α₄β₁ binding sites within a recombinantly produced N-terminal thrombin fragment of human OPN. Initial experiments, using wild-type OPN containing an RGD sequence or an OPN-RGE mutant, showed identical α₄β₁-dependent cell adhesive activity. A strategy to localize α₄β₁binding sites within the thrombin fragment of osteopontin involved performing a series of truncation analyses. Removal of the last 39 amino acids (130) completely eliminated adhesion, indicating all binding activity was present within that portion of the molecule. Combined mutation and deletion analyses of this region revealed the involvement of dual α₄β₁ binding sites. Synthetic peptides for both regions in OPN, ELVTDFPTDLPAT (131) and SVVYGLR (162), were found to block α₄β₁-dependent adhesion. The first peptide when coupled to Sepharose bound the α₄β₁ integrin directly whereas a mutated ELVTEFPTELPAT peptide showed a dramatically reduced ability to bind. These data collectively demonstrate that dual α₄β₁ integrin binding sites are present in a 38 amino acid domain within the N-terminal thrombin fragment of OPN.