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

Abstract

Previous work from our laboratory demonstrates that the α 4β1 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 α4β 1-dependent cell adhesive activity. A strategy to localize α4β1 binding sites within the thrombin fragment of osteopontin involved performing a series of truncation analyses. Removal of the last 39 amino acids (130-168) 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 (131-143) and SVVYGLR (162-168), were found to block α4β1-dependent adhesion. The first peptide when coupled to Sepharose bound the α 4β1 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.