Identification of dual alpha(4)beta(1) 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 alpha (4)beta (1) i ntegrin is an adhesion receptor for OPN and that alpha (4)beta (1) 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 alph a (4)beta (1) binding sites within a recombinantly produced N-terminal thro mbin fragment of human OPN, Initial experiments, using wild-type OPN contai ning an RGD sequence or an OPN-RGE mutant, showed identical alpha (4)beta ( 1)-dependent cell adhesive activity. A strategy to localize alpha (4)beta ( 1) binding sites within the thrombin fragment of osteopontin involved perfo rming a series of truncation analyses, Removal of the last 39 amino acids ( 130-168) completely eliminated adhesion, indicating all binding activity wa s present within that portion of the molecule. Combined mutation and deleti on analyses of this region revealed the involvement of dual alpha (4)beta ( 1) binding sites, Synthetic peptides for both regions in OPN, ELVTDFPTDLPAT (131-143) and SVVYGLR (162-168), were found to block alpha (4)beta (1)-dep endent adhesion. The first peptide when coupled to Sepharose bound the alph a (4)beta (1) integrin directly whereas a mutated ELVTEFPTELPAT peptide sho wed a dramatically reduced ability to bind. These data collectively demonst rate that dual alpha (4)beta (1) integrin binding sites are present in a 38 amino acid domain within the N-terminal thrombin fragment of OPN.