The Integrin α₉β₁ Binds to a Novel Recognition Sequence (SVVYGLR) in the Thrombin-cleaved Amino-terminal Fragment of Osteopontin*

The integrin α₉β₁ mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH₂-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for α₉β₁. In this report, we used site-directed mutagenesis and synthetic peptides to identify the α₉β₁ recognition sequence in osteopontin. α₉-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH₂-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-α₉ monoclonal antibody Y9A2, indicating the presence of a non-RGD α₉β₁recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on α₉β₁-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr¹⁶⁵, blocked α₉β₁-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin β₁ subunit on α₉-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to α₉β₁ and is responsible for α₉β₁-mediated cell adhesion to the NH₂-terminal fragment of osteopontin.