Distinct structural requirements for binding of the integrins αvβ6, αvβ3, αvβ5, α5β1 and α9β1 to osteopontin

The extracellular matrix protein, osteopontin, is a ligand for several members of the integrin family, including α5β1, αvβ3, αvβ5 and α9β1. Osteopontin is a substrate for a number of extracellular proteases, including thrombin and the metalloproteases MMP-3 and MMP-7, which cleave osteopontin at sites close to or within the mapped integrin binding sites. Using affinity chromatography and cell adhesion assays, we now identify the integrin αvβ6 as an additional osteopontin receptor. Utilizing a series of recombinant forms of osteopontin, we compared the structural requirements for αvβ6 binding with those for the 4 other osteopontin-binding integrins. Like α5β1, αvβ3 and αvβ5 (but not α9β1), αvβ6 binds to the RGD site in osteopontin, since RGD peptide or mutation of this site to RAA completely inhibits αvβ6-mediated cell adhesion. For both α9β1 and α5β1, the N-terminal fragment generated by thrombin cleavage is a much better ligand than full length osteopontin, whereas thrombin-cleavage does not appear to be required for optimal adhesion to αvβ3, αvβ5 or αvβ6. A recombinant fragment predicted to be generated by MMP cleavage no longer supported α5β1 or α9β1-mediated adhesion, but adhesion mediated by αvβ5 or αvβ6 was unaffected. Finally, adhesion of αvβ5 or αvβ6 was inhibited by mutation of two aspartic acid residues upstream of the RGD site, whereas adhesion mediated by αvβ3, α5β1 or α9β1 was unaffected by these mutations. These results suggest that the hierarchy of integrin interactions with osteopontin can undergo complex regulation at least in part through the action of extracellular proteases.