A Biochemical Characterization of the Binding of Osteopontin to Integrins α_vβ₁ and α_vβ₅(∗)

Osteopontin (OPN) is an extracellular matrix protein that binds to integrin α_vβ₃. Here we demonstrate that two other integrins, α_vβ₁ and α_vβ₅, are also receptors for OPN. Human embryonic kidney 293 cells adhere to human recombinant osteopontin (glutathione S-transferase-osteopontin; GST-OPN) using integrin α_vβ₁. When the 293 cells are transfected with the β₅ subunit, they can also adhere to GST-OPN using integrin α_vβ₅. Divalent cations regulate the binding of GST-OPN to both α_vβ₁ and α_vβ₅. Mg²⁺ and Mn²⁺ support the binding of GST-OPN to these integrins but Ca²⁺ does not. The highest affinity is observed in Mn²⁺. In the presence of this ion, the affinity of GST-OPN for α_vβ₁ is 18 nM and the affinity for α_vβ₅ is 48 nM. The antibody 8A2, which is an agonist for β₁, promotes the adhesion of 293 cells to GST-OPN even when Ca²⁺ is present. This observation suggests that cellular events could modulate the affinity of α_vβ₁ for OPN. Collectively, these findings prove that integrins α_vβ₁, α_vβ₃, and α_vβ₅ have similar affinity for OPN. Therefore, all three integrins must be considered when evaluating the biological affects of OPN.