A biochemical characterization of the binding of osteopontin to integrins αvβ1 and αvβ5

Abstract

Osteopontin (OPN) is an extracellular matrix protein that binds to integrin αvβ3. Here we demonstrate that two other integrins, αvβ1 and αvβ5, are also receptors for OPN. Human embryonic kidney 293 cells adhere to human recombinant osteopontin (glutathione S-transferase-osteopontin; GST-OPN) using integrin αvβ1. When the 293 cells are transfected with the β5 subunit, they can also adhere to GST-OPN using integrin αvβ5. Divalent cations regulate the binding of GST-OPN to both αvβ1 and αvβ5. 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 αvβ1 is 18 nM and the affinity for αvβ5 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 αvβ1 for OPN. Collectively, these findings prove that integrins αvβ1, αvβ3, and αvβ5 have similar affinity for OPN. Therefore, all three integrins must be considered when evaluating the biological affects of OPN.