CONTROL OF THE ALPHA(5)BETA(1) INTEGRIN/FIBRONECTIN INTERACTION IN-VITRO BY THE SERINE/THREONINE PROTEIN PHOSPHATASE CALCINEURIN

Using Chinese hamster ovary cell lysate, an in vitro assay has been developed to study the interaction of fibronectin with the alpha(5) beta(1) integrin in a cytosolic environment. In our solid phase assay, 96-well microtiter plates were coated with fibronectin in which cell lysate was incubated. A dose-dependent binding of the fibronectin receptor onto the coated plastic was immunodetected by specific polyclonal antibodies raised against the alpha(5) beta(1) integrin. Both soluble fibronectin and PB1, a monoclonal antibody raised against the fibronectin receptor, competed with the alpha(5) beta(1) integrin for binding to the fibronectin-coated plastic. General phosphatase inhibitors used during cell lysis completely abolished the fibronectin/integrin interaction in the assay, indicating that the affinity of the fibronectin receptor might be modulated by a protein phosphatase activity. Furthermore, in this assay, the interaction between the fibronectin receptor and its substrate in a cytosolic environment required intracellular calcium. Additionally, the action of more specific phosphatase inhibitors and the inhibition of the integrin/fibronectin interaction by a monoclonal antibody raised against the calcium/calmodulin-dependent protein phosphatase calcineurin suggested that calcineurin allowed the interaction between the alpha(5) beta(1) integrin and fibronectin. Metabolical labeling experiments showed that alpha(5) beta(1) itself was not the target of phosphorylation/dephosphorylation cascades involving calcineurin and leading to the modulation of integrin affinity. Taken together, these results showed that in vitro one substrate of the serine/threonine protein phosphatase calcineurin regulates the alpha(5) beta(1) integrin affinity by interacting with a yet unidentified effector.