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 de veloped 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 lysa te was incubated. A dose-dependent binding of the fibronectin receptor onto the coated plastic was immunodetected by specific polyclonal ant ibodies raised against the alpha(5) beta(1) integrin. Both soluble fib ronectin 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 du ring cell lysis completely abolished the fibronectin/integrin interact ion in the assay, indicating that the affinity of the fibronectin rece ptor 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 calciu m. Additionally, the action of more specific phosphatase inhibitors an d the inhibition of the integrin/fibronectin interaction by a monoclon al antibody raised against the calcium/calmodulin-dependent protein ph osphatase calcineurin suggested that calcineurin allowed the interacti on between the alpha(5) beta(1) integrin and fibronectin. Metabolical labeling experiments showed that alpha(5) beta(1) itself was not the t arget of phosphorylation/dephosphorylation cascades involving calcineu rin and leading to the modulation of integrin affinity. Taken together , these results showed that in vitro one substrate of the serine/threo nine protein phosphatase calcineurin regulates the alpha(5) beta(1) in tegrin affinity by interacting with a yet unidentified effector.