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.
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Laboratoire d'Etude des Systèmes Adhésifs Cellulaires, URA 1815 du CNRS, Université Joseph Fourier, Grenoble, France.
Frachet
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D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005353:Fibronectins / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011233:Precipitin Tests; D017469:Receptors, Fibronectin / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D005353:Fibronectins; D016023:Integrins; D017469:Receptors, Fibronectin; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium