P. Pomies et al., CONTROL OF THE ALPHA(5)BETA(1) INTEGRIN FIBRONECTIN INTERACTION IN-VITRO BY THE SERINE/THREONINE PROTEIN PHOSPHATASE CALCINEURIN/, Biochemistry, 34(15), 1995, pp. 5104-5112
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.