Interaction of the low-molecular-weight GTP-binding protein rap2 with the platelet cytoskeleton is mediated by direct binding to the actin filaments

The interaction of the low-molecular-weight GTP-binding protein rap2 with t he cytoskeleton from thrombin-aggregated platelets was investigated by indu cing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskele ton was spontaneously restored after one cycle of actin depolymerization an d repolymerization. Exogenous rap2, but not unrelated proteins, added to de polymerized actin and solubilized actin-binding proteins, was also specific ally incorporated into the in vitro reconstituted cytoskeleton. The incorpo ration of exogenous rap:! was also observed when the cytoskeleton from rest ing or thrombin-activated platelets was subjected to actin depolymerization -repolymerization. Moreover, such interaction occurred equally well when ex ogenous rap2 was loaded with either GDP or GTP gamma S. We also found that polyhistidine-tagged rap2 immobilized on Ni2+-Sepharose and loaded with eit her GDP or GTP gamma S, could specifically bind to cytoskeletal actin. More over, when purified monomeric actin was induced to polymerize in vitro in t he presence of rap2, the small G-protein specifically associated with the a ctin filaments. Finally, rap2 loaded with either GDP or GTP gamma S was abl e to bind to purified F-actin immobilized on a plastic surface. These resul ts demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein. (C) 1999 Wiley-Liss, Inc.