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 the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPgammaS. We also found that polyhistidine-tagged rap2 immobilized on Ni(2+)-Sepharose and loaded with either GDP or GTPgammaS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPgammaS was able to bind to purified F-actin immobilized on a plastic surface. These results 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.
Torti
M
M
Department of Biochemistry, University of Pavia, 27100 Pavia, Italy. mtorti@unipv.it
Bertoni
A
A
Canobbio
I
I
Sinigaglia
F
F
Lapetina
E G
EG
Balduini
C
C
D000199:Actins / Q000378:metabolism*; D001710:Biotin; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002919:Chymotrypsinogen / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D017830:Octoxynol; D013917:Thrombin / Q000494:pharmacology; D020668:rap GTP-Binding Proteins / Q000378:metabolism*
D000199:Actins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001710:Biotin; D017830:Octoxynol; D002919:Chymotrypsinogen; D013917:Thrombin; C116756:RAP2A protein, human; D020668:rap GTP-Binding Proteins