Direct binding of the platelet integrin alphaIIbbeta3 (GPIIb-IIIa) to talin. Evidence that interaction is mediated through the cytoplasmic domains of both alphaIIb and beta3.
As a consequence of platelet activation and fibrinogen binding, glycoprotein (GP)IIb-IIIa (integrin alphaIIbbeta3) becomes associated with the cytoskeleton. Although talin has been suggested to act as a linkage protein mediating the attachment of GPIIb-IIIa to actin filaments, direct binding of GPIIb-IIIa to this cytoskeletal protein has not been demonstrated. In the present study, we examined the interaction of GPIIb-IIIa with purified talin using a solid-phase binding assay. Soluble GPIIb-IIIa bound in a time- and dose-dependent manner to microtiter wells coated with talin but not with BSA. Time course studies demonstrated that steady-state binding was achieved after 4-5 h incubation at 37 degrees C. Binding isotherms with varying concentrations of GPIIb-IIIa showed that half-saturation binding was achieved at approximately 15 nM GPIIb-IIIa. At saturation, there was 211 +/- 8 fmol of GPIIb-IIIa bound per well containing 117 +/- 10 fmol of immobilized talin. Besides binding to immobilized talin, GPIIb-IIIa also bound to talin captured by the anti-talin monoclonal antibody 8d4. Moreover, the interaction of GPIIb-IIIa to 8d4-captured talin was blocked by mAb10B2, a monoclonal antibody raised against a synthetic peptide encompassing the entire cytoplasmic sequence of GPIIb. The interaction of talin with the cytoplasmic domain of GPIIb-IIIa was further investigated using peptide-coated wells. Purified talin was found to bind to both synthetic peptides corresponding to the cytoplasmic sequences of GPIIb (P2b) and GPIIIa (P3a). As expected, the binding of talin to P2b-coated wells was specifically blocked by mAb10B2. Thus, these results demonstrate direct binding of GPIIb-IIIa to talin and suggest a role of the cytoplasmic sequences of both GPIIb and GPIIIa in mediating this interaction.
Knezevic
I
I
Department of Pharmacology, University of Illinois, Chicago, Illinois 60612, USA.
Leisner
T M
TM
Lam
S C
SC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016608:Talin / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009842:Oligopeptides; D010446:Peptide Fragments; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D016608:Talin; C047981:arginyl-glycyl-aspartic acid