Direct demonstration of involvement of the adaptor protein ShcA in the regulation of Ca2+-induced platelet aggregation.
Platelet aggregation is mediated by conformational change of integrin alpha(IIb)beta(3). Tyrosine-phosphorylation of cytoplasmic domain of beta(3) upon platelet activation has been demonstrated to play a critical role in this process. Recently, the adaptor protein ShcA has been shown to bind to the tyrosine-phosphorylated beta(3), while it remains open whether ShcA plays any role in platelet aggregation. Here, we show that ShcA bound to tyrosine-phosphorylated beta(3)-tail peptide through its phosphotyrosine-binding domain in vitro. Then, we examined the involvement of ShcA in platelet aggregation by a previously established in vitro assay using platelets permeabilized with streptolysin-O, where exogenous addition of platelet cytosol is required for reconstitution of the Ca(2+)-induced aggregation. When ShcA was specifically depleted with anti-ShcA antibody from the cytosol, this ShcA-depleted cytosol lost the aggregation-supporting activity, which was rescued by addition of purified recombinant ShcA. Thus, ShcA is essential for the Ca(2+)-induced platelet aggregation.
Higashi
Tomohito
T
Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
Yoshioka
Akira
A
Shirakawa
Ryutaro
R
Tabuchi
Arata
A
Nishioka
Hiroaki
H
Kita
Toru
T
Horiuchi
Hisanori
H
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001792:Blood Platelets / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D039661:Integrin beta3 / Q000378:metabolism; D051379:Mice; D010974:Platelet Aggregation / Q000502:physiology*; D020033:Protein Isoforms / Q000235:genetics; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D039661:Integrin beta3; D020033:Protein Isoforms; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D002118:Calcium