Differential effect of the inhibition of Grb2-SH3 interactions in platelet activation induced by thrombin and by Fc receptor engagement.
The adaptor protein Grb2 (growth factor receptor-bound protein 2) is involved in cell proliferation via the Ras signalling pathway. In order to study the role of Grb2 in blood platelet responses, we used a peptide containing two proline-rich sequences derived from Sos (peptidimer), which binds to Grb2-Src homology 3 domain (SH3) with a high affinity, and hence inhibits Grb2-SH3-mediated protein interactions. Platelet aggregation and 5-hydroxytryptamine (serotonin) release measured in the presence of the peptidimer were: (i) significantly decreased when induced by thrombin; and (ii) potentiated when induced by the engagement of the Fc receptor. In thrombin-activated platelets, the Grb2-SH2 domain formed an association with the beta3 subunit of the alphaIIb-beta3 integrin (GPIIb-IIIa), Shc, Syk, Src and SHP1 (SH2-containing phosphotyrosine phosphatase 1), whereas these associations did not occur after the engagement of the receptor for the Fc domain of IgG (FcgammaRIIa) or in resting platelets. Grb2-SH3 domains formed an association with the proline-rich sequences of Sos and Cbl in both resting and activated platelets, since the peptidimer abolished these associations. Inhibition of both fibrinogen binding and platelet aggregation by the peptide RGDS (Arg-Gly-Asp-Ser) had no effect on thrombin-induced Grb2-SH2 domain association with the aforementioned signalling molecules, indicating that these associations occurred during thrombin-induced 'inside-out' signalling. Platelet aggregation induced by direct activation via alphaIIb-beta3 ('outside-in' signalling) was potentiated by the peptidimer. The results show that inhibition of Grb2-SH3 interactions with signal-transduction proteins down-regulates thrombin-induced platelet activation, but also potentiates Fc receptor- and alphaIIb-beta3-mediated platelet activation.
Saci
Abdelhafid
A
INSERM U428, Faculte de Pharmacie, 4 Avenue de l'Observatoire, Paris F-75006, France.
Liu
Wang-Qing
WQ
Vidal
Michel
M
Garbay
Christiane
C
Rendu
Francine
F
Bachelot-Loza
Christilla
C
D048868:Adaptor Proteins, Signal Transducing; D015536:Down-Regulation; D051380:GRB2 Adaptor Protein; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011961:Receptors, Fc / Q000378:metabolism*; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011506:Proteins; D011961:Receptors, Fc; C046617:arginyl-glycyl-aspartyl-serine; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin