TY - JOUR
T1 - A Gi-dependent pathway is required for activation of the small GTPase Rap1B in human platelets
AU - Lova, Paolo
AU - Paganini, Simona
AU - Sinigaglia, Fabiola
AU - Balduini, Cesare
AU - Torti, Mauro
PY - 2002/4/5
Y1 - 2002/4/5
N2 - Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcγRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca2+ concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcγRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the Gi-coupled ADP receptor P2Y12, but was not affected by blockade of the Gq-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca2+ mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca2+ signaling. In platelets stimulated by cross-linking of FcγRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the Gi-coupled α2A-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a Gq-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the Gq-coupled thromboxane A2 receptor by U46619 was completely inhibited by ADP scavengers under conditions in which intracellular Ca2+ mobilization was unaffected. Inhibition of U46619-induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a Gi-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B.
AB - Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcγRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca2+ concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcγRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the Gi-coupled ADP receptor P2Y12, but was not affected by blockade of the Gq-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca2+ mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca2+ signaling. In platelets stimulated by cross-linking of FcγRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the Gi-coupled α2A-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a Gq-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the Gq-coupled thromboxane A2 receptor by U46619 was completely inhibited by ADP scavengers under conditions in which intracellular Ca2+ mobilization was unaffected. Inhibition of U46619-induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a Gi-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B.
UR - http://www.scopus.com/inward/record.url?scp=0037023134&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111803200
DO - 10.1074/jbc.M111803200
M3 - Article
SN - 0021-9258
VL - 277
SP - 12009
EP - 12015
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 14
ER -