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Rapid Ca2+-mediated activation of Rap1 in human platelets.

The EMBO journal (1997-01-15)
B Franke, J W Akkerman, J L Bos
RESUMEN

Rap1 is a small, Ras-like GTPase whose function and regulation are still largely unknown. We have developed a novel assay to monitor the active, GTP-bound form of Rap1 based on the differential affinity of Rap1GTP and Rap1GDP for the Rap binding domain of RalGDS (RBD). Stimulation of blood platelets with alpha-thrombin or other platelet activators caused a rapid and strong induction of Rap1 that associated with RBD in vitro. Binding to RBD increased from undetectable levels in resting platelets to >50% of total Rap1 within 30 s after stimulation. An increase in the intracellular Ca2+ concentration is both necessary and sufficient for Rap1 activation since it was induced by agents that increase intracellular Ca2+ and inhibited by a Ca2+-chelating agent. Neither inhibition of translocation of Rap1 to the cytoskeleton nor inhibition of platelet aggregation affected thrombin-induced activation of Rap1. In contrast, prostaglandin I2 (PGI2), a strong negative regulator of platelet function, inhibited agonist-induced as well as Ca2+-induced activation of Rap1. From our results, we conclude that Rap1 activation in platelets is an important common event in early agonist-induced signalling, and that this activation is mediated by an increased intracellular Ca2+ concentration.

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Sigma-Aldrich
Rap1 Assay Reagent (Ral GDS-RBD, agarose), 650 µg, Reagent (Ral GDS-RBD, agarose). A GST-tagged fusion protein, corresponding to amino acids 788-884 of human Ral GDS-Rap binding domain (RBD), for use in Affinity Binding Assays.
Sigma-Aldrich
Rap1 Activation Assay Kit, Non-radioactive Rap1 Activation Assay Kit that uses Ral GDS RBD, agarose (Catalog # 14-455) to precipitate Rap1-GTP from cell lysates & detection by a Rap1 specific antibody.