Rapid Ca2+-mediated activation of Rap1 in human platelets

Abstract

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 α‐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.

Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells: lack of Rap 1 activation in variant cells

“…Many growth factors and other agents including cAMP activate Rap 1 in various cell types but the precise role of Rap 1 in cellular signaling is unknown (Altschuler et al, 1995;Franke et al, 1997;Posern et al, 1998;M'Rabet et al, 1998;Bos et al, 1997;. Clearly, Rap 1 functions in more ways than as a Ras antagonist: it directly activates B-Raf kinase in PC-12 cells and it is mitogenic and oncogenic in Swiss 3T3 cells (Vossler et al, 1997;Yoshida et al, 1992;Altschuler and Ribeiro-Neto, 1998).…”

“…Rap 1A is the predominant isoform of Rap 1 in human neutrophils (Quilliam et al, 1991) and since we have made similar observations in HL-60 cells (Scheele JS and Boss GR, unpublished data), it seems likely we are observing increased Rap 1A activation. We compared our method for assessing Rap 1 activation with the immunoblotting method described by Franke et al (1997): the immunoblotting method barely detected an increase in Rap´GTP in HL-60 cells treated for 5 min with 8-pCPT-cAMP ( Figure 3c, compare lanes 2 and 4), indicating it was less sensitive than measuring Rap 1 activation quantitatively by the enzymatic method. When we incubated cell extracts with GTP prior to adding electrophoresis sample bu er and generating immunoblots, we found a similar amount of Rap 1 in these samples as in the total cell lysate (Figure 3c, compare lanes 3 and 5 to lane 1) con®rming that the exchange of GTP for GDP was complete and that the GST-tagged RBD peptide quantitatively precipitated Rap 1 from cell extracts.…”

“…The study of the role of Rap 1A/1B in intracellular signaling has been hampered by the lack of a suitable assay to measure their activation state. Franke et al (1997) described an innovative method to assess Rap 1 activation that uses the Rap binding domain (RBD) of Ral GDS as an activation-speci®c probe. This 97 amino acid peptide has an extremely high a nity for Rap´GTP with a K D of 10 nM whereas its a nity for Rap´GDP is undetectably low (Herrmann et al, 1996;Franke et al, 1997).…”

“…Franke et al (1997) described an innovative method to assess Rap 1 activation that uses the Rap binding domain (RBD) of Ral GDS as an activation-speci®c probe. This 97 amino acid peptide has an extremely high a nity for Rap´GTP with a K D of 10 nM whereas its a nity for Rap´GDP is undetectably low (Herrmann et al, 1996;Franke et al, 1997). By adding a tag to the RBD peptide, it can be immobilized on a solid phase allowing Rap´GTP to be removed e ciently from cell extracts; the precipitated Rap´GTP is detected by Western immunoblotting .…”

Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N)

“…It is even more confusing that Rap1 has also been described to act similar to Ras by stimulating DNA synthesis in Swiss 3T3 cells (Yoshida et al, 1992) and activating B-Raf in a cell free system (Ohtsuka et al, 1996). In addition, Ras-independent actions of Rap have been reported, such as the regulation of platelet aggregation (Altschuler et al, 1995;Franke et al, 1997) and the oxidative burst (Maly et al, 1994;Gabig et al, 1995). Thus, although Rap1 is now known to a ect multiple processes, a clear picture of its biological function still needs to be elucidated.…”