title: Activation of the small GTPase Ral in platelets.
abstract: Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including alpha-thrombin. In contrast, the platelet antagonist prostaglandin I2 inhibited alpha-thrombin-induced Ral activation. Activation of Ral by alpha-thrombin could be inhibited by depletion of intracellular Ca2+, whereas the induction of intracellular Ca2+ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca2+ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.
authors.affiliation: Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
authors.forename: R M
authors.initials: RM
authors.lastname: Wolthuis
authors.forename: B
authors.initials: B
authors.lastname: Franke
authors.forename: M
authors.initials: M
authors.lastname: van Triest
authors.forename: B
authors.initials: B
authors.lastname: Bauer
authors.forename: R H
authors.initials: RH
authors.lastname: Cool
authors.forename: J H
authors.initials: JH
authors.lastname: Camonis
authors.forename: J W
authors.initials: JW
authors.lastname: Akkerman
authors.forename: J L
authors.initials: JL
authors.lastname: Bos
mesh_terms: D018528:ATP-Binding Cassette Transporters; D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D011464:Epoprostenol / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D011485:Protein Binding; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D020725:ral GTP-Binding Proteins; D020668:rap GTP-Binding Proteins
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D010446:Peptide Fragments; C095637:RALBP1 protein, human; D006160:Guanosine Triphosphate; D011464:Epoprostenol; D013917:Thrombin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020725:ral GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; D002118:Calcium