Synaptotagmin-like protein 1 interacts with the GTPase-activating protein Rap1GAP2 and regulates dense granule secretion in platelets

Abstract

The small guanine-nucleotide-binding protein Rap1 plays a key role in platelet aggregation and hemostasis, and we recently identified Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. In search of Rap1GAP2-associated proteins, we performed yeast-2-hybrid screening and found synaptotagmin-like protein 1 (Slp1) as a new binding partner. We confirmed the interaction of Rap1GAP2 and Slp1 in transfected COS-1 and HeLa cells and at endogenous level in human platelets. Mapping studies showed that Rap1GAP2 binds through amino acids T524-K525-X-T527 within its C-terminus to the C2A domain of Slp1. IntroductionBlood platelets are essential for hemostasis and play an important role in the development of thrombosis in the vasculature. 1 During primary hemostasis, platelets adhere to sites of endothelial damage and the initial platelet coat is soon reinforced by additional platelets forming a stable aggregate. At the same time, platelets secrete their intracellular granules containing substances that further activate platelets in an autocrine loop and affect local coagulation and endothelial and smooth muscle cell functions. 2 Two main types of secretory granules have been described in platelets, dense granules, and ␣-granules. Dense granules contain small molecules, including adenosine diphosphate, adenosine triphosphate, and 5-hydroxytryptamine (5-HT, serotonin), whereas ␣-granules store many proteins, such as fibrinogen, von Willebrand factor, various cytokines, and growth factors. 3,4 Dense granule secretion is regulated by the small GTPase Rab27 that is localized at the granule membrane. 5 Rab27 probably mediates its action by promoting granule motility or by enhancing tethering and fusion of granules with the plasma membrane. 6,7 For these functions, Rab27 needs to interact with effector proteins, and Munc13-4 was identified as the first Rab27-binding protein in platelets. 8 Other proteins involved in dense granule secretion are the small GTPase Ral 9 and membrane proteins of the soluble NSF attachment protein receptor family either bound to the granule, such as 10 or to the plasma membrane, such as SNAP-23 and syntaxin 2. 11 Granule secretion is accompanied by the development of tight cellular interactions between platelets. This aggregation of platelets is mediated by integrins and other adhesion receptors. 2 Integrin activation is tightly regulated by the small guanine-nucleotidebinding protein Rap1, and deletion of the platelet isoform Rap1b in mouse platelets results in a bleeding phenotype. 12 Rap1 activity is controlled by GTPase-activating proteins (GAPs) and guaninenucleotide exchange factors (GEFs). We recently discovered Rap1GAP2 as the only GTPase-activating protein of Rap1 in platelets. 13 Rap1GAP2 contains a conserved central catalytic GAP domain, an N-terminal 14-3-3-binding site, and a large C-terminal region of unknown function. Platelet activation results in phosphorylation of Rap1GAP2 on serine 9, binding of 14-3-3 protein, and probably inhibition of GAP function. 14 In parallel, ...

Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion

“…Several other C2-domain-containing proteins have also been detected in platelets ( i.e. , Slp4, DOC2, and synaptotagmin (28, 29, 66)), thus there are potential Ca 2+ sensors which could affect secretion. It is interesting to note that many proteins with 2 C2 domains show distinct roles for each.…”

“…phosphatidylserine, PS) (see (27)). Though synaptotagmin-like proteins (Slp1, 4) have been found in platelets (28, 29), a mechanistic understanding of their roles is incomplete. Munc13-4, also contains two C2 domains and appears to play a central role in Ca 2+ -triggered secretion from a number of cells of the hematopoietic lineage (24, 26, 30–33).…”

The nuts and bolts of the platelet release reaction

“…Using a yeast two-hybrid assay, Smolenski and colleagues showed that SLP1 forms a trimeric complex with Rap1, a ras-like GTPase, and the Rap nucleotide exchange factor RAP1GEF2. SLP1 had a negative effect on dense granule release from permeabilized platelets while RAP1GEF2 addition increased release [75]. The same group showed that SLP4/granuphilin interacts with Rab8 in human platelets and its addition to permeabilized platelets enhanced dense granule release [76].…”

MEIS1-mediated transactivation of synaptotagmin-like 1 promotes CXCL12/CXCR4 signaling and leukemogenesis

“…Sytl1 belongs to the synaptotagmin-like or Slp gene family, which encodes a Rab27-binding protein with a 58-kDa molecular weight (39). In association with Rab27, SYTL1 family proteins facilitate transportation and secretion of cytoplasmic granules, such as platelet granules and zymogen granules, to cellular surfaces and transportation of membrane proteins such as receptor tyrosine kinases (24,25,40).…”