title: 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. Slp1 contains a Rab27-binding domain, and we demonstrate that Rap1GAP2, Slp1, and Rab27 form a trimeric complex in transfected cells and in platelets. Purified Slp1 dose-dependently decreased dense granule secretion in streptolysin-O-permeabilized platelets stimulated with calcium or guanosine 5'-O-[gamma-thio] triphosphate. The isolated C2A domain of Slp1 had a stimulatory effect on granule secretion and reversed the inhibitory effect of full-length Slp1. Purified Rap1GAP2 augmented dense granule secretion of permeabilized platelets, whereas deletion of the Slp1-binding TKXT motif abolished the effect of Rap1GAP2. We conclude that Slp1 inhibits dense granule secretion in platelets and that Rap1GAP2 modulates secretion by binding to Slp1.
authors.affiliation: Institute of Biochemistry II, University of Frankfurt Medical School, Frankfurt, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Neumüller
authors.forename: Meike
authors.initials: M
authors.lastname: Hoffmeister
authors.forename: Jan
authors.initials: J
authors.lastname: Babica
authors.forename: Carola
authors.initials: C
authors.lastname: Prelle
authors.forename: Kristina
authors.initials: K
authors.lastname: Gegenbauer
authors.forename: Albert P
authors.initials: AP
authors.lastname: Smolenski
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C500320:RAP1GAP2 protein, human; C431146:SYTL1 protein, human; D033921:Vesicular Transport Proteins; D020827:rap1 GTP-Binding Proteins