Submitted May 1, 2008
Accepted June 1, 2009
Synaptotagmin-like protein 1 interacts with the GTPase-activating protein Rap1GAP2 and regulates dense granule secretion in platelets
Olga Neumuller, Meike Hoffmeister, Jan Babica, Carola Prelle, Kristina Gegenbauer, and Albert P. Smolenski*
Institute of Biochemistry II, University of Frankfurt Medical School, Frankfurt, Germany
UCD Conway Institute, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland
* Corresponding author; email: albert.smolenski{at}ucd.ie.
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-two-hybrid screening and found synaptotagmin-like protein 1 (Slp1) as 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 GTPgammaS. 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.
