The research at the Synaptic Function Unit (SFU) focuses on the elucidation of molecular mechanisms underlying synaptic vesicle exocytosis and its modulation. Current projects include molecular identification and biochemical and physiological characterization of new components of the neurotransmitter release machinery. In past year, we have obtained promising results on the following four projects: (1) Second-messenger regulation of neurotransmitter release via modulation of protein interactions with the exocytotic apparatus has a potentially important role in modulation of neurotransmission and in synaptic plasticity. We have found that Snapin may be a target for cAMP-dependent protein kinase A (PKA) in the neurotransmitter secretory machinery. We found that both recombinant and native Snapin derived from rat brain synaptosomes are phosphorylated by PKA. Deletion mutation and PCR-based site-directed mutagenetic experiments pinpoint the phosphorylation site to serine-50. PKA-phosphorylation of Snapin significantly increases its binding to SNAP-25. A Snapin mutation of serine-50 to aspartic acid (S50D) mimics the effect of PKA phosphorylation of Snapin, suggesting that the introduction of a negatively charged residue at serine-50 is critical to regulate Snapin function. Furthermore, the Snapin S50D mutant enhances the association of synaptotagmin with the SNARE complex. In adrenal chromaffin cells, overexpression of the Snapin S50D mutant leads to an increase in the number of release-competent vesicles. Our results suggest that Snapin is a PKA target for modulating transmitter release and neuronal plasticity via the cAMP-dependent signal transduction pathway. The paper describing our work on Snapin phosphorylation was published at Nature Cell Biology (3, 331-338, 2001). (2) Synaptic vesicle exocytosis is mediated by the assembly of a stable SNARE complex consisting of VAMP, syntaxin, and SNAP-25. Following fusion, synaptic vesicles are recycled by clathrin-dependent endocytosis in a process mediated by a complex of dynamin and amphiphysin. A strict coupling between exo- and endocytotic events is essential at nerve terminals because of the very high rates of vesicle turnover. Even a slight imbalance between of the two processes soon leads to expansion (or shrinkage) of the terminal plasma membrane and to an altered size of the vesicle pool. Identifying proteins that modulate the coupling of exo- and endocytotic processes is critical to elucidating the molecular mechanisms that control neurotransmitter release. We have characterized a functionally significant interaction between syntaphilin, a protein cloned in our lab and known to control SNARE assembly via its binding with syntaxin (Neuron 25, 191-201, 2000), and dynamin. Dynamin was co-immunoprecipitated with syntaphilin in brain homogenate and lysate from transfected HEK 293 cells. Using in vitro biochemical assays, we found that syntaphilin competes with amphiphysin for binding to dynamin and thereby inhibits the formation of the minimal machinery complex required for synaptic vesicle endocytosis. Overexpression of syntaphilin prevents clathrin-mediated endocytosis (uptake) of transferin in cultured COS cells. As shown previously, overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Given its inhibitory effect on synaptic transmission, we suggest that syntaphilin may function as a molecular clamp of both syntaxin and dynamin, thereby acting as a general modulator which couples synaptic vesicle release and recycling. (3) Using the yeast two-hybrid system we have isolated another syntaxin-1A binding protein from the human brain cDNA library. The deduced amino acid sequence is identical to a recently cloned gene called SNAP-29. Synaptosomal fractionation and immunocytochemical staining of hippocampal neurons in culture showed that SNAP-29 is present at synapses and is associated with synaptic vesicles. The interaction of SNAP-29 with syntaxin-1 was further confirmed with immunoprecipitation analysis. Binding competition studies with SNAP-29 demonstrated that it could compete with a-SNAP for binding to synaptic SNAREs and consequently inhibit disassembly of the SNARE complex. Introduction of SNAP-29 into presynaptic superior cervical ganglion neurons (SCGNs) in culture significantly inhibited synaptic transmission in an activity-dependent manner. Although SNAP-29 has been suggested to be a general SNARE component in membrane trafficking, our findings suggest that it may function as a regulator of SNARE complex disassembly and modulate the process of postfusion recycling of the SNARE components. The manuscript describing our work on SNAP-29 was submitted to PNAS and is under revision. (4) Using the yeast two-hybrid selection approach with syntaxin-1A as a bait, we have isolated a cDNA encoding DAP-kinase (Death Associated Protein Kinase), an uncharacterized calcium/calmodulin-regulated protein kinase. Our immunoblotting analysis showed that DAP kinase was found highly enriched in neurons and was localized in the cytosolic fraction of synaptosomes. Association between DAP kinase and syntaxin-1A has been confirmed using in vitro binding assays, coimmunoprecipitation, and pull-down experiments. Recombinant syntaxin-1A could be phosphorylated by DAP kinase both in vitro and in vivo in a calcium/calmodlin-regulated manner. Biochemical and functional significance of this phosphorylation event is currently under evaluation. We believe that the continued application of our current approaches, in combination with newer somatic and germ-cell genetic studies, will allow us to identify SNARE complex regulatory pathways and to uncover additional protein-protein interactions within the synaptic vesicle docking and fusion machinery, thereby allowing us to further elucidate the molecular mechanisms underlying neurotransmitter release and synaptic plasticity.
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