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    The molecular requirements of the G- protein betagamma-SNARE interaction.

    Zurawski, Zack P.
    : https://etd.library.vanderbilt.edu/etd-03252016-105615
    http://hdl.handle.net/1803/11340
    : 2016-04-08

    Abstract

    Gi/o-coupled G-protein coupled receptors (GPCRs) can exert an inhibitory effect on vesicle release through several G-protein driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. G protein betagamma subunits inhibit exocytosis via directly binding to the synaptosomal-associated protein of 25 kDa (SNAP25), competing with the fusogenic calcium sensor synaptotagmin 1 (Syt1) in a calcium-dependent manner for binding sites on SNAP25. Here, we generate several SNAP25 C-terminal mutants that are deficient in G protein betagamma binding while retaining normal vesicle release. The SNAP25-8A mutant features 8 G protein betagamma-binding residues mutated to Ala, and the SNAP25delta3 mutant, in which residue G204 is replaced by a stop codon, both feature a partial reduction in G protein beta1gamma2 binding in vitro. SNAP25-8A exhibits a reduction in the ability of the lamprey serotonin receptor to reduce excitatory postsynaptic current (EPSC) amplitudes, an effect previously shown to be mediated through the G protein betagamma-SNARE interaction. Syt1 binding to these mutants is largely intact. We conclude that the extreme C-terminus of SNAP25 is a critical region for the G protein betagamma-SNARE interaction. To further investigate the physiological relevance of the G protein betagamma-SNARE interaction, we have developed small molecule modulators of the G protein betagamma-SNARE interaction with micromolar potency. A transgenic mouse has been made containing the SNAP25delta3 mutation using the CRISPR-Cas9 reaction. Characterization of the phenotype of this animal is ongoing. In summary, we have identified key residues for the G protein betagamma-SNARE interaction and generated new experimental tools to investigate the importance of this interaction in tissues and disease states where its relevance is not currently known.
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