PT  - JOURNAL ARTICLE
AU  - Christopher A. Wells
AU  - Zack Zurawski
AU  - Katherine M. Betke
AU  - Yun Young Yim
AU  - Karren Hyde
AU  - Shelagh Rodriguez
AU  - Simon Alford
AU  - Heidi E. Hamm
TI  - Gβγ Inhibits Exocytosis via Interaction with Critical Residues on Soluble <em>N</em>-Ethylmaleimide-Sensitive Factor Attachment Protein-25
AID  - 10.1124/mol.112.080507
DP  - 2012 Dec 01
TA  - Molecular Pharmacology
PG  - 1136--1149
VI  - 82
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/82/6/1136.short
4100  - http://molpharm.aspetjournals.org/content/82/6/1136.full
SO  - Mol Pharmacol2012 Dec 01; 82
AB  - Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein βγ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Gβγ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gβγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gβγ binding. Peptides that bound Gβγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gβγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gβγ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to Gβ1γ1. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gβγ, but it still interacted with synaptotagmin-1 in a Ca2+-dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.