RT Journal Article
SR Electronic
T1 Gβγ Inhibits Exocytosis Via Interaction with Critical Residues on SNAP-25
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.112.080507
DO 10.1124/mol.112.080507
A1 Christopher Alexander Wells
A1 Zack P. Zurawski
A1 Katherine M. Betke
A1 Yun Young Yim
A1 Karren Hyde
A1 Shelagh Rodriguez
A1 Simon Alford
A1 Heidi E. Hamm
YR 2012
UL http://molpharm.aspetjournals.org/content/early/2012/09/07/mol.112.080507.abstract
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 SNARE proteins SNAP-25, syntaxin 1A, and synaptobrevin. Gβγ binding to ternary SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors) overlaps with synaptotagmin’s calcium-dependent binding, 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 193-206, significantly inhibited the interaction. Additionally, 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 8 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 BoNT/E-treated neurons. However, the mutant SNAP-25 could no longer support 5-HT-mediated inhibition of exocytosis.