Gβγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.

Wells CA, Zurawski Z, Betke KM, Yim YY, Hyde K, Rodriguez S, Alford S, Hamm HE
Mol Pharmacol. 2012 82 (6): 1136-49

PMID: 22962332 · PMCID: PMC3502621 · DOI:10.1124/mol.112.080507

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.

MeSH Terms (22)

Alanine Animals Binding Sites Botulinum Toxins Calcium Cell Line Exocytosis GTP-Binding Protein beta Subunits GTP-Binding Protein gamma Subunits Lampreys Mutation Neurons Peptides Protein Binding Protein Interaction Domains and Motifs Recombinant Fusion Proteins Sf9 Cells SNARE Proteins Spodoptera Synaptosomal-Associated Protein 25 Synaptotagmin I Syntaxin 1

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