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

Mol Pharmacol. 2012 Dec;82(6):1136-49. doi: 10.1124/mol.112.080507. Epub 2012 Sep 7.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

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

Substances

  • G-protein Beta gamma
  • GTP-Binding Protein beta Subunits
  • GTP-Binding Protein gamma Subunits
  • Peptides
  • Recombinant Fusion Proteins
  • SNARE Proteins
  • Synaptosomal-Associated Protein 25
  • Synaptotagmin I
  • Syntaxin 1
  • Botulinum Toxins
  • Alanine
  • Calcium
  • botulinum toxin type E