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.
Footnotes
This work was supported by the National Institutes of Health National Eye Institute [R01-EY010291]; the National Institutes of Health National Center for Research Resources [UL1-RR024975-01]; and the National Institutes of Health National Heart, Lung, and Blood Institute [T32-HL007411].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- GPCR
- G-protein-coupled receptor
- SNARE
- soluble N-ethylmaleimide-sensitive factor attachment protein receptor
- SNAP
- soluble N-ethylmaleimide-sensitive factor attachment protein
- BoNT/A
- botulinum neurotoxin A
- t-SNARE
- target SNARE
- GST
- glutathione transferase
- TBS
- Tris-buffered saline
- RT
- room temperature
- TFA
- trifluoroacetic acid
- HPLC
- high-performance liquid chromatography
- MIANS
- 2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid
- BoNT/E
- botulinum neurotoxin E
- 5-HT
- 5-hydroxytryptamine
- CI
- confidence interval
- EPSC
- excitatory postsynaptic current
- PDB
- Protein Data Bank.
- Received June 8, 2012.
- Accepted September 7, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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