Gßγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.
Mol Pharmacol
; 82(6): 1136-49, 2012 Dec.
Article
em En
| MEDLINE
| ID: mdl-22962332
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 ßγ 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. Gßγ 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 Gßγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gßγ binding. Peptides that bound Gßγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gßγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gßγ 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 Gß1γ1. 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 Gßγ, but it still interacted with synaptotagmin-1 in a Ca²âº -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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Subunidades beta da Proteína de Ligação ao GTP
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Subunidades gama da Proteína de Ligação ao GTP
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Proteína 25 Associada a Sinaptossoma
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Exocitose
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article