title: Gβγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.
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.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-6600, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Wells
authors.forename: Zack
authors.initials: Z
authors.lastname: Zurawski
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Betke
authors.forename: Yun Young
authors.initials: YY
authors.lastname: Yim
authors.forename: Karren
authors.initials: K
authors.lastname: Hyde
authors.forename: Shelagh
authors.initials: S
authors.lastname: Rodriguez
authors.forename: Simon
authors.initials: S
authors.lastname: Alford
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Hamm
mesh_terms: D000409:Alanine / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005089:Exocytosis / Q000502:physiology*; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D007798:Lampreys; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism
chemical_list: C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D010455:Peptides; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050827:Syntaxin 1; D001905:Botulinum Toxins; D000409:Alanine; D002118:Calcium; C096326:botulinum toxin type E