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Research ArticleArticle

Gβγ Inhibits Exocytosis via Interaction with Critical Residues on Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein-25

Christopher A. Wells, Zack Zurawski, Katherine M. Betke, Yun Young Yim, Karren Hyde, Shelagh Rodriguez, Simon Alford and Heidi E. Hamm
Molecular Pharmacology December 2012, 82 (6) 1136-1149; DOI: https://doi.org/10.1124/mol.112.080507
Christopher A. Wells
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (C.A.W., Z.Z., K.M.B., Y.Y.Y., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois (S.R., S.A.)
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Zack Zurawski
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (C.A.W., Z.Z., K.M.B., Y.Y.Y., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois (S.R., S.A.)
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Katherine M. Betke
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (C.A.W., Z.Z., K.M.B., Y.Y.Y., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois (S.R., S.A.)
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Yun Young Yim
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (C.A.W., Z.Z., K.M.B., Y.Y.Y., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois (S.R., S.A.)
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Karren Hyde
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (C.A.W., Z.Z., K.M.B., Y.Y.Y., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois (S.R., S.A.)
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Shelagh Rodriguez
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (C.A.W., Z.Z., K.M.B., Y.Y.Y., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois (S.R., S.A.)
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Simon Alford
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (C.A.W., Z.Z., K.M.B., Y.Y.Y., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois (S.R., S.A.)
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Heidi E. Hamm
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (C.A.W., Z.Z., K.M.B., Y.Y.Y., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois (S.R., S.A.)
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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.

    http://dx.doi.org/10.1124/mol.112.080507.

  • 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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Molecular Pharmacology: 82 (6)
Molecular Pharmacology
Vol. 82, Issue 6
1 Dec 2012
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Research ArticleArticle

Gβγ Inhibits Exocytosis via Interaction with SNAP-25

Christopher A. Wells, Zack Zurawski, Katherine M. Betke, Yun Young Yim, Karren Hyde, Shelagh Rodriguez, Simon Alford and Heidi E. Hamm
Molecular Pharmacology December 1, 2012, 82 (6) 1136-1149; DOI: https://doi.org/10.1124/mol.112.080507

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Research ArticleArticle

Gβγ Inhibits Exocytosis via Interaction with SNAP-25

Christopher A. Wells, Zack Zurawski, Katherine M. Betke, Yun Young Yim, Karren Hyde, Shelagh Rodriguez, Simon Alford and Heidi E. Hamm
Molecular Pharmacology December 1, 2012, 82 (6) 1136-1149; DOI: https://doi.org/10.1124/mol.112.080507
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