Journal of Biological Chemistry

Neurotransmitter release is a complex process involving tightly controlled co-factors and protein-protein interactions. G-protein coupled receptors negatively regulate exocytosis via the interaction of G-protein βγ (Gβγ) heterodimers with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. The neuronal ternary SNARE complex comprises synaptosomal-associated protein-25 (SNAP25), syntaxin-1A, and synaptobrevin-2. The regions of the SNARE complex that are important for interactions with Gβγ have been extensively characterized, but the critical sites on Gβγ are not well understood. Furthermore, the molecular basis for the specificity of different Gβ and Gγ isoforms for SNARE proteins remains elusive. Thus, we holistically probed the entire family of human Gβ and Gγ isoforms for regions critical for the target-SNARE (tSNARE) interaction using a peptide screening approach. Gβ and γ peptides with high affinities for tSNARE were then subjected to alanine scanning mutagenesis to identify the interaction sites. We found that the N-terminal coiled-coil domain of Gβγ as well as the β-propeller domain of Gβ are hotspots for SNARE interactions. Additionally, we found that the N-terminal Gγ2 peptide is a potent inhibitor of interactions between full-length Gβ1γ2 and SNAP25. We discovered that Gβ1γ2 preferentially interacts with ternary SNARE in the pre-fusion, partially zipped conformation, likely due to increased exposure of the C-terminus of SNAP25. Our combined results suggest that specific Gβγ heterodimers bind to ternary SNARE in the docked and primed state via critical residues of the β-propeller and N-terminal coil-coil domains. We propose that Gβγ binding disrupts zippering up of the SNARE complex and thereby vesicle fusion.

G-protein

Gβγ subunit

SNARE protein

SNAP25

exocytosis, protein-protein interaction

neurotransmission

signal transduction

membrane fusion

GPCR signaling

protein structure

α2a adrenergic receptor

excitatory post-synaptic currents

G-protein coupled inward-rectifying potassium

G-protein coupled receptor

G-protein coupled receptor kinase-2

phospholipase C beta

soluble NSF attachment protein receptor

vesicle associated membrane protein 2

voltage-gated calcium channels

© 2025 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biologyé