Specificity of Gβ and γ subunits to SNARE complex both at rest and after α2aadrenergic receptor stimulation

G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α,β,and γsubunits, with 16 Gα, 5 Gβ and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gβγ by a given GPCR and those that activate each effector in vivo is not clear. In a previous paper, we were able to identify Gβ and Gγ interacting specifically neuronal α2a-adrenergic receptors (α2aARs). However, it still remains unclear how G protein specificity plays out in α2aAR-mediated effector interactions. This receptor is the major autoreceptor that acts as a brake to synaptic transmission in adrenergic neurons and the sympathetic nervous system, and as heteroreceptors on other neurons throughout the brain. Here, we examined the in vivo specificity of Gβγ to the soluble NSF attachment proteins (SNARE) complex upon presynaptic α2aAR activation in both adrenergic (auto-α2aARs) and non-adrenergic (heteroreceptor) neurons for the first time. To understand how this interaction underlies inhibition of synaptic transmission in diverse physiological functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement, we applied a quantitative MRM proteomic analysis of Gβ and Gγ subunits co-immunoprecipitation from transgenic FLAG-α2aARs and wildtype mice. We evaluated Gβ and Gγ subunit binding to the SNARE complex with and without activation of auto-α2aAR and hetero-α2aAR using epinephrine. Without epinephrine stimulation, Gβ 1 and Gγ3 interact with SNARE. When auto-α2aArs are activated, Gβ1, Gβ2, and Gγ3 interact with SNARE. Further understanding of in vivo Gβγ specificity to various effectors offers new insights into the multiplicity of genes for Gβ and Gγ, and the mechanisms underlying GPCR signaling through Gβγ subunits to this interaction as a potential therapeutic target.