The in vivo specificity of synaptic Gβ and Gγ subunits to the α_2a adrenergic receptor at CNS synapses

G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α, β, and γ subunits, with 16 Gα, 5 Gβ and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gβγs activated by a given GPCR and that activate each effector in vivo is not known. Here, we examined the in vivo Gβγ specificity of presynaptic α_2a-adrenergic receptors (α_2aARs) in both adrenergic (auto-α_2aARs) and non-adrenergic neurons (hetero-α_2aARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits, and co-immunoprecipitation of tagged α_2aARs from mouse models including transgenic FLAG-α_2aARs and knock-in HA-α_2aARs, we investigated the in vivo specificity of Gβ and Gγ subunits to auto-α_2aARs and hetero-α_2aARs activated with epinephrine to understand the role of Gβγ specificity in diverse physiological functions such as anesthetic sparing, and working memory enhancement. We detected Gβ₂, Gγ₂, Gγ₃, and Gγ₄ with activated auto α_2aARs, whereas we found Gβ₄ and Gγ₁₂ preferentially interacted with activated hetero-α_2aARs. Further understanding of in vivo Gβγ specificity to various GPCRs offers new insights into the multiplicity of genes for Gβ and Gγ, and the mechanisms underlying GPCR signaling through Gβγ subunits.