Abstract
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 α2a-adrenergic receptors (α2aARs) in both adrenergic (auto-α2aARs) and non-adrenergic neurons (hetero-α2aARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits, and co-immunoprecipitation of tagged α2aARs from mouse models including transgenic FLAG-α2aARs and knock-in HA-α2aARs, we investigated the in vivo specificity of Gβ and Gγ subunits to auto-α2aARs and hetero-α2aARs 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 α2aARs, whereas we found Gβ4 and Gγ12 preferentially interacted with activated hetero-α2aARs. 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.
Highlights
G proteins are major transducers of signals from G-protein coupled receptors (GPCRs)
To study the specificity of neuronal Gβγ subunits to synaptic α2aadrenergic receptors (α2aARs), we used brain synaptosomes from wildtype, α2aAR KO, Hemagglutinin tagged (HA)- and FLAG-α2aAR mice
Wildtype and α2a-ARs KO mice were used as controls for HA- and FLAG-α2aARs mice
Summary
G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). G-protein coupled receptors (GPCRs) are the largest and most diverse superfamily of transmembrane receptors that convey signal transduction across cell membranes, and mediate a vast array of cellular responses necessary for human physiology[1,2,3] Upon their activation, GTP-Gα and Gβγ subunits are released from the GPCR and interact with various effectors to initiate downstream signaling cascades. Given the diversity seen for the expression and affinity of Gβ and Gγ subunits, as well as the affinity of Gβγ-effector interactions, it is likely that specific dimers could permit specialized roles in signal transduction pathways through association with particular GPCRs. Despite many attempts to understand G protein βγ specificity for particular GPCRs, much remains unclear due to a lack of specific antibodies or other methods of confidently assaying such preferences. As the interaction Gβγ dimers with particular GPCRs in the CNS may determine their role in regulating synaptic transmission, or their impact in neurological disease and GPCR targeted drug mechanism, further elucidation of G protein specificities in vivo is necessary
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