The specificity of Gβγ subunits regulating exocytosis through the Gi/o‐coupled α2a adrenergic receptor

Abstract

Modulation of neurotransmitter exocytosis by activated Gi/o‐type G‐protein coupled receptors (GPCRs), such as the α2a adrenergic receptor (α2a‐ARs), is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. Gβγ and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) interaction is known as one of mechanisms that inhibit neurotransmitter release. There are 5 Gβ and 12 Gγ subunits, but protein level of each Gβs and Gγs in brain is not known. It is also not known whether specific Gβγs are activated by a given GPCR in vivo. However, presynaptic α2a‐ARs in both adrenergic (auto α2a‐ARs) and non‐adrenergic neurons (hetero α2a‐ARs) work in a similar manner to inhibit neurotransmitter release and have various physiological functions such as anesthetic sparing and working memory enhancement. In this project, we examine the protein level of each Gβ and Gγ in whole brain synaptosomes and in fractions of synaptosomes. In whole brain synaptosome, we have identified Gβ1 and Gγ2 as dominant subunits present. Although all Gβs and Gγs are highly present in cytosolic fractions, Gγs show interesting changes in protein expression patterns by fractions. We also investigate whether autoreceptors in sympathetic neurons use the same Gβγ subunits as heteroreceptors in other neuronal types. Using several mice models including transgenic Flag‐α2a‐ARs, knock‐in HA‐α2a‐ARs, and wild‐type mice, co‐immunoprecipitation, mass spectrometry analysis, we test our hypothesize that specific Gβγ subunits interact with activated auto‐ and hetero α2a‐ARs and inhibit exocytosis by interacting with SNARE. We have identified Gβ1, Gβ2, Gβ4, Gβ5, Gγ2, Gγ3, Gγ4, Gγ7, Gγ12, and Gγ13 which interact with α2a‐ARs and SNARE. Out of these G proteins, we found Gβ2 and Gγ13 preferentially interacting with activated auto α2a‐ARs. However, we found no difference in Gβγ specificity to SNARE following α2a‐ARs activation. Further understanding Gβγ specificity may offer new insights into the normal functioning of the brain, as well as better understanding of disease progression. The Gβγ‐SNARE interaction may be a new therapeutic target to modulate exocytosis in neural disorders in combination with drugs targeted to Gi/o‐type GPCRs.Support or Funding InformationThis work was supported by the National Institutes of Health (EY10291, MH101679, and T32).