RASA3 identified as a potential effector of the brain G protein G⍺o by mass spectrometry

ABSTRACT: G⍺o, the alpha subunit of the most abundant heterotrimeric G protein in the brain, mediates signaling by opioids and by many neuromodulators to inhibit neural function. An open question is whether activated G⍺o-GTP directly binds to and regulates effector molecules, like all other animal G⍺ proteins, or if it signals solely by releasing Gβ𝛾 subunits. Using mouse brain lysates as native source of G⍺o and its potential effectors, we analyzed immunopurified G⍺o protein complexes by mass spectrometry. Pre-activating G⍺o in the lysates with GTP𝛾S resulted in a ~6 fold increase in the amount of the small G protein GTPase activators RASA3 and RASA2 in the purified complexes, the largest increase among all G⍺o-associated proteins, making RASA2/3 candidate G⍺o effectors. Using purified recombinant proteins, we found that RASA3 binds directly to G⍺o-GTP𝛾S more strongly than it does to G⍺o-GDP. We also found that the addition of Ca2+, a second messenger produced by the G⍺q pathway that opposes G⍺o signaling, strengthened RASA3-G⍺o binding. A C-terminal fragment of RASA3 containing a predicted Ca2+ site was sufficient to bind G⍺o. Binding to G⍺q by this RASA3 fragment was strengthened by Ca2+ but, unlike full-length RASA3, showed a preference for binding G⍺o-GDP instead of G⍺o-GTP𝛾S. We present a model in which RASA3 could mediate G⍺o signaling using two distinct G⍺o-binding sites: one on full-length RASA3 that preferentially binds active G⍺o-GTP via the G⍺o switch regions, and a second on the RASA3 C-terminus that preferentially binds G⍺o-GDP in the presence of Ca2+.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: TuKiet Lam  

LAB HEAD: TuKiet Lam

PROVIDER: PXD069453 | Pride | 2026-01-23

REPOSITORIES: Pride