Binding of the brain G protein G⍺_o to its potential effector RASA3 is promoted by Ca²

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 binding of RASA3 to G⍺_o-GDP. A C-terminal fragment of RASA3 containing a predicted Ca²⁺ site was sufficient to bind G⍺_o, albeit more weakly and without a preference for the activated state of G⍺_o. 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 and a second on the RASA3 C terminus that binds inactive G⍺_o in the presence of Ca²⁺.