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 Ca(2+), 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 Ca(2+) 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 Ca(2+).