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

## TL;DR

This study shows that the brain G protein G⍺o binds to RASA3, a potential effector, and that this binding is enhanced by Ca2+.

## Contribution

The paper identifies RASA3 as a direct effector of G⍺o and reveals a novel Ca2+-dependent binding mechanism.

## Key findings

- Pre-activation of G⍺o with GTPγS increases RASA3 and RASA2 in G⍺o complexes by ∼6-fold.
- RASA3 binds more strongly to G⍺o-GTPγS than to G⍺o-GDP.
- Ca2+ enhances RASA3 binding to G⍺o-GDP and the C-terminal fragment of RASA3 binds G⍺o in a Ca2+-dependent manner.

## 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 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+.