Rapid purification of brain protein complexes containing active and inactive forms of the G protein Gαo.

Abstract

Gαo is the alpha subunit of the most abundant heterotrimeric G protein of the brain and relays signals from G protein-coupled receptors to inhibit neural function. To date, no direct downstream effectors for Gαo have been well-characterized. Active Gα-GTP proteins should form stable complexes with their effectors, but identifying Gαo effectors by isolating such complexes is a challenge since the vast majority of Gαo in the brain is in its inactive, GDP-bound form. In this study, we developed methods to isolate microgram quantities of native Gαo-GTP protein complexes by immunoprecipitation from brain lysates. We found that native Gαo protein in crude detergent lysates of mouse brain rapidly binds and is activated by the slowly-hydrolyzable GTP analog GTPγS. Using size exclusion chromatography and tracking the Gαo-containing complexes by western blotting, we found that native Gαo in crude brain lysates exists in complexes that change size upon activation by GTPγS. We also identified a monoclonal antibody that can efficiently immunoprecipitate Gαo protein complexes from mouse brain lysates for downstream applications such as mass spectrometry and protein-protein interaction assays. Our results and methods enable further research into the Gαo signaling pathway.