The neural G protein Gαo tagged with GFP at an internal loop is functional in Caenorhabditis elegans

Abstract

Gao is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gao remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gao signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here, we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gao protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gao. Transgenic expression of Gao-GFP rescues the defects caused by loss of endogenous Gao in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gao are also rescued by Gao-GFP. The Gao-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gao. Using GFP as an epitope tag, Gao-GFP can be immunoprecipitated from C. elegans lysates to purify Gao protein complexes. The Gao-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gao to compliment the already well-developed genetic analysis of Gao signaling.