Assays for G-protein-coupled receptor signaling using RGS-insensitive Galpha subunits

Regulators of G-protein signaling (RGS) proteins, by their action on Galpha(i/o) proteins, may enhance receptor-effector signaling by physical or kinetic scaffolding mechanisms. However, more than 30 mammalian proteins with RGS activity have been identified so it is difficult to determine which RGS protein is most relevant to a particular receptor system and in any particular cell. To avoid this problem, one approach is to examine agonist-stimulated second messenger signaling in cells expressing Galpha proteins that are insensitive to the GTPase accelerating property of all RGS proteins. This article describes protocols for the preparation and analysis of C6 rat glioma cells stably expressing RGS- and pertussis toxin-insensitive Galpha subunits; pertussis toxin treatment uncouples endogenous Galpha(i/o) proteins and allows for the determination of the expressed RGS-insensitive Galpha activity. Methods to determine signaling at the level of adenylyl cyclase, the extracellular signal-regulated kinase (ERK1/2) mitogen-activated protein kinase pathway, and intracellular Ca2+ levels are described. As a typical G-protein-coupled receptor, we have used the micro-opioid receptor expressed in C6 cells together with RGS-insensitive Galpha(o). In these cells, agonist inhibition of adenylyl cyclase and stimulation of ERK1/2 phosphorylation were enhanced markedly. In contrast, increases in intracellular calcium were less affected. The altered signaling in cells expressing RGS-insensitive Galpha(o) subunits allows for determination of the role of endogenous RGS proteins to limit and/or direct signaling.