Assays for G-Protein-Coupled Receptor Signaling Using RGS-Insensitive Gα Subunits

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