2002. Real-time visualization of a fluorescent Gs: dissociation of the activated G protein from plasma membrane. Mol. Pharmacol. 61:352–359. o n

To study behavior of activated Gs in living cells, green fluo-rescent protein (GFP) was inserted within the internal amino acid sequence of Gs to generate a Gs-GFP fusion protein. The fusion protein maintained a bright green fluorescence and was identified by immunoblotting with antibodies against Gs or GFP. The cellular distribution of Gs-GFP was similar to that of endogenous Gs. Gs-GFP was tightly coupled to the adrenergic receptor to activate the Gs effector, adenylyl cy-clase. Activation of Gs-GFP by cholera toxin caused a gradual displacement of the fusion protein from the plasma membrane throughout the cytoplasm in living cells. Unlike the slow release of Gs-GFP from the membrane induced by cholera toxin, the -adrenergic agonist isoproterenol caused a rapid partial re-lease of the fusion protein into the cytoplasm. At 1 min afte