Abstract

To study behavior of activated Gα_s in living cells, green fluorescent protein (GFP) was inserted within the internal amino acid sequence of Gα_s to generate a Gα_s-GFP fusion protein. The fusion protein maintained a bright green fluorescence and was identified by immunoblotting with antibodies against Gα_s or GFP. The cellular distribution of Gα_s-GFP was similar to that of endogenous Gα_s. Gα_s-GFP was tightly coupled to the β adrenergic receptor to activate the Gα_s effector, adenylyl cyclase. Activation of Gα_s-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 Gα_s-GFP from the membrane induced by cholera toxin, the β-adrenergic agonist isoproterenol caused a rapid partial release of the fusion protein into the cytoplasm. At 1 min after treatment with isoproterenol, the extent of Gα_s-GFP release from plasma membrane sites was maximal; however, insertion of Gα_s-GFP at other membrane sites occurred during the same time period. Translocation of Gα_s-GFP fusion protein induced by isoproterenol suggested that the internalization of Gα_s might play a role in signal transduction by interacting with effector molecules and cytoskeletal elements at multiple cellular sites.