Real-time visualization of a fluorescent G(alpha)(s): dissociation of the activated G protein from plasma membrane.

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