Real-time visualization of a fluorescent GαS:: Dissociation of the activated G protein from plasma membrane

被引:87
作者
Yu, JZ
Rasenick, MM
机构
[1] Univ Illinois, Coll Med, Dept Physiol & Biophys, Chicago, IL 60612 USA
[2] Univ Illinois, Coll Med, Dept Psychiat, Chicago, IL 60612 USA
关键词
D O I
10.1124/mol.61.2.352
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
To study behavior of activated Galpha(s) in living cells, green fluorescent protein (GFP) was inserted within the internal amino acid sequence of Galpha(s) to generate a Galpha(s)-GFP fusion protein. The fusion protein maintained a bright green fluorescence and was identified by immunoblotting with antibodies against Galpha(s) or GFP. The cellular distribution of Galpha(s)-GFP was similar to that of endogenous Galpha(s). Galpha(s)-GFP was tightly coupled to the beta adrenergic receptor to activate the Galpha(s) effector, adenylyl cyclase. Activation of Galpha(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 Galpha(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 Galpha(s)-GFP release from plasma membrane sites was maximal; however, insertion of Galpha(s)-GFP at other membrane sites occurred during the same time period. Translocation of Galpha(s)-GFP fusion protein induced by isoproterenol suggested that the internalization of Galpha(s) might play a role in signal transduction by interacting with effector molecules and cytoskeletal elements at multiple cellular sites.
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页码:352 / 359
页数:8
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