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.
Yu
Jiang-Zhou
JZ
Department of Physiology and Biophysics, University of Illinois at Chicago, College of Medicine, Chicago, Illinois 60612-7342, USA.
Rasenick
Mark M
MM
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D003199:Computer Systems; D004789:Enzyme Activation; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D008223:Lymphoma; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D007545:Isoproterenol