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To investigate the role of subcellular localization in regulating the specificity of G protein βγ signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize βγ dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to β and a carboxyl-terminal yellow fluorescent protein fragment to γ. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on βγ assembly in that it was not obtained using β₂ and γ₁, which do not form a functional dimer. In addition to assembly, BiFC βγ complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent β and γ subunits and by their abilities to potentiate activation of adenylyl cyclase by α_s in COS-7 cells. To …